Note: Descriptions are shown in the official language in which they were submitted.
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DISEASE PREVENTION AND VACCINATION
PRIOR TO THYMIC REACTIVATION
FIELD OF THE INVENTION
The present invention is in the fields of cellular immunology, disease
prevention,
vaccination, and gene therapy. More specifically, the invention is directed to
enhancing bone
marrow (BM) hematopoiesis and functionality, enhancing BM engraftment
following
hematopoietic stem. cell transplant (HSCT), and increasing the functionality
of new and pre-
existing T cells and other cells of the immune system.
BACKGROUND
The Immune S_ sy tem
The major function of the immune system is to distinguish "foreign" (i.e.,
derived
from any source outside the body) antigens from "self ' (i.e., derived from
within the body)
and respond accordingly to protect the body against infection. In more
practical terms, the
immune response has also been described as responding to danger signals. These
danger
signals may be any change in the property of a cell or tissue which alerts
cells of the immune
system that this cell/tissue in question is no longer "normal." Such alerts
may be very
important in causing, for example, rejection of fareign agents such as viral,
bacterial,
parasitic and fungal infections; they may also be used to induce anti-tumor
responses.
However, such danger signals may also be the reason why same autoimmune
diseases start,
due to either inappropriate cell changes in the self cells which are then
become targeted by
the immune system (e.g., the pancreatic (3-islet cells in diabetes mellitus)
Altenratively,
inappropriate stimulation of the immune cells themselves, can lead to the
destruction of
normal self cells, in addition to the foreign cell or microorganism which
induced the initial
response.
In normal immune responses, the sequence of events involves dedicated antigen
presenting cells (APC) capturing foreign antigen and processing it into small
peptide
fragments which are then presented in clefts of major histocompatibility
complex (MHC)
molecules on the APC surface. The MHC molecules can either be of class I
expressed on all
nucleated cells (recognized by cytotoxic T cells (Tc or CTL)) or of class If
expressed
primarily by cells of the immune system (recognized by helper T cells (Th)).
Th cells
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recognize the MHC IIlpeptide complexes on APC and respond. Factors released by
these
cells then promote the activation of either, or both of Tc cells or antibody
producing B cells,
which are specific for the particular antigen. The importance of Th cells in
virtually all
immune responses is best illustrated in HIVIAlDS where their absence through
destruction by
the virus causes severe immune deficiency, which eventually leads to death due
to
opportunistic infections. Inappropriate development of Th (and to a lesser
extent Tc) can also
lead to a variety of other conditions such as allergies, cancer, and
autoimmunity.
The inappropriate development of such cells may be due to an abnormal thymus
in
which the structural organization is markedly altered e.g., in many autoimmune
diseases, the
medullary epithelial cells, which are required for development of mature
thymocytes, are
ectopically expressed in the cortex where immature T cells normally reside.
This could mean
that the developing immature T cells prematurely receive late stage maturation
signals and
in doing so become insensitive to the negative selection signals that would
normally delete
potentially autoreactive cells. Indeed this type of thymic abnormality has
been found in NZB
mice, which develop Lupus-like symptoms (Takeoka et al., (1999) Clin.
Immurcol. 90:388),
and more recently in NOD mice, which develop type I diabetes (Thomas-Vaslin et
al., (1997)
P.N.A.S. USA 94:4598; Atlan-Gepner et al.,(1999) Autoitrarr2uyzity 3:249-260).
It is not known
how or when these forms of thymic abnormality develop, but it could be through
the natural
aging process or from destructive agents such as viral infections (changes in
the thymus have
been described in AIDS patients), stress, chemotherapy and radiation therapy
(Mackall et al.,
(1995) N. Ehg. J. Med. 332:143; Heitger et al., (1997) Blood 99:4053; Mackall
and Gress,
(1997) Immufzol. Rev. 160:91). It is also possible that the defects are
present at birth.
The ability to recognize antigen is encompassed in a plasma membrane receptor
in T
and B lymphocytes. These receptors are randomly generated by a complex series
of
rearrangements of many possible genes, such that each individual T or B cell
has a unique
antigen receptor. This enormous potential diversity means. that for any single
antigen the
body might encounter, multiple lymphocytes will be able to recognize it with
varying degrees
of binding strength (affinity) and elicit varying degrees of responses. Since
antigen receptor
specificity arises by chance, the problem thus arises as to why the body does
not self destruct
through lymphocytes reacting against self antigens. Fortunately there are
several
mechanisms which prevent the T and B cells from doing so. Collectively, these
mechanisms
create a situation where the immune system is tolerant to self.
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The most efficient form of self tolerance is to physically remove or kill any
potentially reactive lymphocytes at the sites where they are produced. These
sites include the
thymus for T cells and the BM for B cells. This is called central tolerance.
An important,
additional method of tolerance is through regulatory Th cells which inhibit
autoreactive cells
either directly or via the production of cytokines. Given that virtually all
immune responses
require initiation and regulation by T helper cells, a major aim of any
tolerance induction
regime would be to target these T helper cells. Similarly, since Tc's are very
important
effector cells, their production is a major aim of strategies for, e.g., anti-
cancer and anti-viral
therapy. In addition, T regulatory cells (Tregs), such as CD4+CD25+ and NKT
cells, provide
a means whereby they can suppress potentially autoreactive cells.
The Thymus
The thymus essentially consists of developing thymocytes (T lymphocytes within
the
thymus) interspersed within the diverse stromal cells (predominantly
epithelial cell subsets)
which constitute the microenvironment and provide the growth factors (GF) and
cellular
interactions necessary for the optimal development of the T cells.
The thymus is an important organ in the immune system because it is the
primary site
of production of T lymphocytes. The role of the thymus is to attract
appropriate BM-derived
precursor cells from the blood, as described below, and induce their
commitment to the T cell
lineage, including the gene rearrangements necessary for the production of the
T cell receptor
(TCR) for antigen. Each T cell has a single TCR type and is unique in its
specificity.
Associated with this TCR production is cell division, which expands the number
of T cells
with that TCR type and hence increases the likelihood that every foreign
antigen will be
recognized and eliminated. However, a unique feature of T cell recognition of
antigen is that,
unlike B cells, the TCR only recognizes peptide fragments physically
associated with MHC
molecules. Normally, this is self MHC, and the ability or a TCR to recognize
the self
MHC/peptide complex is selected for in the thymus. This process is called
positive selection
and is an exclusive feature of cortical epithelial cells. If the TCR fails to
bind to the self
MHClpeptide complexes, the T cell dies by "neglect" because the T cells needs
some degree
of signalling through the TCR for its continued survival and maturation.
Since the outcome of the TCR gene rearrangements is a random event, some T
cells
will develop which, by chance, can recognize self MHC/peptide complexes with
high
affinity. Such T cells are thus potentially self-reactive and could be
involved in autoimmune
diseases, such as multiple sclerosis (MS), rheumatoid arthritis (RA),
diabetes, thyroiditis and
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systemic lupus erythematosus (SLE). Fortunately, if the affinity of the TCR to
self
MHClpeptide complexes is too high, and the T cell encounters this specific
complex in the
thymus, the developing thymocyte is induced to undergo a suicidal activation
and dies by
apoptosis, a process called negative selection. This process is also called
central tolerance.
Such "high affinity for self' T cells die rather than respond because in the
thymus they are
still immature. The most potent inducers of this negative selection in the
thymus are APC
called dendritic cells (DC). DC deliver the strongest signal to the T cells,
which causes
deletion in the thymus. However, in the peripheral lymphoid organs where the T
cells are
more mature, the DC presenting the same MHC/peptide complex to the same TCR
would
cause activation of that T cell bearing the TCR.
Thymus Atrophy and Ale
While the thymus is fundamental for a functional immune system, releasing
about 1 %
of its T cell content into the bloodstream per day, one of the apparent
anomalies of mammals
and other animals is that this organ undergoes severe atrophy as a result of
sex steroid
production. This atrophy occurs gradually over a period of about 5-7 years,
with the nadir
level of T cell output being reached around 20 years of age (Douek et al.,
Nature (1998)
396:690-695) and is in contrast to the reversible atrophy induced during a
stress response to
corticosteroids. Structurally, the thymic atrophy involves a progressive loss
of lymphocyte
content, a collapse of the cortical epithelial network, an increase in
extracellular matrix
material, and an infiltration of the gland with fat cells (adipocytes) and
lipid deposits (Haynes
et al., (1999) J. Clirz. Izzvest. 103: 453). This process may even begin in
young children (e.g.,
around five years of age; Mackall et al., (1995) N. E>zg. J. Med. 332:143),
but it is profound
from the time of puberty when sex steroid levels reach a maximum.
The impact of thymus atrophy is reflected in the periphery, with reduced
thymic input
to the T cell pool, which results in a less diverse TCR repertoire (as this
can only be provided
by the new naive T cells). Altered cytokine profiles (Hobbs et al., (1993) J.
Izzzrzzuzzol.
150:3602; Kurashima et al., (1995) Izzt. Irnmurzol. 7:97), changes in CD4+ and
CD8+ subsets,
biases towards memory as opposed to naive T cells (Mackall et al., (1995) N.
EYZgI. J. Med.
332:143), and a reduced ability to response to antigenic or mitogenic
stimulation are also
observed.
Since the thymus is the primary site for the production and maintenance of the
peripheral T cell pool, this atrophy has been widely postulated as being the
primary cause of
the increased incidence of immune-based disorders in the elderly. In
particular, conditions,
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sucn as genera immunodeficiency, poor responsiveness to opportunistic
infections and
vaccines, and an increase in the frequency of autoinunune diseases, such as
multiple
sclerosis, rheumatoid arthritis, and lupus (Doric et al., (1997) Mecla. Age.
Dev. 95: 131-142;
Weyand et al., (1998) Mecl2. Age. Dev. 102: 131-147; Castle, (2000) Clin
Infect Dis 31(2):
578-585; Murasko et al., (2002) Exp. Gerontol. 37:427-439), increase in
incidence and
severity with age. Such deficiencies of the immune system, are often
illustrated by a
decrease in T cell dependent immune functions (e.g., cytolytic T cell activity
and mitogenic
responses). While homeostatic mechanisms maintain T cell numbers in healthy
individuals,
when there is a major loss of T cells, e.g., in AIDS, and following
chemotherapy or
radiotherapy, adult patients are highly susceptible to opportunistic
infections because all these
conditions involve a loss of T cells and/or other blood cells (see below).
Lymphocyte
recovery is also severely retarded. The atrophic thymus is unable to
reconstitute CD4+ T
cells that are lost during HIV infection (Douek et al. Nature (1998) 396:690-
695) and CD4+
T cells take three to four times longer to return to normal levels following
chemotherapy in
post-pubertal patients as compared to pre-pubertal patients (Mackall et al.
(1995) N. Engl. J.
Med. 332:143-149). As a consequence these patients lack the cells needed to
respond to
infections, and they become severely immune suppressed (Mackall et al., (1995)
N. Eng. J.
Med. 332:143; Heitger et al., (2002) Blood 99:4053). There is also an increase
in cancers
and tumor load in later life (Hirokawa, (1998) "Immunity and Ageing," in
PRINCIPLES AND
PRACTICE OF GERIATRIC MEDICINE, (M. Pathy, ed.) John Wiley and Sons Ltd; Doric
et al.,
(1997) Mech. Age. Dev. 95: 131; Castle, (2000) Clin. Infect. Dis. 31:578).
However, recent work by Douek et al., ((1998) Nature 396:690) has shown thymic
output occurs even if only very slight (about 5°l0 of the young
levels), in older humans (e.g.,
even sixty-five years old and above, and after anti-retroviral treatment in
older HIVpatients).
This was exemplified by the presence of T cells with T Cell Receptor Excision
Circles
(TRECs); TRECs are formed as part of the generation of the TCR for antigen and
are only
found in newly produced T cells). Furthermore Tirnm and Thoman ((1999) J.
Imnzunol.
162:711) have shown that although CD4+ T cells are regenerated in old mice
post-bone
marrow transplant (BMT), they appear to show a bias towards memory cells due
to the aged
peripheral microenvironment coupled to poor thymic production of naive T
cells.TREC
levels has also been analysed following hematopoietic stem cell
transplantation (Douek et al.,
(2000) Lancet 355:1875).
Thymus and the neuroendocrine axis
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The thymus is influenced to a great extent by its bidirectional communication
with the
neuroendocrine system (Kendall, (1988) "Anatomical and physiological factors
influencing
the thymic microenvironment," in THYMUS UPDATE I, Vol. 1. (M. D. Kendall, and
M. A.
Bitter, eds.) Harwood Academic Publishers, p. 27). Of particular importance is
the interplay
between the pituitary, adrenals, and gonads on thymic function, including both
trophic
(thyroid stimulating hormone or TSH, and growth hormone or GH) and atrophic
effects
(luteinizing hormone or LH, follicle stimulating hormone or FSH, and
adrenocorticotropic
hormone or ACTH) (Kendall, (1988) "Anatomical and physiological factors
influencing the
thymic microenvironment," in THYMUS UPDATE I, Vol. 1. (M. D. Kendall, and M.
A. Bitter,
eds.) Harwood Academic Publishers, p. 27; Homo-Delarche et al., (1993)
SprirZger Sem.
Imf~aurcopathol. 14:221) Indeed, one of the characteristic features of thymic
physiology is the
progressive decline in structure and function, which is commensurate with the
increase in
circulating sex steroid production around puberty, which in humans generally
occurs from the
age of 12-14 onwards (Hirokawa and Makinodan, (1975) J. hrcrnufzol. 114:1659;
Tosi et al.,
(1982) Clih. Exp. Immureol. 47:497; and Hirokawa, et al., (1994) Imf~aunoL.
Lett. 40:269).
The thymus essentially consists of developing thymocytes interspersed within
the
diverse stromal cells (predominantly epithelial cell subsets) which constitute
the
microenvironment and provide the growth factors and cellular interactions
necessary for the
optimal development of the T cells. The precise target of the hormones, as
well as the
mechanism by which they induce thymus atrophy and improved immune responses,
has yet
to be determined. Examination of testicular feminised mutant mice, however,
indicates that
functional sex steroid receptors must be expressed on the stromal cells of the
thymus for
atrophy to occur. The symbiotic developmental relationship between thymocytes
and the
epithelial subsets that controls their differentiation and maturation (Boyd et
aL., (1993)
Imnaunol. Today 14:445) means that sex-steroid inhibition could occur at the
level of either
cell type, which would then influence the status of the other cell type. Bone
marrow stem
cells are reduced in number and are qualitatively different in aged patients.
HSC are able to
repopulate the thymus, although to a lesser degree than in the young. Thus,
the major factor
influencing thymic atrophy is appears to be intrathymic. Furthermore,
thymocytes in older
aged animals (e.g., those >18 months) retain their ability to differentiate to
at least some
degree (George and Bitter, (1996) Immuraol. Today 17:267; Hirokawa et aL.,
(1994)
Immunology Letters 40:269; Mackall et al., (1998) Eur. J. Immuf2ol. 28: 1886).
However,
recent work by Aspinall has shown that in aged mice there is a defect in
thymocyte
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production, which is manifested as a block within the precursor triple
negative population,
namely the CD44+CD25+ (TN2) stage. (Aspinall et al., (1997) J. Imnaunol.
158:3037).
In the particular case for AIDS, the primary defect in the immune system is
the
destruction of CD4+cells and to a lesser extent the cells of the myeloid
lineages of
macrophages and dendritic cells (DC). Without these the immune system is
paralysed and
the patient is extremely susceptible to opportunistic infection with death a
common
consequence. The present treatment for AIDS is based on a multitude of anti-
viral drugs to
kill or deplete the HIV virus. Such therapies are now becoming more effective
with viral
loads being reduced dramatically to the point where the patient can be deemed
as being in
remission. The major problem of immune deficiency still exists, however,
because there are
still very few functional T cells, and those which do recover, do so very
slowly. The period
of immune deficiency is thus still a very long time and in some cases immune
defense
mechanisms may never recover sufficiently.
Hematopoiesis
Bone marrow and hematopoietic stem cells
The major cells of the immune system are the B and T lymphocytes (a major
class of
white blood cells), and the antigen presenting cells (APC). All immune cells
are basically
derived from hematopoietic stem cells (HSC) and their progeny, the Common
Lymphoid
Progenitor (CLP) and the Common Myeloid Progenitor (CMP), which are produced
in the
BM. Some of the precursor cells migrate to the thymus and are converted into T
cells and
thymic DC. DC play a role in inducing self-tolerance.
Only a small proportion (approximately 0.01% in young animals) of the HSC are
released from the BM and find their way to the thymus via the blood supply,
where they
undergo division and maturation to form T cells, which are then returned into
the general
circulation. These new recent thymic emigrant (RTE) cells form a major part of
the immune
system, and are primarily Th and Tc and are important in maintaining a
constant supply of
new T cells with a diverse TCR repertoire for the initiation of almost all
immune responses.
Prior to leaving the thymus, Th and Tc cells can effectively distinguish
foreign antigen
because, as described above, T cells are "selected" in the thymus so that
those T cells that
leave recognize all of the cells in the body as self and, under normal
circumstances, do not
respond against them.
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B cells are also ultimately derived from HSC and develop in the BM before
exiting
into the peripheral immune system. Following interactions with T cells, and
other cells of the
immune system, B cells develop into plasma cells that produce and release
large amounts of
antibodies, which help the body destroy infective organisms and abnormal
cells.
Other HSC produced by the BM are also utilized for the production of all other
blood
cells, such as NK cells, regulatory cells, common myeloid progenitor derived
cells,
neutrophils, basophils and eosinophils, dendritic cells, monocytes,
macrophages, platelets and
red blood cells.
Hemat~oietic stem cell tran~lantation
Hematopoietic stem cell transplantation (HSCT) - also commonly known as bone
marrow transplantation (BMT)) - is a treatment used to enhance the recovery of
the immune
system in, e.g., certain critical cancer conditions. "HSCT" and "BMT" and
"transplant" are
used interchangeably and are herein defined as a transplant into a recipitent,
containing or
enriched for HSC, BM cells, stem cells, and/or any other cells which gives
rise to blood,
thymus, BM and/ or any other immune cells, including, but not limited to, HSC,
epithelial
cells, common lymphoid progenitors (CLP), common myelolymphoid progenitors
(CMLP),
multilineage progenitors (MLP), and/or mesenchymal stem cells in the BM. In
some
embodiments, the transplant may be a peripheral blood stem cell transplant
(PBSCT). The
HSC maybe be mobilized from the BM and then harvested from the blood, or
contained
within BM physically extracted from the donor. The HSC may be either purified,
enriched, or
simply part of the collected BM or blood, and are then injected into a
recipient. Transplants
may be allogeneic, autologous, syngeneic, or xenogenic, and may involve the
transplant of
any number of cells, including "mini-transplants," Which involve smaller
numbers of cells.
In some embodiments, HSCT is given prior to, concurrently with, or after sex
steroid
inhibion.
HSC is a nonlimiting exemplary type of cell, which may be transplanted and/or
genetically modified, as used throughout this application. However, as will be
readily
understood by one skilled in the art, in practicing the inventions provided
herein, HSC may
be replaced with any one (or more) of a number of substitute cell types
without undue
experimentation, including, but not limited to BM cells, stem cells, andJor
any other cell
which gives rise to blood, thymus, BM and/ or any other immune cells,
including, but not
limited to, HSC, epithelial stem cells, CLP, CMLP, MLP, and/or mesenchymal
stem cells in
the BM. In some embodiments, HSC are derived from a fetal liver and/or spleen.
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Both chemotherapy and radiation therapy can destroy cancer cells. However,
because
of the lack of specificity, these therapies also kill healthy cells, including
virtually all white
blood cells (WBC), as well as the HSC in the BM. It is this destruction of WBC
and HSC
that leads to the patient's need for HSCT. HSCT allows, for example, stem
cells and their
progeny cells that were damaged by, e.g., chemotherapy or radiation treatment
to be replaced
with healthy stem cells that can ultimately produce the blood cells that the
patient needs.
HSCT is the basic treatment for a number of hematological cancers, such as
leukexnias and lymphomas (cancers of the blood and immune system cells), as
well as non
malignant immune disorders such as severe combined immunodeficiency, Fanconi's
anemia,
myelodysplastic syndromes, amyloidosis, aplastic anemia, Diamond Blackfan
anemia,
hemophagocytic lymphohitiocytosis, Kostmann syndrome, Wiskott-Aldrich
syndrome,
thrombocytopenias, and hemoglobinopathologies, such as sickle cell disease and
thalassemia.
Leukemia and lymphoma, which are commonly treated by myeloablation or
myelodepletion
to rid the body of cancerous Bells, are commonly followed by HSCT to recover
immune
function. The ability of the HSC to first colonise the BM and convert to blood
cells
(engraftment) is directly linked to the absolute number and quality of the HSC
injected, and
the functional capacity of the recipient bone marrow microenvironment and the
HSC niches
.The methods of the present invention either alone or in combination
(concurrently or
sequentially) with the administration of HSC mobilizing agents, such as
cytokines (e.g., G-
CSF or GM-CSF), or drugs (e.g., cyclophosphamide), allow faster and/or better
engraftment
and may also allow chemotherapy and radiation therapy to be given at higher
doses andlor
more frequently.
Modern clinical medical procedures often employ a transplantation of HSC
derived
from another donor's blood (an allogeneic HSCT), where advantage is taken of
donor T cells
reacting against the host cancer cells (graft versus leukaemia (GVL)) but this
is
counterbalanced by other T donor T cells reacting against the host in general
(graft versus
host (GVH) disease) which can be fatal. Since the success of HSCT, and hence
patient
survival, is directly related to the number of HSC injected and the speed of
engraftment,
using the methods of the present invention means that current HSCT programs
will be more
successful and that many more patients will be able to receive HSCT than is
currently
possible.
Mechanisms of enhancing HSC mobilization from the BM are important in ensuring
that as many HSC as possible are available for collection from a donor. GM-CSF
and G-CSF
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are presently used for this purpose, but other agents, such as chemotherapy
and cytokines
have also been shown to be effective. The ability to more effectively mobilize
HSC has
application beyond hematological repair. Recent studies have shown that HSC
are
multipotent and may be utilized for repair of damaged tissues, e.g., cardiac
muscle, skeletal
muscle, liver, bone, connective tissue, epithelial tissue, pancreas,
vasculature.
One limitation of current HSCT strategies is associated with infection,
particularly
viral, fungal, and encapsulated bacteria, due to prolonged immunodeficiency
and this remains
a significant cause of post-transplantal morbidity and mortality in adults.
The infections
associated with HSCT are generally very difficult to control,even with modern
antimicrobial
reagents. Children generally recover immune capacity within months after HSCT
(Parkman
et al., (1997) Immunol. Rev. 157:73), in contrast to the delay in lymphoid
recovery in adult
recipients which may last years and even then be a very poor reflection of the
young
optimum. This delay in adults is dependent on a variety of factors, but
the,susceptibility to
infection is primarily due to the well-recognized decline in T and B cell
production with age
(Parkman et al., (1997) Immunol. Rev. 157:73).
Additionally, the rate of engraftment plays a role, wherein the longer the
rate of
engraftment, the more likely opportunistic infection will occur.
A second limitation of current HSCT strategies occurs when the grafted cells
'reject'
the recipient of the cells. This is known clinically as "graft versus host
disease" (GVHD).
An autologous transplant may avoid GVHD. However, the overall anti-cancer
success rates
of autologous transplants are lower as compared to allogeneic transplants. In
cancer patients,
autologous transplants have the disadvantage that they do not produce a Graft
Versus Tumor
(GVT) effect (which is similar to the GVH effect) and there is the risk that
cancerous cells
may be returned to the patient with the transplant. It has been discovered
that sex steroid
inhibition in murine allogeneic HSCT models and castrated recipients of
allogeneic HSCT
improves post-transplant reconstitution of cells of both the myeloid and
lymphoid lineages.
Data presented herein shows a significant increase in T and B cell
reconstitution without an
exacerbation of GVHD or loss of GVT activity (see, e.g., Example 19).
A further limitation of HSCT treatments is the lack of donors to treat all the
potential
candidates. Although umbilical cord blood (UCB) has been utilized to a fairly
limited extent,
there are few cells from each donor and as a consequence this has been mainly
used in
children where the total number of HSC required is lower (HSC number required
is linked to
patient body weight). Other than UCB, donors are in limited supply and there
must be an
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acceptable MHC match or the risk of GVH is high. If less cells were required,
as a result of
improved engraftment or a less rigorous match was required, thus reducing the
risk of
rejection or GVH, potentially HSCT could be used more widely, for example to
treat
autoimmune disease, and sources such as cord blood could be utilized
(e.g.,1.5x10' cells/kg
for recipient engraftment).
T Cells
T cells are the major component of the immune system, and are produced in the
thymus. The most important T cells are Th cells because these are the cells
that initiate
virtually all immune responses. The absence of these Th cells (e.g., caused by
HIV infection,
chemotherapy, radiation, etc.) directly results in immunosuppression and the
consequent
susceptibility to infections and tumors, and death occurs quickly. An
important role of a
subset of Th cells is to regulate immune responses. The balance between
enhancement and
suppression of T and B cell function has a major effect on e.g., whether a
vaccine is
efficacious, whether a cancer or tumor is attacked, or whether a transplant is
tolerated or
rejected.
The thymus, while being very active in the young, progressively declines in
both size
and functional output with age. This is particularly evident at the onset of
puberty. Since
thymocyte export is directly related to the cellularity in the thymus (Scollay
et al., (1980)
Eur. J. Immu~col. 10:210; Berzins et al., (1998) J. Exp. Meel. 187:1839), age-
related thymic
atrophy results in a gradual decrease in recent thymic emigrants (RTES)
(Steffens et al.,
(2000) Clip. Inamuszol. 97:95; Sempowski et al., (2002) Mol. Inzmuhol. 38:841-
848;
Sutherland et al., (submitted)) and a decrease in the naive to memory T cell
ratio (Ernst et al.,
(1990) J. Inarnunol. 145:1295); Kurashima et al., (1995) Int. hrzmunol. 7:97;
Utsuyama et al.,
(1992) Mech. AgeisZg Dev. 63:57) resulting in a restricted TCR repertoire in
both CD4+ and
CD8+ T cells (Mosley et al., (1998) Cell. Immunol. 189:10; LeMaoult et al.,
(2000) J.
ImnZUnol. 165:2367). Consequently, T cell proliferation in response to non-
specific and
receptor-mediated (CD3/TCR) stimulation is severely compromised with age
(Hertogh-
Huijbregts et al., (1990) Mech. Ageing Dev. 53:141-155; Flurkey et al., (1992)
J. Geroratol.
47:B115; Kirschmann etal., (1992) Cell. Immu~zol. 139:426).
With increasing age there is a gradual decline in the immune function of
humans;
children respond very well, younger adults reasonably so, but from middle age
and older, this
response can be very poor. This decline indicates the presence of deficiencies
or alterations
in one of more of the three major cell types involved in virtually all
response: (i) antigen
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presenting cells (which capture antigen and present it to, and thereby
activate, T
lymphocytes); (ii) T lymphocytes, and (iii) B lymphocytes. Deficiencies or
alterations in any
one of these three cell types may explain why the immune response to
stimulation against
antigens may be suboptimal. The deficiency or alterations may be at the level
of the cell or
may refer to quantity or functionality. Of these, the most likely defect is
encompassed within
the T cell compartment because of the dramatic decline in thymus function with
age primarily
due to the impact of sex steroids. This leads to a loss of new or "naive" T
cells exported into
the bloodstream, which are needed for responses to new antigens. In addition
to the
numerical loss of potential responding T cells, the pre-existing T cells may
be suppressed to
some degree by the presence of sex steroids.
Cancer therapy
As indicated above, chemotherapy and radiotherapy used to treat cancers are
often
deleterious to the patient's non-cancerous cells, particularly the blood
cells. The major
limitation to increasing frequency and dose of such treatments is the ability
of the patient to
survive the treatment and avoid susceptibility to opportunistic infection as a
result of the
compromised immune system. Thus it would greatly benefit the patient if the
immune
recovery was more rapid or the damage less severe.
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SUMMARY OF THE INVENTION
The present invention relates to methods for preventing illness or aiding
recovery in a
patient by enhancing BM haemopoieses and functionality, enhancing BM
engraftment
following HSCT, and increasing the functionality of pre-existing T cells and
other immune
cells by disrupting sex steroid and other hormonal signaling. Immune capacity
will also be
enhanced by increased levels of naive T cells produced through renewed thymic
function, and
also B lymphocytes and other cells of the immune system produced via activated
BM
function.
It has been discovered that interruption of sex steroid andlor other hormonal
signaling
enhances the functionality of BM, HSC, T cells and other cells of the immune
system, either
by direct effects or indirect effects. This discovery has been exploited to
produce the present
invention which, in one aspect of the invention, provide methods of enhancing
BM
haemopoiesis and/or functionality. In some embodiments, haemopoiesis and/or
functionality
of pre-existing BM is improved. In another embodiment, a patient receives an
HSCT, and the
HSC haemopoiesis and/or engraftment is improved in the patient.
In one aspect, the invention provides methods of enhancing engraftment
following
HSCT. In one embodiment, engraftment is enhanced in the BM. In another
embodiment,
engraftment and/or reconstitution is enhanced in the thymus, whereby thymic
recovery is
ultimately induced. In yet another embodiment, engraftment and/or
reconstitution is
enhanced in the spleen and/or other lymphoid organs, tissues, and/or blood. In
some
embodiments, the HSC are allogeneic, and in other embodiments, the HSC are
autologous.
In one embodiment, the numbex of T cell precursors is increased as compared to
the number
that would have been present in a patient that had HSCT without undergoing
interruption of
sex steroid signaling. In other embodiments total white blood cells, donor-
derived DC, BM
precursors, HSC, CLP, MLP, lymphocytes, myeloid cells, granulocytes,
neutrophils,
macrophage, NK, NKT, platelets, naive T cells, memory T cells, helper T cells,
effector T
cells, regulatory T cells, RBC, B cells, donor-, andlor host-derived
peripheral T cells, APC,
andlor donor derived peripheral B cells are increased as compared to the
number that would
have been present in a patient that had HSCT without undergoing interruption
of sex steroid
signaling. In yet other embodiments, a patient is also treated with a cytokine
(e.g., IL-7, SCF,
IL-1 l, G-CSF, or GM-CSF) or hormone (e.g., growth hormone, or its mediator
insulin
dependent growth factor (IGF-1) or any member of the fibroblast growth factor
family e.g.,
FGF 7 /Keratinocyte Growth Factor (KGF)) following HSCT to enhance immune
recovery
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and/or engraftment.. In another embodiment, the present invention either
alone, or in
combination (concurrently or sequentially) with the administration of
hemopoietic agents,
such as cytokines (e.g., G-CSF or GM-CSF), allow faster andlor better
engraftment andlor
homing to the target tissue and/or enhance recovery of immune cells.
In a second aspect of the invention, methods of enhancing the functionality of
immune cells in a patient following HSCT are provided. In one embodiment, the
immune
cells are T cells. In another embodiment, the T cell proliferative
responsiveness to T cell
receptor (TCR) stimulation is improved. In another embodiment, the T cell
responsiveness to
an antigen (e.g., tetanus toxoid (TT) or pokeweed mitogen (PWM), or Keyhole
Limpet
Hemocyanin (KLH)) stimulation is improved. In one embodiment, the T cell
responsiveness
is improved to a recall antigen (d.e., an improved T cell memory response). In
yet another
embodiment the T cell proliferative responsiveness in respect of co-
stimulatory or secondary
signaling is improved. In some embodiments, the kinetics of T cell
responsiveness is
improved. In other embodiments, the T cell response to antigen presented by
APC is
improved. In some embodiments of the invention, immune cell responsiveness is
improved
within about five-, four-, three- or two months post transplant. In certain
embodiments of the
invention, immune cell responsiveness is improved within about one month post-
transplant.
In other embodiments of the invention, immune cell responsiveness is improved
within two
weeks post-transplant. In another embodiment of the invention, immune cell
responsiveness
is improved within one week post-transplant. In yet other embodiments of the
invention,
immune cell responsiveness is improved within three days post transplant. In
other
embodiments the immune rpsonse is improved after 3 or more months post
treatment
involving at this time input fxom newly thymic derived T cells in addition to
pre-existing T
cells.
In a third aspect, the invention provides methods of enhancing pre-existing
immune
cell functionality, including, but not limited to, immune cells in the
periphery. In one
embodiment, the cells are T cells. In another embodiment, the cells are DC ox
other APC. In
yet another embodiment, the cells are NK cells or regulatory cells, such as
CD4+CD25+ T
cells and natural killer T (NKT) cells. In one embodiment of the invention, T
cell
proliferative responsiveness to TCR stimulation is improved in the patient. In
another
embodiment, the T cell responsiveness to antigen (e.g., TT, PWM, or KLH)
stimulation is
improved. In yet another embodiment the T cell proliferative responsiveness to
secondary or
co-stimulatory signaling is improved. In other embodiments, the T cell
response to antigen
presented by APC is improved. In one specific embodiment, a LHRH/GnRH analog
has a
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direct effect or indirect effect on the responsiveness of pre-existing immune
cells. In some
embodiments, sex steroid analogs (agonist and antagonists thereto), such as
GnRHILHRH
analogs, axe used in the methods of the invention to disrupt sex steroid-
mediated signaling,
immune cells or BM. In other embodiments, sex steroid analogs directly
stimulate (i.e.,
directly increase the functional activity of) the thymus, BM, and/or pre-
existing cells of the
immune system, such as T cells, B cells, and DC.
In a fourth aspect, the invention provides methods of enhancing HSC
engraftment and
mobilization in a patient, or in a blood, HSC, or BM donor. In one embodiment
disrupting
sex steroid signaling increases the number andlor functionality of peripheral
immune
progenitor cells such as HSC, CD34+ cells, CLP, or CMP. One embodiment
provides a
method for enhancing HSC mobilization comprising disrupting sex steroid
signaling, either
alone or in combination with administration of an HSC mobilizing agent, for
example,
cytokines, GM-CSF, G-CSF, CSF, chemotherapeutics, cyclophosphamide, flt-3
ligand,
KGF/FGF 7 or other members of the FGF familyor IL-7.
In one embodiment, the present invention provides methods to allow
chemotherapy
and radiation therapy to be given at higher doses andlor more frequently
andlor allow faster
recovery of or less damage to the immune system after chemotherapy and
radiation therapy.
In a fifth aspect, the invention provides methods to prevent or treat illness
in a patient.
One embodiment provides a method for preventing or diminishing the risk of an
infection,
illness, or disease in a patient, the method comprising disrupting sex steroid
mediated
signaling in the patient. In a certain embodiment, signaling is disrupted to
the BM. In
another embodiment, signaling is disrupted to the thymus. In yet another
embodiment,
signaling is disrupted to the spleen. In yet another embodiment, signaling is
disrupted to the
peripheral immune cells.
In some embodiments, the methods of the invention are used to prevent or treat
viral
infections, such as HIV, herpes, influenza, and hepatitis. In other
embodiments, the methods
of the invention are used to prevent or treat bacterial infections, such as
pneumonia and
tuberculosis (TB). In yet other embodiments, the methods of the invention are
used to
prevent or treat fungal infections, parasitic infections, allergies, and/or
tumors and other
cancers, whether malignant or benign.
In other embodiments, the patient receives non-genetically modified HSC
transplantation. In some embodiments, BM or HSC are transplanted into the
patient to
CA 02528521 2005-12-06
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provide a reservoir of precursor cells, which may ultimately be used for the
renewed thymic
growth. Some of these HSC have the ability to turn into DC or other APC, which
may have
the effect of providing better antigen presentation to the T cells and
therefore a better immune
response (e.g., increased Ab production and effector T cells number and/or
function). In
other embodiments the atrophic thymus in an aged (post-pubertal) patient is in
the process of
being reactivated by disruption of sex-steroid signaling at the time of HSCT.
The
reactivating thymus becomes capable of taking up HSC, BM cells from the blood,
and other
appropriate progenitors, and converting them in the thymus to both new T cells
and DC.
In a sixth aspect, the invention provides methods to improve the immune
responsiveness of a patient to a vaccine.
In seventh aspect, gene therapy utilizing genetically modified HSC, lymphoid
progenitor, myeloid progenitor or epithelial stem cells, or combinations
thereof (the group
and each member herein referred to as "GM cells"), are delivered to the
patient to create
particular immunities useful in treating or preventing an illness.
In some embodiments. of certain aspects of the invention, the illness is one
that has a
defined genetic basis, such as that caused by a genetic defect. These genetic
diseases are well
known to those in the art, and include autoimmune diseases, diseases resulting
from the over-
or under-production of certain proteins, tumors and cancers, etc. The disease-
causing
genetic defect is repaired by insertion of the normal gene into the HSC, and,
using the
methods of the invention, every cell produced from this HSC will then carry
the gene
correction
In other embodiments, the disease is a T cell disorder selected from the group
consisting of viral infections (such as human immunodeficiency virus (HIV)), T
cell
functional disorders, and any other disease or condition that reduces T cells
numerically or
functionally, either directly or indirectly, or causes T cells to function in
a manner which is
harmful to the individual.
In yet other embodiments, the present invention provides methods for treating
or
preventing infection by an infectious agent, such as HIV, by transplanting GM
cells that have
been genetically modified to resist or prevent infection, activity,
replication, and the like, and
combinations thereof, of the infectious agent may be injected into a patient
prior to, or
concurrently with, thymic reactivation. In one embodiment, the HSC are
modified to include
a gene whose product interferes with HIV infection, function, and/or
replication in the T cells
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(and/or other HSC-derived cells) of the patient. In a particular embodiment,
HSC are
genetically modified with viral resistance gene, such as the RevMlO gene (see,
e.g.,
Bonyhadi et al., (1997) J. Virol. 71:4707) or the CXCR4 or PolyTAR genes
(Strayer et al.,
(2002) Mol. Tlaer. 5:33). This confers a degree of resistance to the virus,
thereby preventing
or treating disease caused by the virus.
In another aspect, the invention provides for disruption of sex steroid
mediated
signaling to, and subsequent reactivation of, the thymus. In one embodiment,
castration is
used to disrupt the sex steroid mediated signaling. In a particular
embodiment, chemical
castration is used. In another embodiment, surgical castration (e.g., by
removal of the testes
or by ovariectomy) is used. In some embodiments, complete inhibition of sex
steroid .
signaling occurs. In another embodiment, partial disruption of sex steroid
signaling occurs.
In one embodiment, castration reverses the state of the thymus towards its pre-
pubertal state,
thereby reactivating it. In another embodiment, castration modifies the level
of other
molecules, which enhance immune cell responsiveness and/or proliferation
andlor activation
state by having, e.g., a direct effect on pre-existing immune cells.
In certain embodiments, sex steroid mediated signaling may be directly or
indirectly
blocked (e.g., inhibited, inactivated or made ineffectual) by the
administration of modifiers of
sex hormone production, action, binding or signaling, including but not
limited to agents
which bind a sex hormone or its receptor, agonists or antagonists of sex
hormones, including,
but not limited to, GnRH/LHRH, anti-estrogenic and anti-androgeuc agents,
SERMs,
SARMs, anti-estrogen antibodies, anti-androgen ligands, anti-estrogen ligands,
LHRH
ligands, passive (antibody) or active (antigen) anti-LHRH (or other sex
steroid) vaccinations,
or combinations thereof ("blockers"). In one embodiment, one or more blocker
is used. In
some embodiments, the one or more Mocker is administered by a sustained
peptide-release
formulation. Examples of sustained peptide-release formulations are provided
in WO
98/08533, the entire contents of which are incorporated herein by reference.
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DESCRIPTION OF THE FIGURES
Figs.1A-B: Castration rapidly regenerates thymus cellularity. Figs. lA-B are
graphic
representations showing that the changes in thymus weight and thymocyte number
pre- and
post-castration. Thymus atrophy results in a significant decrease in thymocyte
numbers with
age, as measured by thymus weight (Fig. 1A) or by the number of cells per
thymus (Fig. 1B).
For these studies, aged (i.e., 2-year old) male mice were surgically
castrated. Thymus weight
in relation to body weight (Fig. 1A) and thymus cellularity (Figs. 1B) were
analyzed in aged
(1 and 2 years) and at 2-4 weeks post-castration (post-cx) male mice. A
significant decrease
in thymus weight and cellularity was seen with age compared to young adult (2-
month) mice.
This decrease in thymus weight and cell number was restored by castration,
although the
decrease in cell number was still evident at 1 week post-castration (Fig. 1C).
By 2 weeks
post-castration, cell numbers were found to increase to approximately those
levels seen in
young adults (Fig. 1B). By 3 weeks post-castration, numbers have significantly
increased
from the young adult and these were stabilized by 4 weeks post-castration
(Fig. 1B). Results
are expressed as mean ~1SD of 4-8 mice per group (Fig. 1A) or 8-12 mice per
group (Fig.
1B). **= p<0.01; *** = p<0.001 compared to young adult (2 month) thymus and
thymus of
2-6 wks post-castrate mice.
Figs. 2A-D: Castration restores the CD4:CD8 T cell ratio in the periphery. For
these
studies, aged (2-year old) mice were surgically castrated and analyzed at 2-6
weeks post-
castration for peripheral lymphocyte populations. Figs. 2A and 2B show the
total
lymphocyte numbers in the spleen. Spleen numbers remain constant with age and
post-
castration because homeostasis maintains total cell numbers within the spleen
(Figs. 2A and
2B). However, cell numbers in the lymph nodes in aged (18-24 months) mice were
depleted
(Fig. 2B). This decrease in lymph node cellularity was restored by castration
(Fig. 2B). Fig.
2C shows that the ratio of B cells to T cells did not change with age or post-
castration in
either the spleen or lymph node, as no change in this ratio was seen with age
or post-
castration. However, a significant decrease (p<0.001) in the CD4+:CD8+ T cell
ratio was
seen with age in both the (pooled) lymph node and the spleen (Fig. 2E). This
decrease was
restored to young adult (i.e., 2 month) levels by 4-6 weeks post-castration
(Fig. 2D).
Results are expressed as mean~1 SD of 4-8 (Figs. 2A, 2C, and 2E) or 8-10
(Figs. 2B, 2D, and
2F) mice per group. * = p<0.05; * * = p_0.01; * * * = p<0.001 compared to
young adult (2-
month) and post-castrate mice.
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Fig. 3: Thymocyte subpopulations are retained in similar proportions despite
thymus
atrophy or regeneration by castration. For these studies, aged (2-year old)
mice were
castrated and the thymocyte subsets analyzed based on the markers CD4 and CDB.
Representative Fluorescence Activated Cell Sorter (FACS) profiles of CD4 (X-
axis) vs. CD8
(Y-axis) for CD4-CD8-DN, CD4+CD8+DP, CD4+CD8- and CD4-CD8+ SP thymocyte
populations are shown for young adult (2 months), aged (2 years) and aged,
post-castrate
animals (2 years, 4 weeks post-cx). Percentages for each quadrant are given
above each plot.
No difference was seen in the proportions of any CD4/CD8 defined subset with
age or post-
castration. Thus, subpopulations of thymocytes remain constant with age and
there was a
synchronous expansion of thymocytes following castration.
Figs. 4A-B: Regeneration of thymocyte proliferation by castration. Mice were
injected with a pulse of BrdU and analyzed for proliferating (BrdU+)
thymocytes. For these
studies, aged (2-year old) mice were castrated and injected with a pulse of
bromodeoxyuridine (BrdU) to determine levels of proliferation. Representative
histogram
profiles of the proportion of BrdU+ cells within the thymus with age and post-
castration are
shown (Fig. 4A). No difference was observed in the total proportion of
proliferation within
the thymus, as this proportion remains constant with age and following
castration (Fig. 4A
and left~graph in Fig. 4B). However, a significant decrease in number of BrdU+
cells was
seen with age (Fig. 4B, right graph). By 2 weeks post-castration, the number
of BrdU+ cells
increased to a number that similar to seen in young adults (a.e., 2 month)
(Fig. 4B, right
graph). Results are expressed as mean~1SD of 4-14 mice per group. **~=p<_0.001
compared
to young adult (2-month) control mice and 2-6 weeks post-castration mice.
Figs. 5A-H: Castration enhances proliferation within all thymocyte subsets.
For these
studies, aged (2-year old) mice were castrated and injected with a pulse of
bromodeoxyuridine (BrdU) to determine levels of proliferation. Analysis of
proliferation
within the different subsets of thymocytes based on CD4 and CD8 expression
within the
thymus was performed. Fig. 5A shows that the proportion of each thymocyte
subset within
the BrdU+ population did not change with age or post-castration. However, as
shown in Fig.
5B, a significant decrease in the proportion of DN (CD4-CD8-) thymocytes
proliferating was
seen with age. Fig. 5C shows that no change in the total proportion of BrdU+
cells (i.e.,
proliferating cells) within the TN subset was seen with age or post-
castration. However, a
significant decrease in proliferation within the TN1 (CD44+CD25-CD3-CD4-CD8-)
subset
(Fig. 5E) and significant increase in proliferation within TN2 (CD44+CD25+CD3-
CD4-
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CD8-) subset (Fig. 5F) was seen with age. This was restored post-castration
(Figs. 5D-F).
Results are expressed as mean~1SD of 4-17 mice per group. *=p<0.05; ** =p<0.01
(significant) ; ~=** = p<_0.001 (highly significant) compared to young adult
(2-month) mice; ~
= significantly different from 2-6 weeks post-castrate mice (Figs. 5E-H).
Figs. 6A-C: Castration increases T cell export from the aged thymus. For these
studies, aged (2-year old) mice were castrated and were injected
intrathymically with FITC to
determine thymic export rates. The number of FITC+ cells in the periphery was
calculated
24 hours later. As shown in Fig. 6A, a significant decrease in recent thymic
emigrant (RTE)
cell numbers detected in the periphery over a 24 hours period was observed
with age.
Following castration, these values had significantly increased by 2 Weeks post-
cx. As shown
in Fig. 6B, the rate of emigration (export/total thymus cellularity) remained
constant with
age, but was significantly reduced at 2 weeks post-castration. With age, a
significant increase
in the ratio of CD4+ to CD8+ RTE was seen; this was normalized by 1-week post-
cx (Fig.
6C).
Results are expressed as mean~1SD of 4-8 mice per group. * = p<_0.05; ** =
p<0.01; *** _
p<0.001 compared to young adult (2-month) mice for (Fig. 6A) and compared to
all other
groups (Figs. 6B and 6C). ~ = p<_0.05 compared to aged (1- and 2-year old) non-
cx mice and
compared to 1-week post-cx, aged mice.
Figs. 7A-B: Castration enhances thyrnocyte regeneration following T cell
depletion.
3-month old mice were either treated with Cyclophosphamide (intraperitoneal
injection with
200 mg/kg body weight cyclophosphamide, twice over 2 days) (Fig. 7A) or
exposed to
sublethal irradiation (625 Rads) (Fig. 7B). For both models of T cell
depletion studied,
castrated (Cx) mice showed a significant increase in the rate of thymus
regeneration
compared to their sham-castrated (ShCx) counterparts. Analysis of total
thymocyte numbers
at 1 and 2-weeks post-T cell depletion (TCD) showed that castration
significantly increases
thymus regeneration rates after treatment with either cyclophosphamide or
sublethal
irradiation (Figs. 7A and 7B, respectively). Data is presented as mean~1SD of
4-8 mice per
group. For Fig. 7A, **~' = p<0.001 compared to control (age-matched,
untreated) mice; ~ _
p_<0.001 compared to both groups of castrated mice. For Fig. 7B, *~'* =
p<_0.001 compared to
control mice; ~ = p<_0.001 compared to mice castrated 1-week prior to
treatment at 1-week
post-irradiation and compared to both groups of castrated mice at 2-weeks post-
irradiation.
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Figs. 8A-B: Total lymphocyte numbers within the spleen and lymph nodes post-
cyclophosphamide treatment. For these studies, (3 month old) mice were
depleted of
lymphocytes using cyclophosphamide (intraperitoneal injection with 200 mg/kg
body weight
cyclophosphamide, twice over 2 days) and either surgically castrated or sham-
castrated on
the same day as the last cyclophosphamide injection. Thymus, spleen and lymph
nodes
(pooled) were isolated and total cellularity evaluated. Sham-castrated mice
had significantly
lower cell numbers in the spleen at 1 and 4-weeks post-treatment compared to
control (age-
matched, untreated) mice (Fig. 8A). A significant decrease in cell number was
obser',~ed
within the lymph nodes at 1 week post-treatment for both treatment groups
(Fig. 8B). At 2-
weeks post-treatment, Cx mice had significantly higher lymph node cell numbers
compared
to ShCx mice (Fig. 8B). Each bar represents the mean~1SD of 7-17 mice per
group. * _
p<_0.05; ** = p'0.01 compared to control (age-matched, untreated). ~=p<0.05
compared to
castrate mice.
Fig. 9: Changes in thymus (open bars), spleen (gray bars) and lymph node
(black
bars) cell numbers following treatment with cyclophosphamide, a chemotherapy
agent, and
surgical or chemical castration performed on the same day. Note the rapid
expansion of the
thymus in castrated animals when compared to the non-castrate
(cyclophosphamide alone)
group at 1 and 2 weeks post-treatment. In addition, spleen and lymph node
numbers of the
castrate group were well increased compared to the cyclophosphamide alone
group. (n = 3-4
per treatment group and time point). Chemical castration is comparable to
surgical castration
in regeneration of the immune system post-cyclophosphamide treatment.
Figs. l0A-C: Changes in thymus (Fig. 10A), spleen (Fig. 10B) and lymph node
(Fig.
11C) cell numbers following irradiation (625 Rads) one week after surgical
castration. For
these studies, young (3-month old) mice were depleted of lymphocytes using
sublethal (625
Rads) irradiation. Mice were either sham-castrated or castrated 1-week prior
to irradiation.
A significant increase in thymus regeneration (i.e., faster rate of thymus
regeneration) was
observed with castration (Fig. ~10A). Note the rapid expansion of the thymus
in castrated
animals when compared to the non-castrate (irradiation alone) group at 1 and 2
weeks post-
treatment. (n = 3-4 per treatment group and time point). No difference in
spleen (Fig. 10B)
or lymph node (Fig. 10C) cell numbers was seen with castrated mice. Lymph node
cell
numbers were still chronically low at 2-weeks post-treatment compared to
control mice (Fig.
10C). Results are expressed as mean~1SD of 4-8 mice per group. ~' = p<0.05;
~~~ = p<0.01
compared to control mice; =~'~'* = p<0.001 compared to control and castrated
mice.
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Figs.11A-C: Changes in thymus (Fig. 11A), spleen (Fig. 11B) and lymph node
(Fig.
11C) cell numbers following irradiation and castration on the same day. For
these studies,
young (3-month old) mice were depleted of lymphocytes using sublethal (625
Rads)
irradiation. Mice were either sham-castrated or castrated on the same day as
irradiation.
Castrated mice showed a significantly faster rate of thymus regeneration
compared to sham-
castrated counterparts (Fig. 11A). Note the rapid expansion of the thymus in
castrated
animals when compared to the non-castrate group at 2 weeks post-treatment. No
difference
in spleen (Fig. 11B) or lymph node (Fig. 11C) cell numbers was seen with
castrated mice.
Lymph node cell numbers were still chronically low at 2-weeks post-treatment
compared to
control mice (Fig. 11C). Results are expressed as mean~1SD of 4-8 mice per
group. * -
p<0.05; ** = p<0.01 compared to control mice; *** = p<0.001 compared to
control and
castrated mice.
Fig.12A-B: Total lymphocyte numbers within the spleen and lymph nodes post-
irradiation treatment. 3-month old mice were either castrated or sham-
castrated 1-week prior
to sublethal irradiation (625Rads). Severe lymphopenia was evident in both the
spleen (Fig.
12A) and (pooled) lymph nodes (Fig. 12B) at 1-week post-treatment. Splenic
lymphocyte
numbers were returned to control levels by 2-weeks post-treatment (Fig. 12A),
while lymph
node cellularity was still significantly reduced compared to control (age-
matched, untreated)
mice (Fig. 12B). No differences were observed between the treatment groups.
Each bar
represents the mean~1SD of 6-8 mice per group. ** - p<0.01; *** - p<0.001
compared to
control mice.
Figs. 13A-B: Castration restores responsiveness to HSV-1 immunization. Mice
were
immunized in the hind foot-hock with 4x105 pfu of HSV. On Day 5 post-
infection, the
draining lymph nodes (popliteal) were analyzed for responding cells. Fig. 13A
shows the
lymph node cellularity following foot-pad immunization with Herpes Simplex
Virus-1 (HSV-
1). Note the increased cellularity in the aged post-castration as compared to
the aged non-
castrated group. Fig. 13B illustrates the overall activated cell number as
gated on CD25 vs.
CD8 cells by FACS (i.e., the activated cells are gated on CD8+CD25+ cells).
Castration of
the aged mice restored the immune response to HSV-1 with CTL numbers
equivalent to
young mice. Results are expressed as mean~1SD of 8-12 mice. **=p<0.01 compared
to both
young (2-month) and castrated mice
Figs.14A-C: V D 10 expression (HS V-specific) on CTL (cytotoxic T lymphocytes)
in
activated LN (lymph nodes) following HSV-1 inoculation. Despite the normal
V(310
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responsiveness in aged (i.e., 18 months) mice overall, in some mice a complete
loss of V(310
expression was observed. Representative histogram profiles are shown. Note the
diminution
of a clonal response in aged mice and the reinstatement of the expected
response post-
castration. .
Figs. 15A-B: Castration enhances activation following HSV-1 infection. Fig.
15A
shows representative FACS profiles of activated (CD8+CD25+) cells in the LN of
HSV-1
infected mice. No difference was seen in proportions of activated CTL with age
or post-
castration. As shown in Castration of the aged mice restored the inunune
response to HSV-1
with CTL numbers equivalent to young mice. Results axe expressed as mean~1SD
of 8-12
mice. *~'=p<0.01 compared to,both young (2-month) and castrated mice.
Fig. 16: Specificity of the immune response to HSV-1. Popliteal lymph node
cells
were removed from mice immunized with HSV-1 (removed 5 days post-HSV-1
infection),
cultured for 3-days, and then examined for their ability to lyse HSV peptide
pulsed EL 4
target cells. CTL assays were performed with non-immunized mice as control for
background levels of lysis (as determined by 5lCr-release). Aged mice showed a
significant
(p50.01, **) reduction in CTL activity at an E:T ratio of both 10:1 and 3:1
indicating a
reduction in the percentage of specific CTL present within the lymph nodes.
Castration of
aged mice xestored the CTL response to young adult levels since the castrated
mice
demonstrated a comparable response to HSV-1 as the young adult (2-month) mice.
Results
are expressed as mean of 8 mice, in triplicate ~1 SD. ** = p<_0.01 compared to
young adult
mice; ~ = significantly different to aged control mice (p<_0.05 for E:T of
3:1; p<_0.01 for E:T
of 0.3:1).
Figs.17A-B: Analysis of V ~ TCR expression and CD4+ T cells in the immune
response to HSV-1. Popliteal lymph nodes were removed 5 days post-HSV-1
infection and
analyzed ex-vivo for the expression of CD25, CD8 and specific TCR V 0 markers
(Fig. 17A)
and CD4lCD8 T cells (Fig. 17B). The percentage of activated (CD25~ CD8+ T
cells
expressing either V~ 10 or V08.1 is shown as mean ~1SD for 8 mice per group in
Fig. 17A.
No difference was observed with age or post-castration. However, a decrease in
CD4/CD8
ratio in the resting LN population was seen with age (Fig. 17B). This decrease
was restored
post-castration. Results are expressed as mean~1SD of 8 mice per group. *** =
p_<0.001
compared to young and post-castrate mice.
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Figs. 18A-D: Castration enhances regeneration of the thymus (Fig. 18A), spleen
(Fig.
18B) and BM (Fig. 18D), but not lymph node (Fig. 18C) following BM
transplantation
(BMT) of Ly5 congenic mice. 3 month old, young adults, C57BL6 Ly5.1+ (CD45.1+)
mice
were irradiated (at 6.25 Gy), castrated, or sham-castrated 1 day prior to
transplantation with
C57BL6 Ly5.2+ (CD45.2+) adult BM cells (106 cells). Mice were killed 2 and 4
weeks later
and the), thymus (Fig. 18A), spleen (Fig. 18B), lymph node (Fig. 18C) and BM
(Fig. 18D)
were analyzed for immune reconstitution. Donor/Host origin was determined with
anti-
CD45.2 (Ly5.2), which only reacts with leukocytes of donor origin. There were
significantly
more donor cells in the thymus of castrated mice 2 and 4 weeks after BMT
compared to
sham-castrated mice (Fig. 18A). Note the rapid expansion of the thymus in
castrated animals
when compared to the non-castrate group at all time points post-treatment.
There were
significantly more cells in these spleen and BM of castrated mice 2 and 4
weeks after BMT
compared to sham-castrated mice (Figs. 18B and 18D). There was no significant
difference
in lymph node cellularity 2, 4, and 6 weeks after BMT (Fig. 18C). Castrated
mice had
significantly increased congenic (Ly5.2) cells compared to non-castrated
animals. Data is
expressed as mean+1SD of 4-5 mice per group. ~==p_<0.05; *~=p<_0.01.
Figs.19A-C: Castration increases BM and thymic cellularity following congenic
BMT. As shown in Fig. 19A, there are significantly more cells in the BM of
castrated mice 2
and 4 weeks after BMT. BM cellularity reached untreated control levels
(1.5x10~ 1.5x106)
in the sham-castrates by 2 weeks. BM cellularity is above control levels in
castrated mice 2
and 4 weeks after congenic BMT. Fig. 19b shows that there are significantly
more cells in
the thymus of castrated mice 2 and 4 weeks after BMT. Thymus cellularity in
the sham-
castrated mice is below untreated control levels (7.6x10 ~ 5.2x106) 2 and 4
weeks after
congenics BMT. 4 weeks after congenic BMT and castration thymic cellularity is
increased
above control levels. Fig. 19C shows that there is no significant difference
in splenic
cellularity 2 and 4 weeks after BMT. Spleen cellularity has reached control
levels (8.5x10 ~
1.1x100 in sham-castrated and castrated mice by 2 weeks. Each group contains 4
to 5
animals. Open bars indicate sham-castration; closed bars indicate castration.
Fig. 20: Castration increases the proportion of HSC following congenic BMT.
Representative FACS dot plots illustrating c-kit (y-axis) versus sca-1 (x-
axis) expression.
HSC are c-kithlsca-1h'. There is a significant increase in the proportion of
donor-derived
HSCs following castration, 2 and 4 weeks after BMT.
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Figs. 21A-B: Castration increases the proportion and number of HSC following
congenic BMT. As shown in Fig. 21A, there was a significant increase in the
proportion of
HSCs following castration, 2 and 4 weeks after BMT (* p<0.05). Fig. 21B shows
that the
number of HSCs is significantly increased in castrated mice compared to sham-
castrated
controls, 2 and 4 weeks after BMT (* p<0.05 ** p<0.01). Each group contains 4
to 5
animals. Open bars indicate sham-castration; closed bars indicate castration.
Figs. 22A-B: There are significantly more donor-derived B cell precursors and
B
cells in the BM of castrated mice following BMT. As shown in Fig. 22A, there
were
significantly more donor-derived CD45.1+B220~IgM-B cell precursors in the BM
of castrated
mice compared to the sham-castrated controls (* p<0.05). Fig. 22B shows that
there were
significantly more donor-derived B220~IgM+B cells in the BM of castrated mice
compared
to the sham-castrated controls (* p<0.05). Each group contains 4 to 5 animals.
Open bars
indicate sham-castration; closed bars indicate castration.
Fig. 23: Castration does not effect the donor-derived thymocyte proportions
following congenic BMT. 2 weeks after sham-castration and castration there is
an increase in
the proportion of donor-derived double negative (CD45.1+CD4-CD8-) early
thymocytes.
There are very few donor-derived (CD45.1+) CD4 and CD8 single positive cells
at this early
time point. 4 weeks after BMT, donor-derived thymocyte profiles of sham-
castrated and
castrated mice are similar to the untreated control.
Fig. 24: Castration does not increase peripheral B cell proportions following
congenic
BMT. There is no difference in splenic B220 expression comparing castrated and
sham-
castrated mice, 2 and 4 weeks after congenic BMT.
Fig. 25: Castration does not increase peripheral B cell numbers following
congenics
BMT. There is no significant difference in B cell numbers 2 and 4 weeks after
BMT. 2 weeks
after congenic BMT B cell numbers in the spleen of sham-castrated and
castrated mice are
approaching untreated control levels (5.0 x 10'~ 4.5x106). Each group contains
4 to 5
animals. Open bars indicate sham-castration; closed bars indicate castration.
Fig. 26: Donor-derived triple negative, double positive and CD4 and CD8 single
positive thymocyte numbers are increased in castrated mice following BMT. Fig.
26A shows
that there were significantly more donor-derived triple negative (CD45.1+CD3-
CD4-CD8-)
thymocytes in the castrated mice compared to the sham-castrated controls 2 and
4 weeks after
BMT (* p<0.05 ~'*p<0.01). Fig. 26B shows there were significantly more double
positive
CA 02528521 2005-12-06
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(CD45.1+CD4+CD8+) thymocytes in the castrated mice compared to the sham-
castrated
controls 2 and 4 weeks after BMT (* p<0.05 *~p<0.01). As shown in Fig. 26C,
there were
significantly more CD4 single positive (CD45.l~CD3~CD4~CD8-) thymocytes in the
castrated mice compared to the sham-castrated controls 2 and 4 weeks after BMT
(x' p<0.05
**p<0.01). Fig. 26D shows there were significantly more CD8 single positive
(CD45.1~CD3~CD4-CD8'~) thymocytes in the castrated mice compared to the sham-
castrated
controls 4 weeks after BMT (* p<0.05 *~=p<0.01). Each group contains 4 to 5
animals. Open
bars indicate sham-castration; closed bars indicate castration.
Fig. 27: There are very few donor-derived, peripheral T cells 2 and 4 weeks
after
congenic BMT. As shown in Fig. 27A, there was a very small proportion of donor-
derived
CD4+ and CD8+ T cells in the spleens of sham-castrated and castrated mice 2
and 4 weeks
after congenic BMT. Fig. 27B shows that there was no significant difference in
donor-
derived T cell numbers 2 and 4 weeks after BMT. 4 weeks after congenics BMT
there are
significantly less CD4+ and CD8+ T cells in both sham-castrated and castrated
mice compared
to untreated age-matched controls (CD4+-1.1x10 ~ 1.4x106, CD8+ - 6.0x106 ~
1.0x105) Each
group contains 4 to 5 animals. Open bars indicate sham-castration; closed bars
indicate
castration.
Fig. 2~: Castration increases the number of donor-derived DC in the thymus 4
weeks
after congenics BMT. As shown in Fig. 28A, donor-derived DC were CD45.1+CDllc+
MHCITF. Fig. 28B shows there were significantly more donor-derived thymic DC
in the
castrated mice 4 weeks after congenic BMT (* p<0.05). Dendritic cell numbers
are at
untreated control levels 2 weeks aftex congenic BMT (1.4x105~ 2.8x104). 4
weeks after
congenic BMT dendritic cell numbers are above control levels in castrated
mice. Each group
contains 4 to 5 animals. Open bars indicate sham-castration; closed bars
indicate castration.
Figs. 29A-C: Castration enhances immune cell reconstitution in allogeneic HSCT
recipients. Lethally irradiated (1300 cGy) CBA/J recipients-(3 month old)
received
transplants with B lO.BR TCD BM (5 x 106). Recipients were either castrated or
sham-
castrated one day before transplant. Animals were humanely killed on days 14,
28 and 42
and BM (Fig. 29A), thymus (Fig. 29B), and spleen (Fig. 29C) organ cellularity
was assessed.
~' ( p< 0.05). Each group contained 4 to 5 animals.
Figs. 30A-C: Castration enhances donor-derived HSC and B cells in allogeneic
HSCT recipients. Castrated and sham-castrated recipients were transplanted as
in described
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in Fig. 29. As shown in Fig. 30A, 14 days after HSCT there were very few donor-
derived
HSCs (Ly9.1-Liri Sca-1+c-kit+) in both sham-castrated and castrated mice;
however, by day
28, donor HSC numbers were 4-fold higher in the castrated group. Additionally,
as shown in
Figs. 30B-C, there are significantly more donor-derived B cells in the BM
(Fig. 30B) and
spleen (Fig. 30C) of castrated mice. Central and peripheral B cell populations
were analyzed
using total BM or splenic cell counts and multicolor flow cytometry. B cells
were separated
into developmental stages based on CD45R, IgM and CD43 expression. Total B
cells
(CD45R+), pro-B cells (CD43+CD45R+IgM-), pre-B cells (CD43-CD45R +IgM-),
immature
B cells (CD43-CD45R+IgM+). Donor/host origin was determined with anti-Ly9.l,
which
only xeacts with leukocytes of host origin. Each group contained 4 to 5
animals. Open bars
indicate sham-castrated animals, and closed bars represent castrated animals.
* ( p< 0.05)
represents a significant increase in cell number in the castrated group
compared to the sham-
castrated control.
Figs. 31A-E: Castration enhances thymocyte and peripheral T cell
reconstitution as
well as the number of host and donor-derived DC in allogeneic HSCT recipients.
Castrated
and sham-castrated recipients were transplanted as described in Fig. 29.
Animals were
humanely killed on days 14, 28 and 42, and thymocyte and T cell populations
were analyzed
using total thymic (Fig. 31A-F) or splenic (Fig. 31G) cell counts and
multicolor flow
cytometry. DC were defined as CDllchl Ia-khl. Fig. 31A depicts numbers of TN
(CD3-
CD4-CD8-) thymocytes. Fig. 31B depicts numbers of DP (CD4+CD8+) thymocytes.
Fig.
31C depicts numbers of CD4+ SP (CD3+CD4+CD8-) thymocytes. Fig. 31D depicts
numbers of CD8+ SP (CD3+CD4-CD8+) thymocytes. As shown in Fig. 331E, there are
significantly more host-derived DC in castrated recipients at both 14 and 28
days after
allogeneic HSCT as compared to sham-castrated control recipients.
Additionally, as shown
in Fig. 31F, there are significantly more donor-derived DC in castrated
recipients 28 days
following allogeneic HSCT, as compared to sham-castrated controls. Fig. 31G
depicts
numbers of peripheral T cells, which were identified using anti-CD3, anti-CD4
and anti-CD8.
Donor/host origin was determined with anti-Ly9.l, which only reacts with
leukocytes of host
origin. Donor CD4 T cells were Ly9.1-CD3+CD4+CD8- and donor CD8 T cells were
Ly9.1-
CD3+CD4-CD8+. Each group contained 4 to 5 animals. Open bars indicate sham-
castrated
animals, and closed bars represent castrated animals. a' ( p< 0.05) and k~'
(p<0.01) represent a
significant increase in cell number in the castrated group compared to the
sham-caste ated
control.
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Figs. 32A-G: Castration does not alter the function of donor-derived T cells
following allogeneic HSCT. Castrated and sham-castrated recipients were
transplanted as in
Figs. 29. T cell functionality was assessed 42 days after transplantation.
Fig. 32A shows the
number of donor-derived T cells (CD3+CD4+ and CD3+CD8+) six weeks after allo-
HSCT.
Fig. 32B shows that castration has no effect on the proliferative capability
of T cells after
allogeneic HSCT. Fig. 32C shows no difference in alloreactive T-cell
proliferation in an
MLR. Splenic T cells (4 x105 cells/well) from each group (n=5) were incubated
with
irradiated (20 Gy) BALB/c splenic stimulator cells (2 x105 cells/well) in 96-
well plates for 5
days and [3H]-thymidine was added during the final 20 hours of culture. Fig.
32D shows no
difference in cytolytic activity of donor-derived T cells. Fig.32E shows
intracellular IFNy
expression of alloreactive T cells. Splenic B6 T cells were harvested on day
42 from sham-
castrated or castrated recipients as described above and incubated with
irradiated (20 Gy)
(BALB/C - third party) splenic stimulator cells in 24-well plates for 5 days.
Cells were
harvested, and restimulated with TCD, irradiated (20 Gy) (BALB/C or B10.BR
internal
biological control) splenic stimulator cells for 16 hours. Brefeldin A (10
mg/mL) was added
after the first hour of incubation. Intracellular IFNy expression in donor-
derived CD3+CD8+
cells was measured by flow cytometric analysis. Representative plots are shown
in Fig. 32E
and graphically represented as the percentage of donor-derived CD8+ T cells
that express
IFN-y in Fig. 32F. Fig. 32G shows that T cell functionality was significantly
enhanced 48
hrs. after challenge when mice were castrated at the time of alto-HSCT. The
DTH assay was
performed at week 6 following allogeneic HSCT in sham-castrated and castrated
mice, and
the swelling was measured by subtracting left hind footpad swell from right
hind one. Open
bars indicate sham-castrated animals, and closed bars represent castrated
animals.
Figs. 33A-B: Castration does not aggravate GVHD or decrease GVL activity in
allogeneic HSCT recipients. For the experiments depicted in Fig. 33A, lethally
irradiated
(1300 cGy) (B6 x C3H) F1 recipients (3 months old) received transplants with
B6 TCD BM
cells (5 x 106) + splenic T cells (0.5 x 106). Survival is depicted as a
Kaplan-Meier
curve. Open circles represent a TCD-BM only (no T cells) control group (n=4).
Closed
circles represent sham-castrated recipients; and open squares represent
castrated recipients.
Each group contained 8 animals. For the experiments depicted in Fig. 33B,
lethally
irradiated, 3 month old B6D2F1/J recipients received P815 (H-2d) cells (1 x
103), C57BL6
TCD BM cells (5 x 106) and C571BL6 T cells (5 x 105). Survival is depicted as
a Kaplan-
Meier curve. Open circles indicate a TCD-BM only (no T cells) control group
(n=4). Closed
28
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circles represent sham-castrated recipients, and open squares represent
castrated recipients.
Each test group contained 8-9 animals.
Figs. 34A-I: Castration and IL-7 treatment have an additive effect in the
thymus
following allogeneic HSCT. Castrated and sham-castrated recipients were
transplanted
described in Fig. 1. Recipients killed on day 14 (Fig. 34A) received, in
addition, 10 g/day IL,-
7 or PBS (control) by intraperitoneal injection from day 0 to day 13.
Recipients killed on day
28 (Fig. 34B) received 10 g/day IL-7 or PBS (control) from day 21 to day 28.
Thymic
cellularity was calculated from total cell counts. * ( p< 0.05) represents a
significant increase
in cell number in the castrated group compared to the sham-castrated control.
Control: sham-
castrated, PBS injected recipients; CX: castrated, PBS injected recipients; IL-
7: sham-
castrated, IL-7 injected recipients; and IL-7 & CX: castrated, IL-7 injected
recipients.
Semiquantitative R.T-PCR was performed on whole BM 2 weeks after allo-HSCT and
castration. After HPRT equilibration templates from castrated and sham-
castrated mice were
compared for the expression of TGF(31 and KGF (Fig. 34C). KGF-~- and IL7-~-
mice were
castrated and 2 weeks later thymic, BM and splenic cellularity were analysed.
Fig. 34D-F
shows the results from the thymus (Fig. 34D), BM (Fig. 34E), spleen (Fig. 34F)
of KGF-/-
mice . Fig. 34G-I shows the results from the thymus (Fig. 34G), BM (Fig. 34H),
and spleen
(Fig. 34I) of IL7-l- mice.
Fig. 35: Castration enhances engraftment in. the BM, thymus, and spleen
following
HSCT. Mice were castrated 1 day before congenic HSCT. 5x106 Ly5.1+ BM cells
were
injected intravenously into irradiated (800 rads) C57BL6 mice. The BM, spleen
and thymus
were analyzed by flow cytometry at various time points (2-6 weeks) post-
transplant. As
shown in Fig. 35B, two weeks after castration and HSCT, there are
significantly more cells in
the BM of castrated mice as compared to sham-castrated controls. Similarly, as
shown in Fig.
35C, there is a significant increase in thymic cell number 2, 4 and 6 weeks
post-transplant as
compared to sham castrated controls. As shown in Fig. 35C, in the periphery,
splenic cell
numbers are also significantly higher than controls 4 and 6 weeks post-
transplant in the
castrated recipients. Gray bars represent castrated recipients. Black bars
represent sham-
castrafied controls.
Fig. 36A-B: Castration enhances engraftment of HSC in the BM following
congenic
HSCT. Mice were castrated 1 day before congenic HSCT. 5x106 Ly5.1+ BM cells
were
injected intravenously into irradiated (800 rads) C57BL6 mice. The BM was
analyzed for
lin-c-kit+sca-1+ HSC by flow cytometry at two weeks post-transplant (Fig.
36A). Two
29
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weeks after BMT transplantation and castration there are significantly more
donor-derived
HSCs in the BM of castrated mice compared to sham castrated controls (Fig.
36B).
Figs. 37A-B: Castration enhances engraftment of HSC in the BM following
congenic
HSCT (2.5x106 cells and 5x106 cells). Mice were castrated 1 day before
congenic HSCT.
2.5x106 (Fig. 37A-B) or 5x106 (Fig. 37C-D) Ly5.1+BM cells were injected
intravenously
into irradiated (800 rails) C57/BL6 mice. The BM was analyzed for lin-c-
kit+sca-1+ HSC by
flow cytometry at two weeks post-transplant. Figs. 37A-D depict percent of
common
lymphoid precursors in the BM. Two weeks after BMT transplantation and
castration there is
a significantly increased proportion of donor-derived HSCs in the BM of
castrated mice
compared to sham castrated controls.
Figs. 38A-B: Castration enhances the rate of engraftment of donor-derived DC
in the
thymus following congenic HSCT (2.5x106 cells and 5x106 cells). 5x106 Ly5.1+
BM cells
were injected intravenously into irradiated (800 rails) C57lBL6 mice.
Thymocytes were
analyzed by flow cytometry at two weeks post-transplant (Fig. 38A). Donor-
derived DC
were defined as CD45.1+CDllc+MHC class II+ CDllb+~r-_ Donor-derived CD1 1b+
and
CDllb- DC are significantly increased'in the thymii of castrated mice compared
to sham-
castrated controls 2 weeks after BMT (Fig. 38B).
Figs. 39A-D: Castration enhances the rate of engraftment of donor-derived B
cells in
the spleen following congenic HSCT. 5x106 Ly5.1+ BM cells were injected
intravenously
into irradiated (800 rails) C57/BL6 mice. Splenocytes were analyzed by flow
cytometry at
two weeks post-transplant (Fig. 39A-C). There are significantly more B220+ B
cells in the
spleens of castrated mice, as compared the sham-castrated controls, 2 weeks
after congenics
BMT (Fig. 39D).
Fig. 40: The phenotypic composition of peripheral blood lymphocytes was
analyzed
in human patients (all >60 years) undergoing LHRH agonist treatment for
prostate cancer.
Patient samples were analyzed before treatment and 4 months after beginning
LHRH agonist
treatment. Total lymphocyte cell numbers per ml of blood were at the lower end
of control
values before treatment in all patients. Following treatment, six out of nine
patients showed
substantial increases in total lymphocyte counts (in some cases a doubling of
total cells was
observed). Correlating with this was an increase in total T cell numbers in
six out of nine
patients. Within the CD4~" subset, this increase was even more pronounced with
eight out of
nine patients demonstrating increased levels of CD4 T cells. A less
distinctive trend was seen
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within the CD8'~ subset with four out of nine patients showing increased
levels, albeit
generally to a smaller extent than CD4+ T cells.
Fig. 41: Analysis of human patient blood before and after LHRH-agonist
treatment
demonstrated no substantial changes in the overall proportion of T cells, CD4
or CD8 T cells,
and a variable change in the CD4:CD8 ratio following treatment. This indicates
the minimal
effect of treatment on the homeostatic maintenance of T cell subsets despite
the substantial
increase in overall T cell numbers following treatment. All values were
comparative to
control values.
Fig. 42: Analysis of the proportions of B cells and myeloid cells (NK, NKT and
macrophages) within the peripheral blood of human patients undergoing LHRH
agonist
treatment demonstrated a varying degree of change within subsets. While NK,
NKT and
macrophage proportions remained relatively constant following treatment, the
proportion of
B cells was decreased in four out of nine patients.
Fig. 43: Analysis of the total cell numbers of B and myeloid cells within the
peripheral blood of human patients post-treatment showed clearly increased
levels of NK.
(five out of nine patients), NKT (four out of nine patients) and macrophage
(three out of nine
patients) cell numbers post-treatment. B cell numbers showed no distinct trend
with two out
of nine patients showing increased levels; four out of nine patients showing
no change and
three out of nine patients showing decreased levels.
Figs. 44A-B: Chemical castration in humans enhances nai.'ve and memory T
cells. As
shown in Fig. 44A, a significant increase in naive (CD62L+CD45RA+CD45R0-) CD4+
T
cells was observed following LHRH-A treatment. As shown in Fig. 44B, both
naive and
memory (CD62L-CD45RA-CD45R0+) CD8+ T cells numbers were enhanced following the
LHRH agonist treatment. Each bar represents the mean~1SD of 16 patients. * =
p,<0.05; ** _
p<_0.01 compared to pre-treatment values.
Fig. 45A-B: Chemical castration in humans enhances peripheral blood lymphocyte
numbers. The phenotypic composition of peripheral blood was analyzed in human
patients
(all >60years of age) undergoing chemical castration with a LHRH-A as part of
their routine
treatment for prostate cancer. Patients were analysed prior to treatment and
at 4-months of
treatment. As shown in Fig. 45A, total lymphocyte number per p,1 peripheral
blood was
significantly increased following LHRH-A treatment. T his was reflected by a
significant
increase in total T cells, CD4+ and CD8~" T cells (Fig 45B).
31
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Fig. 46A-B: LHRH-A treatment effectively depletes serum testosterone, and
increases thymic function and T cell export. In the Fig. 46A experiment,
prostate cancer
patients were treated with LHRH-A for 4 months. Blood was analyzed by FACS and
serum
was analyzed by RIA both prior to treatment and following 4-months of LHRH-A
treatment.
As shown in Fig. 83A, no testosterone was detected in patient sera at 4-months
of LHRH-A
treatment. The bar represents the mean of 13 patients analyzed. In the Fig 46B
experiment,
direct evidence for an increase in thymic function and T cell export was found
following
analysis of TREC levels in 10 patients. Within both the CD4+ and CD8+ T cell
population,
five out of ten patients showed an increase (>25% above initial presentation
values) in
absolute TREC levels (per ml of blood) by 4 months of LHRH-A treatment. This
was also
reflected in a proportional increase (per 1x105 cells; data not shown). This
correlated with six
out of ten patients showing an overall increase in total TREC levels. Only 1
patient showed a
decrease in total TRECs (about 30°lo decrease).
Fig. 47: Chemical castration in humans enhances NK numbers. Analysis was
performed prior to LHRH-A treatment and at 4-months of treatment. A
significant increase
in NK cells, but not B cells, was observed with LHRH-A treatment. Results are
presented as
the mean~1SD of 13 patients. ** = p_<0.01 compared to pre-treatment values.
Figs. 48A-B: Chemical castration in humans does not increase proliferation of
T
cells. Figs. 48A-B depict analyses of cellular proliferation performed using
Ki-67 antigen
detection. In all patients, levels of proliferation within naive, activated
and memory cell
subsets for both CD4+ (Fig. 48A) and CD8+ T cells (Fig. 48B), was not altered
with LHRH-A
treatment.
Figs. 49A-C: Analysis of natural killer (NK) cell recovery at various time
points (2-8
weeks) following HSCT in control patients and LHRH-A treated patients. As
shown in Figs.
49A-B, respectively, a similar trend was observed for both control allogeneic
and autologous
transplant recipients. In contrast, allogeneic patients who were given LHRH-A
treatment 3
weeks prior to HSCT showed a significantly higher number of NKT cells from D14-
5M post-
transplant (Fig. 49C; data is expressed as mean ~ 1 SBM of 6-20 patients;
*=p_0.05).
Fig. 50: FACS analysis of NKT cell reconstitution at various time points (day
14, 21,
28 and 35) following HSCT in control patients. An early recovery was observed
in
allogeneic patients, and was seen predominantly within the CD8+ population
early post-
transplant, which indicated extrathymic routes of regeneration. Also, CD4+NKT
cells were
evident from 1 month post-transplant.
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Figs. 51A-B: B cell reconstitution following HSCT at various time points (2-12
months) following HSCT in control patients. As shown in Fig. 51B, B cell
regeneration
occurs occurring relatively faster in autologous transplant patients as
compared to that of
allogeneic patients (Fig. 51A). However, a return to control values (shaded)
was not evident
until at least 6 months post-transplant in both groups.
Figs. 52A-B: CD4+ reconstitution following HSCT at various time points (2-12
months) following HSCT in control patients. While B cell numbers were
returning to control
values by 6 months post-transplant (see Figs. 48A-B), CD4+ T cell numbers were
severely
reduced, even at 12 months post-transplant, in both autologous (Fig. 52B) and
allogeneic
(Fig. 52A) recipients.
Figs. 53A-C: CD8+ regeneration following HSCT at various time points (2-12
months) following HSCT in control patients. As shown in Fig. 53A-B, CD8+ T
cell
numbers regenerated quite rapidly post-transplant in both allogeneic and
autologous
recipients, respectively. However, as shown in Fig. 53C, the CD8+ T cells are
mainly of
a
extrathymic origin as indicated by the increase in TCRyB+ T CD8+T cells, CDBaa
T cells,
and CD28-CD8+ T cells.
Figs. 54A-B: FAGS analysis of proliferation in various populations of CD4+ and
CD8+ T cells before (Fig. 54A) and 28 days after (Fig. 54B) HSCT in control
patients using
the marker Ki-67. Cells were analyzed on the basis of naive, memory and
activated
phenotypes using the markers CD45R0 and CD27. The majority of proliferation
occurred in
CD8+ T cell subset, which further indicated that these cells were
extrathymically derived and
that the predominance of proliferation occurred within peripheral T cell
subsets.
Figs. SSA-D: Naive CD4+ T cell regeneration at various time points (2-12
months)
following HSCT in control patients and LHRH-A treated patients. Fig. 55A
depicts FACS
analysis of naive CD4+ T cells (CD45RA+CD45R0-CD62L+) in control (no LHRH-A
treatment) patients, and shows a severe loss of these cells throughout the
study. As shown in
Figs. 55B-C, naive CD4+ T cell began to regenerate in the control patients by
12 months
post-HSCT in autologous transplant patients (Fig. 55C) but were still
considerably lower than
the control values in allogeneic control patients (Fig. 55B). These results
indicated that the
thymus was unable to restore adequate numbers of naive T cells in control
patients post-
transplant due to the age of the patients. In contrast, in patients that were
given LHRH-A 3-
weeks prior to allogeneic HSCT showed a significantly higher number of naive
CD4+ T cells
at both 9 & 12 months post-transplant compared to controls (Fig. 55D). This
indicates
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enhanced regeneration of the thymic-dependent T cell pathway with sex steroid
ablation
therapy. Data is expressed as mean~1SEM of 6-20 patients. *=p<0.05.
Figs. 56A-D: TREC levels at various time points (1-12 months) following HSCT
in
control patients and LHRH-A treated patients. Analysis of TREC levels, which
are only seen
in recent thymic emigrants (RTE), emphasized the inability of the thymus to
restore levels
following transplant in both allogeneic (Fig. 52A) and autologous (Fig. 52B)
patients. Again,
this was due to the age of the patients, as well as the lack of thymic
function due to thymic
atrophy, which has considerable implications in the morbidity and mortality of
these patients.
In contrast, patients undergoing allogeneic peripheral blood stem cell
transplantation
demonstrated a significant increase in CD4+TREC+ cells/ml blood when treated
with an
LHRH-A prior to allogeneic transplantation (p<_0.01 at 9 months post-
transplant compared to
control (non-LHRH-A treated). Allogeneic patients who were given LHRH-A
treatment
showed a significantly higher number of CD4+TREC+ cells/ml blood at 9 months
post-
transplant (Fig. 56C) compared to controls. Autologous LHRH-A treated patients
also
showed significantly higher levels at 12 months post-transplant (Fig. 56D).
This indicates
enhanced regeneration of the thymus with sex steroid ablation therapy. Data is
expressed as
mean ~ 1 SEM of 5-18 patients. *=p_0.01.
Fig. 57A-C: LHRH-A administration enhances responsiveness to TCR specific
stimulation following allogeneic (Fig. 57A-B) and autologous (Fig. 57C) HSCT.
Three
weeks prior to HSCT, patients were given LHRH-A. Patients who did not receive
the agonist
were used as control patients. Analysis of TCR specific stimulation was
performed using 5 p,g
anti-CD3 and 10 p,g anti-CD28 cross-linking at various time points (1-12
months) post-
transplant. As shown in Figs. 57A-B, allogeneic LHRH-A treated patients showed
enhanced
proliferative responses (assessed by 3H-Thynudine incorporation) compared to
control
patients at all time-points except 6 and 9 months (due to low patient numbers
analyzed at this
time). At 6 and 9 months post-transplant control patients had similar
responsiveness to pre-
treatment values. However at all other time-points, they were considerably
lower. In
contrast, LHRH-A treated patients had equivalent responsiveness at all time-
points except 6
months compared to pre-treatment. LHRH-A treated patients showed enhanced
proliferative
responses (assessed by 3H-Thymidine incorporation) compared to control
patients at 1, 3 and
4 months post-transplant. This indicates a contribution of direct peripheral T
cell effects, as
new CD4+ T cells are not evident until at least 1-2 months post-transplant
(Fig. 57B; Data is
expressed as mean~1SEM of 5-12 patients. *=p<_0.05; 'f'*=p<0.01). As shown in
Fig. 57C,
autologous LHRH-A treated patients showed enhanced proliferative responses
(assessed by
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'H-Thymidine incorporation) compared to control patients at all time-points
except 5 months.
Restoration to pre-treatment values was observed by 12 months post-transplant
in both
control and LHRH-A treated patients.
Fig. 5$A-B: LHRH-A administration enhances responsiveness to PWM and TT
mitogenic stimulation following allogeneic HSCT. Three weeks prior to HSCT,
patients
were treated with LHRH-A. Patients who did not receive the agonist were used
as control
patients. Analysis of mitogenic responsiveness was performed using pokeweed
mitogen
(PWM) or tetanus toxoid (TT) at various time points (1-12 months) post-
transplant. Patients
treated with LHRH-A prior to HSCT showed an enhanced responsiveness to PWM
(Fig.
58A) and TT (Fig. 58B) stimulation at all time-points studied compared to
control patients.
Fig. 59A-B: LHRH-A administration enhances responsiveness to PWM and TT
mitogenic stimulation following autologous HSCT. Three weeks prior to HSCT,
patients
were treated with LHRH-A. Patients who did not receive the agonist were used
as control
patients. Analysis of mitogenic responsiveness was performed using PWM or TT
at various
time point (1-12 months) post-transplant. Patients treated with LHRH-A prior
to HSCT
showed an enhanced responsiveness to PWM (Fig. 59A) and TT (Fig. 59B)
stimulation at the
majority of time-points studied compared to control patients (p<_0.001 at 3
months). By 12-
months post-transplant, LHRH-A treated patients had restored responsiveness.
Figs. 60A-D. LHRH-A treatment enhances the rate of engraftment in autologous
HSCT patients. Three weeks prior to HSCT, patients were treated with LHRH-A
(Figs. 60A,
C and D). Patients who did not receive the agonist were used as control
patients (Figs. 60B).
Total white blood cell (WBC) counts and granulocyte (G) counts per ~,1 of
blood were
determined at days 14, 28, and 35 post transplant. As shown in Fig. 60A,
autologous
patients who were given LHRH-A treatment showed a significantly higher number
of WBC
at D14 post-transplant compared to controls (Fig. 60B) (p<0.05), with 87%
showing
granulocyte engraftment (?500 cellslwl blood) compared to 45% of controls
(p<0.05) at this
time point. Autologous patients who were given LHRH-A treatment also showed a
significantly higher number of neutrophils at D10-12 post-transplant compared
to controls
(Fig. 60C; data is expressed as mean~1SEM of 8-20 patients. ~'=p<0.05). In
addition,
although not significant, autologous patients had higher lymphocyte counts
throughout the
time-points analyzed in LHRH-A treated compared to control group (Fig. 60D).
Figs. 61A-D: LHRH-A treatment enhances the rate of engraftment in allogeneic
HSCT patients. Three weeks prior to HSCT, patients were treated with LHRH-A
(Figs. 61A,
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C and D). Patients who did not receive the agonist were used as control
patients (Fig. 61B).
Total white blood cell (WBC) counts and granulocyte (G) counts per ~,1 of
blood were
determined at day 14, 28, and 35 post transplant. As shown in Fig. 61A,
allogeneic patients
who were given LHRH-A treatment showed a significantly higher number of WBC at
D14
post-transplant compared to controls (Fig. 61B) (p_0.05) with 64~o showing
granulocyte
engraftment (>_500 cellslwl blood) compared to 44°70 of controls at
this time point. In
addition, allogeneic patients who were given LHRH-A treatment showed a
significantly
higher number of neutrophils at D9, 12 & 19 post-transplant compared to
controls (Fig. 61C;
data is expressed as mean~1SEM of 8-20 patients. ~=p<_0.05). Additionally,
analysis of
patients undergoing peripheral blood stem cell transplantation demonstrated a
significant
increase in lymphocyte counts when treated with an LHRH-A prior to allogeneic
transplantation (p<0.05 at days 10, 12, 13 & 17-21 post-transplant) (Fig.
61D).
Figs. 62A-F. TCR specific peripheral T cell proliferative responses are
enhanced
within one week of castration. Eight week-old mice were castrated and analyzed
for anti-
CD3/anti-CD28 stimulated T cell proliferative response 3 days (Figs. 62A, C,
and E) and 7
days (Figs. 628, D, and F) after surgery. Peripheral (cervical, axillary,
brachial and inguinal)
lymph node (Figs. 62A and B), mesenteric lymph node (Figs. 62C and D), and
spleen cells
(Figs. 62E and F) were stimulated with varying concentrations of anti-CD3 and
co-stimulated
with anti-CD28 at a constant concentration of 10 ~tglml for 48 hours. Cells
were then pulsed
with tritiated thymidine for 18 hours and proliferation was measured as 3H-T
incorporation.
Diamonds indicate castrated animals. Squares indicate sham-castrated control
mice. n=4,
*p<0.05 (non-parametric, unpaired, Mann-Whitney statistical test).
Fig. 63: LHRH-A administration enhances responsiveness to TCR specific
stimulation following treatment for chronic cancer sufferers. Patients with
chronic
malignancies were treated with LHRH-A. Analysis of TCR specific stimulation
was
performed using anti-CD3 and anti-CD28 cross-linking from at various time
points (day 7 -
12 months) following LHRH-A administration. LHRH-A treated patients showed
enhanced
proliferative responses (assessed by 3H-Thymidine incorporation) compared to
pre-treatment
levels in a cyclical fashion. This reflected the administration of the agonist
with monthly
depot injections. These results indicate a direct influence on peripheral T
cells. However, the
enhanced response seen at 12-months post-treatment reflect changes in thymic-
derived T
cells as well, since agonist administration was ceased from 4-months for all
patients.
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Figure 64 is a line graph showing that while 60% of the sham-operated mice had
diabetes, fewer than 20% of the castrated group had diabetes.
Figure 65 is a bar graph showing that castrated NOD mice had a marked increase
in
total thymocyte number but no differences in total spleen cells.
Figures 66A-C are bar graphs showing that there was a significant increase in
all
thymocyte subclasses (Fig. 66A) in castrated NOD mice. There no change in B
cells
compared to sham-castrated NOD mice (Fig. 66C) nor in the total T or B cells
in the spleen
(Fig. 66B).
Figures 67A and 67B show a marked in total thymocytes (Fig. 67A) and spleen
cells
(Fig. 68B) in castrated NZB mice.
Figure 68 is a graph showing decreased tumor incidence in mice that have been
castrated and immunized as compared to controls.
Figures 69A-C are bar graphs showing that castrated and immunized mice have
increased splenic cellularity as compared to controls.
Figures 70A-B are graphs showing increased yIFN production in mice that have
castrated and immunized as compared to controls.
Figures 71A-B are graphs showing that castrated and immunized mice exhibit
enhanced antigen-specific CTL responses as compared to controls.
Figures 72A-E are graphs showing that thymectomy does not impact the effect of
sex
steroid inhibitionBMT on common lymphoid progenitors in the BM (Fig. 72A),
total BM B
cells (Fig. 72B), immature B cells in the BM (Fig. 64C), total cell numbers in
the spleen (Fig.
72D) or on total B cells in the spleen (Fig. 72E).
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DETAILED DESCRIPTION OF THE INVENTION
The patent and scientific literature referred to herein establishes knowledge
that is
available to those with skill in the art. The issued U.S. patents,
applications, published
foreign applications, and references, including GenBank database sequences,
that are cited
herein are hereby incorporated by reference in its entirety to the same extent
as if each was
specifically and individually indicated to be incorporated by reference.
The present invention comprises methods for increasing the BM functionality
following sex steroid ablation and/or interruption of sex steroid signalling,
either without,
prior to, or in combination with, thymus regeneration. "Increasing the
function of BM" and
"enhancing BM functionality" is herein defined as an improvement in the
production andlor
output of immune cells, including precursors, for example HSC (and consequent
inci°eases in
blood cells) from the BM, including improvement in haemopoiesis and/or
enhancement of
engraftment following HSCT. An improvement in output may include, but is not
limited to,
an improved ability to mobilize immune cells, including HSC into the periphery
or to a target
tissue, in particular, immune or damaged tissue). In one embodiment, HSC
haemopoiesis is
improved. In another embodiment, HSC output is improved. In certain
embodiments, blood
and/or immune cell numbers are increased. In yet another embodiment, HSC
engraftment is
improved following HSCT. In another embodiment HSC mobilization into the
periphery or
homing to target tissue is improved. In yet another embodiment, proliferative
ability, andlor
the ability to differentiate into haematopoietic or non-haemopoitetic progeny
is improved.
The present invention also comprises methods for increasing the function of T
cells
and other immune cells following sex steroid ablation and/or interruption of
sex steroid
signaling, either without, prior to, or in combination with, thymus
regeneration. The terms
"immune cells" and "cells of the immune system" are used interchangeably and
are herein
defined as HSC, T cells, B cells, DC, and/or other blood cells, including, but
not limited to
HSC progeny, CLP, MLP, lymphocytes, myeloid cells, neutrophils, granulocytes,
basophils,
eosinophils, NK, NKT, platelets, red blood cells, monocytes, macrophage, naive
T cells, and
precursors of the aforementioned. The cells may or may not be peripheral, and
the cells may
be found in any one or more of the BM, blood, spleen, lymph nodes, thymus.
mucosal
membranes, skin, or other tissues.
"Increased" or "enhanced functionality" of immune cells means that the immune
cells
are more able to provide an adequate required immune response, when compared
to the
immune response normally expected without sex steroid ablation. In one case,
the immune
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cells are T cells. In other examples, the immune cells are B cells, DC, and/or
HSC.
"Increased functionality" includes, but is not limited to, improved killing of
target cells;
increased lymphocyte proliferative response; improved signaling ability;
improved homing
ability; improved APC activation, increased levels or activity of receptors,
cell adhesion
molecules, or co-stimulatory molecules; decreased apoptosis; increased release
of cytokines,
interleukins, and other growth factors; increased levels of antibody (Ab) in
the plasma; and
increased levels of innate immunity (e.g., natural killer (NIA) cells, DC,
neutrophils,
macrophages, etc.) in the blood and throughout the body. Each may either
directly or
indirectly assist in combating disease and infection, thereby increasing
responsiveness to,
resistance to, treatment for, and prevention of, e.g" infection by various
foreign agents, and
increasing immune responsiveness to vaccines.
The present invention further comprises methods for preventing, diminishing
the risk,
or treating illness or disease in a patient. In one embodiment, the disease is
a T cell disorder.
In another embodiment, the disease is an autoimmune disease or allergy.
Additionally, the
present disclosure also provides methods for improving a patient's immune
response to a
vaccine antigen (e.g., that of an agent) by disrupting sex steroid mediated
signaling and
causing the thymus to reactivate. In both cases, the functional status of the
peripheral T cells
may be improved and may be accomplished by quantitatively and qualitatively
restoring the
peripheral T cell pool, particularly at the level of naive T cells. These
naive T cells are then
able to respond to a greater degree to presented foreign antigen.
As described above, the aged (post-pubertal) thymus causes the body's immune
system to function at less than peak levels (such as that found in the young,
pre-pubertal
thymus). "Post-pubertal" is herein defined as the period in which the thymus
has reached
substantial atrophy. In humans, this occurs by about 20-25 years of age, but
may occur
earlier or later in a given individual. "Pubertal" is herein defined as the
time during which
the thymus begins to atrophy, but may be before it is fully atrophied. In
humans this occurs
from about 10-20 years of age, but may occur earlier or later in a Given
individual. "Pre-
pubertal" is herein defined as the time prior to the increase in sex steroids
in an individual. In
humans, this occurs at about 0-10 years of age, but may occur earlier or later
in a given
individual.
The terms "vaccinating," "vaccination," "vaccine, " "immunizing,"
"immunization,"
are herein defined as administration to a patient of a preparation to elicit
an immune response
to an antigen. Vaccination may include both prophylactic and therapeutic
vaccines. As will
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be understood by those skilled in the art, infection by, e.g., a virus or
other agent is also a
method of vaccinating an individual.
The terms "improving," "enhancing," or "increasing" "vaccine responses" or
"vaccine
responsiveness" in a patient or "improving," "enhancing," or "increasing" the
"immune
responsiveness of a patient to a vaccine" and other similar language is used
interchangeably
and herein defined as meaning that a patient's immune response to the vaccine
or vaccine
antigen is improved compared to the immune response which would have otherwise
occurred
in a patient without disruption of sex steroid signaling.
"Illness" and "disease" are used interchangeably and are herein defined as any
disease,
infection or medical condition (symptomatic or asymptomatic) in which an
immune response,
defence or modified immune system would be beneficial to the patient. The
illness may be
caused by an infectious agent, cancer, drug treatment (e.g., chemotherapy),
irradiation,
chemical poisoning, genetic defect or other disorder.
As herein defined, "prevention" of or "preventing" an illness is herein
defined as
complete as well as partial protection including without limitation reduced
severity of clinical
symptoms than would have otherwise occurred in the patient. With an improved
or modified
immune system the individual will have a reduced likelihood of succumbing to,
or suffering
from, a tumor or cancer, allergy, autoimmune diseases, a prevailing infection
(e.g., viral,
bacterial, fungal, or parasitic) or illness, and/or will show better responses
to a vaccination
(e.g., increased levels of antibody (Ab) specific to that vaccine or antigen,
and development
of effector T cells). Prevention of an illness may occur by activating or
modifying immune
defense mechanisms to inhibit or reduce the development of clinical symptoms,
such as to a
point where only reduced or minimal medical care is required. Preventing an
infection also
encompasses defending the body against infectious agents, such as viruses,
bacteria,
parasites, fungi, etc. or against non-infectious agents. This may take the
form of preventing
such agents from entering the cells in the body and/or the efficient removal
of the agents by
cells of the broad immune system (e.g., NK, DC, macrophages, neutrophils,
etc.). In some
instances complete prevention of illness is not achieved, and instead partial
prevention is
achieved in which a stronger, more resilient or more effective immune system
will aid the
body in decreasing the extent, severity and duration of illness or clinical
symptoms of illness
or recovery time or delay the onset of clinical symptoms.
"Treatment" of or "treating" an illness encompasses completely or partially
reducing
the symptoms of the illness in the patients, as compared to those symptoms
that would have
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otherwise occurred in the patient without sex steroid ablation or interruption
of sex steroid
mediated signaling. Treatment of an illness may occur by activating immune
defense
mechanisms to inhibit, delay or reduce the development of clinical symptoms.
In one
example, the patient has already contacted the agent, or is at a high risk of
doing so.
The ability to have improved response to, respond better to, or to overcome, a
new (by
prevention) or existing (by treatment) illness involves improving the immune
system of the
body, which includes increasing the number and/or functionality of the BM
cells and/or
thymic-derived factors, and/or increasing the number and/or functionality of
immune cells.
Activation of the immune system also increases the number of lymphocytes
capable of
responding to the antigen of the agent in question, which leads to the
elimination (complete
or partial) of the antigen and/or foreign agent creating a situation where the
host is treated for
or resistant to the infection or disease. With an improved or modified immune
system the
individual will have a reduced likelihood of succumbing to or suffering from a
tumor or
cancer, allergy, autoimmune diseases, a prevailing infection (e.g., viral,
bacterial, fungal, or
parasitic) or illness, and/or will show better responses to a vaccination
(e.g., increased levels
of antibody (Ab) specific to that vaccine or antigen, and development of
effector T cells).
This increase in the immune defense was exemplified in the dramatic
improvement of
aged mice to the human herpes simplex virus infection (see Example 3, and
Figs. 13-17).
The castrated aged mice initially showed a marked increase of lymphocyte
infiltration into
the draining lymph node. This infiltration is the first step in an immune
response, and is
generally required to increase the likelihood of an antigen-specific
lymphocyte contacting the
antigen. The next step is the activation of the lymphocytes by antigen and the
development
of Ab and/or CTL and release of cytokines from lymphocytes, all of which
combine to
destroy the agent.
"Infectious agents," "foreign agents," and "agents" are used interchangeably
and
include any cause of disease or illness in an individual. Agents include, but
are not limited
to viruses, bacteria, fungi, parasites, prions, cancers, precancerous cells,
chemical or
biological toxins, allergens, asthma-inducing agents, self proteins and
antigens which
contribute to autoimmune disease, etc.
. In one case, the agent is a virus, bacteria, fungi, or parasite e.g., from
the coat protein
of a human papilloma virus (HPV), which causes uterine cancer; or an influenza
peptide (e.g.,
hemagglutinin (HA), nucleoprotein (NP), or neuraminidase (N)).
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Non-limiting examples of infectious viruses include: Retroviridae (e.g., human
immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or
HTLV-
III/LAV, or HIV-III) and other isolates, such as HIV-LP; Picornaviridae (e.g.,
polio viruses,
hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses,
echoviruses);
Calciviridae (e.g., strains that cause gastroenteritis); Togavixidae (e.g.,
equine encephalitis
viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis
viruses, yellow fever
viruses); Coronaviridae (e.g., coronaviruses, severe acute respiratory
syndrome (SARS)
virus); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses);
Filoviridae (e.g.,
ebola vimses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus,
measles virus,
respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses);
Bungaviridae (e.g.,
Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena
viridae
(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and
rotaviruses);
Birnaviridae; Hepadnaviridae (e.g., Hepatitis B virus); Parvoviridae
(parvoviruses);
Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most
adenoviruses);
Herpesviridae (e.g., herpes simplex virus (HSV) 1 and 2, varicella zoster
virus,
cytomegalovirus (CMV), herpes viruses); Poxviridae (e.g., variola viruses,
vaccinia viruses,
pox viruses); and Iridoviridae (e.g., African swine fever virus); and
unclassified viruses (e.g.,
the etiological agents of Spangiform encephalopathies, the agent of delta
hepatitis (thought to
be a defective satellite of hepatitis B virus), the agents of non-A, non-B
hepatitis (class
1=intetually transmitted; class 2=parenterally transmitted (i.e., Hepatitis
C); Norwalk and
related viruses, and astroviruses).
Non-limiting.examples of infectious bacteria include: Helicobacter pyloric,
Bnrelia
burgdorferi, Legionella pneumoplailia, Mycobacteria sporozoites (cp.) (e.g.,
M. tuberculosis,
M. avium, M. intracellulare, lVl. kansaii, M. gordonae), Staphylococcus
aureus, Neisseria
gonorrhoeae, Neisseria meuingitidis, Listeria morcocytogenes, Streptococcus
pyogenes
(Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus
(anaerobic sps.),
Streptococcus pnemnoniae, pathogenic Campylobacter cp., Enterococcus cp.,
Haemophilus
influenzae, Bacillus afitracis, Cofynebacterium diphtheri.ae,
Corynebacteriurrz sp.,
Erysipelothrix rhusiopathiae, Clostridium perfrifagens, Clostridium teta~i,
Enterobacter
aerogenes, Klebsiella pneumoiaiae, Pasturella multocida, Bacteroides sp.,
Fusobacteriunt
rzucleatum, Streptobacillus mofiilifornais, Treponema pallidium, Trepotaema
pertefaue,
Leptospira, and Actinomyces israelli.
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WO 2004/103271 PCT/US2004/011921
Non-limiting examples of infectious fungi include: Cryptococcus raeoformah.s,
Histoplasma capsulatuna, Coccidioides immitis, Blastonayces dernaatitidis,
Chlamydia
trachomatis, Cahdida albicans.
Other infectious organisms (i.e., protists) include, but are not limited to,
Plasi7aodium
falciparum and Toxoplasrraa gorZdii.
In other embodiments, the agent is an allergen. Allergic conditions include,
but are
not limited to, eczema, allergic rhinitis or coryza, hay fever, bronchial
asthma, urticaria
(hives) and food allergies, and other atopic conditions.
In yet another embodiment, the agent is a cancer or tumor. The cancer or tumor
may
be malignant or non-malignant. As used herein, a tumor or cancer includes,
e.g., tumors of
the brain, lung (e.g., small cell and non-small cell), and pleura,
gynecological, urogenital and
endocrine system, ( e.g., cervix, uterus, endometrium, bladder, renal organs,
ovary, breast,
and/or prostate), gastrointestinal tract (e.g., anal, bile duct, carcinoid
tumor, gallbladder,
gastric or stomach, liver, esophagus, pancreas, rectum, small intestine,
and/or colon), as well
as other carcinomas, and bone, skin and connective tissue (e.g., melanomas
and/or sarcomas),
andlor the hematological system (e.g., blood, myelodysplastic syndromes,
myeloproliferative
disorders, plasma cell neoplasm, lymphomas andlor leukemias).
This invention may be used with any animal species (including humans) having
sex
steroid driven maturation and an immune system, such as mammals and
marsupials. In some
examples, the invention is used with large mammals, such as humans.
The terms thymus "regeneration," "reactivation" and "reconstitution" and their
derivatives are used interchangeably herein, and are herein defined as the
recovery of an
atrophied or damaged (e.g., by chemicals, radiation, graft versus host
disease, infections,
genetic predisposition) thymus to its active state. "Active state" is herein
defined as meaning
a thymus in a patient whose sex steroid hormone mediated signaling has been
disrupted,
achieves an output of T cells that is at least 10%, or at least 20%, or at
least 40%, or at least
60%, or at least 80%, or at least 90% of the output of a pre-pubertal thymus
(i.e., a thymus in
a patient who has not reached puberty).
"Recipient," "patient" and "host" are used interchangeably and are herein
defined as
a subject receiving sex steroid ablation therapy and/or therapy to interrupt
sex steroid
mediated signaling and/or, when appropriate, the subject receiving the HSC
transplant.
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WO 2004/103271 PCT/US2004/011921
"Donor" is herein defined as the source of the transplant, which may be
syngeneic, allogeneic
or xenogeneic. In some instances, the patient may provide, e.g., his or her
own autologous
cells for transplant into the patient at a later time point Allogeneic HSC
grafts may be used,
and such allogeneic grafts are those that occur between unmatched members of
the same
species, while in xenogeneic HSC grafts the donor and recipient are of
different species.
Syngeneic HSC grafts, between matched animals, may also be used. The terms
"matched,"
"unmatched," "mismatched," and "non-identical" with reference to HSC grafts
are herein
defined as the MHC ard/er minor histocompatibility markers of the donor and
the recipient
are (matched) or are not (unmatched, mismatched and non-identical) the same.
Throughout this specification the word "comprise", or variations such as
"comprises"
or "comprising", will be understood to imply the inclusion of a stated
element, integer or
step, or group of elements, integers or steps, but not the exclusion of any
other element,
integer or step, or group of elements, integers or steps.
DISRUPTION OF SEX STEROID MEDIATED SIGNALLING
The present invention further provides methods of disruption of sex steroid-
mediated
signaling in a patient, wherein the patient's thymus may or may not be
subsequently
reactivated. Additionally, the present invention provides methods of improving
the
functional status of immune cells (e.g., T cells) of the patient. With respect
to T cells, the
thymus begins to increase the rate of proliferation of the early precursor
cells (CD3-CD4-
CD8- cells) and converts them into CD4+CD8+, and subsequently new mature
CD3h'CD4+CD8- (T helper (Th) lymphocytes) or CD3h'CD4-CD8+ (cytotoxic T
lymphocytes
(CTL)). The rejuvenating thymus also increases its uptake of HSC, or other
stem cells or
progenitor cells capable of forming into T cells, from the blood stream and
converts them
into new T cells and intrathymic DC. The increased activity in the thymus
resembles in
many ways that found in a normal younger thymus (e.g., a prepubertal patient).
The result of
this renewed thymic output is increased levels of naive T cells (those T cells
which have not
yet encountered antigen) in the blood: There is also an increase in the
ability of the
peripheral T cells to respond to stimulation, e.g., by cross-linking with anti-
CD28 Abs, or by
TCR stimulation with, e.g., anti-CD3 antibodies, or stimulation with mitogens,
such as
pokeweed mitogen (PWM) and this increased T cell responsiveness can occur
before thymic
regeneration, such as within 2, 3, 4, 5 6, 7, 14 or 21 days. This combination
of events results
in the body becoming better able to defend against infection and other immune
system
challenges (e.g., cancers), or recover from immune system challenges (e.g.,
becoming better
44
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WO 2004/103271 PCT/US2004/011921
able to recover from chemotherapy and radiotherapy). As a result, the methods
of the
invention may be used to prevent or treat an illness or infection, increase a
patient's immune
responsiveness to a vaccine, and for optional gene therapy.
As used herein, "sex steroid ablation," "inhibition of sex steroid-mediated
signaling,"
"sex steroid disruption" "interruption of sex steroid signaling" and other
similar terms are
herein defined as at least partial disruption of sex steroid (and/or other
hormonal) production
and/or sex steroid (and/or other hormonal) signaling, whether by direct or
indirect action. In
one embodiment, sex steroid signaling to the thymus is interrupted. As will be
readily
understood, sex steroid-mediated signaling can be disrupted in a range of ways
well known to
those of skill in the art, some of which are described herein. For example,
inhibition of sex
hormone production or blocking of one or more sex hormone receptors will
accomplish the
desired disruption, as will administration of sex steroid agonists and/or
antagonists, or active
(antigen) or passive (antibody) anti-sex steroid vaccinations.
A non-limiting method for creating disruption of sex steroid-mediated
signalling is
through castration. Methods for castration include, but are not limited to,
chemical castration
and surgical castration.
"Castration" is herein defined as the reduction or elimination of sex steroid
production, action and/or distribution in the body. This effectively
eventually returns the
patient to a pre-pubertal status when the thymus is more fully functioning
than immediately
prior to castration. Surgical castration removes the patient's gonads. Methods
for surgical
castration are well known to routinely trained veterinarians and physicians.
One non-limiting
method for castrating a male animal is described in the examples below. Other
non-limiting
methods for castrating human patients include a hysterectomy or ovariectomy
procedure (to
castrate women) and surgical castration to remove the testes (to castrate
men). In some
clinical cases, permanent removal of the gonads via physical castration may be
appropriate.
Chemical castration is a less permanent version of castration. As herein
defined,
"chemical castration" is the administration of a chemical for a period of
time, which results
in the reduction or elimination of sex steroid production, action andlor
distribution in the
body. A variety of chemicals are capable of functioning in this manner. Non-
limiting
examples of such chemicals are the sex steroid inhibitors and/or analogs
described below.
During the chemical delivery, and for a period of time afterwards, the
patient's hormone
production may be turned off or reduced. The castration may be reversed upon
termination
of chemical delivery or by delivery of the relevant sex hormones.
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WO 2004/103271 PCT/US2004/011921
The terms "sex steroid analog," "sex steroid ablating agent," "sex steroid
inhibitor,"
"inhibitor of sex steroid signalling," "modifier of sex steroid signalling,"
and other similar
terms are herein defined as any one or more pharmaceutical agent that will
decrease, disrupt,
prevent, or abolish sex steroid (and/or other hormone) mediated signalling.
GnRH (also
called LHRH or GnRH/LHRH herein), and analogs thereof, are nonlimiting
exemplary
inhibitors of sex steroid signalling used throughout this application.
However, as will be
readily understood by one skilled in the art, in practicing the inventions
provided herein,
GnRH/LHRH, or analogs thereof, may be replaced with any one (or more) of a
number of
substitute sex steroid inhibitors or analogs (or other blocker(s) or physical
castration) which
are described herein, without undue experimentation.
Any pharmaceutical drug, or other method of castration, that ablates sex
steroids or
interrupts sex steroid-mediated signaling may be used in the methods of the
invention. For
example, one nonlimiting method of, inhibiting sex steroid signaling,
reactivating the thymus
andlor enhancing the functionality of BM and immune cells is by modifying the
normal
action of GnRH on the pituitary (i. e., the release of gonadotrophins, FSH and
LH) and
consequently reducing normal sex steroid production or release from the
gonads. Thus, in
one case, sex steroid ablation is accomplished by administering one or more
sex hormone
analogs, such as a GnRH analog. GnRH is a hypothalamic decapeptide that
stimulates the
secretion of the pituitary gonadotropins, leutinizing hormone (LH) and
follicle-stimulating
hormone (FSH). Thus, GnRH agonists (e.g., in the form of Synarel° or
Lupron~) initially
result in over stimulation of the receptor and through feedback mechanisms
will suppress the
pituitary production of FSH and LH by desensitization of LHRH Receptors. These
gonadotrophins normally act on the gonads to release sex steroids, in
particular estrogens in
females and testosterone in males; the release of which is significantly
reduced by the
absence of FSH and LH. The direct consequences of this are a drop in the
plasma levels of
sex steroids, and as a result, progressive.release of the inhibitory signals
on the thymus. A
more rapid drop in circulating sex steroid levels can be achieved for example
by the use of a
GnRH antagonist.
In some embodiments, the sex steroid mediated signaling is disrupted by
administration of a sex steroid analog, such as an analog of leutinizing
hormone-releasing
hormone (LHRH). Sex steroid analogs and their use in therapies and chemical
castration are
well known. Sex steroid analogs are commercially and their use in therapies
and chemical
castration are well known. Such analogs include, but are not limited to, the
following
agonists of the LHRH receptor (LHRH-R): buserelin (e.g., buserelin acetate,
trade names
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WO 2004/103271 PCT/US2004/011921
Suprefact0 (e.g., 0.5-02 mg s.c./day), Suprefact DepotO, and Suprefact~ Nasal
Spray (e.g., 2
p.g per nostril, every 8 hrs.), Hoechst, also described in U.S. Patent Nos.
4,003,884,
4,118,483, and 4,275,001); Cystorelin0 (e.g., gonadorelin diacetate
tetrahydrate, Hoechst);
deslorelin (e.g., desorelin acetate, Deslorell~, Balance Pharmaceuticals);
gonadorelin (e.g.,
gonadorelin hydrocholoride, trade name Factrel~ (100 p,g i.v. or s.c.), Ayerst
Laboratories);
goserelin (goserelin acetate, trade name Zoladex~, AstraZeneca, Aukland, NZ,
also
described in U.S. Patent Nos. 4,100,274 and 4,128,638; GB 9112859 and GB
9112825);
histrelin (e.g., histerelin acetate, Supprelin~, (s.c.,l0 ~,g/kg.day), Ortho,
also described in EP
217659); leuprolide (leuprolide acetate, trade name Lupron~ or Lupron Depot;
Abbott/TAP, Lake Forest, IL, also described in U.S. Patent Nos. 4,490,291
3,972,859,
4,008,209, 4,992,421, and 4,005,063; DE 2509783); leuprorelin (e.g.,
leuprorelin acetate,
trade name Prostap SRO (e.g., single 3.75 mg dose s.c. or i.m.lmonth),
Prostap3~ (e.g.,
single 11.25mg dose s.c. every 3 months), Wyeth, USA, also described in
Plosker et al.,
(1994) Drugs 48:930); lutrelin (Wyeth, USA, also described in U.S. Patent No.
4,089,946);
Meterelin~ (e.g., Avorelina (e.g., 10-15 mg slow-release formulation), also
described in EP
23904 and WO 91/18016); nafarelin (e.g., trade name Synarel~ (i.n. 200-1800
p,g/day),
Syntex, also described in U.S. Patent No. 4,234,571; WO 93/15722; and EP
52510); and
triptorelin (e.g., triptorelin pamoate; trade names Trelstar LA~ (11.25 mg
over 3 months),
Trelstar LA Debioclip~ (pre-filled, single dose delivery), LA Trelstar Depot~
(3.75 mg over
one month), and Decapeptyl~, Debiopharm S.A., Switzerland, also described in
U.S. Patent
Nos. 4,010,125., 4,018,726, 4,024,121, and 5,258,492; EP 364819). LHRH analogs
also
include, but are not limited to, the following antagonists of the LHRH-R:
abarelix (trade
name PlenaxisTM (e.g., 100 mg i.m. on days 1, 15 and 29, then every 4 weeks
thereafter),
Praecis Pharmaceuticals, W c., Cambridge, MA) and cetrorelix (e.g., cetrorelix
acetate, trade
name CetrotideTM (e.g., 0.25 or 3 mg s.c.), Zentaris, Frankfurt, Germany).
Additional sex
steroid analogs include Eulexin0 (e.g., flutamide (e.g., 2 capsules 2x/day,
total 750 mg/day),
Schering-Plough Corp., also described in FR 7923545, WO 86/01105 and PT
100899), and
dioxane derivatives (e.g., those described in EP 413209), and other LHRH
analogs such as
are described in EP 181236, U.S. Patent Nos. 4,608,251, 4,656,247, 4,642,332,
4,010,149,
3,992,365, and 4,010,149. Combinations of agonists, combinations of
antagonists, and
combinations of agonists and antagonists are also included. One non-limiting
analog of the
invention is deslorelin (described in U.S. Patent No. 4,218,439). For a more
extensive list, of
analogs, see Vickery et al. (1984) LHRH AND ITS ANALOGS: CONTRACEPTIVE &
THERAPEUTIC APPLICATIONS (Vickery et al., eds.) MTP Press Ltd., Lancaster, PA.
Each
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WO 2004/103271 PCT/US2004/011921
analog may also be used in modified form, such as acetates, citrates and other
salts thereof,
which are well known to those in the art.
One non-limiting example of administration of a sex steroid ablating agent is
a
subcutaneous/intradermal injection of a "slow-release" depot of GnRH agonist
(e.g., one,
three, or four month Lupron~ injections) or a subcutaneouslintradermal
injection of a "slow-
release" GnRH-containing implant (e.g., one or three month Zoladex0, e.g., 3.6
mg or 10.8
mg implant). These could also be given intramuscular (i.m.), intravenously
(i.v.) or orally,
depending on the appropriate formulation. Another example is by subcutaneous
injection of
a "depot" or "impregnated implant" containing, for example, about 30 mg of
Lupron~ (e.g.,
Lupron DepotO '(leuprolide acetate for depot suspension) TAP Pharmaceuticals
Products,
Inc., Lake Forest, IL). A 30 mg Lupron~ injection is sufficient for four
months of sex
steroid ablation to allow the thymus to rejuvenate and export new naive T
cells into the blood
stream.
Many of the mechanisms of inhibiting sex steroid signaling described herein
are well
known and some of these drugs, in particular the GnRH angonists, have been
used for many
years in the treatment of disorders of the reproductive organs, such as some
hormone
sensitive cancers including. breast and prostate cancer, endometriosis,
reproductive disorders,
hirsuitism, precocuis puberty, sexual deviancy and in the control of
fertility.
Tn certain examples, the thymus of the patient is ultimately reactivated by
sex steroid
ablation andlor interruption or disruption of sex steroid-mediated signalling.
In some cases,
disruption reverses the hormonal status of the patient. According to the
methods of the
invention, the hormonal status of the recipient is reversed such that the
hormones of the
recipient approach pre-pubertal levels. By lowering the level of sex steroid
hormones in the
recipient, the signalling of these hormones to the thymus is lowered, thereby
allowing the
thymus to be reactivated. The patient may be pubertal or post-pubertal, or the
patient has (or
has had) a disease that at least in part atrophied the thymus. Alternatively,
the patient has (or
has had) a treatment of a disease, wherein the treatment of the disease at
least in part
atrophied the thymus of the patient. Such treatment may be anti-viral,
immunosuppression,
chemotherapy, andlor radiation treatment. In other embodiments, the patient is
menopausal
or has had sex steroid (or other hormonal levels) decreased by another means,
e.g., trauma,
drugs, etc.
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Sex steroid ablation or interruption of sex steroid mediated signaling has one
or more
direct effect on the BM and/or cells of the immune system, wherein
functionality is
improved. The effects may occur prior to, or concurrently with, thymic
reactivation.
In some embodiments, sex steroid ablation or inhibition of sex steroid
signaling is
accomplished by administering an anti-androgen such as an androgen blocker
(e.g.,
bicalutamide, trade names Cosudex~ or Casodex0, 5-500 mg, e.g., 50 mg po Q)D,
AstraZeneca, Aukland, NZ), either alone or in combination with an LHRH analog
or any
other method of castration. Sex steroid ablation or interruption of sex
steroid signaling may
also be accomplished by administering cyproterone acetate (trade name,
Androcor0, Shering
AG, Germany; e.g., 10-1000 mg, 100 mg bd or tds, or 300 mg IM weekly, a 17-
hydroxyprogesterone acetate, which acts as a progestin, either alone or in
combination with
an LHRH analog or any other method of castration. Other anti-androgens rnay be
used (e.g.,
antifungal agents of the imidazole class, such as liarozole (Liazol~ e.g., 150
mg/day, an
aromatase inhibitor) and ketoconazole, flutamide (trade names Euflex~ and
Eulexin~,
Shering Plough Corp; N.J.; 50-500 mg e.g., 250 or 750 mg po QID), megestrol
acetate
(Megace~ e.g., 480-840 mg/day or nilutamide (trade names Anandron~, and
Nilandron~,
Roussel, France e.g., orally, 150-300 mg/day)). Antiandrogens are often
important in
therapy, since they are commonly utilized to address flare by GnRH analogs.
Some
antiandrogens act by inhibiting androgen receptor translocation, which
interrupts negative
feedback resulting in increased testosterone levels and minimal loss of
libido/potency.
Another class of anti-androgens useful in the present invention are the
selective androgen
receptor modulators (SARMS) (e.g., quinoline derivatives, bicalutamide (trade
name
Cosudex~ or Casodex~, as above), and flutamide (trade name Eulexin~, e.g.,
orally, 250
mglday)). Other well known anti-androgens include 5 alpha reductase inhibitors
(e.g.,
dutasteride,(e.g., po 0.5 mg/day)) which inhibits both 5 alpha reductase
isoenzymes and
results in greater and more rapid DHT suppression; finasteride (trade name
Proscar0; 0.5-500
mg, e.g." 5 mg po daily), which inhibits 5alpha reductase 2 and consequent DHT
production,
but has little or no effect on testosterone or LH levels);
In other embodiments, sex steroid ablation or inhibition of sex steroid
signaling is
accomplished by administering anti-estrogens either alone or in combination
with an LHRH
analog or any other method of castration. Some anti-estrogens (e.g.,
anastrozole (trade name
Arimidex0), and fulvestrant (trade name Faslodex0, 10-1000 mg, e.g., 250 mg 1M
monthly)
act by binding the estrogen receptor (ER) with high affinity similar to
estradiol and
consequently inhibiting estrogen from binding. Faslodex0 binding also triggers
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WO 2004/103271 PCT/US2004/011921
conformational change to the receptor and down-regulation of estrogen
receptors, without
significant change in FSH or LH levels. Other non-limiting examples of anti-
estrogens are
tamoxifen (trade name Nolvadex0); Clomiphene (trade name Clomid0) e.g., 50-250
mglday,
a non-steroidal ER ligand with mixed agonist/antagonist properties, which
stimulates release
of gonadotrophins; diethylstilbestrol ((DES), trade name Stilphostrol~) e.g.,
1-3 mg/day,
which shows estrogenic activity similar to, but greater than, that of estrone,
and is therefore
considered an estrogen agonist, but binds both androgen and estrogen receptors
to induce
feedback inhibition on FSH and LH production by the pituitary,
diethylstilbestrol diphosphate
e.g., 50 to 200 mg/day; as well as danazol, , droloxifene, and iodoxyfene,
which each act as
antagonists. Another class of anti-estrogens which may be used either alone or
in
combination with other methods of castration, are the selective estrogen
receptor modulators
(SERMS) (e.g., toremifene (trade name Fareston~, 5-1000 mg, e.g., 60 mg po
Q>17),
raloxofene (trade name Evista0), and tamoxifen (trade name Nolvadex~, 1-1000
mg, e.g., 20
mg po bd), which behaves as an agonist at estrogen receptors in bone and the
cardiovascular
system, and as an antagonist at estrogen receptors in the mammary gland).
Estrogen receptor
downregulators (ERDs) (e.g., tamoxifen (trade name, Nolvadex0)) may also be
used in the
present invention.
Other non-limiting examples of methods of inhibiting sex steroid signalling
which
may be used either alone or in combination with other methods of castration,
include
aromatase inhibitors and other adrenal gland blockers (e.g.,
Aminoglutethimide, formestane,
vorazole, exemestane, anastrozole (trade name Arimidex~, 0.1-100 mg, e.g., 1
mg po QTD),
which lowers estradiol and increases LH and testosterone), letrozole (trade
name Femara~,
0.2-500 mg, e.g., 2.5 mg po Q1D), and exemestane (trade name AromasinC~)1-2000
mg, e.g.,
mg/day); aldosterone antagonists (e.g., spironolactone (trade name,
Aldactone0) e.g., 100
25 to 400 mglday), which blocks the androgen cytochrome P-450 receptor;) and
eplerenone, a
selective aldosterone-receptor antagonist) antiprogestogens (e.g.,
medroxypregesterone
acetate, e.g. 5 mg/day, which inhibits testosterone syntheses and LH
synthesis); and
progestins and anti-progestins such as the selective progesterone response
modulators
(SPRM) (e.g., megestrol acetate e.g., 160 mglday, mifepristone (RU 4g6,
Mifeprex0, e.g.
200 mg/day); and other compounds with estrogen/antiestrogenic activity, (e.g.,
phytoestrogens, flavones, isoflavones and coumestan derivatives, lignans, and
industrial
compounds with phenolic ring (e.g., DDT)).. Also, anti-GnRH vaccines (see,
e.g., Hsu et al.,
(2000) Cancer Res. 60:3701; Talwar, (1999) Ini~nunol. Rev. 171:173-92), or any
other
pharmaceutical which mimics the effects produced by the aforementioned drugs,
may also be
CA 02528521 2005-12-06
WO 2004/103271 PCT/US2004/011921
used. In addition, steroid receptor based modulators, which may be targeted to
be thymic
and/or BM specific, may also be developed and used. Many of these mechanisms
of
inhibiting sex steroid signaling are well known. Each drugs may also be used
in modified
form, such as acetates, citrates and other salts thereof, which are well known
to those in the
art.
Because of the complex and interwoven feedback mechanisms of the hormonal
system, administration of sex steroids may result in inhibition of sex steroid
signalling. For
example, estradiol decreases gonadotropin production and sensitivity to GnRH
action.
However, higher levels of estradiol result in gonadotropin surge. Likewise,
progesterone
influences frequency and amount of LH release. In men, testosterone inhibits
gonadotropin
production. Estrogen administered to men decreases LH and testosterone, and
anti-estrogen
increases LH.
In other embodiments, prolactin is inhibited in the patient. Another means of
inhibiting sex steroid mediated signaling may be by means of direct or
indirect modulation of
prolactin levels. Prolactin is a single-chain protein hormone synthesized as a
prohormone.
The normal values for prolactin are males and nonpregnant females typically
range from
about 0 to 20 ng/ml, but in pregnancy the range is typically about 10 to 300
ng/ml . Overall,
several hundred different actions have been reported for prolactin. Prolactin
stimulates breast
development and milk production in females. Abnormal prolactin is known to be
involved in
pituitary tumors, menstrual irregularities, infertility, impotence, and
galactorrhea (breast milk
production). A considerable amount of research is in progress to delineate the
role of
prolactin in normal and pathologic immune responses. It appears that prolactin
has a
modulatory role in several aspects of immune function, yet there is evidence
to suggest that
hyperprolactinemia is immunosuppressive (Matera L, Neuf~oif~zmuhomodulation.
1997 Jul-
Aug;4(4):171-80). Administration of prolactin in pharmacological doses is
associated with a
decreased survival and an inhibition of cellular immune functions in septic
mice. (Oberbeck
R , J Surg Res. 2003 Aug;113(2):248-56). There are also a large number of
drugs which
impair dopaminergic inhibition of prolactin and give rise to
hyperprolactinemia.
Antidopaminergic agents include haloperidol, fluphenazine, sulpiride,
metoclopramide and
gastrointestinal prokinetics (e.g.., bromopride, clebopride, domperidone and
levosulpiride )
which have been exploited clinically for the management of motor disorders of
the upper
gastrointestinal tract.
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Inhibin A aald B peptides made in the gonads in response to gonadotropins,
down
regulates the pituitary and suppress FSH. Activin normally up regulates GnRH
receptors and
stimulate FSH synthesis, however over production may shut down sex steroid
production.
Thus these hormones may also be the target of inhibition of sex steroid-
mediated signalling.
In certain embodiments, an LHRH-R antagonist is delivered to the patient,
followed
by an LHRH-R agonist. For example, the antagonist can be administered as a
single injection
of sufficient dose to cause castration within 5-8 days (this is normal for,
e.g., Abarelix).
When the sex steroids have reached this castrate level, the agonist is given.
This protocol
abolishes or limits any spike of sex steroid production, before the decrease
in sex steroid
production, that might be produced by the administration of the agonist, In an
alternate
embodiment, an LHRH-R agonist that creates little or no sex steroid production
spike is used,
with or without the prior administration of an LHRH-R antagonist.
Inhibition of sex steroid si nag
Sex steroids comprise a large number of the androgen, estrogen and progestin
family
of hormone molecules. Non-limiting members of the progestin family of C21
steroids include
progesterone, l7or,-hydroxy progesterone, 20oc-hydroxy progesterone,
pregnanedione,
pregnanediol and pregnenolone. Non-limiting members of the androgen family of
C19
steroids include testosterone, androstenedione, dihydrotesterone (DHT),
androstanedione,
androstandiol, dehydroepiandrosterone and l7oc-hydroxy androstenedione. Non-
limiting
members of the estrogen family of C17 steroids include estrone, estradiol-17a,
and estradiol-
17[3.
Signalling by sex steroids is the net result of complex outcomes of the
components of
the pathway that includes biosynthesis, secretion, metabolism,
compartmentalization and
action. Parts of this pathway are not fully understood; nevertheless, there
are numerous
existing and potential mechanisms for achieving inhibition of sex steroid
signalling. In one
aspect of the present invention, inhibition of sex steroid signalling is
achieved by modifying
the bioavailable sex steroid hormone levels at the cellular level, the so
called 'free' levels, by
altering biosynthesis or metabolism, the binding to sex steroid receptors on
or in target cells,
and/or intracellular signalling of sex steroids.
It is possible to influence the signalling pathways either directly or
indirectly. The
direct methods include methods of influencing sex steroid biosynthesis and
metabolism,
binding to the respective receptor and intracellular modification of the
signal. The indirect
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methods include those methods known to influence sex steroid hormone
production and
action such as the peptide hormone and growth factors present in the pituitary
gland and the
gonad. The latter include but not be limited to follicle stimulating hormone
(FSH),
luteinizing hormone (LH) and activin made by the pituitary gland, and inhibin,
activin and
insulin-like growth factor-1 (IGF-1) made by the gonad.
The person skilled in the art will appreciate that inhibition of sex steroid
signaling
may take place by making the aforementioned modifications at the level of the
relevant
hormone, enzyme, receptor, binding molecule and/or ligand, either by direct
action upon that
molecule or by action upon a precursor of that molecule, including a nucleic
acid that
encodes or regulates it, or a molecule that can modify the action of sex
steroid.
Direct methods of inhibiting si n
Biosynthesis
The rate of biosynthesis is the major rate determining step in the production
of steroid
hormones and hence the bioavailability of 'free' hormone in serum. Inhibition
of a key
enzyme such as P450 cholesterol side chain cleavage (P450scc), early in the
pathway, will
reduce production of all the major sex steroids. On the other hand, inhibition
of enzymes
later in the pathway, such as P450 aromatase (P450arom) that converts
androgens to
estrogens, or 5cx-reductase that converts testosterone to DHT, will only
effect the production
of estrogens or DHT, respectively. Another important facet of sex steroid
hormone
biosynthesis is the family of oxidoreductase enzymes that catalyze the
interconversion of
inactive to bioactive steroids, for example, androstenedione to testosterone
or estrone to
estradiol-1?~by 1-?~-hydroxysteroid dehydrogenase (17-HSD). These enzymes are
tissue and
cell specific and generally catalyze either the reduction or oxidation
reaction e.g., 17[3 HSD
type 3 is found exclusively in the Leydig cells of the testes, whereas 17(3
HSD type 1 is found
in the ovary. They therefore offer the possibility of specifically reducing
production of the
active forms of androgens or estrogens.
There are many known inhibitors of the enzymes in the steroid biosynthesis
pathway
that are either already in clinical use or are under development. Some
examples of these
together with their treatment modalities are listed above. It is important
that the action of
these enzyme inhibitors does not unduly influence production of other steroids
such as
glucocorticoids and mineralocorticoids from the adrenal gland that are
essential for metabolic
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stability. When using such inhibitors, it may be necessary to provide the
patient with
replacement glucocorticoids and sometimes mineralocorticoids.
Sex steroid biosynthesis occurs in varied sites and utilizing multiple
pathways,
predominantly produced the ovaries and testes, but there is some production in
the adrenals,
as well as synthesis of derivatives in other tissues, such as fat. Thus,
multiple mechanisms of
inhibiting sex steroid signaling may be required to ensure adequate inhibition
to achieve the
present invention. '
Metabolism and compartmentalization
Sex steroid hormones have a short half-life in blood, generally only several
minutes,
due to the rapid metabolism, particularly by the liver, and clearance by the
kidney and fat.
Metabolism includes conjugation by glycosylation and sulphation, as well as
reduction. Some
of these metabolites retain biological activity either as prohormones, for
example estrone
sulphate, or through intrinsic bioactivity such as the reduced androgens. Any
interference in
the rate of metabolism can influence the 'free' levels of sex steroid
hormones., however
methods of achieving this are not currently available as are methods of
influencing
biosynthesis.
Another method of reducing the level of 'free' sex steroid hormone is by
compartmentalization by binding of the sex steroid hormone to proteins present
in the serum
such as sex hormone binding globulin, corticosteroid-binding globulin, albunun
and
testosterone-estradiol binding globulin. Binding to sex steroid ligands, such
as carrier
molecules may make sex steroids unavailable for receptor binding. Increased
binding may
result from increased levels of carriers, such-as SHBG or introduction of
other ligands which
bind the sex steroids, such as soluble receptors. Alternatively decreased
levels of carrier
molecules may make sex steroids more susceptible to degradation.
Active or passive immunization against a particular sex steroid hormone is a
form of
compartmentalization. There are examples in the literature of this approach
successfully
increasing ovulation rates in animals after immunization against estrogen or
androgen. Sex
steroids are secreted from cells in secretory vesicles. Inhibition or
modification of the
secretory mechanism is another method of inhibiting sex steroid signaling
Receptors and intracellular signalling
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The sex steroids act on cells via specific receptors that can be either
intracellular, or,
as shown more recently, on the target cell membrane.
The intracellular receptors are members of the nuclear receptor superfamily.
They are
located in the cytoplasm of the cell and are transported to the nucleus after
binding with the
sex steroid hormone where they alter the transcription of specific genes.
Receptors for the
sex steroid hormones exist in several forms. Well known in the literature are
two forms of
the progesterone receptor, PRA and PRB, and three forms of the estrogen
receptor, ERoc,
ER(31 and ER(32. Transcription of genes in response to the binding of the sex
steroid
hormone receptor to the steroid response element in the promoter region of the
gene can be
modified in a number of ways. Co-activators and co-repressors exist within the
nucleus of
the target cell that can modify binding of the steroid-receptor complex to the
DNA and
thereby effect transcription. The identity of many of these co-activators and
co-repressors are
known and methods of modifying their actions on steroid receptors are the
topic of current
research. Examples of the transcription factors involved in sex steroid
hormone action are
NF-1, SP1, Oct-land TFIID. These co-regulators are required for the full
action of. the
steroids. Methods of modifying the actions of these nuclear regulators could
involve the
balance between activator and repressor by the use of antagonists or through
control of
expression of the genes encoding the regulators. Additionally, c-AMP
More recently, specific receptors for estrogens and progesterone have been
identified
on the membranes of cells whose structures are different from the
intracellular PR. Unlike
the classical steroid receptors that act on the genome, these receptors
deliver a rapid, non-
genomic action via intracellular pathways that are not yet fully understood.
One report
suggests.that estrogens interacting with membrane receptors activate the
sphingosine pathway
that is related to cell proliferation.
There are methods available or in development to alter the action of steroids
via their
cytoplasmic receptors. In this case, antiandrogens, antiestrogens, and
antiprogestins that
interact with the specific steroid receptors, are well known in the literature
and are in clinical
use, as described above. Their action may be to compete for, or block the
receptor, to modify
receptor levels, sensitivity, conformation, associations or signaling. These
drugs come in a
variety of forms, steroidal and non-steroidal, competitive and non-
competitive. Of particular
interest are the selective receptor modulators, SARMS, SERMS and SPRM, which
are
targeted to particular tissues and are exemplified above.
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Down regulation of receptors can be achieved in 2 ways; first, by excess
agonist
(steroid ligand), and second, by inhibiting transcription of the respective
gene that encodes
the receptor. The first method can be achieved through the use of selective
agonists such as
tamoxifen. The second method is not yet in clinical use.
Indirect methods of inhibiting si nalin
Biosynthesis
One of the indirect methods of inhibiting sex steroid signalling involves down
regulation of the biosynthesis of the respective steroid by a modification to
the availability or
action of the pituitary gonadotrophins, FSH and LH, that are responsible for
driving the
biosynthesis of the sex steroid hormones in the gonad. One established
inhibitor of FSH
secretion is inhibin, a hormone produced by the gonads in response to FSH.
Administration
of inhibin to animals has been shown to reduce FSH levels in serum due to a
decrease in the
pituitary secretion of FSH. The best known way of accomplishing a reduction in
both
gonadotrophins is via the hypothalamic hormone, GnRH/LHRH, which drives the
pituitary
synthesis and secretion of FSH and LH. Agonists and antagonists of GnRH that
reduce the
secretion of FSH and LH, and hence gonadal sex steroid production, are now
available for
clinical use, as described herein.
Another indirect method of reducing the biosynthesis of sex steroid hormones
is to
modify the action of FSH and LH at the level of the gonad. This could be
achieved by using
antibodies directed against FSH and LH, or molecules designed to compete with
FSH and LH
'for their respective receptors on gonadal cells that produce the sex steroid
hormones.
Another method of modifying the action of FSH and LH on gonadal cells is by a
co-regulator
of gonadotrophin action. For example, activin can reduce the capacity of the
theca cells of
the ovary and the Leydig cells of the testis to produce androgen in response
to LH.
Modification may take place at the level of hormone precursors such as
inhibition of
cleavage of a signal peptide, for example the signal peptide of GnRH.
Receptors and intracellular si nallin.g
Indirect methods of altering the signalling action of the sex steroid hormones
include
down-regulation of the receptor pathways leading to the genomic or non-genomic
actions of
the steroids. An example of this is the capacity of progesterone to down
regulate the level of
ER in target tissues. Future methods include treatment with molecules known to
influence
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the co-regulators of the receptors in the cell nucleus leading to a decrease
in the capacity of
the cell to respond to the steroid.
Additional factors
While the stimulus for the direct and indirect effects on BM functionality, BM
lymphopoiesis, and immune cell functionality is fundamentally based an the
inhibition of the
effects of sex steroids andlor the direct effects of the LHRH analogs, it may
be useful to
include additional substances which can act in concert to enhance or increase
(additive,
synergistic, or complementary) the thymic, BM, and/or immune cell effects and
functionality.
Additional substances may or may not be used. Such compounds include, but are
not limited
to, cytokines and growth factors, such as interleukin-2 (IL-2; 100,000 to
1,000,000 ILT, e.g.,
600,000 ILT/Kg every 8 hours by IV repeat doses), interleukin-7 (IL-7;
lOng/kg/day to
100mcg/kg/day subject to therapeutic discretion), interleukin-15 (IL-15; 0.1-
20 mug/kg IL-15
per day), interleukin 11 (IL-11; 1-1000 p,g/kg) members of the epithelial and
fibroblast
growth factor families, stem cell factor (SCF; also known as steel factor or c-
kit ligand; 0.25-
12..5 mg/ml), granulocyte colony stimulating factor (G-CSF; 1 and 15
p,g/kg/day IV or SC),
granulocyte macrophage stimulating factor (GM-CSF; 50-1000 p.g/sq meter/day SC
or IV),
insulin dependent growth factor (IGF-1), and keratinocyte growth factor (KGF;
1 p,g/kg to
100 mgJkg/day) (see, e.g., Sempowski et al., (2000) J. Immunol. 164:2180;
Andrew and
Aspinall, (2001) J. Immuhol. 166:1524-1530; Rossi et al., (2002) Blood
100:682);
erythropoietin (EPO; 10-500units/kg IV or SC). A non-exclusive list of other
appropriate
hematopoietins, CSFs, cytokines, lymphokines, hematopoietic growth factors and
interleukins for simultaneous or serial co-administration with the present
invention includes,
Meg-CSF (Megakaryocyte-Colony Stimulating Factor, more recently referred to as
c-mpl
ligand), MIF (Macrophage Inhibitory Factor), LIF (Leukemia Inhibitory Factor),
TNF
(Tumor Necrosis Factor), IGF, platelet derived growth factor (PDGF), M-CSF, IL-
1, IL-4,
IL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, LIF, flt3/flk2, human growth
hormone, B-cell
growth factor, B-cell differentiation factor and eosinophil differentiation
factor, or
combinations thereof.
One or more of these additional compounds) may be given once at the initial
LHRH
analog (or other castration method) application. Each treatment may be given
in combination
with the agonist, antagonist or any other form of sex steroid disruption.
Since the growth
factors have a relatively rapid half-life (e.g., in the hours) they may need
to be given each day
(e.g., every day for 7 days or longer). The growth factors/cytokines may be
given in the
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optimal form to preserve their biological activities, as prescribed by the
manufacturer, e.g., in
the form of purified proteins. However, additional doses of any one or
combination of these
substances may be given at any time to further stimulate the functionality of
the BM and
other immune cells. In certain cases, sex steroid ablation or interruption of
sex steroid
signalling is done concurrently with the administration of additional
cytokines, growth
factors, or combinations thereof. In other cases, sex steroid ablation or
interruption of sex
steroid signalling is done sequentially with the administration of additional
cytokines, growth
factors, or combinations thereof.
The term "mobilizing agent" is herein defined as agents such as SDF-1 (e.g.,
AMD3100), growth hormone, GM-CSF, G-CSF and chemotherapeutics (e.g.,
cyclophosphamide), which enhance mobilisation of stem cells from the BM.
G-CSF and GM-CSF are known to mobilize the production of granulocytes
(primarily
neutrophils) and macrophages, respectively, and also result in increased
production of DC
from the BM, which help provide a non-specific immune response.in the patient
to antigenic
challenge (Janeway et al., (2001) Irninunobiolo~y 5th Ed., p. 325).
Clinically, G-CSF and
GM-CSF are used, for example, to decrease the incidence of infection (as
manifested by
febrile neutropenia) in patients with non-myeloid malignancies receiving
myelosuppressive
anti-cancer drugs, which are typically associated with a significant incidence
of severe
neutropenia and fever. Additionally, both of these drugs are approved
clinically to prevent
infections in. patients receiving HSCT. Both G-CSF and GM-CSF are currently
used in
patients undergoing peripheral blood progenitor cell collection or therapy.
Colony
stimulating factors (CSFs), which stimulate the differentiation and/or
proliferation of BM
stem cells, have generated much interest because of their therapeutic
potential for restoring
depressed levels of hematopoietic stem cell-derived cells. CSFs in both human
and murine
systems have been identified and distinguished according to their activities.
For example,
granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro
formation of
neutrophilic granulocyte and macrophage colonies, respectively while GM-CSF
and
interleukin-3 (IL-3) have broader activities and stimulate the formation of
both macrophage,
neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulates the
formation of
mast, megakaryocyte and pure and mixed erythroid colonies (when erythropoietin
is added).
GM-CSF accelerates recovery of neutrophils and maintains functional capacity,
yet has little
demonstrable effect on platelet recovery. In contrast 1I,-3 promotes a slower
increase
recovery in neutrophils and monocytes while accelerating the recovery of
platelets.
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Thus, G-CSF and/or GM-CSF are used in some of the methods of the invention.
Sex
steroid ablation together (sequentially or concurrently) with G-CSF and/or GM-
CSF therapy
results in an increase in the output from the BM of both lymphoid and myeloid
cell, which in
turn significantly improves both the short and long term outcomes for patients
suffering, or
likely to suffer from, infections. In another method, the CSFs are
administered 3-4 days after
chemotherapy or radiation therapy. Clinical outcomes already associated with
the use of the
CSFs are also greatly enhanced by an interruption to sex steroid signaling. In
particular,
using the methods of the instant invention together with CSF's, allows for
much greater
infection control in patients receiving e.g., cancer radiation or
chemotherapy. Additionally, if
the immune system can be effectively a~ld promptly "rebooted," increased
dosages andlor
frequency of chemotherapy drugs or radiation therapy may be used. This may
occur with or
without the introduction of allogeneic or autogenic HSC, which would further
enhance the
timely return of immune system functionality. Castration will also result in a
lower number
of HSC that have to be transplanted, which will be useful when only a limited
number of
HSC can be obtained from a donor or when cord blood stem cells are used for
transplant.
For instance, the concurrent use of two separate classes of drugs (e.g., a
GnRH
analog, such as Lupron~, and an androgen blocker, such as Cosudex0) may allow
for the
same immune system regeneration but may require a reduced dosage of the G-CSF
or GM-
CSF. Similarly, the concurrent use of these two separate classes of drugs may
allow for a
greater, or more prolonged rejuvenation of immune system cells, while
utilizing the same
dosage of G-CSF or GM-CSF. Additionally, the concurrent use of two separate
classes of
drugs may allows for the same rejuvenation of immune system cells, while
utilizing a
reduced dosage (i.e., a reduction compared to the "normally" used dosages used
for the
treatment of prostate cancer, endometriosis, or breast cancer] of the drug, or
combination of
drugs, used to ablate or interrupt sex steroid signaling. Further, the
concurrent use of these
two separate classes of drugs allows for a greater, or prolonged rejuvenation
of immune
system cells, while utilizing a reduced dosage of the drug, or combination of
drugs, used to
ablate or interrupt sex steroid signaling.
Indications
The use of drugs known to cause sex steroid ablation, or which interrupt sex
steroid
signaling, either alone or in combination, with or without the aforementioned
growth factors
and cytokines may be used for the following: reduction of infections
associated with a
number of treatment regimens; rejuvenation of the BM following ablative
therapy (see, e.g.,
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Example 19); as an adjunct in enabling HSC engraftment (see, e.g., Example
22); as an
adjunct in the effective management of allogeneic or autologous organ or cell
transplants
(see, e.g., Examples 21 and 22, and co-pending, co-owned U.S. Serial Nos.
10/419,039 and
10/749,119); vaccination protocols (see, e.g., Examples 10-13, 29, and co-
owned, co-pending
U.S. Serial Nos. 10/418,747 and 10/748,450); management of various autoimmune
diseases
(see, e.g., Examples 32-35 and co-owned, co-pending U.S. Serial Nos.
10/419,066 and
10/749,118); treatment or management of the consequences of various infectious
diseases
(see, e.g., Examples 3 and 14); and improvement in the prevention and
treatment of various
cancers (see, e.g., Example 13, and co-owned, co-pending U.S. Serial Nos.
10/418,727 and
10/749,122, and various gene therapy protocols (see Example 14 and co-owned,
co-pending
U.S. Serial No. 10/419,068 and 10/748,851).
The use of these drugs in these diseases will either result in more effective
treatment
outcomes or will result in the overall treatment protocols being more
efficient. Additionally,
as described in, e.g., Examples 25 and 26, the doses or administration of the
various
chemotherapy drugs (or doses of radiation therapy) may be altered such that
they now
produce less side effects and/or result in better quality of life outcomes for
the patients.
Moreover, the coadministration of the various cytokines and growth factors may
allow for a
reduced number of HSC that need to be transplanted. For example, using the
method of the
invention, it may now be possible to use human cord blood for adult HSCT,
since a reduced
number of cells is required to obtain engraftment.
PHARMACEUTICAL COMPOSITIONS
The compounds used in this invention may be supplied i.n any pharmaceutically
acceptable carrier or may be supplied without a carrier. Formulations of
pharmaceutical
compositions can be prepared according to standard methods (see, e.g.,
Remin~ton, The
Science and Practice of Pharmacy, Gennaro A.R., ed., 20th edition, Williams &
Wilkins PA,
USA (2000)). Non-limiting examples of pharmaceutically acceptable carriers
include
physiologically compatible coatings, solvents and diluents. For parenteral,
subcutaneous,
intravenous, and intramuscular adnunistration, the compositions may be
protected such as by
encapsulation. Alternatively, the compositions may be provided with carriers
that protect the
active ingredient(s), while allowing a slow release of those ingredients.
Numerous polymers
and copolymers are known in the art for preparing time-release preparations,
such as various
versions of lactic acid/glycolic acid copolymers. See, for example, U.S.
Patent No.
CA 02528521 2005-12-06
WO 2004/103271 PCT/US2004/011921
5,410,016, which uses modified polymers of polyethylene glycol (PEG) as a
biodegradable
coating.
Formulations intended to be delivered orally can be prepared as liquids,
capsules,
tablets, and the like. These compositions can include, for example,
excipients, diluents,
and/or coverings that protect the active ingredients) from decomposition. Such
formulations
are well known (see, e.g., Remington, The Science and Practice of Pharmacy,
Gennaro A.R.,
ed., 20th edition, Williams & Wilkins PA, USA (2000)).
In any of the formulations of the invention, other compounds that do not
negatively
affect the activity of the LHRH analogs (i.e., compounds that do not block the
ability of an
LHRH analog to disrupt sex steroid hormone signalling) may be included.
Examples are
various growth factors and other cytokines as described herein.
DOSE
Doses of a sex steroid analog or inhibitor used, in according with the
invention, to
disrupt sex steroid hormone signaling, can be readily determined by a
routinely trained
physician or veterinarian, and may be also be determined by consulting medical
literature
(e. g., THE PHYSICIAN'S DESK REFERENCE, 52ND EDITION, Medical Economics
Company,
1998).
The dosage regimen involved in a method for treating the above-described
conditions
will be deternuned by the attending physician considering various factors
which modify the
action of drugs, e.g., the condition, body weight, sex and diet of the
patient, the severity of
any illness, time of administration and other clinical factors. Progress of
the treated patient
can be monitored by periodic assessment of the hematological profile, e.g.,
differential cell
count and the like.
The dosing recited above is adjusted to compensate for additional components
in the
therapeutic composition. These include co-administration with other CSF,
cytokine,
lymphokine, interleukin, hematopoietic growth factor; co-administration with
chemotherapeutic drugs andlor radiation; and various patient-related issues as
identified by
the attending physician such as factors which modify the action of drugs,
e.g., the condition,
body weight, sex and diet of the patient, the severity of any illness, time of
administration and
other clinical factors.
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In addition to dosing described above, for example, LHRH analogs and other sex
steroid analogs can be administered in a one-time dose that will last for a
period of time (e.g.,
3 to 6 months). In certain cases, the formulation will be effective for one to
two months. The
standard dose varies with type of analog used, but is readily determinable by
those skilled in
the art without undue experimentation. In general, the dose is between about
0.01 mg/kg and
about 10 mglkg, or between about 0.01 mglkg and about 5 mg/kg.
The length of time of sex steroid inhibition or LHRHIGnRH analog treatment
varies
with the degree of thymic atrophy and damage, and is readily determinably by
those skilled in
the art without undue experimentation. For example, the older the patient, or
the more the
patient has been exposed to T cell depleting reagents such as chemotherapy or
radiotherapy,
the longer it is likely that they will require treatment, for example with
GnRH. Four months
is generally considered long enough to detect new T cells in the blood.
Methods of detecting
new T cells in the blood are known in the art. For instance, one method of T
cell detection is
by determining the existence of T cell receptor excision circles (TREC's),
which are formed
when the TCR is being formed and are lost in the cell after it divides. Hence,
TREC's are
only found in new (naive) T cells. TREC levels are an indicator of thymic
function in
humans. These and other methods are described in detail in WO/00 230,256.
Dose varies with the sex steroid inhibitor or, e.g. anti-sex steroid vaccine
or other
blocker used. In certain cases, a dose may be prepared to last as long as a
periodic epidemic
lasis. For example, "flu season" occurs usually during the winter months. A
formulation of
an LHRH analog can be made and delivered as described herein to protect a
patient for a
period of two or more months starting at the beginning of the flu season, with
additional
doses delivered every two or more months until the risk of infection decreases
or disappears.
The formulation can be made to enhance the immune system. Alternatively, the
formulation can be prepared to specifically deter infection by e.g., influenza
(flu) viruses
while also enhancing the immune system. This latter formulation may include
genetically
modified (GM) cells that have been engineered to create resistance to flu
viruses (see below).
The GM cells can be administered with the sex steroid analog or LHRH analog
formulation
or separately, both spatially and/or in time. As with the non-GM cells,
multiple doses over
time can be administered to a patient to create protection and prevent
infection with the flu
virus over the length of the flu season.
As will be understood by persons skilled in the art, at least some of the
means for
disrupting sex steroid signalling will only be effective as long as the
appropriate compound is
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administered. As a result, an advantage of certain embodiments of the present
invention is
that once the desired immunological affects of the present invention have been
achieved, (2-3
months) the treatment can be stopped and thee subjects reproductive system
will return to
normal.
DELIVERY OF AGENTS FOR CHEMICAL CASTRATION
Administration of sex steroid ablating agents may be by any method which
delivers
the agent into the body. Thus, the sex steroid ablating agent maybe be
administered, in
accordance with the invention, by any route including, without limitation,
intravenous,
subdermal, subcutaneous, intramuscular, topical, and oral routes of
administration.
In addition to the methods described above, delivery of the compounds for use
in the
methods of this invention may be accomplished via a number of methods known to
persons
skilled in the art. One standard procedure for administering chemical
inhibitors to inhibit sex
steroid mediated signalling utilizes a single dose of an LHRH agonist that is
effective for
three months. For this, a simple one-time i.v. or i.m. injection would not be
sufficient as the
agonist would be cleared from the patient's body well before the three months
are over.
Instead, a depot injection or an implant may be used, or any other means of
delivery of the
inhibitor that will allow slow release of the inhibitor. Likewise, a method
for increasing the
half life of the inhibitor within the body, such as by modification of the
chemical, while
retaining the function required herein, may be used.
Useful delivery mechanisms include, but are not limited to, laser irradiation
of the
skin. This embodiment is described in more detail in co-owned, co-pending U.S.
Serial No.
10/418,727 and also in U.S. Patent Nos. 4,775,361, 5,643,252, 5,839,446,
6,056,738,
6,315,772, and 6,251,099. Another useful delivery mechanism includes the
creation of high
pressure impulse transients (also called stress waves or impulse transients)
on the skin. This
embodiment is described in more detail in co-owned, co-pending U.S. Serial No.
10/418,727
and also U.S. Patent Nos. 5,614,502 and 5,658,822. Each method may be
accompanied or
followed by placement of the compounds) with or without carrier at the same
locus. One
method of this placement is in a patch placed and maintained on the skin for
the duration of
the treatment.
Timin
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In one case, the administration of agents (or other methods of castration)
that ablate
sex steroids or interrupt to sex steroid signaling occurs prior to a, e.g., a
chemotherapy or
radiation regimen that is likely to cause some BM marrow cell ablation andlor
damage to
circulating immune cells.
CELLS
Injection of hematopoietic progenitor cells, e.g., broadly defined as CD34+
hematopoietic cells (ideally autologous) can enhance the degree and kinetics
of thymic
regrowth andlor increases in immune cell and BM functionality and engraftment
without,
prior to or concurrently with thymic regeneration. HSC may also be further
defined as Thy-1
low and CD38- ; CD34+CD38-; Thy-1 low cells also lack markexs of other cell
lineages (lin
-ve) are the more primitive HSC being longer lasting or having longer-term
repopulating
capacity.
The methods of the various inventions described herein can be supplemented by
the
addition of, e.g., CD34+ HSC andlor epithelial stem cells. In one instance,
these cells are
autologous or syngeneic and have been obtained from the patient or twin prior
to thymus
reactivation. The HSC can be obtained by sorting CD34+ or CD341° cells
from the patient's
blood and/or BM. The number of HSC can be enhanced in several ways, including
(but not
limited to) by administering G-CSF (Neupogen, Amgen) to the patient prior to
collecting
cells, culturing the collected cells in SCGF, and/or administering G-CSF to
the patient after
CD34+ cell supplementation. Alternatively, the CD34+ cells need not be sorted
from the
blood or. BM if their population is enhanced by prior injection of G-CSF into
the patient
. . HSC may be used for genetic modification. These may be derived from BM,
peripheral blood, or umbilical cord, or any other source of HSC, and may be
either
autologous or nonautologous. Also useful are lymphoid and myeloid progenitor
cells,
mesenchymal stem cells also found in the bone marrow and epithelial stem
cells, also either
autologous or nonautologous. The stem cells may also include umbilical cord
blood. They
may also include stem cells which have the potential to form into many
different cell types eg
embryonic stem cells and adult stem cells now found in may tissues, e.g., BM,
pancreas,
brain, and the olfactory system.
In the event that nonautologous (donor) cells are used, tolerance to these
cells is
created during or after thymus reactivation. During or after the initiation of
blockage of sex
steroid mediated signaling, the relevant (genetically modified (GM) or non-
genetically
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modified) donor cells are transplanted into the recipient. These cells,
ideally stem or
progenitor cells, are incorporated into and accepted by the thymus wherein
they create
tolerance to the donor by eliminating any newly produced T cells which by
chance could be
eactive against them. They are then "belonging to the recipient" and may
become part of the
production of new T cells and DC by the thymus. The resulting population of T
cells
recognize both the recipient and donor as self, thereby creating tolerance for
a graft from the
donor (see co-owned, co-pending U.S. Serial No. 10/419, 039 and
PCT/IBOl/02740).
In another embodiment the administration of stem or precursor donor cells
(genetically modified or not genetically modified) comprises cells from more
than one
individual, so that the recipient develops tolerance to a range of MHC types,
enabling tree
recipient to be considered a suitable candidate for a cell, tissue or organs
transplant more
easily or quickly, since they are an MHC match to a wider range of donors.
The present invention also provides methods for incorporation of foreign DC
into a
patient's thymus. This may be accomplished by the administration of donor
cells to a
recipient to create tolerance in the recipient. The donor cells may be HSC,
epithelial stem
cells, adult or embryonic stem cells, or hematopoietic progenitor cells. The
donor cells may
be CD34+ HSC, lymphoid progenitor cells, or myeloid progenitor cells. In some
cases, the
donor cells are CD34+ or CD341o HSC. The donor HSC may develop into DC in the
recipient. . The donor cells may be administered to the recipient and migrate
through the
peripheral blood system to the reactivating thymus either directly or via the
BM. To enhance
thymic incorporation for tolerance induction, the stem cells may also be
injected
intrathymically in combination with activation of thymic regrowth through use
of sex steroid
inhibitors, e.g., LHRH/GnRH analogues. Even non-HSC are likely to be induced
to form into
DC within the thymic microenvironment and its content of appropriate growth
factors for
such cells.
The uptake into the thymus of the hematopoietic precursor cells is
substantially
increased in the inhibition or absence of sex steroids. These cells become
integrated into the
thymus and produce DC, NK, NKT, and T cells in the same manner as do the
recipient's
cells. The result is a chimera of T cells, DC and the other cells. The
incorporation of donor
DC in the recipient's thymus means that T cells produced by this thymus will
be selected
such that they are tolerant to donor cells. Such tolerance allows for a
further transplant from
the donor (or closely matched to the donor) of cells, tissues and organs with
a reduced need
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for immonusuppressive drugs since the transplanted material will be recognized
by the
recipient's immune system as self.
OPTIONAL GENETIC MODIFICATION OF STEM OR PROGENITOR CELLS
The present disclosure also comprises methods for optionally altering the
immune
system of an individual and methods of gene therapy. This is accomplished by
the
administration of GM cells to a recipient and through disruption of sex
steroid mediated
signaling. The invention further comprises methods of gene therapy through
enhancing the
functionality of BM and/or immune cells in conjunction with a regenerating
thymus, or
alternatively, prior to, or Without reactivation of the thymus.
The genetically modified cells may be HSC, epithelial stem cells, embryonic or
adult
stem cells, or myeloid or lymphoid progenitor cells. In one embodiment, the
genetically
modified cells are CD34+ or CD341o HSC, lymphoid progenitor cells, or myeloid
progenitor
cells. In another embodiment, the genetically modified cells are CD34+ or
CD341o HSC.
The genetically modified cells are administered to the patient and migrate
through the
peripheral blood system to the thymus. The uptake into the thymus of these
hematopoietic
precursor cells is substantially increased in the absence of sex steroids.
These cells become
integrated into the thymus and produce dendritic cells and T cells carrying
the genetic
modification from the altered cells. The results are a population of T cells
with the desired
genetic change that circulate in the peripheral blood of the recipient, and
the accompanying
increase in the population of cells, tissues and organs caused by reactivation
of the patient's
thymus.
Within 3-4 weeks of the start of blockage of sex steroid mediated signaling
(approximately 2-3 weeks after the initiation of LHRH treatment), the first
new T cells are
present in the blood stream. Full development of the T cell pool, however, may
take 3-4 (or
more) months.
The present disclosure also comprises methods for gene therapy using
genetically
modified hematopoietic stem cells, lymphoid progenitor cells, myeloid
progenitor cells,
epithelial stem cells, or combinations thereof (GM cells). Previous attempts
by others to
deliver such cells as gene therapy have been unsuccessful, resulting in
negligible levels of the
modified cells. The present disclosure provides a new method for delivery of
these cells
which promotes uptake and differentiation of the cells into the desired T
cells. The modified
cells are injected into a patient. The modified stem and progenitor cells are
taken up by the
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thymus and converted into T cells, dendritic cells, and other cells produced
in the thymus.
Each of these new cells contains the genetic modification of the parent
stem/progenitor cell.
In one embodiment, the methods of the invention use genetically modified HSC,
lymphoid progenitor cells, myeloid progenitor cells, epithelial stem cells or
combinations
thereof (collectively referred to as GM cells) to produce an immune system
resistant to attack
by particular antigens.
An appropriate gene or polynucleotide (i.e., the nucleic acid sequence
defining a
specific protein) that will create or induce resistance to one or more
infectious or other agents
is engineered into the stem and/or progenitor cells. By introducing the
specific gene into the
HSC, the cell differentiates into, e.g., an APC, it expresses the protein as a
peptide expressed
in the context of MHC class I or II. This expression will greatly increase the
number of APC
"presenting" the desired antigen than would normally occur, thereby increasing
the chance of
the appropriate T cell recognizing the specific antigen and responding.
Many antineoplastic or cytotoxic anticancer drugs used in the clinic today
cause
moderate to severe bone marrow toxicity (e.g., vinblastine, cisplatin,
methotrexate, alkylating
agents, anti-folate, a vinca alkaloid and anthracyclines). In another
embodiment of the
present invention, drug resistance genes can be introduced into HSC to confer
resistance to
anticancer drugs. Such genes include for example dihydrofolate reductase. The
use of the
present invention to provide HSC which are resistant to the cytotoxic effects
of these
chemotherapeutics may allow for the greater use of these drugs and/or less
side effects by
reducing the incidence and severity of myelosupporession. (Podda et al.,
(1992) Pf-oc. lVatl.
Acad. Sci. USA 89:9676; Banerjee et al., (1994) Ste~ra Cells 12:378)
The modified stem and progenitor cells are taken up by the thymus and
converted into
T cells, DC, and other cells produced in the thymus. Each of these new cells
contains the
genetic modification of the parent stem/progenitor cell, and is thereby
completely or partially
resistant to infection or damage by the agent or agents. B cells are also
increased. in number
in the bone marrow, blood and peripheral lymphoid organs, such as the spleen
and lymph
nodes, within e.g., two weeks of castration. In one embodiment, a patient has
already been in
contact with an agent, or is at a high risk of doing so.
The person may be given a sex steroid analog to activate their thymus, andlor
to
improve their bone marrow function, which includes the increased ability to
take up and
produce HSC. The person rnay be injected with their own HSC, or may be
injected with
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HSC from an appropriate donor, which has, e.g., treatment with G-CSF for 3
days (2
injections, subcutaneously per day) followed by collection of HSC from the
blood on days 4
and 5. The HSC may be transfected or transduced with a gene (e.g., encoding
the protein,
peptide, or antigen from the agent) to produce to the required protein or
antigen. Following
injection into the patient, the HSC enter the bone marrow and eventually some
evolve into
antigen presenting cells (APC) throughout the body. The antigen is expressed
in the context
of MHC class I and/or MHC class II molecules on the surface of these APC. By
expressing
the desired antigen, the APC improve the activation of T and B lymphocytes.
The
transplanted HSC may also enter the thymus, develop into DC, and present the
antigen in
question to developing T lymphocytes. If present in low numbers (e.g., <0.1%
of thymus
cells) the DC can bias the selection of new T cells to those reactive to the
antigen. If the
particular DC are present in high numbers, the same principle can be used to
delete the new T
cells which are potentially reactive to the antigen, which may be used in the
prevention or
treatment of autoimrnune diseases.
W one embodiment, a patient is infected with HIV. In a specific embodiment,
the
method for treating this patient includes the following steps, which are
provided in more
detail below: (1) treatment with Highly Active Anti-Retrovirus Therapy (HAARTI
to lower
the viral titer, which treatment continues throughout the procedure to prevent
or reduce
infection of new T cells; (2) ablation of T cells (immunosuppression); (3)
blockage of sex
steroid mediated signaling, for example, by administering a sex steroid
analog, such as an
LHRH analog; (4) at the time the thymus begins reactivating, administration of
GM cells that
have been modified to contain a gene that expresses a protein that will
prevent HIV infection,
prevent HIV replication, disable the HIV virus, or other action that will stop
the infection of
T cells by HIV; (5) if the GM cells are not autologous, administration of the
donor cells
before or concurrently with thymus reactivation will prime the immune system
to recognize
the donor cells as self; and (6) when the thymic chimera is established and
the new cohort of
mature T cells have begun exiting the thymus, reduction and eventual
elimination of
immunosuppression.
During or after the castration step, hematopoietic stem or progenitor cells,
or
epithelial stem cells, from the donor may be transplanted into the recipient
patient. These
cells are accepted by the thymus as belonging to the recipient and become part
of the
production of new T cells and DC by the thymus. The resulting population of T
cells
recognize both the recipient (and donor, in the case of nonautologus
transplants) as self.
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Tolerance for a graft from the donor may also be created in the recipient. The
graft may be
cells, tissues or organs of the donor, or combinations thereof.
In one embodiment, thymic grafts can be used in the methods of the invention
to
improve engraftment of the donor cells or tolerance to the donor graft. In
some
embodiments, thymic grafts are when the patient is athymic, when the patient's
thymus is
resistant to regeneration, or to hasten regeneration. In certain embodiments,
a thymic
xenograft to induce tolerance is used (see .e.g., U.S. Patent No. 5,658,564).
In other
embodiments, an allogenic thymic graft is used.
As herein defined, the phrase "creating tolerance" or "inducing tolerance" in
a patient,
and other similar phrases, refers to complete, as well as partial tolerance
induction (e.g" a
patient may become either more tolerant, or completely tolerant, to the graft,
as compared to
a patient that has not been treated according to the methods of the
invention). Tolerance
induction can be tested, e.g., by an MLR reaction, using methods known in the
art.
Thus, in one method, a patient receives a HSCT during or after castration. In
one
case, the patient is injected with their own HSC. In another case, the patient
is injected with
HSC from an appropriate donor. The patient or donor may or may not be
pretreated with G-
CSF (e.g., 2 s.c. injections per day for three days, followed by collection of
HSC from the
blood on days 4 and 5). In one case, hematopoietic cells are supplied to the
patient before or
concurrently with thymic reactivation, which increases the immune capabilities
of the
patient's body. The transplanted cells may or may not be genetically modified.
The
transplanted cells may be HSC, epithelial stem cells, or hematopoietic
progenitor cells. The
transplanted cells may be CD34+ HSC, lymphoid progenitor cells, or myeloid
progenitor
cells. In certain cases, the transplanted cells are CD34+ or CD341o HSC. The
HSC may or
may not be genetically modified.
In certain methods, the HSC are transfected or transduced with a gene (e.g.,
encoding
the protein, peptide, or antigen from the agent or other gene of interest) to
produce a protein
or antigen of interest. In one example, the methods of the invention use
genetically modified
HSC, lymphoid progenitor cells, myeloid progenitor cells, epithelial stem
cells or
combinations thereof (collectively referred to as "GM cells") to produce an
immune system
resistant to attack by particular antigens (see, e.g., Example 14). This
method is described in
more detail in co-owned U.S. Serial Nos. 09/758,910, 10/419,068, and
10/399,213.
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The uptake into the thymus of HSC is substantially increased in the absence of
sex
steroids. These cells become integrated into the thymus and produce DC and T
cells carrying
the genetic modification from the altered cells. The results are a population
of T cells with
the desired genetic change that circulate in the peripheral blood of the
recipient, and the
accompanying increase in the population of cells, tissues and organs caused by
a reactivating
thymus, which are capable of rapid, specific responses to antigen. Within
three to four weeks
of the start of blockage of sex steroid mediated signaling (approximately two
to three weeks
after the initiation of LHRH treatment), the first new T cells may be preset
in the blood
stream. Full development of the T cell pool may take three to four months.
Following injection into the patient, the HSC enter the bone and bone marrow
from
the the blood and then some exit back to the blood to be eventually converted
into T cells,
DC, APC throughout the body. Antigens are expressed in the context of MHC
class I and/or
MHC class II molecules on the surface of these APC. In the case of GM cells,
by expressing
the desired antigen, the APC improve the activation of T and B lymphocytes.
The
transplanted HSC may also enter the thymus, develop into DC, and present the
antigen in
question to developing T lymphocytes.
If present in low numbers (e.g., < 0.1% of thymus cells) the DC can bias the
selection
of new T cells to those reactive to the antigen. If the particular DC are
present in high
numbers, the same principle can be used to delete the new T cells which are
potentially
reactive to the antigen, which may be used in the prevention of autoimmune
diseases. B cells
are also increased in number in the BM, blood and peripheral lymphoid organs,
such as the
spleen and lymph nodes, within e.g., two weeks of castration.
In the case of the HSC being GM for a specific trait, each of the new cells
contains
the genetic modification of the parent stem/progenitor cell, and is thereby
completely or
partially resistant to infection or damage by the agent or agents
Methods for isolating and transducing stems cells and progenitor cells are
well known
to those skilled in the art. Examples of these types of processes are
described, for example, in
PCT Publication Nos. WO 95/08105, WO 96133281, WO 96133282, U.S. Patent Nos.
5,681,559, 5,199,942, 5,559,703, 5,399,493, 5,061,620.
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Antisense Polynucleotides
The term "antisense" is herein defined as a polynucleotide sequence which is
complementary to a polynucleotide of the present invention. The polynucleotide
may be
DNA or RNA. Antisense molecules may be produced by any method, including
synthesis by
ligating the genes) of interest in a reverse orientation to a viral promoter
which permits the
synthesis of a complementary strand. Once introduced into a cell, this
transcribed strand
combines with natural sequences produced by the cell to form duplexes. These
duplexes then
block either the further transcription or translation. In this manner, mutant
phenotypes may
be generated.
Catalytic Nucleic Acids
The term "catalytic nucleic acid" is herein defined as a DNA molecule or DNA
containing molecule (also known in the art as a "deoxyribozyme" or "DNAzyme")
or an
RNA or RNA-containing molecule (also known as a "ribozyme") which specifically
recognizes a distinct substrate and catalyzes the chemical modification of
this substrate. The
nucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T and
U, as well as
derivatives thereof. Derivatives of these bases are well known in the art.
Typically, the catalytic nucleic acid contains an antisense sequence for
specific
recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic
activity. The
catalytic strand cleaves a specific site in a target nucleic acid. The types
of ribozymes that
are particularly useful in this invention are the hammerhead ribozyme
(Haseloff and Gerlach
(1988) Nature 334:585), Perriman et al., (1992) Gene 113:157) and the hairpin
ribozynle
(Shippy et al., (1999) Mol. Biotechhoh 12:117).
dsRNA
Double stranded RNA (dsRNA) is particularly useful for specifically inhibiting
the
production of a particular protein. Although not wishing to be limited by
theory, one group
has provided a model for the mechanism by which dsRNA can be used to reduce
protein
production (Dougherty and Parks, (1995), Curr. Opira. Cell Biol. 7:399). This
model has
more recently been modified and expanded (Waterhouse et al., (1998) Proc.
Natl. Acad.. Sci.
USA 95:13959). This technology relies on the presence of dsRNA molecules that
contain a
sequence that is essentially identical to the mRNA of the gene of interest, in
this case an
mRNA encoding a polypeptide according to the first aspect of the invention.
Conveniently,
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the dsRNA can be produced in a single open reading frame in a recombinant
vector or host
cell, where the sense and antisense sequences are flanked by an unrelated
sequence which
enables the sense and anti-sense sequences to hybridize to form the dsRNA
molecule with the
unrelated sequence forming a loop structure. The design and production of
suitable dsRNA
molecules for the present invention are well within the capacity of a person
skilled in the art,
particularly considering Dougherty and Parks, (1995), Curr. Opin. Cell Biol.
7:399;
Waterhouse et a.1., (1998) Proc. Nat!. Acad. Sci. USA 95:13959; and PCT
Publication Nos.
WO 99/32619, WO 99/53050, WO 99149029, and WO 01/34815.
Anti-HIV Constructs
Those skilled in the art would be able to develop suitable anti-HIV constructs
for use
in the present invention. Indeed, a number of anti-HIV antisense constructs
and ribozymes
have already been developed and are described, for example; in U.S. Patent
Nos. 5,811,275,
5,741,706, and 5,144,019, and PCT Publication No. WO 94/26877 and Australian
Patent
Application No. 56394/94.
Genes
Useful genes and gene fragments (polynucleotides) for use in the methods of
the
invention involving GM HSCTs include those that code for resistance to
infection of T cells
by a particular infectious agent or agents. Such infectious agents include,
but are not limited
to, HIV, T cell leukemia virus, and other viruses that cause
lymphoproliferative diseases.
With respect to HIV/AI17S, a number of genes and/or gene fragments may be
used,
including, but not limited to, the nef transcription factor; a gene that codes
for a ribozyme that
specifically cuts HIV genes, such as tat and f~ev (Bauer et al., (1997) Blood
89:2259); the
' trans-dominant mutant form of HIV-1 rev gene, RevMlO, which has been shown
to inhibit
HIV replication (Bonyhadi et al., (1997) J. Virol. 71:4707); an overexpression
construct of
the HIV-1 rev-responsive element (RRE) (Kohn et al., (1.999) Blood 94:368);
any gene that
codes for an RNA or protein whose expression is inhibitory to HIV infection of
the cell or
replication; and fragments and combinations thereof.
These genes or gene fragments may be used in a stably expressible form. The
term
"stably expressible" is herein defined to mean that the product (RNA and/or
protein) of the
gene or gene fragment ("functional fragment") is capable of being expressed on
at least a
semi-permanent basis in a host cell after transfer of the gene or gene
fragment to that cell, as
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well as in that cell's progeny after division andlor differentiation. This
requires that the gene
or gene fragment, whether or not contained in a vector, has appropriate
signaling sequences
for transcription of the DNA to RNA. Additionally, when a protein coded for by
the gene or
gene fragment is the active molecule that affects the patient's condition, the
DNA also codes
for translation signals.
In most cases the genes or gene fragments are contained in vectors. Those of
ordinary
skill in the art are aware of expression vectors that may be used to express
the desired RNA
or protein.
Expression vectors are vectors that are capable of directing transcription of
DNA
sequences contained therein and translation of the resulting RNA. Expression
vectors are
capable of replication in the cells to be genetically modified, and include
plasmids,
bacteriophage, viruses, and minichromosomes. Alternatively the gene or gene
fragment may
become an integral part of the cell's chromosomal DNA. Recombinant vectors and
methodology are well-known to those skilled in the art.
Expression vectors useful for expressing the proteins of the present invention
may
comprise an origin of replication. Suitably constructed expression vectors
comprise an origin
of replication for autonomous replication in the cells, or are capable of
integrating into the
host cell chromosomes. Such vectors may also contain selective markers, a
limited number
of useful restriction enzyme sites, a high copy number, and strong promoters.
Promoters are
DNA sequences that direct RNA polymerase to bind to DNA and initiate RNA
synthesis;
strong promoters cause such initiation at high frequency.
- In one example, the DNA vector construct comprises a promoter, enhancer, and
a
polyadenylation signal. The promoter may be selected from the non-limiting
group
consisting of HIV, such as the Long Terminal Repeat (LTR), Simian Virus 40
(SV40),
Epstein Barr virus (EBV), cytomegalovirus (CMV), Rous sarcoma virus (RSV),
Moloney
virus, mouse mammary tumor virus (MMTV), human actin, human myosin, human
hemoglobin, human muscle creatine, human metalothionein. In another example,
an
inducible promoter is used so that the amount and timing of expression of the
inserted gene or
polynucleotide can be controlled.
The enhancer may be selected from the group including, but not limited to,
human
actin, human myosin, human hemoglobin, human muscle creatine and viral
enhancers such as
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those from CMV, RSV and EBV. The promoter and enhancer may be fiom the same or
different gene.
The polyadenylation signal may be selected, for example, from the group
consisting
of: LTR polyadenylation signal and SV40 polyadenylation signal, particularly
the SV40
minor polyadenylation signal among others.
The expression vectors of the present invention may be operably linked to DNA
coding for an RNA or protein to be used in this invention, i.e., the vectors
are capable of
directing both replication of the attached DNA molecule and expression of the
RNA. or
protein encoded by the DNA molecule. Thus, for proteins, the expression vector
must have
an appropriate transcription start signal upstream of the attached DNA
molecule, maintaining
the correct reading frame to permit expression of the DNA molecule under the
control of the
control sequences and production of the desired protein encoded by the DNA
molecule.
Expression vectors may include, but are not limited to, cloning vectors,
modified cloning
vectors and specifically designed plasmids or viruses. An inducible promoter
may be used so
that the amount and timing of expression of the inserted gene or
polynucleotide can be
controlled.
One having ordinary skill in the art can produce DNA constructs which are
functional
in cells. In order to test expression, genetic constructs can be tested for
expression levels ih
vitro using tissue culture of cells of the same type of those to be
genetically modified.
Methods Of Genetic Modification
Standard recombinant methods can be used to introduce genetic modifications
into the
cells being used for gene therapy. For example, retroviral vector transduction
of cultured
HSC is one successful method known in the art (Belmont and Jurecic (1997)
"Methods for
Efficient Retrovirus-Mediated Gene Transfer to Mouse Hematopoietic Stem
Cells," in Gene
Therapy Protocols (P:D. Robbins, ed.), Humana Press, pp.223-240; Bahnson et
al., (1997)
"Method for Retrovirus-Mediated Gene Transfer to CD34+-Enriched Cells," in
Gene Therapy
Protocols (P.D. Robbins, ed.), Humana Press, pp.249-263). Additional vectors
include, but
are not limited to, those that are adenovirus derived or lentivirus derived,
and Moloney
murine leukemia virus-derived vectors.
Also useful for genetic modification of HSC are the following methods:
particle-
mediated gene transfer such as with the gene gun (Yang, N.-S. and P.
Ziegelhoffer, (1994)
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"The Particle Bombardment System for Mammalian Gene Transfer," In PARTICLE
BOMBARDMENT TECHNOLOGY FOR GENE TRANSFER (Yang, N.-S. and Christou, P., eds.),
Oxford University Press, New York, pp. 117-141), liposome-mediated gene
transfer (Nabel
et a.l., (1992) Hmn. Geyae Thef°. 3:649), coprecipitation of
genetically modified vectors with
calcium phosphate (Graham and Van Der Eb, (1973) Vif~ol. 52:456),
electroporation (Potter et
al., (1984) Proc. Natl. Acad. Sci. USA 81:7161), and microinjection (Capecchi,
(1980) Cell
22:479), as well as any other method that can stably transfer a gene or
oligonucleotide, which
may be in a vector, into the HSC and other cells to be genetically modified
such that the gene
will be expressed at least part of the time.
SKEWING OF DEVELOPING TCR REPERTOIRE TOWARDS, OR AWAY FROM,
SPECIFIC ANTIGENS: ALLERGIES AND AUTOIMMUNE DISEASES
The ability to enhance the uptake into the thymus of hematopoietic stem cells
means
that the nature and type of dendritic cells can be manipulated. For example,
the stem cells
can be transfected with specific genes) which eventually become expressed in
the dendritic
cells in the thymus (and elsewhere in the body). Such genes can include those
which encode
specific antigens for which an immune response would be detrimental, as in
autoiminune
diseases and allergies.
This aspect of the invention stems from the discovery that direct effects of
sex steroid
inhibition on the BM functionality and immune cell functionality, and on the
eventual
reactivation of the thymus of an autoimmune patient will facilitate in
overcoming an
autoimmune disease suffered by that patient. This same principle also applies
to patients
suffering from allergies. As the thymus is reactivating, a new or modified
immune system is
created, one that no longer recognizes and/or responds to a self antigen.
In accordance with the invention, the following protocol may be applied. A.
patient
diagnosed with an autoimmune disease (e.g., type I diabetes) is first
immunosuppressed to
stop disease progression. .This may be done by administering an
immunosuppressant (e.g.,
cyclosporine or rapamycin) alone or together with anti-T and B cell
antibodies, such as anti-
CD3 or anti-T cell gamma globulin to get rid of T cells and anti-CD 19, CD20,
or CD21 to get
rid of B cells. At the same time that the patient is being immunosuppressed,
sex steroid
analogs (or other methods of castration) may be administered. His own T cells
may then be
mobilized with GCSF. If his autoimmunity arose as a result of a cross-reaction
of his T cells
with a pathogen he had previously encountered, the ablation of the T cells
will remove the
auto-reactive T cells, and the newly developed T cells will not continue to
recognize his cells
CA 02528521 2005-12-06
WO 2004/103271 PCT/US2004/011921
(e.g., his 0-islet cells) as foreign. In this manner, his autoimmune disease
is alleviated.
Moreover, once his autoinunune disease has been alleviated, the sex steroid
ablation therapy
can be stopped, thereby restoring the patient's fertility.
In another non-limiting example of the invention, the autoimmune patient is
reconstituted with allogeneic stem cells. In some embodiments, these
allogeneic stem cells
are umbilical cord blood cells, which do not include mature T cells.
In some embodiments, the transplanted HSC may follow full myeloablation or
myelodepletion, and thus result in a full HSC transplant (e.g., 5x106 cells/kg
body weight per
transplant). In some embodiments, only minor myeloablation need be achieved,
for example,
2-3 Gy irradiation (or 300 rads) followed by administration of about 3-4 x105
cellslkg body
weight. W some embodiments, T cell depletion (TCD), and/or another method of
immune
cell depletion, is used (see, e.g., Example 2). It may be that as little as
10% chimerism may
be sufficient to alleviate the symptoms of the patient's allergy or autoimmune
disease. In
some embodiments, the donor HSC are from umbilical cord blood (e.g.,1.5x10~
cells/kg for
recipient engraftment).
In other embodiments, patients begin to receive Lupron up to 45 days before
myelo-
ablative chemotherapy and continue on the Lupron concurrently with the BMT
such that the
total length of exposure to the drug is around 9 months (equivalent to 3
injections as each
Lupron injection delivers drug over a 3 month period). At various intervals
over the course of
study, blood samples are collected for analysis of T cell numbers
(particularly of new thymic
emigrants) and functions (specifically, response to T cell stimuli in vitro).
This embodiment
is also generally applicable to HSCT for other purposes described herein
(e.g., HSCT
following cancer radiation or chemotherapy).
In other embodiments, the transplanted HSC may follow lymphoablation. In some
embodiemtns, T cells and/or B cells may be selectively ablated, to remove
cells, as needed
(e.g., those cells involved in autoimmunity or allergy). The selection can
involve deletion of
cells that are activated, or of a cell type involved in the autoimmune or
allergic response. The
cells may be selected based upon cell surface markers, such as CD4, CDB, B220,
thyl, TCR ,
CD3, CDS, CD7, CD25, CD26, CD23, CD30, CD38, CD49b, CD69, CD70, CD71, CD95,
CD96, antibody specificity or Ig chain, or upregulated cytokine receptors
e.g., IL2-R B chain,
TGF(3. One well known method for depletion is the use of antilymphocyte
globulin. Other
methods of selecting and sorting cells are well known and include magnetic and
fluorescent
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cell separation, centrifugation, and more specifically, hemapheresis,
leukopheresis, and
lymphopheresis.
In some embodiments, HSCT is performed without myeloablation, myelodepletion,
lymphodepletion, T cell ablation, and/or other selective immune cell ablation.
In other embodiments, the methods of the invention further comprise
immunosuppressing the patient by e.g., administration of an immunosuppressing
agent
(e.g.,cyclosporin, prednisone, ozothioprine, FK506, Imunran, and/or
methotrexate) (see, e.g.,
U.S. Patent No. 5,876,708). In an embodiment, immunosuppression is performed
in the
absence of HSCT. In one embodiment, immunosuppression is performed in
conjunction
with (e.g., prior to, concurrently with, or after) HSCT. In another
embodiment,
immunosuppresion is performed in the absence of myeloablation, lymphoablation,
T cell
ablation and/or other selective immune cell ablation, deletion, or depletion.
In yet another
embodiment, immunosuppression is performed in conjunction with (e.g., prior
to,
concurrently with, or after) myeloablation, lymphoablation, T cell ablation,
andlor other
selective immune cell ablation, deletion, or depletion.
As described above, myeloablation, myelodepletion, lymphoablation,
immunosuppression, T cell ablation, and/or other selective immune cell
ablation, are
nonlimiting exemplary types of immune cell ablation, which are used throughout
this
application. The general term "immune cell depletion" is defined herein as
encompassing
each of these methods, i. e., myeloablation, myelodepletion, lymphoablation, T
cell ablation,
and/or other selective immune cell ablation (e.g., B cell or NK cell
depletion). As will be
readily understood by one skilled in the art, in practicing the inventions
provided herein, any
one of these "depletion" methods may be replaced with any one (or more) of the
other
"depletion" methods.
In one embodiment, NK cells are depleted. NK antibodies usefule for depleting
the
NK populations are known in the art. For example, one source of anti-NK
antibody is anti-
human thymocyte polyclonal anti-serum. U.S. Patent No. 6,296,846 describes NK
and T cell
depletion methodsm, as well as non-myeloablative therapy and formation of a
chimeric
lymphohematopoietic population, all of which may be used in the methods of the
invention.
In some embodiments, the methods of the invention further comprise, e.g.,
prior to
HSCT, absorbing natural antibodies from the blood of the recipient by
hemoperfusing an
organ (e.g.., the liver or kidney) obtained from the donor.
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In another embodiment the present invention further includes a T cell help-
reducing
treatment, such as increasing the level of the activity of a cytokine which
directly or
indirectly (e.g., by the stimulation or inhibition of the level of activity of
a second cytokine)
promotes tolerance to a graft (e.g., IL-10, IL-4, or TGF-.beta.), or which
decreased the level
of the activity of a cytokine which promotes rejection of a graft (i.e., a
cytokine which is
antagonistic to or inhibits tolerance (e.g., IFN.beta., IL,-1, IL,-2, or IL-
12)). In some
embodiments, a cytokine is administered to promote tolerance. The cytokine may
be derived
from the donor species or from the recipient species (see, e.g., U.S. Patent
No. 5,624,823,
which describes DNA encoding porcine interleukin-10 for such use). The
duration of the
help-reducing treatment may be approximately equal to, or is less than, the
period required
for mature T cells of the recipient species to initiate rejection of an
antigen after first being
stimulated by the antigen (in humans this is usually 8-12 days). In other
embodiments, the
duration is approximately equal to or is less than two-, three-, four-, five-,
or ten times the
period required for mature T cells of the recipient to initiate rejection of
an antigen after fit~st
being stimulated by the antigen. The short course of help-reducing treatment
may be
administered in the presence or absence of a treatment which~may stimulate the
release of a
cytokine by mature T cells in the recipient, e.g., in the absence of
Prednisone (17,21-
dihydroxypregna-1,4-dime-3,11,20-trione). The help-reducing treatment may be
begun
before or about the time the graft is introduced. The short course of help-
reducing treatment
may be pre-operative or post-operative. Tn some embodiments, the donor and
recipient are
class I matched.
In yet further embodiments, where the antigen is not an auto-antigen but,
rather, an
external antigen (e.g., pollen or seafood), similar strategies can be
employed. If the allergy
arose from some chance activation of an aberrant T or B cell clone,
immunosuppression to
remove T cells and B cells, followed by (or concurrent with) thymus
regeneration will
remove the cells causing the allergic response. Since the allergy arose from
the chance
activation of an aberrant T or B cell clone, it is unlikely to arise again
and, the newly
regenerated thymus may also create regulatory T cells. While there may be auto-
reactive IgE
still circulating in the patient, these will eventually disappear, since the
cells secreting them
are effectively depleted. Once the immune system has been re-established, the
sex steroid
ablation therapy can be stopped, and the patient's fertility restored.
The present invention provides methods for treating autoimmune disease without
a
BMT, with BMT, or with GM cells as described herein. The methods of the
invention may
further comprise an organ or cell transplant to repair or replace damaged
cells, tissues or
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organs. For example, in IDDM, a patient may require an islet cell transplant
to replace islet
cells damaged. Prevention of clinical symptoms of autoimmune disease may be
achieved
using the methods of the present invention, where a patient has pre-clinical
symptoms or
familial predisposition.
In further embodiments of the invention, genetic modification of the HSC may
be
employed if the antigen involved in the autoimmune disease or allergy is
known. For
example, in multiple sclerosis, the antigen may be myelinglycoprotein (MOG)
myelin
oligodendroglial protein, myelin basic protein or proteolipid protein. In
pernicious anemia,
the antigen may be the gastric proton pump. In type I diabetes, the antigen
may be pro-
insulin ( J Clih Invest. (2003) 111:1365., GAD or an islet cell antigen. T
cell epitopes of type
II collagen have been described with rheumatoid arthritis in (Ohnishi et al.
(2003) Iht. J. Mol.
Med. 1:331). For Hashimoto's thyroiditis, an antigen is thyroid peroxidase,
and for Graves
disease an antigen is the thyroid-stimulating hormone receptor, (Dawe et al.,
(1993) Spf°inger
Semin. Imnaureopathol. 14:285. Systemic lupus erythematosus antigens include
DNA,
histones, ribosomes, snRNP, scRNP e.g., Hl histone protein. In particlar Ro
(SS-A) and La
(SS-B) ribonucleo-protein antigens (e.g., Ro60 and Ro52).are associated with
patients
systemic lupus erythematosus (SLE) and rheumatoid arthritis. Myasthenia gravis
antigens
include acetylcholine receptor alpha chain, and some T cell epitopes are
described in Atassi
et al., (2001) Crit. Rev. Imrnut2ol. 21:1. Likewise, certain allergic
reactions are in response to
known antigens (e.g., allergy to feline saliva antigen in cat allergies). In
these situations, the
donor HSC may first be genetically modified to express the antigen prior to
being
administered to the recipient. HSC may be isolated based on their expression
of CD34.
These cells can then be adnunistered to the patient together with inhibitors
of sex steroid
mediated signaling, such as GnRH analogs, which enhances the functionality of
the BM.
Accordingly, the genetically-modified HSC not only develop into DC, and so
tolerize the
newly formed T cells, but they also enter the BM as DC and delete new,
autoreactive or
allergic B cells. Thus, central tolerance to the auto-antigen or allergen is
achieved in both the
thymus and the bone marrow, thereby alleviating the patient's autoimmune
disease or allergic
symptoms. In some embodiments, immune cell depletion or suppression is also
used.
In another example for the depletion of hyperreactive T cells, for which the
target
antigen is known, thymic epithelial stem cells (e.g., autologous epithelial
stem cells) can be
transfected with the gene encoding the specific antigen for which tolerance is
desired.
Thymic epithelial progenitor cells can be isolated from the thymus itself
(especially in the
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embryo) by their labeling with the Ab MTS 24 or its human counterpart (see
Gill et al.,
(2002) Nat. Immunol. 3:635).
Thus, in accordance with the invention, the basic principle is stop ongoing
autoimtnune disease or prevent one developing in highly predictive cases
(e.g., in familial
distribution) with T cell and/or B cell (as appropriate) depletion, followed
by rebuilding a
new tolerant immune system. First, the autoimmune disease is diagnosed, and a
determination is made as to whether or not there is a familial (genetic)
predisposition. Next,
a determination is made as to whether or not there had been a recent prolonged
infection in
the patient which may have lead to the autoimmune disease through antigen
mimicry or
inadvertent clonal activation. In practice it may not be possible to determine
the cause of the
disease. Next, T cell depletion is performed and, as appropriate, B cell
depletion is
performed (or other immune cell depletion), combined with chemotherapy,
radiation therapy
andlor anti-B cell reagents (e.g., CD19, CD20, and CD21) or antibodies to
specific Ig
subclasses (anti IgE). The bone marrow and immune cell functionality is
improved by
administering GnRH to the patient. Simultaneous with this is the injection of
HSC which
have been in vitro transfected with a gene encoding the autoantigen to enter
the rejuvenating
thymus and convert to DC for presentation of the autoantigen to developing T
cells thereby
inducing tolerance. The transfected HSC will also produce the antigen in the
bone marrow,
and present the antigen to developing immature B cells, thereby causing their
deletion,
similar to that occurring to T cells in the thymus. Use of the
immunosuppressive regimes
(anti-T, -B therapy) would overcome any untoward activation of pre-existing
potentially
autoreactive T and B cells. Moreover, in the case of non-obvious genetic
predisposition,
GnRI-I may be combined with G-CSF injection to increase blood levels of
autologous HSC to
enhance the thymic regrowth. - '
In some embodiments, hematopoietic or lymphoid stem and/or progenitor cells
from a
donor (e.g., an MHC-matched donor) are transplanted into the recipient to
increase the speed
of ultimate regeneration of the thymus. In another embodiment these cells are
transplanted
from a healthy donor, without autoimmune disease or allergies, to replace
aberrant stem
andlor progenitor cells in the patient.
In one embodiment, a patient's autoimmune disease is eliminated at least in
part by
clearance of the patient's T cell population. Sex steroid mediated signaling
is disrupted.
Upon repopulation of the peripheral blood with new T cells, the aberrant T
cells that failed
tolerance induction to self remain eliminated from the T cell population.??
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In another embodiment, a patient's immune system cells causing allergies are
eliminated by the same lymphocyte ablation treatements accompanied by
disruption of sex
steroid mediated signaling to enhance thymic T cell development, to allow
repopulation of
the peripheral blood stream with a "clean" population of T cells. In other
cases, allergies and
S autoimmune diseases are alleviated following sex steroid signaling
disruption due to
increased functionality of the BM and other immune cells.
PREVENTION
The invention further provides methods for preventing, increasing resistance
to, or
treating an infection of a patient through enhancing the functionality of BM
and/or immune
cells in conjunction with a regenerating thymus, or alternatively, prior to,
or without
reactivation of the thymus. At this stage, the patient's immune system is
enhanced,
rejuvenated and reactivated, thereby increasing its response to foreign
antigens such as
viruses and bacteria. This is shown, for example, in Figs. 13-17, which show
the effects of
thymic reactivation on the mouse immune system, as demonstrated with viral
(HSV)
challenge. The mice having prior reactivation of the thymus demonstrate
resistance to HSV
infection, while those not having thymic reactivation (aged thymus) have
higher levels of
HSV infection. It is well known that the mouse immune system is very similar
to the human
inunune system and is used as a model for human disease. Thus, results in mice
can be
projected to show human responses. This is reinforced by the data showing the
effects of
thymic reactivation in humans. The ability to increase the functionality of
immune cells is
exemplified in Example 23, in which TCR responsiveness and proliferation is
increased in
castrated mice as early as 3-7 days following castration, and before thymus
regeneration.
IMPROVEMENT OF VACCINE RESPONSE
The present disclosure is in the field of "active vaccinations," where an
antigen is
administered to a patient whose immune system then responds to the antigen by
forming an
immune response against the antigen. Vaccination may include both prophylactic
and
therapeutic vaccines. As will be appreciated by those in the art, the methods
of the invention
may be used with virtually any method of vaccination in combination with sex
steroid
inhibition without undue experimentation. In some embodiments, the vaccination
is given
prior to or concurrently with, thymic reactivation. Multiple doses (e.g.,
booster
immunizations) may also be given as needed.
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In one embodiment, the vaccine is a killed or inactivated vaccine (e.g., by
heat or
other chemicals). In another embodiment, the vaccine is an attenuated vaccine
(e.g.,
poliovirus and smallpox vaccines). In an embodiment, the vaccine is a subunit
vaccine (e.g.,
hepatitis B vaccine, in which hepatitis B surface antigen (HBsAg) is the agent-
specific
protein).
In another embodiment, the vaccine is a recombinant vaccine. One type of
recombinant vaccine is an attenuated vaccine in which the agent (e.g., a
virus) has specific
virulence-causing genes deleted, which renders the virus non-virulent. Another
type of
recombinant vaccine employs the use of infective, but non-virulent, vectors
which are
genetically modified to insert a gene encoding target antigens. Examples of a
recombinant
vaccines is a vaccinia virus vaccines.
lii yet another embodiment, the vaccine is a DNA vaccine. DNA-based vaccines
generally use bacterial plasmids to express protein immunogens in vaccinated
hosts.
Recombinant DNA technology is used to clone cDNAs encoding immunogens of
interest into
eukaryotic expression vectors. Vaccine plasmids are then amplified in
bacteria, purified, and
directly inoculated into the hosts being vaccinated. DNA can be inoculated by
a needle
injection of DNA in saline, or by a gene gun device which delivers DNA-coated
gold beads
into the skin. Methods for preparation and use of such vaccines will be well-
known to, or
may be readily ascertained by, those of ordinary skill in the art.
There are several parameters that can influence the nature and extent of
irtunune
responses: the level and type of antigen, the site of vaccination, the
availability of appropriate
APC, the general health of the individual, and the status of the T and B cell
pools. Of these,
T cells are the most vulnerable because of the marked sex steroid-induced
shutdown i.n
thymic export that becomes profound from the upset of puberty and the global
suppresson of
T cell responses by sex steroids. Any vaccination program should therefore
only be logically
undertaken when the level of potential responder T cells is optimal with
respect to both the
existence of naive T cells representing a broad repertoire of specificity, and
the presence of
normal ratios of Thl to Th2 cells and Th to Tc cells. The type of T cell help
that supports an
immune response determines whether the raised antibody will be C'-dependent
and
phagocyte-mediated defenses will be mobilized (a type 1 response), or whether
the raised
antibody will be C'-independent and phagocyte-independent defenses will be
mobilized (a
type 2 response) (for reviews, see Fearon and Locksley (1996) Science 272:50;
Seder and
Paul (1994) Annu. Rev. Immunol. 12:635). Historically type 1 responses have
been
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associated with the raising of cytotoxic T cells and type 2 responses with the
raising of
antibody. Thus, the level and type of cytokines generated may also be
manipulated to be
appropriate for the desired response (e.g., some diseases require Thl
responses, and some
require Th2 responses, for protective immunity). This includes codelivery of
Thl- or Th2-
type cytokines (e.g., delivery of recombinant cytokines or DNA encoding
cytokines) to shift
the immune response patterns in the patient. Immunostimulatory CpG
oligonucletides have
also been utilized to shift immune response to various vaccine formulations to
a more Thl-
type response.
By the methods described herein, sex steroid inhibition results in BM and
immune
cell functionality is increased without, prior to, or concurrently with thymic
regrowth, which
allows for improved immune responsiveness to vaccines.
This procedure can be combined with any other form of immune system
stimulation,
including adjuvant, accessory molecules, and cytokine therapies. For example,
useful
cytokines include, but are not limited to, interleukin 2 (IL-2) and IL-15 as a
general immune
growth factor, IL-4 to skew the response to Th2 (humoral immunity), and IFNy
to skew the
response to Thl (cell mediated, inflammatory responses), IL 12 to promote Thl
and IL,10 to
promote Th2 cells. Accessory molecules include but are not limited to
inhibitors of CTLA4,
which enhance the general immune response by facilitating the CD28/B7.1,B7.2
stimulation
pathway, which is normally inhibited by CTLA4.
Recombinant gene expression vectors may be used for the vaccination methods of
the
invention. The recombinant vectors may be plasmids or cosmids, which include
the antigen
coding polynucleotides of the invention, but may also be viruses or
retroviruses. The vectors
used in the polynucleotide vaccines may be "naked" (i.e., not associated with
a delivery
vehicle such as liposomes, colloidal particles, etc.). For convenience, the
term "plasmid" as
used in this disclosure will refer to plasmids or cosmids, depending on which
is appropriate to
use for expression of the peptide of interest (where the choice between the
two is dictated by
the size of the gene encoding the peptide of interest). A commonly used
plasmid vector
which may be used is pBR322.
Various viral vectors that can be utilized in the vaccination methods of the
invention
include adenovirus, herpes virus, vaccinia, or an RNA virus such as a
retrovirus. Retroviral
vectors may be derivatives of a marine or avian retrovirus. Examples of
retroviral include,
but are not limited to: Moloney marine leukemia virus (MoMuLV), Harvey rnurine
sarcoma
virus (HaMuS-V), marine mammary tumor virus (MuMTV), and Rous Sarcoma Virus
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(RS V). Other plasmids and viral vectors useful in the vaccination methods of
the
invention are well known in the vaccine art.
EFFECTS ON B1VI AND HSC
The present invention provides methods for increasing the function of BM in a
patient, including increasing production of HSC and enhancing haemopoiesis.
These
methods are useful in a number of applications. For example, one of the
difficult side effects
of chemotherapy or radiotherapy , whether given for cancer or for another
purpose, can be its
negative impact on the patient's BM. Depending on the dose of chemotherapy,
the BM may
be damaged or ablated and production of blood cells may be impeded.
Administration of a
dose of a sex steroid analog (such as an LHRH analog) according to this
invention after
chemotherapy treatment aids in recovery from the damage done by the
chemotherapy to the
BM and blood cells. Alternatively, administration of the LHRH analog in the
weeks prior to
delivery of chemotherapy increases the population of HSC and other blood cells
so that some
of the deleterious effects of chemotherapy will be decreased.
The improvement in BM function may be applicable to, for example, patients
with
blood disorders. The term "blood disorders" is herein defined as any disorder
or malady that
involves the cells of the blood system in a patient, either directly or
indirectly, including, but
not limited to, disorders associated with hematopoiesis, e.g., leukemia. Thus,
for example,
the methods of the present invention are useful to replace the cancerous blood
system cells
with new cells from a donor (matched or unmatched) in an allogenic HSCT, or
following
autologous HSCT with the patient's own cells.
Increased HSC production by the BM causes consequential increase in red blood
cells, which are, in turn, useful for management of RBC production. This can
be easily
determined by looking for, e.g., increased hematocrit. '
In some examples, the increased HSC are CD34+ or CD341o HSC. Mobilized HSCs
(e.g., using G-CSF) can assist in the "repair" or rejuvenation of tissues,
such as with heart
tissue and lung tissue. HSC have the potential to generate non-hematopoietic
tissue. While
much of the work has been carried out if2 vitf°o, a study at the Mayo
Clinic, Rochester has
shown that after BMT a small number of cardiomyocytes are donor derived.
Similarly, the
Beuamont Hospital, Michigan have used HSC to repair damaged heart muscle,
although it is
unclear whether the HSC become myocytes or vasculature. Mice experiments have
also
shown the potential of HSC to become insulin producing (3 - cells. Other work
has shown
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HSC are capable of becoming skeletal muscle (myocytes), liver (hepatocytes),
bone,
connective tissue, epithelial tissue (e.g. of lung, gut and skin),
vaseulature, neurons, and islet
0 cells.
The methods described herein are useful to repair damage to the BM and/or
assist in
the replacement of blood cells that may have been injured or destroyed by
various therapies
(e.g., cancer chemotherapy drugs, radiation therapy ) or diseases (e.g., HIV,
chronic renal
failure).
In some chemotherapy :regimens, such as high dose chemotherapy to treat any of
the
blood cancers, ablation of the BM is a desired effect. The methods of the
invention may be
used immediately after ablation occurs to stimulate the BM and increase the
production of
HSC and their progeny blood cells, so as to decrease the patient's recovery
time. Following
administration of the chemotherapy, usually allowing one or more days for the
chemotherapy
to clear from the patient's body, a dose of LHRH analog according to the
methods described
herein is administered to the patient. This can be in conjunction with the
administration of
autologous or heterologous BM or hematopoietic stem or progenitor cells, as
well as other
factors such as colony stimulating factors (CSFs) and stem cell factor (SCF).
Alternatively, a patient may have suboptimal (or "tired) BM function and may
not be
producing sufficient or normal numbers of,HSC arid other blood cells. This can
be caused
by a variety of conditions, including normal ageing, prolonged infection, post-
chemotherapy,
post-radiation therapy, chronic disease states including cancer, genetic
abnormalities, and
imrnunosuppression induced in transplantation. Further, radiation, such as
whole-body
radiation, can have a major impact on the BM productivity. These conditions
can also be
either pre-treated to minimize the negative effects (such as for chemotherapy
and/or radiation
therapy, or treated after occurrence to reverse the effects.
Effects On T Cells
Sex steroids in males and females can be inhibited temporarily by taking
disruptors of
sex steroid mediated signaling (e.g., GnRH agonists). It has been shown that
loss of steroids
causes a reaetivation of thymus function and enhanced production of naive T
cells.
Furthermore, even before those new T cells have had a chance to leave the
thymus, the pre-
existing T cells are much more sensitive to stimulation, which results in a
much more
effective immune response. This increased responsiveness is evident within
days of the loss
of sex steroids (see, e.g., Example 23). This may be because there are no
inhibitory effects of
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sex steroids. This may be because of the production of "helper" or "adjuvant"
factors by the
reactivating or reactivated thymus, which are able to costimulate the T cells
in conjunction
with the foreign stimulus. Also, since many cells of the immune system have
surface
receptors for GnRH, the GnRH itself may provide an additional stimulation for
the T cells.
Since the effect of the loss of sex steroids on peripheral T cells is so
rapid, GnRH can be
given as a single treatment simultaneously with the delivery of, for example,
a vaccine. A
one month formulation is useful which has the beneficial effects of
stimulating immune
responses but without the side effects of longer loss of sex steroids. A
subsequent "booster"
injection of the antigen can also be administered.
Sex steroid inhibitors (e.g., GnRH analogs) are useful to boost all forms of
immunotherapy in cancer patient, particularly for the removal of cancer cells
with have
escaped chemotherapy or surgery, but also for the defense against
opportunistic infections.
These analogs may also be used prophylacticly to improve immune response to
vaccination
programs designed to prevent, e.g., infections or cancer.
The methods of the invention utilize inhibion of sex steroid signaling. Sex
steroids
suppress the function of the thymus, BM, and also T and B lymphocytes
throughout the body,
which are concentrated in the major lymphoid areas of the body including, but
not limited to,
the blood, lymph nodes, mucosal tissue (e.g., respiratory, gastrointestinal,
genital). It has
surprisingly been discovered that ablation of sex steroids and/or interruption
of sex steroid-
mediated signaling may be used not only to regenerate the thymus (and thus the
number and
'quality' of T cells), but also to improve the functionality of pre-existing
and newly produce
T cells (and other cells of the immune system) either without, prior to, or
concurrently with,
thymus regeneration.
A poor immune response can have immediate and clinically important
consequences.
It can mean an increased susceptibility to common infections (e.g.,
influenza), increased
susceptibility to cancers and tumors, and/or poor responsiveness to
vaccinations.
An increase in the number and/or proportion of naive T cells in the total T
cell pool
has a positive immediate therapeutic effect on a number of clinical (or
potentially clinical)
conditions and diseases, including, but not limited to, cancer,
immunodeficiency (particularly
viral infections, e.g., Acquired Immune Deficiency Syndrome (Aff~S) and Severe
Acute
Respiratory Syndrome (SARS) or influenz), autoimmunity, transplantation,
allergies, as well
as improving the general efficacy of vaccination programs. Each of these
applications is
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described in detail in co-owned and co-pending U.S. Application Ser. Nos.
10/418,747,
10/419,039, 10/418,953, 10/418,727, 10/419,066, and 10/419,068.
With respect to the application of the methods of the instant invention to the
prevention of viral infections, a recent example of the effects of a poorly
functioning immune
system is observed with the advent of the SARS virus. This virus, although
related to the
common cold virus, is different enough so that the mature immune system cannot
recognize
it. Only naive T cells are able to deal with previous unknown infections, such
as SARS.
However, with few naive T cells, and an inability to create reasonable
additional quantities,
the average adult is highly susceptible to this disease, whereas children seem
able to cope
with it. This demographic is reflected in the mouality Figs. - the average age
for death is
approximately 50 years, with no pre-pubescent deaths being recorded.
In one example, inhibion of sex steroid signaling (e.g., using LHRH/GnRH
analogs)
is used to boost the responsiveness of T and B lymphocytes to stimulation with
antigen. This
stimulation may be a microorganism (e.g., bacteria, virus, fungi, parasites,
etc.) entering the
body.
In another example, inhibion of sex steroid signaling (e.g., using LHRH/GnRH
analogs) is used to enhance the immune response to a vaccine antigen.
In one nonlimiting example, inhibion of sex steroid signaling (e.g., using
LHRH/GnRH analogs) occurs prior to immune system challenge. This allows time
.for the
loss or sex steroids to occur. The inhibion of sex steroid signaling (e.g.,
using LHRH/GnRH
analogs) may also be accomplished simultaneously or sequentially with the
administration of
the stimulant to act as an "adjuvant" for enhancing the immure response
directly (which
could be mediated via direct signaling at the cell surface, increase in
cytokines, decrease in
inhibitors, etc.). The inhibion of sex steroid signaling (e.g., using
LHRH/GnRH analogs)
may also be given on multiple occasions. The immediate effects are due to
enhancing the
functionality of pre-existing lymphoid (and non-lymphoid) cells. With time,
the reduction of
sex steroids increases the production and functionality of T cells, B cells
and APC, which
additively, synergistically, or complementarily continue to enhance the
response. Increases
of new T cells, B cells, and other immune cells, such as APC, together with
increased
sensitivity of pre-existing T cells, B cells, and APC to stimulation, may then
be used for the
generation of more efficacious immune responses to primary infection,
secondary infection,
vaccination, etc.
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Thus, disruptors of sex steroid signaling are used according to the methods of
the
instant invention, to cause a clinically positive effect by initiating an
increased activation or
functionality of these immune cells even before these drugs have been able to
cause
significant thymic regrowth.
These drugs, according to the invention, also may be used to assist in the
replacement
of blood cells that may have been injured or destroyed by various other
therapies or diseases,
including but not limited to cancer chemotherapy drugs andlor cancer radiation
therapy, as
wells as diseases, such as chronic renal failure. As described in more detail
elsewhere herein,
the use of sex steroid inhibition eli-ugs in combination with G-CSF, GM-CSF,
erythropoietin
(EPO), SCF, or other hormones or cytokines may also be used to further improve
the
enhancement of the production of blood cells by those compounds.
The phrase "modifying the T-cell population makeup" is herein defined as
altering the
nature and/or ratio of T cell subsets defined functionally and by expression
of characteristic
molecules. Examples of these characteristic molecules include, but are not
limited to, the T
cell receptor, CD4, CDB, CD3, CD25, CD28, CD44, CD45, CD62L and CD69.
"Increasing the number of cells" e.g., T-cells, is herein defined as an
absolute increase
in the number of T cells in a subject in the thymus and/or in circulation
and/or in the spleen
andlor in the BM and/or in peripheral tissues such as lymph nodes,
gastrointestinal,
urogenital and respiratory tracts. This phrase also refers to a relative
increase in T cells, for
instance when compared to B cells.
A "subject having a depressed or abnormal T-cell population or function"
includes
an individual infected with. the human immunodeficiency virus, especially one
who has
AIDS, or any other virus or infection which attacks T cells or any T cell
disease for which a
defective gene has been identified. Furthermore, this phrase includes any post-
pubertal
individual, especially an aged person who has decreased immune responsiveness
and
increased incidence of disease as a consequence of post-pubeztal thymic
atrophy.
In cases where the subject is infected with HIV, it is useful that the HSC are
genetically modified such that they and their progeny, in particular T cells,
macrophages and
DC, are resistant to infection and/or destruction with the HIV virus. The
genetic modification
may involve introduction into HSC one or more nucleic acid molecules which
prevent viral
replication, assembly and/or infection. The nucleic acid molecule may be a
gene which
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~~encodes an antiviral protein, an antisense construct, a ribozyme, a dsRNA
and a catalytic
nucleic acid molecule.
In cases where the subject has defective T cells, the HSC may be genetically
modified
to normalize the defect. For diseases such as T cell leukaemias, the
modification may include
the introduction of nucleic acid constructs or genes which normalise the HSC
and inhibit or
reduce its likelihood of becoming a cancer cell.
It will be appreciated by those skilled in the art that the present methods
may be useful
in treating any T cell disorder which has a defined genetic basis.
The methods of the present invention are also useful for the treatment of
AIDS, where
the treatment involves reduction of viral load, increases in T cell number and
functionality,
reactivation of thymic function through inhibition of sex steroids signalling.
The patients
may receive HSC which have been genetically modified such that all progeny
(e.g., T cells
and DC) are resistant to further HIV infection. This means that not only will
the patient be
depleted of HIV virus and no longer susceptible to general infections because
the T cells have
returned to normal levels, but the new T cells being resistant to HIV will be
able to remove
any remnant viral infected cells. In principle a similar strategy could be
applied to gene
therapy in HSC for any T cell defect or any viral infection which targets T
cells.
Effect on B cells
As with other cells of the immune system, B cell function is also diminished
with age,
which is in part due to the decline in T cell production and consequent lack
of T cell help.
However, there are also significant age-associated changes in B cell function
(Hu et al.,
(1993) Izzt., Immuzzol. 5:.1035-1039). Despite B cell numbers remaining
relatively constant
throughout life due to tightly regulated homeostatic mechanisms, there is a
decrease in export
from the BM, clonal expansion of peripheral B cells, and a narrowing of the
antibody
response (LeMaoult et al., (1999) J. Inzzzzurcol. 162:6384-6391). Decreased
antibody response
to foreign antigens in the aged are thought to be primarily due to a decline
in T cell help (Hu
et a1.,(1993) Int. Inzzzzurzol. 5:1035-1039; LeMaoult et al., (1999) J.
Imnzuhol.. 162:6384-
6391). However, defective class switching (Weksler et al., (2000) Uacciyze
18:1624-1628)
and a preferential loss of high affinity antibodies may play a role (Nicoletti
et al., (1993) J.
Inzmurzol. 150:543-549).
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Castration of aged mice results in an increase in IL-7 responsive B cell
progenitors,
including late pro-B cells, pre- B cells, and immature B cells (Ellis et al.,
(2001) Iht. Immunol.
13:553-558). The absolute number of B cells in the periphery is also increased
(Elks et al.,
(20001) Iht. ImnZUUOI. 13:553-558). This increase in circulating B cells is
largely due to an
increase in the number of recent BM emigrants (CD45R1°CD24h1) and these
cells remain at an
elevated level for up to 54 days after castration (Ellis et al., (2001) lyat
Imrnunol. 13:553-558).
Thus, the methods of the invention may be used to increase the number and
functionality of B cells without, prior to. or concurrently with thymic
regeneration. This
may be useful for, e.g., increased control (by prevention or treatment) of
bacterial infections
in normal patients, in patients with compromised immune systems, such as those
patients
receiving a disease treatment regimen. Increased B cell number and
functionality may also
be useful following surgery andlor in burn victims, and in other instances
wherein the
patient's immune system is compromised.
Effects On DC
The present invention also provides methods for increasing DC functionality
and/or
DC number. Following sex steroid ablation (e.g., following delivery of an LHRH
analog)
DC are increased in the thymus, and in the periphery, which may also assist
the T cell
stimulation. DC are important antigen presenting cells and increased numbers
and/or
function may be useful in improving responsiveness to agents, e.g., cancers.
Enhanced DC
functionality may also be useful in achieving resistance to agents such as
allergens or self-
antigens (in the case of autoimmune disorders).
Effects on Platelets
The present invention also provides methods for increasing platelet cell
number
and/or functionality. Thrombocytopenia is common and has a variety of causes,
including,
but not. limited to, poor BM and splenomegaly. The condition generally results
in bleeding
disorders that are very difficult to treat. The most common diseases
associated with
thrombocytopenia include leukemia, aplastic anemia, cirrhosis, and Gauchers
disease.
Massive blood replacement is often needed because platelets have a short half
life in stored
blood which used for transfusions.
Effect on NK, NKT, and Treg cells
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The present invention also provides methods for increasing NK and NKT cell
number
and/or functionality (see, e.g., Example 17 and Figures 43 and 49). This may
be useful in the
treatment of diseases displaying NK deficiencies, e.g., Crohn's disease,
Chediak-Highashi
syndrome. Impaired NK cell function has been reported in patients with
connective tissue
diseases including lupus and rheumatoid arthritis. NK cells are important in
defending against
cancer and infectious agents. Other non-limiting diseases associated with NK
cell deficiency
(numerical and/or functional), which may benefit from the methods of the
invention include
herpes virus infections (e.g.,' varicella-zoster virus (VZV, chicken pox,
shingles), HSV CMV,
and EBV), other vitral infections (e.g., measles, mumps, influenza and HIV,
which are now
thought to be controlled, at least in part, by NK cells), mycobacterial
infections (e.g., M.
tuberculosis, M.avium, which are apparently controlled by both NK and
macrophage), other
malignancies (e.g., solid tumours, including melanoma, breast cancer and
rectal cancer), and
BMT for acute myelogenous leukemia with haploidentical/mismatch donors. NKT
cells are
important regulators of the immune response because they are very
highproducers of
cytokines without the need for prior activation. They have a role in
preventing autoimmune
disease but also promote anti-cancer effects. Tregs (commonly defined as being
CD4+CD25+) are also major regulators of the immune response primarily through
their
cytokine production.
Macrophage.
The present invention also provides methods for increasing macrophage number
and
functionality (see, e.g., Example 17 and Figure 43). which would have a
primary role in
helping remove infectious agents particularly bacteria.
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EXAMPLES
The following Examples provide specific examples of methods of the invention,
and
are not to be construed as limiting the invention to their content.
EXAMPLE 1
REVERSAL OF AGED-INDUCED THYMIC ATROPHY
Materials and Methods
Animals. CBA/CAH and C57B16/J male mice were obtained from Central
Animal Services, Monash University and were housed under conventional
conditions.
C57B16/J Ly5.1+ were obtained from the Central Animal Services Monash
University, the
Walterand Eliza Hall Institute for Medical Research (Parkville, Vicotoria) and
the A.R.C.
(Perth, Western Australia) and were housed under conventional conditions. Ages
ranged
from 4-6 weeks to 26 months of age and are indicated where relevant.
Surgical castration. Animals were anesthetized by intraperitoneal injection of
0.3 ml
of 0.3 mg xylazine (Rompun0; Bayer Australia Ltd., Botany NSW, Australia) and
1.5 mg
ketamine hycliochloride (Ketalar~; Parke-Davis, Caringbah, NSW, Australia) in
saline.
Surgical castration was performed by a scrotal incision, revealing the testes,
which were tied
with suture and then removed along with surrounding fatty tissue. The wound
was closed
using surgical staples. Sham-castration followed the above procedure without
removal of the
testes and was used as controls for all studies.
Bromodeoxyuridine (BrdU) incorporation. Mice received two intraperitoneal
injections of BrdU (Sigma Chemical Co., St. Louis, MO) at a dose of 100 mglkg
body weight
in 100 u1 of PBS, 4-hours apart (i.e., at 4 hour intervals). Control mice
received vehicle
alone injections. One hour after the second injection, thymuses were dissected
and either a
cell suspension made for FACS analysis, or immediately embedded in Tissue Tek
(O.C.T.
compound, Miles Inc., Indiana), snap frozen in liquid nitrogen, and stored at -
70°C until use.
Flow Cytometric analysis. Mice were killed by COZ asphyxiation and thymus,
spleen, and mesenteric lymph nodes were removed. Organs were pushed gently
through a
200 ~m sieve in cold PBS/1% FCS/0.02% Azide, centrifuged (650 g, 5 min,
4°C), and
resuspended in either PBS/FCS/Az. Spleen cells were incubated in red cell
lysis buffer (8.9
g/liter ammonium chloride) for 10 min at 4°C, washed and resuspended in
PBS/FCS/Az. Cell
concentration and viability were determined in duplicate using a hemocytometer
and
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ethidium bromide/acridine orange and viewed under a fluorescence microscope
(Axioskop;
Carl Zeiss, Oberkochen, Germany).
For 3-color immunofluorescence, cells were labeled with anti-a(3TCR-FITC, anti-
CD4-PE and anti-CD8-APC (all obtained from Pharmingen, San Diego, CA) followed
by
flow cytometry analysis. Spleen and lymph node suspensions were labeled with
either
a(3TCR-FITC/CD4-PE/CD8-APC or B220-B (Sigma) with CD4-PE and CD8-APC. B220-B
was revealed with streptavidin-Tri-color conjugate purchased from Caltag
Laboratories, Inc.,
Burlingame, CA.
For BrdU detection of cells, cells were surface labeled with CD4-PE and CD8-
APC,
followed by fixation and permeabilization as previously described (Carayon and
Bord, (1989)
J. Imm. Meth. 147:225). Briefly, stained cells were fixed overnight at
4°C in 1%
paraformaldehyde (PFA)/0.01% Tween-20. Washed cells were incubated in 500 ~ul
DNase
(100 Kunitz units, Roche, USA) for 30 mins at 37°C in order to denature
the DNA. Finally,
cells were incubated with anti-BrdU-FITC (Becton-Dickinson) for 30min at room
temperature, washed and resuspended for FACS analysis.
For BrdL1 analysis of TN subsets, cells were collectively gated out on Lin-
cells in
APC, followed by detection for CD44-biotin and CD25-PE prior to Brdl,T
detection. All .
antibodies were obtained from Pharmingen (San Diego, CA).
For 4-color Immunofluorescence, thymocytes were labeled for CD3, CD4, CDB,
B220 and Mac-1, collectively detected by anti-rat Ig-Cy5 (Amersham, U.K.), and
the
negative cells (TN) gated for analysis. They were fuuher stained for CD25-PE
(Pharmingen,
San Diego, CA) and CD44-B (Pharmingen, San Diego, CA) followed by Streptavidin-
'Tri-
color (Caltag, CA) as previously described (Godfrey and Zlotnik, (1993)
Immunol. Today
14:547). BrdU detection was then performed as described above.
Samples were analyzed on a FACSCaliburTM (Becton-Dickinson). Viable
lymphocytes were gated according to 0° and 90° light scatter
profiles and data was analyzed
using CellQuestTM software (Becton-Dickinson).
Immunohistology. Frozen thymus sections (4 ~,m) were cut using a cryostat
(Leica) and immediately fixed in 100% acetone.
For two-color irnmunofluorescence, sections were double-labeled with a panel
of
monoclonal antibodies: MTS6, 10, 12, 15, 16, 20, 24, 32, 33, 35 and 44
(Godfrey et al.,
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(1990) Iznnzunol. 70:66; Table 1) produced in this laboratory and the co-
expression of
epithelial cell determinants was assessed with a polyvalent rabbit anti-
cytokeratin Ab (Dako,
Carpinteria, CA). Bound mAb was revealed with FITC-conjugated sheep anti-rat
Ig (Silenus
Laboratories, Victoria, Australia) and anti-cytokeratin was revealed with
TRTTC-conjugated
goat anti-rabbit Ig (Silenus Laboratories, Victoria, Australia).
For BrdU detection of sections, sections were stained with either anti-
cytokeratin
followed by anti-rabbit-TRITC or a specific mAb, which was then revealed with
anti-rat Ig-
Cy3 (Amersham, Uppsala, Sweden). BrdU detection was then performed as
previously
described (Penit et al., (1996) Proc. Natl. Acad. Sci, USA 86:5547). Briefly,
sections were
fixed in 70% Ethanol for 30 mins. Semi-dried sections were incubated in 4M
HCI,
neutralized by washing in Borate Buffer (Sigma), followed by two washes in
PBS. BrdU was
detected using anti-BrdU-FITC (Becton-Dickinson).
For three-color irmnunofluorescence, sections were labeled for a specific MTS
mAb
together with anti-cytokeratin. BrdU detection was then performed as described
above.
Sections were analyzed using a Leica fluorescent and Nikon confocal
microscopes.
Migration studies (i.e., Analysis of recent thymic emigrants (RTE)). Animals
were anesthetized by intraperitoneal injection of 0.3 ml of 0.3 mg xylazine
(Rompun~; Bayer
Australia Ltd., Botany NSW, Australia) and 1.5 mg ketamine hydrochloride
(Ketalar~;
Parke-Davis, Caringbah, NSW, Australia) in saline.
Details of the FTTC labeling of thymocytes technique are similar to those
described
elsewhere (Scollay et al., (1980) Pz-oc. Natl. Acad. Sci, USA 86:5547; Berzins
et al., (1998) J.
Exp. Med. 187:1839). Briefly, thymic lobes were exposed and each lobe was
injected with
approximately 10 ~m of 350 ~ug/ml FTTC (in PBS). The wound was closed with a
surgical
staple, and the mouse was warmed until fully recovered from anesthesia. Mice
were killed
by C02 asphyxiation approximately 24 hours after injection and lymphoid organs
were
removed for analysis.
After cell counts, samples were stained with anti-CD4-PE and anti-CD8-APC,
then
analyzed by flow cytometry. Migrant cells were identified as live-gated FITC+
cells
expressing either CD4 or CD8 (to omit autofluorescing cells and doublets). The
percentages
of FITC+ CD4 and CD8 cells were added to provide the total migrant percentage
for lymph
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nodes and spleen, respectively. Calculation of daily export rates was
performed as described
by Berzins et al., ((1998) J. Ex~. Med. 187:1839).
Data analyzed using the unpaired student 't' test or nonparametrical Mann-
Whitney U-
test was used to determine the statistical significance between control and
test results for
experiments performed at least in triplicate. Experimental values
significantly differing from
control values are indicated as follows: *p< 0.05, **p< 0.01 and ***p< 0.001.
Results
I. The effect of age on thymocyte populations.
(i) Thymic weight and thymocyte number
With increasing age there is a highly significant (p<_0.0001) decrease in both
thymic
weight (Fig. 1A) and total thymocyte number (Figs. 1B) in mice. Relative
thymic weight
(mg thymus/g body) in the young adult has a mean value of 3.34 which decreases
to 0.66 at
18-24 months of age (adipose deposition limits accurate calculation). The
decrease in thymic
weight can be attributed to a decrease in total thymocyte numbers: the 1-2
month (i.e., young
adult) thymus contains about 6.7 x 10~ thymocytes, decreasing to about 4.5 x
106 cells by 24
months. By removing the effects of sex steroids on the thymus by castration,
thymocyte cell
numbers are regenerated and by 4 weeks post-castration, the thymus is
equivalent to that of
the young adult in both weight (Fig. 1A) and cellularity (Fig. 1B).
Interestingly, there was a
significant (p<0.001) increase in thymocyte numbers at 2 weeks post-castration
(1.2 x 10s),
which is restored to normal young levels by 4 weeks post-castration.
The decrease in T cell numbers produced by the thymus is not reflected in the
periphery, with spleen cell numbers remaining constant with age (Fig. 2A and
2B).
Homeostatic mechanisms in the periphery were evident since the B cell to T
cell ratio in
spleen and lymph nodes was not affected with age and the subsequent decrease
in T cell
numbers reaching the periphery (Fig. 2C). However, the ratio of CD4+ to CD8+ T
cell
significantly decreased (p<_ 0.001) with age from 2:1 at 2 months of age, to a
ratio of 1:1 at 2
years of age (Figs. 2D). Following castration and the subsequent rise in T
cell numbers
reaching the periphery, no change in peripheral T cell numbers was observed:
splenic T cell
numbers and the ratio of B:T cells in both spleen and lymph nodes was not
altered following
castration (Figs. 2A-C). The reduced CD4:CD8 ratio in the periphery with age
was still
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evident at 2 weeks post-castration but was completely reversed by 4 weeks post-
castration
(Fig. 2D)
(ii) Thymocyte subpopulations with age and post-castration.
To determine if the decrease in thymocyte numbers seen with age was the result
of the
depletion of specific cell populations, thymocytes were labeled with defining
markers in
order to analyze the separate subpopulations. In addition, this allowed
analysis of the kinetics
of thymus repopulation post-castration. The proportion of the main thymocyte
subpopulations was compared with those of the young adult (2-4 months) thymus
(Fig. 3) and
found to remain uniform with age. In addition, further subdivision of
thymocytes by the
expression of a(3TCR revealed no change in the proportions of these
populations with age.
At 2 and 4 weeks post-castration, thymocyte subpopulations remained in the
same
proportions and, since thymocyte numbers increase by up to 100-fold post-
castration, this
indicates a synchronous expansion of all thymoeyte subsets rather than a
developmental
progression of expansion.
The decrease in cell numbers seen in the thymus of aged (2 year old) animals
this
appears to be the result of a balanced reduction in all cell phenotypes, with
no significant
changes in T cell populations being detected. Thymus regeneration occurs in a
synchronous
fashion, replenishing all T cell subpopulations simultaneously rather than
sequentially.
II. Proliferation of thyni~ocytes
As shown in Figs. 4A-4B, 15-20% of thymocytes were proliferating at 2-4 months
of
age. The majority (about 80%) of these are double positive (DP) i.e., CD4+,
CD8+) with the
triple negative (TN) (i.e., CD3-CD4-CD8-) subset making up the second largest
population at
about 6% (Figs. 5A). These TN cells are the most immature cells in the thymus
and
encompass the intrathymic precursor cells. Accordingly, most division is seen
in the
subcapsule and cortex by immunohistology. Some division is seen in the
medullary regions
aligning with FACS analysis which revealed a proportion of single positive
(i.e.,
CD4+CD8- or CD4-CD8+) cells (9% of CD4+ T cells and 25% of CD8+ T cells) in
the
young (2 months) thymus, dividing (Fig. 5B).
Although cell numbers were significantly decreased in the aged mouse thymus (2
years old), the total proportion of proliferating thymocytes remained constant
(Figs. 4B and
5C), but there was a decrease in the proportion of dividing cells in the CD4-
CD8- and
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proliferation of CD4-CD8+ T cells was also significantly (pc 0.001) decreased
(data not
shown). Immunohistology revealed the distribution of dividing cells at 1 year
of age to
reflect that seen in the young adult (2-4 months); however, at 2 years,
proliferation is mainly
seen in the outer cortex and surrounding the vasculature with very little
division in the
medulla.
As early as one week post-castration there was a marked increase in the
proportion of
proliferating CD4-CD8- cells and the CD4-CD8+ cells (data not shown).
Castration clearly
overcomes the block in proliferation of these cells with age. There was a
corresponding
proportional decrease in proliferating CD4+CD8- cells post-castration (data
not shown). At 2
weeks post-castration, although thymocyte numbers significantly increase,
there was no
change in the overall proportion of thymocytes that were proliferating, again
indicating a
synchronous expansion of cells (Figs. 4A, 4B, and 5C). Immunohistology
revealed the
localization of thymocyte proliferation and the extent of dividing cells to
resemble the
situation in the 2-month-old thymus by 2 weeks post-castration.
The DN subpopulation, in addition to the thymocyte precursors, contains
' , ~ ~TCR, +CD4-CD8- thymocytes, which are thought to have down-regulated
both co-
receptors at the transition to SP cells (Godfrey and Zlotnik, (1993)
Irfamunol. T~day 14:547).
By gating on these mature cells, it was possible to analyze the true TN
compartment (CD3-
CD4-CD8-) and their subpopulations expressing CD44 and CD25. Figs. 5E-H
illustrate the
extent of proliferation within each subset of TN cells in young, old and
castrated mice. This
showed a significant (p<0.001) decrease in proliferation of the TN1 subset
(CD44~CD25-
CD3-CD4-CD8-), from about 10%% in the normal young to around 2% at 18 months
of age
(Fig. SE) which was restored by 1 week post-castration.
III. Thymocyte emigration
Approximately 1 % of T cells migrate from the thymus daily in the young mouse
(Scollay et al., (1980) Proc. Natl. Acad. Sci, USA 86:5547). Migration in
castrated mice was
found to occur at a proportional rate equivalent to the normal young mouse at
14 months and
even 2 years of age, although significantly (p<_ 0.0001) reduced in number
(Figs. 6A and 6B).
There was an increase in the CD4:CD8 ratio of the recent thymic emigrants from
about 3:1 at
2 months to about 7:1 at 26 months (Fig. 6C). By 1 week post-castration, this
ratio had
normalized (Fig. 6C). By 2- weeks post-castration, cell number migrating to
the periphery
had substantially increased with the overall rate of migration reduced to 0.4%
reflecting the
expansion of the thymus (Fig. 6B).
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Discussion
It has been shown that aged thymus, although severely atrophic, maintains its
functional capacity with age, with T cell proliferation, differentiation and
migration occurring
at levels equivalent to the young adult mouse. Although thymic function is
regulated by
several complex interactions between the neuro-endocrine-immune axes, the
atrophy induced
by sex steroid production exerts the most significant and prolonged effects
illustrated by the
extent of thymus regeneration post-castration.
Thymus weight is significantly reduced with age as shown previously (Hirokawa
and
Makinodan, (1975) J. Irnm.unol. 114:1659, Aspinall, (1997) J. Immurzol.
158:3037) and
correlates with a significant decrease in thymocyte numbers. The stress
induced by the
castration technique, which may result in further thymus atrophy due to the
actions of
corticosteroids, is overridden by the removal of sex steroid influences with
the 2-week
castrate thymus increasing in cellularity by 20-30 fold from the pre-castrate
thymus. By 3
weeks post-castration, the aged thymus shows a significant increase in both
thymic size and
cell number, surpassing that of the young adult thymus presumably due to the
actions of sex
steroids already exerting themselves in the 2 month old mouse.
The data confirms previous findings that emphasise the continued ability of
thymocytes to differentiate and maintain constant subset proportions with age
(Aspinah,
(1997) J. InznzuJZOI. 158:3037). In addition, thymocyte differentiation has
been shown to
occur simultaneously post-castration indicative of a synchronous expansion in
thymocyte
subsets. Since thym.ocyte numbers are decreased significantly with age,
proliferation of
thymocytes was analysed to determine if this was a contributing factor in
thymus atrophy.
Proliferation of thymocytes was not affected by age-induced thymic atrophy or
by
removal of sex-steroid influences post-castration with about 14% of all
thymocytes
proliferating. However, the localisation of this division differed with age:
the 2 month mouse
thymus shows abundant division throughout the subcapsular and cortical areas
(TN and DP T
cells) with some division also occurring in the medulla. Due to thymic
epithelial
disorganisation with age, localisation of proliferation was difficult to
distinguish but appeared
to be less uniform in pattern than the young and relegated to the outer
cortex. By 2 weeks
post-castration, dividing thymocytes were detected throughout the cortex and
were evident in
the medulla with similar distribution to the 2 month thymus.
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The phenotype of the proliferating population as determined by CD4 and CD8
analysis, was not altered with age or following castration. However, analysis
of proliferation
within thymocyte subpopulations, revealed a significant decrease in
proliferation of both the
TN and CD8'~ cells With age. Further analysis within the TN subset on the
basis of the
markers CD44 and CD25, revealed a significant decrease in proliferation of the
TN1
(CD44~CD25-) population which was compensated for by an increase in the TN2
(CD44-
CD25+) population. These abnormalities within the TN population, reflect the
findings by
Aspinall ((1997) d. Inamureol. 158:3037). Surprisingly, the TN subset was
proliferating at
normal levels by 2 weeks post-castration indicative of the immediate response
of this
population to the inhibition of sex-steroid action. Additionally, at both 2
weeks and 4 weeks
post-castration, the proportion of CD8+ T cells that were proliferating was
markedly
increased from the control thymus, possibly indicating a role in the re-
establishment of the
peripheral T cell pool.
Thymocyte migration was shown to occur at a constant proportion of thymocytes
with
age conflicting with previous data by Scollay et al., ( (1980) Proc. Natl.
Acad. Sci, USA
86:5547) who showed a ten-fold reduction in the rate of thymocyte migration to
the
periphery. The difference in these results may be due to the difficulties in
intrathymic FITC
labelling of 2 year old thymuses or the effects .of adipose deposition on FITC
uptake.
However, the absolute numbers of T cells migrating was decreased significantly
as found by
Scollay resulting in a significant reduction in ratio of RTEs to the
peripheral T cell pool. This
will result in changes in the periphery predominantly affecting the T cell
repertoire (Mackall
et al., (1995) N. E~zg. J. Med. 332:143). Previous papers (e.g., Mackall et
al., (1995) N. E'ng.
J. Med. 332:143) have shown a skewing of the T cell repertoire to a memory
rather than
naive T cell phenotype with age. The diminished T cell repertoire however, may
not cope if
the individual encounters new pathogens, possibly accounting for the rise in
immunodeficiency in the aged. Obviously, there is a need to re-establish the T
cell pool in
immunocompromised individuals. Castration allows the thymus to repopulate the
periphery
through significantly increasing the production of naive T cells.
In the periphery, T cell numbers remained at a constant level as evidenced in
the B:T
cell ratios of spleen and lymph nodes, presumably due to peripheral
homeostasis (Mackall et
al., (1995) N. Eng. J. Med. 332:143; Berzins et al., (1998) J. Exp. Med.
187:1839). However,
disruption of cellular composition in the periphery was evident with the aged
thymus
showing a significant decrease in CD4:CD8 ratios from 2:1 in the young adult
to 1:1 in the 2
year mouse, possibly indicative of the more susceptible nature of CD4+ T cells
to age or an
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increase in production of CD8+ T cells from extrathymic sources. By 2 weeks
post-
castration, this ratio has been normalised, again reflecting the immediate
response of the
immune system to surgical castration.
The above findings have shown firstly that the aged thymus is capable of
functioning
in a nature equivalent to the pre-pubertal thymus. In this respect, T cell
numbers are
significantly decreased but the ability of thymocytes to differentiate is not
disturbed. Their
overall ability to proliferate and eventually migrate to the periphery is
again not influenced by
the age-associated atrophy of the thymus. However, two important findings were
noted.
Firstly, there appears to be an adverse affect on the TN cells in their
ability to proliferate,
correlating with findings by Aspinall ((1997) J. Immuraol. 158:3037). This
defect could be
attributed to an inherent defect in the thymocytes themselves. Yet the data
shown here, and
previous work has shown thymocyte differentiation, although diminished, still
occurs and
stem cell entry from the BM is also not affected with age (Hirokawa, (1998),
"Immunity and
Ageing," in PRINCIPLES AND PRACTICE OF GERIATRIC MEDICINE, (M. Pathy, ed.)
John W iley
and Sons Ltd; Mackall and Gress, (1997) Immuuol. Rev. 160:91). Secondly, the
CD8+ T cells
were significantly diminished in their proliferative capacity with age and,
following
castration, a significantly increased proportion of CD8+ T cells proliferated
as compared to
the 2 month mouse. The proliferation of mature T cells is thought to be a
final step before
migration. (Such and Zlotnik, (1991) J. Imrrauraol. 146:3068), such that a
significant decrease
in CD8+ proliferation would indicate a decrease in their migrational
potential. This
hypothesis is. supported by the finding that the ratio of CD4:CD8 T cells in
RTES increased
with age, indicative of a decrease in CD8 T cells migrating. Alternatively, if
the thymic
epithelium is providing the key factor for the CD8 T cell maintenance, whether
a
lymphostromal molecule or cytokine influence, this factor may be disturbed
with increased
sex-steroid production. By removing the influence of sex-steroids, the CD8 T
cell population
can again proliferate optimally.
The defect in proliferation of the TN1 subset which was observed indicates
that loss of
cortical epithelium affects thymocyte development at the crucial stage of TCR
gene
rearrangement whereby the cortical epithelium provides factors such as II,-7
and SCF
necessary for thymopoiesis (Godfrey and Zlotnik, (1993) Irrarnunol. Today
14:547; Aspinall,
(1997) J. Irramunol. 158:3037). Indeed, IL-7-l- and IL-7R-r- mice show similar
thymic
morphology to that seen in aged mice (Wiles et al., (1992) Eur. J. Irnrr2unol.
22:1037; Zlotnik
and Moore, (1995) Curr. Opin. InZrnurcol. 7:206); von Freeden-Jeffry, (1995)
J. Exp. Med.
181:1519).
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In conclusion, the aged thymus still maintains its functional capacity,
however, the
thymocytes that develop in the aged mouse are not under the stringent control
by thymic
epithelial cells as seen in the normal young mouse due to the lack of
structural integrity of the
thymic microenvironment. Thus the proliferation, differentiation and migration
of these cells
will not be under optimal regulation and may result in the increased release
of
autoreactive/imlnunodysfunctional T cells in the periphery. The defects within
both the TN
and particularly, CD8+ populations, may result in the changes seen within the
peripheral T
cell pool with age. Restoration of thymus function by castration will provide
an essential
means for regenerating the peripheral T cell pool and thus in re-establishing
immunity in
irninunosuppressed, immunodeficient, or immunocompromised individuals.
EXAMPLE 2
REVERSAL OF CHEMOTHERAPY- OR RADIATION-INDUCED THYMIC
ATROPHY
Materials and Methods
Materials and methods were as described in Example 1. W addition, the
following
methods were used.
BM reconstitution. Recipient mice (3-4 month-old C57BL6/J) were subjected to
5.SGy irradiation twice over a 3-hour interval. One hour following the second
irradiation
dose, mice were injected intravenously with 5x106 donor BM cells. BM cells
were obtained
by passing RPMI-1640 media through the tibias and femurs of donor (2-month old
congenic
C57BL6/J LyS.I+) mice, and then harvesting the cells collected in the media.
Irradiation. 3-4 month old mice were subjected to 625Rads of whole body CJ-
irradiation.
T cell Depletion Using Cyclophosphamide. Old mice (e.g., 2 years old) were
injected with cyclophosphamide (200 mg/kg body wt over two days) and
castrated.
Results
Castration enhanced regeneration following severe T cell depletion (TCD). For
both
models of T cell depletion studied (chemotherapy using cyclophosphamide or
sublethal
irradiation using 625Rads), castrated (Cx) mice showed a significant increase
in the rate of
thymus regeneration compared to their sham-castrated (ShCx) counterparts
(Figs. 7A and
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7B). By 1 week post-treatment castrated mice showed significant thymic
regeneration even
at this early stage (Figs. 7 and 9-11). In comparison, non-castrated animals,
showed severe
loss of DN and DP thymocytes (rapidly-dividing cells) and subsequent increase
in proportion
of CD4 and CD8 cells (radio-resistant). This is best illustrated by the
differences in
thymocyte numbers with castrated animals showing at least a 4-fold increase in
thymus size
even at 1 week post-treatment. By 2 weeks, the non-castrated animals showed
relative
thymocyte normality with regeneration of both DN and DP thymocytes. However,
proportions of thymocytes are not yet equivalent to the young adult control
thymus. Indeed,
at 2 weeks, the vast difference in regulation rates between castrated and non-
castrated mice
was maximal (by 4 weeks thymocyte numbers were equivalent between treatment
groups).
Thymus cellularity was significantly reduced in ShCx mice 1-week post-
cyclophosphamide treatment compared to both control (untreated, aged-matched;
p<_0.001)
and Cx mice (p<_0.05) (Fig. 7A). No difference in thymus regeneration rates
was observed at
this time-point between mice castrated 1-week earlier or on the same day as
treatment, with-
both groups displaying at least a doubling in the numbers of cells compared to
ShCx mice
(Figs. 7A and 8A). Similarly, at 2-weeks post-cyclophosphamide treatment, both
groups of
Cx mice had significantly (5-6 fold) greater thymocyte numbers (p<_U.001) than
the ShCx
mice (Fig. 7A). In control mice there was a gradual recovery of thymocyte
number aver 4
weeks but this was markedly enhanced by castration, even within one week (data
not shown).
Similarly spleen and lymph node numbers were increased in the castrate mice
after one week
(data not shown).
The effect of the timing of castration on thymic recovery was examined by
castration
one week prior to either irradiation (Fig. 10) or on the same day as
irradiation (Fig. 11).
When performed one week prior, castration had a more rapid impact on thymic
recovery (Fig.
IOA compared to Fig 11 A). By two weeks the same day castration had "caught
up" with the
thymic regeneration in mice castrated one week prior to treatment. In both
cases there were
no major effects on spleen or lymph nodes (Figs. lOB and 10C, and Figs. 11B
and 11C)
respectively.
Following irradiation treatment, both ShCx and mice castrated on the same day
as
treatment (SDCx) showed a significant reduction in thymus cellularity compared
to control
mice (p<_0.001) (Figs. 7B and 11A) and mice castrated 1-week prior to
treatment (p<_0.01)
(Fig. 7B). At 2 weeks post-treatment, the castration regime played no part in
the restoration
of thymus cell numbers with both groups of castrated mice displaying a
significant
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enhancement of thymus cellularity post-irradiation (PIrr) compared to ShCx
mice (p_0.001)
(Figs. 7B, 10A, and 11A). Therefore, castration significantly enhances thymus
regeneration
post-severe T cell depletion, and it can be performed at least 1-week prior to
immune system
insult.
Interestingly, thymus size appears to 'overshoot' the baseline of the control
thymus.
Indicative of rapid expansion within the thymus, the migration of these newly
derived
thymocytes does not yet (it takes about 3-4 weeks for thymocytes to migrate
through and out
into the periphery). Therefore, although proportions within each subpopulation
are equal,
numbers of thymocytes are building before being released into the periphery.
Following cyclophosphamide treatment of young mice (about 2-3 months), total
lymphocyte numbers within the spleen of Cx mice, although reduced, were not
significantly
different from control mice throughout the time-course of analysis (data not
shown).
However, ShCx mice showed a significant decrease in total splenocyte numbers
at 1- and 4-
weeks post-treatment (p<_0.05) (Fig. 8A). Within the lymph nodes, a
significant decrease in
cellularity was observed at 1-week post-treatment for both sham-castrated and
castrated mice
(p<_0.01) (Fig. 8B), possibly reflecting the influence of stress steroids. By
2-weeks post-
treatment, lymph node cellularity of castrated mice was comparable to control
mice however
sham-castrated mice did not restore their lymph node cell numbers until 4-
weeks post-
treatment, with a significant (p<_0.05) reduction in cellularity compared to
both control and
Cx mice at 2-weeks post-treatment (Fig. 8B). These results indicate that
castration may
enhance the rate of recovery of total lymphocyte numbers following
cyclophosphamide
treatment.
Sublethal irradiation (625 Rads) induced a profound lymphopenia such that at 1-
week
post-treatment, both treatment groups (Cx and ShCx), showed a significant
reduction in the
cellularity of both spleen and lymph nodes (p_0.001) compared to control mice
(Figs. 12A
and 12B). By 2 weeks post-irradiation, spleen cell numbers were similar to
control values for
both castrated and sham-castrated mice (Fig. 12A), whilst lymph node cell
numbers were still
significantly lower than control values (p<_0.001 for sham-castrated mice;
p<_0.01 for
castrated mice) (Fig. 12B). No significant difference was observed between the
Cx and ShCx
mice.
Fig. 9 illustrates the use of chemical castration compared to surgical
castration in
enhancement of T cell regeneration. The chemical used in this example,
Deslorelin (an
LHRH-A), was injected for four weeks, and showed a comparable rate of
regeneration post-
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cyclophosphamide treatment compared to surgical castration. The enhancing
effects were
equivalent on thymic expansion and also the recovery of spleen and lymph node.
The
kinetics of chemical castration are slower than surgical, that is, mice take
about 3 weeks
longer to decrease their circulating sex steroid levels. However, chemical
castration is still as
effective as surgical castration and can be considered to have an equivalent
effect.
Discussion
The impact of castration on thymic structure and T cell production was
investigated in
animal models of immunodepletion. Specifically, Example 2 examined the effect
of
castration on the recovery of the immune system after sublethal irradiation
and
cyclophosphamide treatment. These farms of immunodepletion act to inhibit DNA
synthesis
and therefore target rapidly dividing cells. In the thymus these cells axe
predominantly
immature cortical thymocytes, but all subsets are effected (Fredrickson and
Basch, (1994)
Dev. C~m~. I~amurt.ol: 18:251). In normal healthy aged animals, the
qualitative and
quantitative deviations in peripheral T cells seldom lead to pathological
states. However,
major problems arise following severe depletion of T cells because of the
reduced capacity of
the thymus for T cell regeneration. Such insults occur in HIV/AIDS, and
particularly
following chemotherapy and radiotherapy in cancer treatment (Mackall et al.,
(1995) N. Erag.
J. Med. 332:143).
In both sublethally irradiated and cyclophosphamide treated mice, castration
markedly
enhanced thymic regeneration. Castration was carried out on the same day as
and seven days
prior to immunodepletion in order to appraise the effect of the predominantly
corticosteroid
induced, stress response to surgical castration on thymic regeneration.
Although increases in
thymus cellularit~r and architecture were seen as early as one week after
immunodepletion,
the major differences were observed two weeks after castration. This was the
case whether
castration was penormed on the same day or one week prior to immunodepletion.
Immunohistology demonstrated that in all instances, two weeks after castration
the
thymic architecture appeared phenotypically normal, while that of noncastrated
mice was
disorganised. Pan epithelial markers demonstrated that immunodepletion caused
a collapse
in cortical epithelium and a genexal disruption of thymic architecture in the
thymii of
noncastrated mice. Medullary markers supported this finding. Interestingly,
one of the first
features of castration-induced thymic regeneration was a marked upregulation
in the
extracellular matrix, identified by MTS 16.
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Flow cytometry analysis data illustrated a significant increase in the number
of cells in
all thymocyte subsets in castrated mice. At each time point, there was a
synchronous
increase in all CD4, CD8 and ec(3-TCR - defined subsets following
immunodepletion and
castration. This is an unusual but consistent result, since T cell development
is a progressive
process it was expected that there would be an initial increase in precursor
cells (contained
within the CD4- CD8- gate) and this may have occurred before the first time
point.
Moreover, since precursors represent a very small proportion of total
thymocytes, a shift in
their number may not have been detectable. The effects of castration on other
cells, including
macrophages and granulocytes were also analysed. In general there was little
alteration in
macrophage and granulocyte numbers within the thymus.
W both irradiation and cyclophosphamide models of immunodepletion thymocyte
numbers peaked at every two weeks and decreased four weeks after treatment.
Almost
immediately after irradiation or chemotherapy, thymus weight and cellularity
decreased
dramatically and approximately 5 days later the first phase of thymic
regeneration begun.
The first wave of reconstitution (days 5-14) was brought about by the
proliferation of
radioresistant thymocytes (predominantly double negatives) which gave rise to
all thymocyte
subsets (Penit and Ezine, (1989) Proc. Natl. Acad. Sci, USA 86:5547). The
second decrease,
observed between days 16 and 22 was due to the limited proliferative ability
of the
radioresistant cells coupled with a decreased production of thymic precursors
by the BM
(also effected by irradiation). The second regenerative phase was due to the
replenishment of
the thymus with BM derived precursors (Huiskamp et al., (1983) Radiat. Res.
95:370).
In adult mice the development from a HSC to a mature T cell takes
approximately 28
days (Shortman et.al., (1990) Sem. Irramurcol. 2:3). Therefore, it is not
surprising that little
change was seen in peripheral T cells up to four weeks after treatment. The
periphery would
be supported by some thymic export, but the majority of the T cells found in
the periphery up
to four weeks after treatment would be expected to be proliferating
cyclophosphamide or
irradiation resistant clones expanding in the absence of depleted cells.
Several long term
changes in the periphery would be expected post-castration including, most
importantly, a
diversification of the TCR repertoire due to an increase in thymic export.
EXAMPLE 3
THYMIC REGENERATION FOLLOWING INHIBITION OF SEX STEROIDS
RESULTS IN RESTORATION OF DEFICIENT PERIPHERAL T CELL FUNCTION
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Materials and Methods
Materials and methods were as described in Examples 1 and 2. In addition, the
following methods were used.
HSV-1 immunization. Aged (>_18 months) mice were surgically castrated. 6 weeks
after castration (following thymus reactivation). Following anesthetic, mice
were injected in
the hind leg (foot-hock) with 4x105 plaque forming units (pfu) of HSV-1(KOS
strain) in
sterile PBS using a 20-gauge needle. Infected mice were housed in isolated
cages and
humanely killed on D5 post-immunization at which time the popliteal (draining)
lymph nodes
were removed for analysis.
Virus was obtained from Assoc. Prof. .Frank Carbone (Melbourne University).
Virus
stocks were grown and titrated on VERO cell nionolayers in MEM supplemented
with 5%
FCS (Gibco-BRL, Australia).
Analysis of the draining (popliteal) lymph nodes was performed on D5 post-
infection.
For HSV-1 studies, popliteal lymph node cells were stained for anti-CD25-PE,
anti-CD8-
APC and anti-V(310-biotin. For detection of DC, an FcR block was used prior to
staining for
CD45.1-FTTC, I-Ab-PE and CDllc-biotin. All biotinylated antibodies were
detected with
streptavidin-PerCP. For detection of HSC, BM cells were gated on Liri cells by
collectively
staining with anti-CD3, CD4, CDB, Gr-l, B220 and Mac-1 (all conjugated to
FITC). HSC
were detected by staining with CD117-APC and Sca-1-PE. For TN thymocyte
analysis; cells
were gated .on the Liri population and detected by staining with CD44-biotin,
CD25-PE and
c-kit-APC.
Cytotoxicity assay of lymph node cells. Lymph node cells were incubated for
three
days at 37°C, 6.5% C02. Specificity was determined using a non-
transfected cell line (EL4)
pulsed with gB49s-sos peptide (gBp) and EL4 cells alone as a control. A
starting effectoraarget
ratio of 30:1 was used. The plates were incubated at 37°C, 6.5% C02 for
four hours and then
centrifuged 650gmaX for 5 minutes. Supernatant (1001) was harvested from each
well and
transferred into glass fermentation tubes for measurement by a Packard Cobra
auto-gamma
counter.
Results
To determine the functional consequences of thymus regeneration (e.g., whether
castration can enhance the immune response, herpes simplex virus (HSV)
immunization was
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examined as it allows the study of disease progression and role of CTL.
Castrated mice were
found to have a qualitatively and quantitatively improved responsiveness to
the virus.
Mice were immunized in the footpad and the popliteal (draining) lymph node
analyzed at D5 post-immunization. In addition, the footpad was removed and
homogenized
to determine the virus titer at particular time-points throughout the
experiment. The regional
(popliteal) lymph node response to HSV-1 infection (Figs. 13-17) was examined.
A significant decrease in lymph node cellularity was observed with age (Figs.
13A,
13B, and 14). At D5 (d.e., 5 days) post-immunization, the castrated mice have
a significantly
larger lymph node cellularity than the aged mice (data not shown). Although no
differ°ence in
the proportion of activated (CD8+CD25+) cells was seen with age or post-
castration (Fig. 15),
activated cell numbers within the lymph nodes were significantly increased
with castration
when compared to the aged controls (data not shown). Further, activated cell
numbers
correlated with that found for the young adult (data not shown), indicating
that CTLs were
being activated to a greater extent in the castrated mice, but the young adult
may have an
enlarged lymph node due to B cell activation. This was confirmed with a CTL
assay
detecting the proportion of specific lysis occurring with age and post-
castration (Fig. 16).
Aged mice showed a significantly reduced target cell lysis at effectoraarget
ratios of 10:1 and
3:1 compared to young adult (2-month) mice (Fig. 16). Castration restored the
ability of
mice to generate specific CTL responses post-HSV infection (Fig. 16).
In addition, while overall expression of V(310 by the activated cells remained
constant
with age (data not shown), a subgroup of aged ( 18-month) mice showed a
diminution of this
clonal response (Figs. 14A-C). By six weeks post-castration, the total number
of infiltrating
lymph node cells and the number of activated CD25+CD8+ cells had increased to
young adult
levels (data not shown). More importantly however, castration significantly
enhanced the
CTL responsiveness to HSV-infected target cells, which was greatly reduced in
the aged mice
(Fig. 16) and restored the CD4:CD8 ratio in the lymph nodes (Fig. 17B).
Indeed, a decrease
in CD4+ T cells in the draining lymph nodes was seen with age compared to both
young
adult and castrated mice (Fig. 17B), thus illustrating the vital need for
increased production
of T cells from the thymus throughout life, in order to get maximal immune
responsiveness.
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EXAMPLE 4
INHIBITION OF SEX STEROIDS ENHANCES UPTAKE OF NEW
HEMATOPOIETIC PRECURSOR CELLS INTO THE THYMUS WHICH ENABLES
CHIMERIC MIXTURES OF HOST AND DONOR LYMPHOID CELLS
(T, B, AND DC)
Materials and methods were as described in Examples 1-3. In addition, the
following
techniques were used:
Previous experiments have shown that microchimera formation plays an important
role in organ transplant acceptance. DC have also been shown to play an
integral role in
tolerance to graft antigens. Therefore, the effects of castration on thymic
chimera formation
and dendritic cell number was studied.
In order to assess the role of stem cell uptake in thymus regeneration, BM
reconstitution was performed as described in Example 2
For the syngeneic experiments, three month old mice (n=4) were used per
treatment
group. All controls were age matched and untreated.
The total thymus cell numbers of castrated and noncastrated reconstituted mice
were
compared to untreated age matched controls and are summarized in Fig. 18A. In
mice
castrated 1 day prior to reconstitution, there was a significant increase
(p<0.01) in the .rate of
thymus regeneration compared to sham-castrated (ShCx) control mice. Thymus
cellularity in
the sham-castrated mice was below untreated control levels (7.6x10 ~ 5.2x106)
2 and 4
weeks after congenic BMT, while thymus cellularity of castrated mice had
increased above
control levels at 4-weeks post-BMT (Fig. 18A). At 6 weeks, cell numbers
remained below
control levels. However, those of castrated mice were three fold higher than
the noncastrated
mice (p<0.05) (Fig. 18A).
There were also significantly more cells (p_<0.05) in the BM of castrated mice
4 weeks
after BMT (Fig. 18D). BM cellularity reached untreated control levels (1.5x10~
1.5x106) in
the sham-castrates by 2 weeks, whereas BM cellularity was increased above
control levels in
castrated mice at both 2 and 4 weeks after congenic BMT (Fig. 18D). Mesenteric
lymph
node cell numbers were decreased 2-weeks after irradiation and reconstitution,
in both
castrated and noncastrated mice; however, by the 4 week time point cell
numbers had reached
control levels. There was no statistically significant difference in lymph
node cell number
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between castrated and noncastrated treatment groups (Fig. 18C). Spleen
cellularity reached
untreated control levels (1.5x10 ~ 1.5x106) in the sham-castrates and
castrates by 2 weeks,
but dropped off in the sham group over 4-6 weeks, whereas the castrated mice
still had high
levels of spleen cells (Fig. 18B). Again, castrated mice showed increased
lymphocyte
numbers at these time points (i.e., 4 and 6 weeks post-reconstitution)
compared to non-
castrated mice (p<0.05) although no difference in total spleen cell number
between castrated
and noncastrated treatment groups was seen at 2-weeks (Fig. 18B).
Thus, in mice castrated 1 day prior to reconstitution, there was a significant
increase
(p<_0.01) in the rate of thymus regeneration compared to sham-castrated (ShCx)
control mice
(Fig. I8A). Thymus cellularity in the sham-castrated mice was below untreated
control
levels (7.6x10 ~ 5.2x106) 2 and 4 weeks after congenic BMT, while thymus
cellularity of
castrated mice had increased above control levels at 4-weeks post-BMT (Fig.
18A).
Castrated mice had significantly increased congenic (Ly5.2) cells compared to
non-castrated
animals.
In noncastrated mice, there was a profound decrease in thymocyte number over
the 4
week time period, with little or no evidence of regeneration (data not shown).
In the castrated
group, however, by two weeks there was already extensive thymopoiesis which by
four
weeks had returned to control levels, being 10 fold higher than in
noncastrated mice. Flow
cytometric analysis of the thymuses with respect to CD45.2 (donor-derived
antigen)
demonstrated ~ that no donor derived cells were detectable in the noncastrated
group at 4
weeks, but remarkably, virtually all the thymocytes in the castrated mice were
donor-derived
at this time point (data not shown). Given this extensive enhancement of
thymopoiesis from
donor-derived hemopoietic precursors, it was important to determine whether T
cell
differentiation had proceeded normally. CD4, CD8 and TCR defined subsets were
analyzed
by flow cytometry. There were no proportional differences in thymocytes subset
proportions
2 weeks after reconstitution (data not shown). This observation was not
possible at 4 weeks,
because the noncastrated mice were not reconstituted with donor-derived cells.
However, at
this time point the thymocyte proportions in castrated mice appear normal.
In a parallel set of experiments, 3 month old, young adults, C571BL6 mice were
castrated or sham-castrated 1 day prior to BMT. For congenic BMT, the mice
were subjected
to 800RADS TBI and IV injected with 5 x 106 Ly5.1+ BM cells. Mice were killed
2 and 4
weeks later and the BM, thymus and spleen were analyzed for immune
reconstitution.
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Donor/Host origin was determined with anti-CD45.1 antibody, which only reacts
with
leukocytes of donor origin.
The results from this parallel set of experiments are shown in Figs. 19-28.
Figures 20 and 21 show an increase in the number and proportion of donor
derived
HSC in the BM of castrated animals. This indicates improved engraftment and
suggests faster
recovery from BMT.
Figure 22 shows an increase in donor derived B cell precursors and B cells in
the BM
of castrated mice. However, Figure 24 and 25 show castration does not alter
the number or
proportion of B cells in the periphery at 2 and 4 weeks post castration.
Figure 26 shows castration increased numbers of donor derived TN, DP, CD4 and
CD8 cells in the thymus. However Figure 23 shows castration does not alter the
donor
thymocyte proportions of CD4 and CD8 cells. In the periphery, there are very
few CD4 or
CD8 cells and at the time points considered, there was no increase in these
cells with
castration.
Importantly, Figure 28 shows and increased number of donor DC in the thymus by
4
weeks post castration.
Discussion
Example 4 shows the influence of castration on syngeneic and congenic BM
transplantation. Starzl et al., (1992) Lancet 339:1579 reported that
microchimeras evident in
lymphoid and nonlymphoid tissue were a good prognostic indicator for allograft
transplantation. That is it was postulated that they were necessary for the
induction of
tolerance to the graft (Starzl et a7.., (1992) Lancet 339:1579). Donor-derived
DC were present
in these chimeras and were thought to play an integral role in the avoidance
of graft rejection
(Thamson and Lu, (1999) Im~rzunol. Today 20:20). DC are known to be key
players in the
negative selection processes of thymus and if donor-derived DC were present in
the recipient
thymus, graft reactive T cells may be deleted.
In order to determine if castration would enable increased chimera formation,
a study
was performed using syngeneic foetal liver transplantation. The results showed
an enhanced
regeneration of thymii of castrated mice. These trends were again seen when
the experiments
were repeated using congenic (Ly5) mice. Due to the presence of congenic
markers, it was
possible to assess the chimeric status of the mice. As early as two weeks
after foetal liver
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reconstitution there were donor-derived dendritic cells detectable in the
thymus, the number
in castrated mice being four-fold higher than that in noncastrated mice. Four
weeks after
reconstitution the noncastrated mice did not appear to be reconstituted with
donor derived
cells, suggesting that castration may in fact increase the probability of
chimera formation.
Given that castration not only increases thymic regeneration after lethal
irradiation and fetal
liver reconstitution and that it also increases the number of donor-derived
dendritic cells in
the thymus, along-side stem cell transplantation this approach increases the
probability of
graft acceptance.
EXAMPLE 5
IMMUNE CELL DEPLETION
In order to prevent interference with the graft by the existing T cells in the
potential
graft recipient patient, the patient underwent T cell depletion (ablation).
One standard
procedure for this step is as follows. The human patient received anti-T cell
antibodies in the
form of a daily injection of 15 mg/kg of Atgam (xeno anti-T cell globulin,
Pharmacia
Upjohn) for a period of 10 days in combination with an inhibitor of T cell
activation,
cyclosporin A, 3 mg/kg, as a continuous infusion for 3-4 weeks followed by
daily tablets at 9
mg/kg as needed. This treatment did not affect early T cell development in the
patient's
thymus; as the amount of antibody necessary to have such an affect cannot be
delivered due
to the size and configuration of the human thymus. The treatment was
maintained for
approximately 4-6 weeks to allow the loss of sex steroids followed by the
reconstitution of
the thymus.
The prevention of T cell reactivity may also be combined with inhibitors of
second
level signals such as interleukins, accessory molecules (e.g., antibodies
blocking, e.g., CD28),
signal transduction molecules or cell adhesion molecules to enhance the T cell
ablation or
other immune cell depletion. The thymic reconstitution phase would be linked
to injection of
donor HSC (obtained at the same time as the organ or tissue in question either
from blood,
pre-mobilized from the blood with G-CSF (2 intradermal injections/day for 3
days) or
collected directly from the BM of the donor. The enhanced levels of
circulating HSC would
promote uptake by the thymus (activated by the absence of sex steroids and/or
the elevated
levels of GnRH). These donor HSC would develop into intrathymic DC and cause
deletion
of any newly formed T cells which by chance would be "donor-reactive". This
would
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establish central tolerance to the donor cells and tissues and thereby prevent
or greatly
minimize any rejection by the host. The development of a new repertoire of T
cells would
also overcome the immunodeficiency caused by the T cell-depletion regime.
The depletion of peripheral T cells minimizes the risk of graft rejection
because it
depletes non-specifically all T cells including those potentially reactive
against a foreign
donor. Simultaneously, however, because of the lack of T cells the procedure
induces a state
of generalized immunodeficiency which means that the patient is highly
susceptible to
infection, particularly viral infection.
EXAMPLE 6
SEX STEROID ABLATION THERAPY
The patient was given sex steroid ablation therapy in the form of delivery of
an
LHRH agonist. This was given in the form of either Leucrin (depot injection;
22.5 mg) or
Zoladex0 (implant; 10.8 mg), either one as a single dose effective for 3
months. This was
effective in reducing sex steroid levels sufficiently to reactivate the
thymus. W some cases it
is also necessary to deliver a suppresser of adrenal gland production of sex
steroids.
Cosudex0 (5 mglday or 50 mg/day) may also be given as one tablet per day for
the duration .
of the sex steroid ablation therapy. Alternatively, the patient is given a
GnRH antagonist,
e.g.,Cetrorelix or Abarelix as a subcutaneous injection
Reduction of sex steroids in the blood to minimal values takes about 1-3 weeks
post
surgical castration, and about 3-4 weeks following chemical castration. In
some cases it is
necessary to extend the treatment to a second 3 month injection/implant. The
thymic
expansion may be increased by simultaneous enhancement of blood HSC either as
an
allogeneic donor (in the case of grafts of foreign tissue) or autologous HSC
(by injecting the
host with G-CSF to mobilize these HSC from the BM to the thymus.
EXAMPLE 7
ALTERNATIVE DELIVERY METHOD
In place of the 3 month depot or implant administration of the LHRH agonist,
alternative methods can be used. In one example the patient's skin may be
irradiated by a
laser such as an Er:YAG laser, to ablate or alter the skin so as to reduce the
impeding effect
of the stratum corneum.
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Laser ablation or alteration is described in U.S. Patent Nos. 6,251,100,
6,419,642 and
4,775,361.
In another example, delivery is by means of laser generated pressure waves. A
dose of
LHRH agonist is placed on the skin in a suitable container, such as a plastic
flexible washer
(about 1 inch in diameter and about 1/16 inch thick), at the site where the
pressure wave is to
be created. The site is then' covered with target material such as a black
polystyrene sheet
about 1 mm thick. A Q-switched solid state ruby laser (20 ns pulse duration,
capable of
generating up to 2 joules per pulse) is used to generate a single impulse
transient, which hits
the target material. The black polystyrene target completely absorbs the laser
radiation so
that the skin is exposed only to the impulse transient, and not laser
radiation. The procedure
can be repeated daily, or as often as required, to maintain the circulating
blood levels of the
agonist.
EXAMPLE 8
ADMINISTRATION OF DONOR CELLS
Where practical, the level of hematopoietic stem cells (HSC) in the donor
blood is
enhanced by injecting into the donor granulocyte-colony stimulating factor (G-
CSF) at 10
p.g/kg for 2-5 days prior to cell collection (e.g., one or two injections of
10 p.g/kg per day for
each of 2-5 days). The donor may also be injected with LHRH agonist and/or a
cytokine,
such as G-CSF or GM-CSF, prior to (e.g., 7-14 days before) collection to
enhance the level or
. quality of stem cells in the blood. CD34+ donor cells are purified from the
donor blood or
BM, such as by using a flow cytometer or immunomagnetic beading. Antibodies
that
specifically bind to human CD34 are commercially available (from, e.g.,
Research
Diagnostics Inc., Flanders, NJ; Miltenyi-Biotec, Germany). Donor-derived HSC
are
identified by flow cytometry as being CD34+. These CD34+ HSC may also be
expanded by
in vitro culture using feeder cells (e.g., fibroblasts), growth factors such
as stem cell factor
(SCF), and LIF to prevent differentiation into specific cell types. At
approximately 3-4
weeks post LHRH agonist delivery (i.e., just before or at the time the thymus
begins to
regenerate) the patient is injected with the donor HSC, optimally at a dose of
about 2-4 x 106
cells/kg. G-CSF may also be injected into the recipient to assist in expansion
of the donor
HSC. If this timing schedule is not possible because of the critical nature of
clinical
condition, the HSC could be administered at the same time as the GnRH. It may
be necessary
to give a second dose of HSC approximately 2-3 weeks later to assist in the
thymic regrowth
and the development of donor DC (particularly in the thymus). Once the HSC
have engrafted
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(i.e., incorporated into) and/or migrated to the BM and thymus, the effects
should be
permanent since HSC are self-renewing.
The reactivating or reactivated thymus takes up the donor HSC and converts
them
into donor-type T cells and DC, while converting the recipient's HSC into
recipient-type T
cells and DC. By inducing deletion by cell death, or by inducing tolerance
through
immunoregulatory cells, the donor and host DC tolerize any new T or NK cells
that are
potentially reactive with donor or recipient cells.
EXAMPLE 9
TRANSPLANTATION OF GRAFT HSC
While the recipient is still undergoing continuous T cell depletion and/or
other
immune cell depletion and/or immunosuppressive therapy, the HSC are
transplanted from the
donor to the recipient patient. The recipient thymus has been activated by
GnRH treatment
and infiltrated by exogenous HSC.
Within about 3-4 weeks of LHRH therapy the first new T cells are present in
the
blood stream of the recipient. However, in order to allow production of a
stable chimera of
host and donor hematopoietic cells, immunosuppressive therapy may be
maintained for about
3-4 months. The new T cells are purged of potentially donor reactive and host
reactive cells,
due to the presence of both donor and host DC in the reactivating thymus.
Having been
positively selected by the host thymic epithelium, the T cells retain the
ability to respond to
normal infections by recognizing peptides presented by host APC in the
peripheral blood of
the recipient. The incorporation of donor DC into the recipient's lymphoid
organs establishes
an immune system situation virtually identical to that of the host alone,
other than the
tolerance of donor cells, tissue and organs. Hence, normal immunoregulatory
mechanisms
are present. These may also include the development of regulatory T cells
which switch on
or off immune responses using cytokines such as IL4, 5, 10, TGF-beta, TNF-
alpha.
EXAMPLE 10
IMMUNIZATION AND PREVENTION OF VIRAL INFECTION (INFLUENZA)
Influenza viruses are segmented RNA viruses that cause highly contagious acute
respiratory infections. The major problem associated with vaccine development
against
influenza is that these viruses have the ability to escape immune surveillance
and remain in a
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host population by altering antigenic sites on the hemagglutinin (HA) and
neuraminidase (N)
envelope glycoproteins by phenomena termed antigenic drift and antigenic
shift. The
primary correlate for protection against influenza virus is neutralizing
antibody against HA
protein that undergoes strong selection for antigenic drift and shift.
However, much more
conserved antigenic cross-reactivities for different strains of influenza
virus occur between
internal proteins, such as the nucleoprotein (NP) (Shu et al., (1993) J.
Virol. 67:2723). CTL
and protection from influenza challenge following immunization with a
polynucleotide
encoding NP has previously been shown (Ulmer et al. (1993) Science 259:1745).
Materials and Methods
Surgical Castration. BALB/c mice are anesthetized by intraperitoneal injection
of
30-40 p,1 of a mixture of 5 ml of 100 mg/ml ketamine hydrochloride (Ketalar~;
Parke-Davis,
Caringbah, NSW, Australia) plus 1 ml of 20 mg/ml xylazine (Rompun~; Bayer
Australia
Ltd., Botany NSW, Australia) in saline. Surgical castration is performed as
described
elsewhere herein by a scrotal incision, revealing the testes, which are tied
with suture and
then removed along with surrounding fatty tissue. The wound is closed using
surgical
staples. Sham-castrated mice prepared following the above procedure without
removal of the
testes are used as controls.
Chemical castration. Mice are injected i.m. with 10 mg/kg LupronC7 (a GnRH
agonist) as a 1 month slow release formulation. Alternatively mice are
injected with a GnRH
antagonist (e.g., Cetrorelix or Abarelix). Confirmation of loss of sex
steroids is performed by
standard radioimmunoassay of plasma samples following manufacturer's
instructions.
Castrate levels (<0.5 ng testosterone or estrogen /ml) should normally be
achieved by 3-4
weeks post injection.
Preparation of influenza A/PR/8/34 subunit vaccine. Purified influenza
A/PR/8/34
(H1N1) subunit vaccine preparation is prepared following methods known in the
art. Briefly,
the surface hemagglutinin (HA) and neuraminidase (NA) antigens from influenza
A/PR/8/34
particles are extracted using a non-ionic detergent (7.5% N-octyl-~-o-
thioglucopyranoside).
After centrifugation, the HANNA-rich supernatant (55% HA) is used as the
subunit vaccine.
Influenza A/PR/8/34 subunit immunization. Approximately 6 weeks following
surgical castration or about 8 weeks following chemical castration, mice are
immunized with
100 p,1 of formalin-inactivated influenza AIPR/8/34 virus (about 7000 HAU)
injected
subcutaneously.
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Booster immunizations can optionally be performed at about 4 weeks (or later)
following the primary immunization. Freund's complete adjuvant (CFA) is used
for the
primary immunization and Freund's incomplete adjuvant is used for the optional
booster
immunizations.
Alternatively, the influenza A/PR/8/34 subunit vaccine preparation (see above)
may
be intramuscularly injected directly into, e.g., the quadriceps muscle, at a
dose of about 1 p,g
to about 10 p,g dilute in a volume of 40 ~.1 sterile 0.9% saline.
Plasmid DNA. Preparation of plasmid DNA expression vectors are readily known
in
the art (see, e.g., Current Protocols In Immunolo~y, Unit 2.14, John E.
Coligan et al., (eds),
Wiley and Sons, New York, NY (1994), and yearly updates including 2002).
Briefly, the
complete influenza A/PR/8/34 nucleoprotein (NP) gene or hemagglutinin (HA)
coding
sequence is cloned into an expression vector, such as, pCMV, which is under
the
transcriptional control of the cytomegalovirus (CMV) immediate early promoter.
Empty plasmid (e.g., pCMV with no insert) is used as a negative control.
Plasmids
are grown in Escherichia coli DHSa or HB 101 cells using standard techniques
and purified
using Qiagen0 Ultra-Pure~-100 columns (Chatsworth, CA) according to
manufacturer's =
instructions. All plasmids are verified by appropriate restriction enzyme
digestion and
agarose gel electrophoresis. Purity of DNA preparations is determined by
optical density
readings at 260 and 280 nm. All plasmids are resuspended in TE buffer and
stored at -20°C
until use.
DNA immunization. Methods of DNA immunization are well known in the art. For
instance, methods of intradermal, intramuscular, and particle-mediated (''gene
gun") DNA
immunizations are described in detail in, e.g., Current Protocols In
Immunolo~y, Unit 2.14,
John E. Coligan et al., (eds), Wiley and Sons, New York, NY (1994), and yearly
updates
including 2002).
Cytokine-encoding DNAs are optionally administered to shift the immune
response to
a desired Thl- or a Th2-type immune response. Thl-inducing genetic adjuvants
include, e.g.,
IFN-y and IL-12. Th2-inducing genetic adjuvants include, e.g., IL-4, IL-5, and
IL-10. For
review of the preparation and use of Thl- and Th2- inducing genetic adjuvants
in the
induction of immune response (see, e.g., Robinson, et al., (2000) Adv. Virus
Res. 55:1).
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Influenza AJPR/8/34 virus challenge. W an effort to determine if castrated
mice are
better protected from influenza virus challenge (with and without vaccination)
as compared to
their sham-castrated counterparts, metofane-anesthetized mice are challenged
by intranasal
inoculation of 50 D1 of influenza A/PR/8/34 (H1N1) influenza virus containing
allantoic fluid
diluted 104 in PBS/2% BSA (50-100 LDSO; 0.25 HAU). Mice axe weighed daily and
sacrificed following >20% loss of pre-challenge weight. At this dose of
challenge virus,
100% of naive mice should succumb to influenza infection by 4-6 days.
Sublethal infections are optionally done to activate memory T cells, but use a
10-~
dilution of virus. Sublethal infections may also be optionally done to
determine if non-
immunized, castrated mice have better immune responses than the sham castrated
controls, as
determined by ELISA, cytokine assays (Th), CTL assays, etc. outlined below.
Viral titers for
lethal and sublethal infections may be optimized prior to use in these
experiments.
Enzyme-linked immunosorbant assays. At various time periods pre- and post-
immunization (or pre- and post- infection), mice from each group are bled, and
individual
mouse serum is tested using standard quantitative enzyme-linked immunosorbant
assays
(ELISA) to assess anti-HA or -NP specific IgG levels in the serum. IgGl and
IgG2a levels
may optionally be tested, which are known to correlate with Th2 and Thl-type
antibody
responses, respectively.
Preparation and stimulation of splenocytes for cytokine production. Spleens
are
aseptically harvested from the various groups of mice (n=2-3) and pooled in
p60 petri dishes
containing about 4 ml RPMI-10 media (RPMI-1640, 10% fetal bovine serum, 50
p,g/ml
gentamycin). Spleens are prepared and RBC lysed using standard procedures.
Cells are then
counted, and resuspended in RPMI-1U containing 80 L1/ml rat IL-2 (Sigma, St.
Louis, MO) to
a final cell concentration of 2x10 cells/ml. One hundred microliters of cells
are dispensed
into wells of a 96-well tissue culture plate for a final concentration of
2x106 cells/well.
Stimulations are conducted by adding 100 ~,l of the appropriate peptide or
inactivated
influenza virus diluted in RPMI-10. CD8+ T cells are stimulated with either
the Kd-restricted
HAsss-sai peptide (IYSTVASSL; SEQ ID NO:1) (Winter, Fields, and Brownlee,
(1981)
Nature 292:72) or the Kd-restricted NP147-iss peptide (TYQRTRALV; SEQ ID N0:2)
(Rotzschke et al., (1990) Nature 348:252). CD4+ T cells are stimulated with
inactivated
influenza virus (13,000 HAU per well of boiled influenza virus plus 13,000 HAU
per well of
formalin-inactivated influenza virus) plus anti-CD28 (1 ~.g/ml) and anti-CD49d
(1 ~,g/ml)
(Waldrop et al., (1998) J. Immuraol. 161:5284). Negative control stimulations
are done with
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media alone. Cells are then incubated as described below to detect
extracellular cytokines by
ELISA or intracellular cytokines by FACS staining.
Chromium release assay for CTL. CTL responses to influenza HA and NP are
measured using procedures well known to those in the art (see, e.g., Current
Protocols In
Immunolo~y, John E. Coligan et al., (eds), Unit 3, Wiley and Sons, New York,
NY (1994),
and yearly updates including 2002). The synthetic peptide HAsss-541 IYSTVASSL
(SEQ ID
N0:1) (Winter, Fields, and Brownlee, (1981) Nature 292:72) or NP14~-iss
TYQRTRALV
(SEQ ID N0:2) (Rotzschke et al., (1990) Nature 348:252) are used as the
peptide in the
target preparation step. Responder splenocytes from each animal are washed
with RPMI-10
and resuspended to a final concentration of 6.3x106 cells/ml in RPMI-10
containing 10 U/mI
rat IL-2 (Sigma, St. Louis, MO). Stimulator splenocytes are prepared from
naive, syngeneic
mice and suspended in RPMI-10 at a concentration of 1x10' cells/ml. Mitomycin
C is added
to a final concentration of 25 p,g/ml. Cells are incubated at
37°C/5%COZ for 30 minutes and
then. washed 3 times with RPMI-10. The stimulator cells are then resuspended
to a
concentration of 2.4x106 cells/ml and pulsed with HA peptide at a final
concentration of
9x 10-6M or with NP peptide at a final concentration of 2x 10-6M in RPMI-10
and 10 U/ml IL-
2 for 2 hours at 37°C/5% CO2. The peptide-pulsed stimulator cells
(2.4x106) and responder
cells (6.3x106) are then co-incubated in 24-well plates in a volume of 2 ml SM
media (RFMI-
10, 1 mM non-essential amino acids, 1 mM sodium pyruvate) for 5 days at
37°Cl5aloCO2. A
chronuum-..release assay is used to measure the ability of the in vitro
stimulated responders
(now called effectors) to lyre peptide-pulsed mouse mastocytoma P815 cells
(MHC matched,
H-2d). P815 cells are labeled with slCr by taking 0.1 ml aliquots of p815 in
RPMI-10 and
adding 25 p.1 FBS and 0.1 mCi radiolabeled sodium chromate (NEN, Boston, MA)
in 0.2 ml
normal saline. Target cells are incubated for 2 hours at 37°C/5%COz,
washed 3 times with
RPMI-10 and resuspended in 15 ml polypropylene tubes containing RPMI-10 plus
HA (9x10-
6M) or NP (1x10-6) peptide. Targets are incubated for 2 hours at
37°C/5%C02 . The
radiolabeled., peptide-pulsed targets are added to individual wells of a 96-
well plate at 5x104
cells per well in RPMI-10. Stimulated responder cells from individual
immunization groups
(now effector cells) are collected, washed 3 times with RPMI-10, and added to
individual
wells of the 96-well plate containing the target cells for a final volume of
0.2 ml/well.
Effector to target ratios are 50:1, 25:1, 12.5:1 and 6.25:1. Cells are
incubated for 5 hours at
37°Cl5%COa and cell lysis is measured by liquid scintillation counting
of 25 ~.1 aliquots of
supernatants. Percent specific lysis of labeled target cells for a given
effector cell sample is
[100 x (Cr release in sample-spontaneous release sample) / (maximum Cr release-
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spontaneous release sample)]. Spontaneous chromium release is the amount of
radioactive
released from targets without the addition of effector cells. Maximum chromium
release is
the amount of radioactivity released following lysis of target cells after the
addition of
TritonX-100 to a final concentration of 1%. Spontaneous release should not
exceed 15%.
Detection of IFN~y or IL-5 in bulk culture supernatants by ELISA. Bulk culture
supernatants may be tested for IFNy and IL-5 cytokine levels, which are known
to correlate
with Th1 and Th2-type response, respectively. Pooled splenocytes are incubated
for 2 days at
37°C/ 5% C02, and then supernatants are harvested and pooled All ELISA
antibodies and
purified cytokines are purchased from Pharmingen (San Diego, CA). Fifty
microliters of
purified anti-cytokine monoclonal antibody diluted to 5 p.ghnl (rat anti-mouse
IFNy) or 3
p,g/ml (rat anti-mouse IL-5) in coating buffer (0.1 M NaHC03, pH 8.2) is
distributed per well
of a 96-well ELISA plate (Corning, Corning, NY) and incubated overnight at
4°C. Plates are
washed, blocked, and rewashed with PBS-T. Standards (recombinant mouse
cytokine) and
samples are added to wells at vaxious dilutions in RPMI-10 and incubated
overnight at 4°C
for maximum sensitivity. Plates are washed 6 times with PBS-T. Biotinylated
rat anti-mouse
cytokine detecting antibody is diluted in PBS-T to a final concentration of 2
~,g/ml and 100 ~.1
was distributed per well. Plates are incubated for 1 hr. at 37°C and
then washed 6 times with
PBS-T. Streptavidin-AP (Gibco BRL, Grand Island, NY) is diluted 1:2000
according to
manufacturer's instructions, and 100 p.1 is distributed per well. Plates are
incubated for 30
min. and washed an additional 6 times with PBS-T. Plates are developed by
adding 100
p.l/well of AP developing solution (BioRad, Hercules, CA) and incubating at
room
temperature for 50 miilutes. Reactions are stopped by addition of 100 ~,1 0.4
M NaOH and
read at. OD4o5. Data are analyzed using Softmax Pro Version 2.21 computer
software
(Molecular Devices, Sunnyvale, CA).
Intracellular cytokine staining and FRCS analysis. Splenocytes may be tested
for
intracellular IFNy and IL-5 cytokine levels, which are known to correlate with
Thl and Th2-
type response, respectively. Pooled splenocytes are incubated for 5-6 hours at
37°C in a
humidified atmosphere containing 5% C02. A Golgi transport inhibitor, Monensin
(Pharmingen, San Diego, CA), is added at 0.14 ~,l/well according to the
manufacturer's
instructions, and the cells are incubated for an additional 5-6 hours (Waldrop
et al., (1998) J.
Inzmunol. 161:5284). Cells are thoroughly resuspended and transferred to a 96-
well U-
bottom plate. All reagents (GolgiStop kit and antibodies) are purchased from
Pharmingen
(San Diego, CA) unless otherwise noted, and all FACS staining steps are done
on ice with
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ice-cold reagents. Plates are washed 2 times with FACS buffer (lx PBS, 2% BSA,
0.1% wlv
sodium azide). Cells are surface stained with 50 p.1 of a solution of 1:100
dilutions of rat anti-
mouse CD8[3-APC, -CD69-PE, and -CD16/CD32 (FcyIII/RII; 'Fc Block') in FAGS
buffer.
For tetramer staining (see below), cells were similarly stained with CD8~3-
Tricolor, CD69-
PE, CD 16/CD32, and HA- or NP-tetramer-APC in FACS buffer. CeIIs are incubated
in the
dark for 30 min. and washed 3 times with FACS buffer. Cells are permeabilized
by
thoroughly resuspending in 100 p1 of CytofixlCytoperm solution per well and
incubating in
the dark for 20 minutes. Cells are washed 3 times with Permwash solution.
Intracellular
staining is completed by incubating 50 p,1 per well of a 1:100 dilution of rat
anti-mouse IFNy
FITC in Permwash solution in the dark for 30 min. Cells are washed 2 times
with Permwash
solution and 1 time with FAGS buffer. Cells are fixed in 200 p,1 of 1 %
paraformaldehyde
solution and transferred to microtubes arranged in a 96-well format. Tubes are
wrapped in
foil and stored at 4°C until analysis (less than 2 days). Samples are
analyzed on a FACScan°
flow cytometer (Becton Dickenson, San Jose, CA). Compensations are done using
single-
stained control cells stained with rat anti-mouse CD8-FITC, -PE, -Tricolor, or
-APC.
Results are analyzed using FIowJo Version 2.7 software (Tree Star, San Carlos,
CA).
Tetramers. HA and NP tetramers may be used to quantitate HA- and NP-specific
CD8+ T cell responses ,following HA or NP immunization. Tetramers are prepared
essentially as described previously (Flynn et al., (1998) Immunity 8:683). The
present
example utilizes the H-2Kd MHC elass I glycoprotein complexed the synthetic
influenza
A/PR/8/34 virus peptide HAg33-541 (~'STVASSL; SEQ ID NO:1) (Winter, Fields,
and
Brownlee, (1981) Nature 292:72) or NPi~~_iss (TYQRTRALV; SEQ ll~ N0:2)
(Rotzschke et
al., (1990) Nature 348:252).
It is noted that the methods described in this example are applicable to a
wide array
agents, with only minor variations, which would be readily determinable by
those skilled in
the art.
EXAMPLE 11
IMMUNIZATION AND PREVENTION OF PARASITIC INFECTION (MALARIA)
The circumsporozoite protein (CSP) is a target of this pre-erythocytic
immunity
(Hoffman et al., (1991) Science 252:520. In the Plasmodium, yoelii (P. yoelii)
rodent model
system, passive transfer P. yoelii CSP-specific monoclonal antibodies
(Charoenvit et al.,
(1991) J. Imnaurzol. 146:1020), as well as adoptive transfer of P. yoelii CSP-
specific CDB~" T
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cells (Rodrigues et al., (1991) Izzt. Izzzmunol. 3:579, Weiss et al., (1992)
J. Inzznunol.
149:2103) and CD4+ T cells (Renia et al., (1993) J. Immunol. 150:1471) are
protective.
Numerous vaccines designed to protect mice against sporozoites by inducing
immune
responses against the P. yoelii CSP have been evaluated.
Any Plasznodiufn spof~o,zoite proteins known in the art capable of inducing
protection
against malaria usable in this invention may be used, such as P. falciparum,
P. vivax, P.
malariae, and P. ovale CSP; SSP2(TRAP); Pfsl6 (Sheba); LSA-1; LSA-2; LSA-3;
MSA-1
(PMMSA, PSA, p185, p190); MSA-2 (Gymmnsa, gp56, 38-45 kDa antigen); RESA
(Pf155);
EBA-175; AMA-1 (Pf83); SERA (p113, p126, SERP, Pf140); RAP-1; RAP-2; RhopH3;
PfHRP-II; Pf55; Pf35; GBP (96-R); ABRA (p101); Exp-1 (CRA, Ag5.1); Aldolase;
Duffy
binding protein of P, vivax; Reticulocyte binding proteins; HSP70-1 (p75);
Pfg25; Pfg28;
Pfg48/45; and Pfg230.
Materials and Methods
Castration. Surgical an.d/or chemical castration is performed as above.
Parasites. The 17XNL (nonlethal) strain of P. yoelii is used as described
previously
(U.S. Patent No. 5,814,617).
Preparation of irradiated P. yoelii sporozoites. Preparation of irradiated P.
yoelii
sporozoites for immunization has been described previously (see, e.g., Franke
et al., (2000)
Infect. hzzmufz. 68:3403). Briefly, sporozoites are isolated by the
discontinuous gradient
technique (Pacheco et al., (1979) J. Parisitol. 65:414) from infected
Anopheles stephens
mosquitoes that have been irradiated at 10,000 rads (l3~Ce).
Immunization with irradiated P. yoelii sporozoites. Mice are intravenously
immunized with 50,000 sporozoites at approximately 6 weeks following surgical
castration or
about 8 weeks following chemical castration via the tail vein. Booster
immunizations of
20,000 to 30,000 sporozoites are optionally given at 4 weeks and 6 weeks
following the
primary immunization (see, e.g., Franke et al., (2000) Infect Irnmun.
68:3403).
Plasmid DNA and DNA immunization. Plasmid DNA encoding the full length P.
yoelli CSP are known in the art. For instance, the pyCSP vector described in
detail in
Sedegah et al., ((1998) Proc. Natl. Acad. Sci. USA 95:7648) may be used.
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Methods of DNA immunization are also well known in the art. For instance,
methods
of intradermal, intramuscular, and particle-mediated ("gene gun") DNA
immunizations are
described in detail in, e.g., Current Protocols In Immunology, Unit 2.14, John
E. Coligan et
al., (eds), Wiley and Sons, New York, NY (1994), and yearly updates including
2002).
Peptide Immunization. Methods of P. yoelii CSP peptide preparation are known
in
the art (see, e.g., Franke et al., (2000) Infect Imznun. 68:3403).
Chromium release assay for CTL. Since CD8+ CTL against the P. yoelii CSP have
been shown to adoptively transfer protection (Weiss et al., (1992) J.
Immurcol. 149:2103), and
CD8+ T cells are required for the protection against P. yoelii induced by
immunization with
irradiated sporozoites (Weiss et al., (1988) Proc. Natl. Acad. Sci USA
85:573), it must be
determined if P. yoelii CSP vaccination (e.g., irradiated sporozoite, CSP
peptide, or CSP
DNA immunizations) elicits a CSP-specific CTL.
CTL responses are measured using procedures well known to those- in the art
(see,
e.g., Current Protocols Tn Inununolo~y, John E. Coligan et al., (eds), Unit 3,
Wiley and Sons,
New York, NY (1994_, and yearly updates including 2002). The general procedure
described elsewhere herein for influenza HA and NP is used except that the
cells are pulsed
with the synthetic P. yoelli CSP peptide (281-296; SYVPSAEQILEFVI~.QI; SEQ ID
N0:3).
Inhibition of liver stage development assay. The liver stage development assay
and acquisition of mouse hepatocytes from mouse livers by in situ collagenase
perfusion. have
been described previously (Franke et al., (1999) Vaccirr.e 17:1201; Franke et
a1.,(2000) Infect.
Ir~znzuf2. 68:3403). Hepatocyte cultures are seeded onto eight-chamber Lab-Tek
plastic slides
at 1x105 cells/chamber and incubated with 7.5 x 104 P. yoelli sporozoites for
3 hours. The
cultures are then washed and cultured for and additional 24 hours at 37 C/5%
C02. Effector
cells are obtained as described above for the chromium release assay for CTL
and are added
and cultured with the infected hepatocytes for about 24-48 hours. The cultures
are then
washed, and the chamber slides are fixed for 10 min. in ice-cold absolute
methanol. The
chamber slides are then incubated with a monoclonal antibody (NYLS 1 or NYLS3,
both
described previously in U.S. Patent No. 5,814,617) directed against liver
stage parasites of P.
yoelii before incubating with FITC-labeled goat anti-mouse Ig. The number of
liver-stage
schizonts in triplicate cultures are then counted using an epifluorescence
microscope. Percent
inhibition is calculated using the formula [(control-test)/control) x100].
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Infection and challenge. For a lethal challenge dose, the IDso of P. yoelli
sporozoites must be determined prior to experimental challenge. However, it is
also initially
possible to inject mice intravenously in the tail vein with a dose of about 50
to 100 P. yoelii
sporozoites (nonlethel, strain 17XNL). Forty-two hours after intravenous
inoculation, mice
are sacrificed and livers are removed. Single cell suspensions of hepatocytes
in medium are
prepared, and 2x105 hepatocytes are placed into each of 10 wells of a mufti-
chamber slide.
Slides may be dried and .frozen at -70°C until analysis. To count the
number of schizonts,
slides are dried and incubated with NYLS 1 before incubating with FITC-labeled
goat anti-
mouse Ig, and the numbers of liver-stage schizonts in each chamber are counted
using
fluorescence microscopy.
Once it is demonstrated that castration and/or immunization reduces the
numbers of
infected hepatocytes, blood smears are obtained to determine if immunization
protect against
blood stage infection. Mice are considered protected if no parasites are found
in the blood
smears at days 5-14 days post-challenge.
To test the preventative efficacy of castration alone (no vaccination) from a
P. yoelli
sporozoite primary infection, castrated mice are infected and analyzed as
described above.
Sham-castrated mice are used as controls.
Human studies. After establishing the efficacy in mice, large numbers of
humans are
immunized in a double blind placebo controlled field trial.
EXAMPLE 12
IMMUNIZATION AND PREVENTION OF BACTERIAL INFECTION (TB A~85~
Tuberculosis (TB) is a chronic infectious disease of the lung caused by the
pathogen
Mycobacteriufn tuberculosis, and is one of the most clinically significant
infections
worldwide. (see, e.g., U.S. Patent No. 5,736,524; for review see Bloom and
Murray, (1:993),
Science 257, 1055.
M. tuberculosis is an intracellular pathogen that infects macrophages.
Immunity to
TB involves several types of effector cells. Activation of macrophages by
cytokines, such as
IFNy, is an effective means of minimizing intracellular mycobacterial
multiplication.
Acquisition of protection against TB requires both CD8+ and CD4+T cells (see,
e.g., Orme et
al., (1993) J. Infect. Dis. 167:1481). These cells are known to secrete Thl-
type cytokines,
such as IFNy, in response to infection, and possess antigen-specific cytotoxic
activity. In
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fact, it is known in the art that CTL responses are useful for protection
against M.
tuberculosis (see, e.g., Flynn et al., (1992) Proc. Natl. Acad. Sci. USA
89:12013).
Predominant T cell antigens of TB are those proteins that are secreted by
mycobacteria during their residence in macrophages. These T cell antigens
include, but are
not limited to, the antigen 85 complex of proteins (85A, 85B, 85C) (Wiker and
Harboe,
((1992) Microbiol. Rev. 56: 648) and ESAT-6 (Andersen, (1994) Infect.
Immurzity, 62:2536).
Other T cell antigens have also been described in the art (see, e.g., Young
and Garbe, (1991)
Res. Microbiol. 142:55; Andersen, (1992) J. Infect. Dis. 166:874; Siva and
Lowrie, (1994)
Immurzol. 82:244; Romain et a." (1993) Proc. Natl. Acad. Sci. ZJSA 90:5322;
and Faith et al.,
(1991) Irrznzurzol. 74:1).
The genes for each of the three antigen 85 proteins (A, B, and C) have been
cloned
and sequenced (see, e.g., Borremans et al., (1989) Infect. Immunity 57:3123);
DeWit et al.,
(1994) DNA Seq. 4:267), and have been shown to elicit strong T cell responses
following
both infection and vaccination.
Materials and Methods
Castration of mice. Surgical and/or chemical castration. of BALB/c or C57BL/6
mice is peuormed as above.
Protein immunization. General methods for Mycobacterium tuberculosis (TB)
bacilli purification and immunization are known in the art (see, e.g., Current
Protocols In
Immunology, Unit 2.4, John E. Coligan et al., (eds), Wiley and Sons, New York,
NY (1994),
and yearly updates including 2002). The purified TB may be prepare using
preparative SDS-
PAGE. Approximately 2 mg of the TB protein is loaded across the wells of a
standard 1.5
rnm slab gel using a large-tooth comb. An edge of the gel may be removed and
stained
following electrophoresis to identify the TB protein band on the gel. The gel
region that
contains the TB protein band is then sliced out of the gel, placed in PBS at a
final
concentration 0.5 mg purified TB protein per ml, and stored at 4°C
until use. The purified
TB protein may then be emulsified with an equal volume of complete Freund's
adjuvant
(CFA) for immunization.
Approximately 6 weeks following surgical castration or about 8 weeks following
chemical castration, 2 ml of the purified TB (0.5 mg/ml in PBS) is emulsified
2 ml CFA and
stored at 4°C. The TB/CFA mixture is slowly drawn into and expelled
through a 3-ml glass
syringe attached to a 19 gauge needle, being certain to avoid excessive air
bubbles. Once the
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emulsion is at a homogenous concentration, the needle is replaced by a 22
gauge needle, and
all air bubbles are removed. The castrated and sham-castrated mice are
injected
intramuscularly with a 50 p.1 volume of the TB/CFA emulsion (immunization may
also be
done via the intradermal or subcutaneous routes). M. bovis BCG may also be
used in a
vaccine preparation.
A booster immunization can optionally be performed 4-8 weeks (or later)
following
the primary immunization. The TB adjuvant emulsion is prepared in the same
manner
described above, except that incomplete Freund's adjuvant (IFA) is used in
place of CFA for
all booster immunizations. Further booster immunizations can be performed at 2-
4 week (or
later intervals) thereafter.
Plasmid DNA. Suitable Ag85-encoding DNA sequences and vectors have been
described previously (see, e.g., U.S. Patent No. 5,736,524). Other suitable
expression vectors
would be readily ascertainably by hose skilled in the art.
Antigen 85 DNA Immunization. Methods of DNA immunization are well known
in the art. For instance, methods of intradermal, intramuscular, and particle-
mediated ("gene
gun") DNA immunizations are described in detail in, e.g., Current Protocols In
Itnmunolo~y,
Unit 2.14, John E. Coligan et al., (eds), Wiley and Sons, New York, NY (1994),
and yearly
updates including 2002).
Cytokine-encoding DNAs are optionally administered to shift the immune
response to
a desired Thl- or a Th2-type immune response. Thl-inducing genetic adjuvants
include, e.g.,
IFN-y and IL-12. Th2-inducing genetic adjuvants include, e.g., IL-4, IL-5; and
IL-10. For
review of the preparation and use of Thl- and Th2- inducing genetic adjuvants
in the
induction of immune response, see, e.g., Robinson, et al., (2000) Adv. Virus
Res. 55:1-74.
Approximately 6 weeks following surgical castration or about 8 weeks following
chemical castration, mice are intramuscularly injected with 200 p,g of DNA
diluted in 100 p,1
saline.
Booster DNA immunizations are optionally administered at 4 weeks post-prime
and 2
weeks post-boost.
Enzyme-linked immunosorbant assays. At various time periods pre- and post-
immunization, mice from each group are bled, and individual mouse serum is
tested using
standard quantitative ELISA to assess anti-Ag85 specific IgG levels in the
serum. IgGl and
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IgG2a levels may optionally be tested, which are known to correlate with Th2
and Th-type
antibody responses, respectively.
Serum is collected at various time points pre- and post-prime and post boost,
arid
analyzed for the presence of anti-Ag85 specific antibodies in serum. Basic
ELISA methods
are described elsewhere herein, except purified Ag85 protein is used.
Cytokine assays. Spleen cells from vaccinated mice are analyzed for cytokine
secretion in response to specific Ag85 restimulation, as described, e.g., in
Huygen et al.,
(1992) Ifafect. Immunity 60:2880, and in U.S. Patent No. 5,736,524. Briefly,
spleen cells are
incubated with culture filtrate (CF) proteins from M, bovis BCG puxified Ag85A
or the
C57BL/6 T cell epitope peptide (amino acids 241-260).
Four weeks post-prime and 2 weeks post boost (or later), cytokines are assayed
using
standard bio-assays for IL-2,IFNy and IL-6, and by ELISA for IL-4 and 1L-10
using methods
well known to those in the art. See, e.g., Current Protocols In Immunolo~y,
Unit 6, John E.
Coligan et al., (eds), Wiley and Sons, New York, NY (1994), and yearly updates
including
2002.
Mycobacterial infection and challenge. To test the efficacy of the
vaccinations,
mice are challenged by intravenous injection of live M. bovis BCG (0.5 mg). At
various time
points post-challenge, BCG multiplication is analyzed in both mouse spleens
and lungs.
Positive controls are naive mice (castrated and/or sham castrated as
appropriate) receiving a
challenge dose.
To test the efficacy of sex steroid ablation to prevent primary infection,
Iive M. bovis
BCG are injected similarly to that described in the challenge experiment
above. Sham
castrated mice are used as controls.
The number of colony-forming units (CFU) in the spleen and lungs of the
challenged,
vaccinated mice, as well as in the lungs of the castrated, primary infected
mice is expected to
be substantially lower than in negative control animals, which is indicative
with protection in
the live M. bovis challenge model.
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EXAMPLE 13
IMMUNIZATION AND PREVENTION OF CANCER
To determine if sex steroid ablation is effective in preventing cancer and/or
in
eliciting a protective immune response following vaccination with a cancer
antigen, the
following studies are performed.
Materials and Methods
Castration. C57BL/6 mice surgical andlor chemical castration is performed as
above.
CEA immunization. Approximately 6 weeks following surgical castration or about
8
weeks following chemical castration, mice are inoculated with an adenovirus
vector encoding
the human carcinoembiyonic antigen (CEA) gene (MC38-CEA-2) (Conry ex al.,
(1995)
Cancer Gene Then. 2:33), such as AdCMV-hcea described in U.S. Patent Noe
6,348,450.
Alternatively, a plasmid DNA encoding the human CEA gene is injected into the
mouse (e.g.,
intramuscularly into the quadriceps muscle) utilizing one of the various
methods of DNA
vaccination described elsewhere herein.
Tumor challenge. To asses the efficacy of sex steroid ablation on anti-tamor
activity of mice immunized with CEA, mice are subjected to'a tumor challenge.
At various
time points post immunization, syngeneic tumor cells expressing the human CEA
gene
(MC38-CEA-2) (Conry et al., (1995) Cahcen Gene Ther. 2:33) are inoculated into
the mice.
Mice are observed every other day for development of palpable tumor nodules.
Mice are
sacrificed when the tumor nodules exceed 1 cm in diameter. The time between
inoculation
and sacrifice is the survival time.
To test the efficacy of sex steroid ablation preventing tumors, tumor cells
expressing
the human CEA gene are inoculated into castrated, non-vaccinated mice as
outlined above.
Sham castrated mice are used as controls.
EXAMPLE 14
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TRANSPLANTATION OF GENETICALLY MODIFIED HSC (GENE THERAPY)
I. SCID-hu Mouse Model
Materials and Methods
Mice. SCID-hu mice are prepared essentially as described previously (see,
e.g.,
Namikawa et al., (1990) J. Exp. Med. 172:1055 and Bonyhadi et al., (1997) J.
Virol.
71:4707) by surgical transplantation of human fetal liver and thymus fragments
into CB-17
scidlscid mice. Methods for the construction of SCm-hu Thy/Liv mice can also
be found,
e.g., in Current Protocols In Immunolo~y, Unit 4.8, John E. Coligan et al.,
(eds), Wiley and
Sons, New York, NY (1994), and yearly updates including 2002.
Surgical castration of mice. The SCID-hu mice are anesthetized by
intraperitoneal
injection of 30-40 ~,l of a mixture of 5 ml of 100 mg/ml ketamine
hydrochloride (Ketalar~;
Parke-Davis, Caringbah, NSW, Australia) plus 1 ml of 20 mg/ml xylazine
(Rompun0; Bayer
Australia Ltd., Botany NSW, Australia) in saline. Surgical castration is
performed as
described above by a scrotal incision, revealing the testes, which are tied
with suture and then
removed along with surrounding fatty tissue. The wound is closed using
surgical staples.
Sham-castrated mice prepared following the above procedure without removal of
the testes
are used as controls.
Chemical castration. Chemical castration is performed as above.
Isolation of human CD34 + HSC. Human cord blood (CB) HSC are collected axed
processed using techniques well known to those skilled in the art (see, e.g.,
DiGusto et al.,
w '-"' (1997) Blood,' 87:1261 (1997), Bonyhadi et al., (1997) J. Viral.
71:4707). A portion of each
CB sample is HLA phonotyped for the MA2.1 surface molecule. CD34+ cells are
enriched
using immunomagnetic beads using the method described in Bonyhadi et al.
((1997) J. Virol.
71:4707). Briefly, CB cells are incubated with anti-CD34 antibody (QBEND-10,
Immunotech) and then washed and resuspended at a final concentration of 2x10'
cells/ml.
CD34+ cells are then enriched using goat-anti-mouse IgGl magnetic beads
(Dynal) following
manufacturer's instructions. The CD34+ cells are then incubated with 50 ~.1 of
glycoprotease
(O-sialoglycoprotein endopeptidase), which causes release of the CD34+ cells
from the
immunomagnetic beads. The beads are removed using a magnet, and the cells are
then
subjected to flow cytometry using conjugated anti-CD34-PE to determine the
total level of
CD34+ cells present in the population. Alternatively, the cells are
magnetically labeled with
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anti-CD34 and sorted on an autoMACSTM. The autoMACSTM may be used for magnetic
pre-
sorting of cells before further flow cytometric sorting. For example, anti-
FITC- or anti-PE
MACS~ MicroBeads, may be added to the FITC or PE stained cells. Then the cells
are
sorted on the autoMACST"~ according to their magnetic labeling. The positive
and negative
fractions may then be collected for sorting by flow cytometry.
Optionally, HSC are expanded ex vivo with IL-3, IL-6, and either SCF or LIF
(10
ng/ml each).
RevMlO vectors and preparation of genetically modified (GM) HSC. RevMlO is
known in the art, and has been described extensively in studies of GM HSC for
the survival
of T cells in HIV-infected patients (see, e.g., Woffendin et al., (1996) Proc.
Natl. Acad. Sci.
USA, 93:2889; for review, see Amado et al., (1999) Front. Biosci. 4:d468). The
H1V Rev
protein is known to affect viral latency in HIV infected cells and is
essential for HIV
replication. RevMlO is a derivative of Rev because of mutations within the
leucine-rich
domain of Rev that interacts with cell factors. RevMlO has a substitution of
aspartic acid for
I5 leucine at position 78 and of Leucine for glutamic acid at position 79. The
result of these
mutations is that RevMlO is able to compete effectively with the wild-type HIV
Rev for
binding to the Rev-responsive element (RRE).
Any of the RevMlO gene transfer vectors known and described in the art may be
used. For example, the retroviral RevMlO vector, pLJ-RevMlO is used to
transducer the
HSC. The pLJ-RevMlO vector has been shown to enhance T cell engraftment after
delivery
into HIV-infected individuals (Ranga et al., (1998) Proc. Natl. Acad. Sca. USA
95:1201).
Other methods of construction and retroviral vectors suitable for the
preparation of GM HSC
are well known in the art (see, e.g., Bonyhadi et al., (1997) J.
Vii°ol. 71:4707).
In another example, the pRSV/TAR RevMlO plasmid is used for non-viral vector
delivery using particle-mediated gene transfer into the isolated target HSC
essentially as
described in Woffendin et al., (1994) Proc. Natl.. Acad. Sci. USA, 91:11581.
The pRSV/TAR
RevMlO plasmid contains the Rous sarcoma virus (RSV) promoter and tat-
activation
response element (TAR) from -18 to +72 of HIV is used to express the RevMlO
open
reading frame may also be used (Woffendin et al., (1994) Proc. Natl. Acad.
Sci. USA,
91:11581; Liu et al., (1997) Geae Ther. 1:32). Ifz vitro transfection of this
plasmid into
human PBL has previously been shown to provide resistance to HIV infection
(Woffendin et
al., (1994) Proc. Natl. Acad. Sci. USA, 91:11581).
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A marker gene, such as the Lyt-2a (murine CDBa) gene, may also be incorporated
into the RevMlO vector for ease of purification and analysis of GM HSC by FAGS
analysis
in subsequent steps (see, a g., Bonyhadi et al., (1997) J. Virol. 71:4707).
A ~RevlO, which contains a deletion of the methionine (Met) initiation codon
(ATG),
as well as a linker comprising a series of stop codons inserted in-frame into
the BgIII site of
the RevMlO gene, is constructed and used as a negative control (see, a g.,
Bonyhadi et al.,
(1997) J. Vdrol. 71:4707).
Injection of GM HSC into rxiice. SC1D-hu mice are analyzed, and the mice
determined to be HLA mismatched (MA2.1) with respect to the human donor HSC
are give
approximately 400 rads of total body irradiation (TBI) about four months
following the
thymic and liver grafts in an effort to eliminate the cell population. After
TBI, mice are
reconstituted with the RevMlO GM HSC (see above) as described previously (see,
e.g.,
DiGusto et al., (1997) Blood, 87:1261, Bonyhadi et al., (1997) J. Vdrol.
71:4707). Control
mice are injected with unmodified HSC or with HSC that have been modified
v~rith the
ORevM 10 gene or an irrelevant gene.
Analysis of CTM HSC by flow cytometry. Approximately 8 to 12 weeks after GM
HSC reconstitution, the Thy/Liv grafts are removed, and the thymocytes are
obtained and
analyzed for the HL.A pheonotype (MA2.1) and the distribution of CD4+, CD8+,
and Lyt2 ".
(the "marker" murine homolog of CDBa) surface expression using methods of flow
cytometry and FACS analysis readily known to those skilled in the art (see,
e.g., Bonyhadi et
al., (1997) J. Virol. 71:4707; see also Current Protocols In hnmunology, Units
4.8 and 5,
John E. Coligan et al., (eds), Wiley and Sons, New York, NY (1994), and yearly
updates
including 2002). Thymocytes are also tested for transgenic DNA with primers
specific for
the RevMlO gene using standard PCR methods.
Analysis of GM HSC resistance to HIV infection. Approximately 8 to .12 weeks
(or later) after GM HSC reconstitution, the Thy/Liv grafts are removed and the
thymocytes
are obtained from the GM HSC reconstituted SCID-hu mice. The thymocytes are
stimulated
ira vitro and infected with the JR-CSF molecular isolate of HIV-1 as described
previously
(Bonyhadi et al., (1997) J. Vir-ol. 71:4707). Briefly, the thymocytes are
stimulated irz vitro in
the presence of irradiated allogeneic feeder cells (106 peripheral blood
mononuclear cells/ml
and 105 JY cells/ml) in RPMI medium containing 10% FCS, 50 ~glml streptomycin,
50 U/G
penicillin G, 1x MEM vitamin solution, lx insulin transferring-sodium selenite
medium
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supplement (Sigma), 40 U human rIL-2/ml, and 2 p.g/ml phytohemagglutinin (PHA)
(Sigma).
About every 10 days, cells are restimulated with feeder cells and PHA as
described
previously in Vandekerckhove et al., (1992) J. ExB. Med. 1:1033. Approximately
5 days
after stimulation, cells were sorted on the basis of donor HLA phenotype
(MA2.1) and Lyt2
(the "marker" murine homolog of CDBa). Sorted cells are restimulated and may
be expanded
to increase the cell composition to greater than about 90% purity. CD4+/Lyt2+
cells are then
sorted out and an aliquot of approximately 5x104 of the sorted cells are place
in multiple
wells of a 96-well U bottom tissue culture plate. About 200 TCmSO of EW, an
HIV-1
primary isolate, or 1000 TCIDSO of JR-CSF, an HIV-1 molecular isolate, are
added to each
well. Methods of virus stock preparation have been described previously
(Bonyhadi et al.,
(1993) Nature, 363:728). Medium is changed every day from days 3 to 12.
Aliquots of
supernatant are collected every other day and stored at -80° C until
use. Tissue culture
supernatants are then analyzed using a p24 ELISA following manufacturer's
instructions
(Coulter).
II. Therapy of HIV Infected Individual
Materials and Methods.
Isolation of human CD34 + HSC. As most HIV infected patients have very low
titers of HSC, it is possible to use a donor to supply cells. Where practical,
the level of HSC
in the donor blood is enhanced by injecting into the donor granulocyte-colony
stimulating
factor (G-CSF) at 10 p.g/kg for 2-5 days prior to cell collection.
In this example, human cord blood (CB) HSC are collected and processed using
techniques well known to those skilled in the art (see, e.g., DiGusto et al.,
(1997) Bload,
87:1261; Bonyhadi et al., (1997) J. Virol. 71:4707). A portion of each CB
sample is HLA
phonotyped, and the CD34+ donor cells are purified from the donor blood (or
BM), such as
by using a flow cytometer or immunomagnetic beading, essentially as described
above.
Donor-derived HSC are identified by flow cytometry as being CD34+.
Optionally, HSC are expanded ex vivo with IL-3, IL-6, and either SCF or LIF
(10
ng/ml each).
RevMlO vectors and preparation of genetically modified (GM) HSC. Any of the
RevMlO gene transfer vectors known and described in the art, including those
described in
the mouse studies above, may be used. Methods of gene transduction using GM
retroviral
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vectors or gene transfection using particle-mediated delivery are also well
known in the art,
and are described elsewhere herein.
As described above, a retroviral vector may be constructed to contain the
trans-
dominant mutant form of HIV-1 t~ev gene, RevMlO, which has been shown to
inhibit HIV
replication (Bonyhadi et al., (1997) J. Vir~ol. 71:4707). Amphotropic vector-
containing
supernatants are generated by infection with filtered supernatants from
ecotropic producer
cells that were transfected with the vector.
The collected CD34+ cells are optionally pre-stimulated for 24 hours in LCTM
media
supplemented with IL-3, IL,-6 and SCF or LIF (10 ng/ml each) to induce entry
of the cells
into the cell cycle.
In this example, CD34+-enriched HSC undergo transfection by a linearized
RevMlO
plasmid utilizing particle-mediated ("gene gun" transfer) essentially as
described in
Woffendin et al., (1996) Proc. Natl. Acad. Sci. USA, 93:2889. However, if
retroviral
transduction is done, supernatants containing the vectors are repeatedly added
to the cells for
2-3 days to allow transduction of the vectors into the cells.
HAART Treatment of HIV-infected patients. HAART therapy is begun before T
cell depletion and sex steroid ablation, and therapy is maintained throughout
the procedure to -
reduce the viral titer.
T cell depletion. T cell depletion is performed as given in Example 5 to
remove as
many HIV infected cells as possible.
Sex steroid ablation therapy. The HIV-infected patient is given sex steroid
ablation
therapy as described in Example 6.
Injection of GM HSC into patients. Prior to injection, the GM HSC are expanded
in culture for approximately 10 days in X-Vivo 15 medium comprising ll-2
(Chiron, 300
IU/ml). At approximately 1-3 weeks post LHRH agonist delivery, just before or
at the time
the thymus begins to reactivate, the patient is injected with the genetically
modified HSC,
optimally at a dose of about 2-4 x 106 cells/kg. Optionally G-CSF may also be
injected into
the recipient to assist in expansion of the GM HSC.
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T_m_m__ediately prior to patient infusion, the GM HSC are washed four times
with
Dulbecco's PBS. Cells are resuspended in 100 ml of saline comprising 1.25%
human
albumin and 4500 U/ml IL-2, and infused into the patient over a course of 30
minutes.
Following sex steroid ablation and injection of the GM HSC in the HIV-infected
patient, all new T cells (as well as DC, macrophages, etc. ) are resistant to
subsequent
infection by this virus. Injection of allogeneic HSC into a patient undergoing
thymic
reactivation means that the HSC will enter the thymus. The reactivating or
reactivated
thymus takes up the genetically modified HSC and converts them into donor-type
T cells and
DC, while converting the HSC of the recipient into recipient-type T cells and
DC. By
inducing deletion by cell death, or by inducing tolerance through
immunoregulatory cells, the
donor DC will tolerize any T cells that are potentially reactive with
recipient.
When the thymic chimera is established, and the new cohort of mature T cells
have
begun exiting the thymus, reduction and eventual elimination of
immunosuppression occurs.
Post-infusion studies. Following infusion, the persistence and half life of GM
HSC
in the HIV-infected patient is tested periodically using limiting dilution PCR
of PBL samples
obtained from the patient essentially as described in Woffendin et al., (1996)
Proc. l~atl.
Acad. Sci. USA, 93:2889. The relative level of GM HSC in the infected patient
is compared
to the negative control patient that received the ORevMlO vector.
Various standard hematologic (e.g., CD4+ T cell counts), inununologic (e.g.,
neutralizing antibody titers), and virologic (e.g., viral titer) studies are
also performed using
methods well known to those skilled in the art.
Termination of immunosuppression. Termination of immunosuppression i.s
performed as given in Example 16.
EXAMPLE 15
ALTERNATIVE PROTOCOLS
In the event of a shortened time available for transplantation of donor cells,
tissue or
organs, the timeline as used in Examples 1-14 is modified. T cell ablation or
other immune
cell depletion and sex steroid ablation are begun at the same time. T cell
ablation or other
immune cell depletion is maintained for about 10 days, while sex steroid
ablation is
maintained for around 3 months. In one embodiment, HSC transplantation is
performed
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when the thymus starts to reactivate, at around 10-12 days after start of the
combined
treatment.
In an even more shortened time table, the two types of ablation and the HSC
transplant are started at the same time. In this event, T cell ablation or
other immune cell
depletion is maintained 3-12 months, for example, for 3-4 months.
EXAMPLE 16
TERMINATION OF IMMUNOSUPPRESSION
When the thymic chimera is established and the new cohort of mature T cells
have
begun exiting the thymus, blood is taken from the patient and the T cells
examined irz vitro
for their lack of responsiveness to donor cells in a standard mixed lymphocyte
reaction (see,
e.g., Current Protocols In Immunolo~y, John E. Coligan et al., (eds), Wiley
and Sons, New
York, NY (1994), and yearly updates including 2002). If there is no response,
the
immunosuppressive therapy is gradually reduced to allow defense against
infection. If there
is no sign of rejection, as indicated in part by the presence of activated T
cells in the blood,
the immunosuppressive therapy is eventually stopped completely. Because the
HSC have a
strong self-renewal capacity, the hematopoietic chimera so formed will be
stable theoretically
for the life of the patient (that is a normal, non-tolerized and non-grafted
person).
EXAMPLE 17
USE OF LHRH AGONIST TO REACTIVATE THE THYMUS IN HUMANS
Materials and Methods:
In order to show that a human thymus can be reactivated by the methods of this
invention, these methods were used on patients who had been treated with
chemotherapy for
prostate cancer.
Patients. Sixteen patients with Stage I-III prostate cancer (assessed by their
prostate
specific antigen (PSA) score) were chosen for analysis. All subjects were
males aged between
60 and 77 who underwent standard combined androgen blockade (CAB) based on
monthly
injections of GnIZH agonist 3.6 mg goserelin acetate (Zoladex0) or 7.5 mg
leuprolide
(Lupron0) treatment per month for 4-6 months prior to localized radiation
therapy for prostate
cancer as necessary.
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FRCS analysis. The appropriate antibody cocktail (20 ~1) was added to 200 ~1
whole
blood and incubated in the dark at room temperature (RT) for 30min. RBC, were
lysed and
remaining cells washed and resuspended in 1%PFA for FACS analysis. Samples
were stained
with antibodies to CD19-FITC, CD4-FITC, CD8-APC, CD27-F1TC, CD45RA-PE, CD45R0-
CyChrome, CD62L-F1TC and CD56-PE (all from Pharmingen, San Diego, CA).
Statistical analysis. Each patient acted as an internal control by comparing
pre- and
post-treatment results and were analyzed using paired student t-tests or
Wilcoxon signed rank
tests.
Results:
Prostate cancer patients were evaluated before and 4 months after sex steroid
ablation
therapy. The results are summarized in Figs. 19-23. Collectively the data
demonstrate
qualitative and quantitative improvement of the status of T cells in many
patients.
I. The Effect of LHRH Therapy on Total Numbers of Lymphocytes and T cells
Subsets Thereof:
The phenotypic composition of peripheral blood lymphocytes was analyzed in
patients (all >60 years) undergoing LHRH agonist treatment for prostate cancer
(Fig. 40).
Patient samples were analyzed before treatment and 4 months after beginning
LHRH agonist
treatment. Total lymphocyte cell numbers per ml of blood were at the lower end
of control
values before treatment in all patients.
Following treatment, six out of nine patients showed substantial increases in
total
lymphocyte counts (in some cases a doubling of total cells was observed).
Correlating with
this was an increase in total T cell numbers in six out of nine patients.
Within the CD4+
subset, this increase was even more pronounced with eight out of nine patients
demonstrating
increased levels of CD4+ T cells. A less distinctive trend was seen within the
CD8+ subset
with four out of nine patients showing increased levels albeit generally to a
smaller extent
than CD4+ T cells.
II. The Effect Of LHRH Therapy On The Proportion Of T Cells Subsets:
Analysis of patient blood before and after LHRH agonist treatment demonstrated
no
substantial changes in the overall proportion of T cells, CD4+ or CD8+ T cells
and a variable
change in the CD4+:CD8+ ratio following treatment (Fig. 41). This indicates
that there was
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little effect of treatment on the homeostatic maintenance of T cell subsets
despite the
substantial increase in overall T cell numbers following treatment. All values
were
comparative to control values.
III. The Effect Of LHRH Therapy On The Proportion Of B Cells And Myeloid
Cells:
Analysis of the proportions of B cells and myeloid cells (NK, NKT and
macrophages)
within the peripheral blood of patients undergoing LHRH agonist treatment
demonstrated a.
varying degree of change within subsets (Fig. 42). While NK, NKT and
macrophage
proportions remained relatively constant following treatment, the proportion
of B cells was
decreased in four out of nine.
IV. The Effect Of LHRH Agonist Therapy On The Total Number Of B Cells And
Myeloid Cells:
Analysis of the total cell numbers of B and myeloid cells within the
peripheral blood
post-treatment showed clearly increased levels of NK (five out of nine
patients), NKT (four
out of nine patients) and macrophage (three out of nine patients) cell numbers
post-treatment
(Fig. 43). B cell numbers showed no distinct trend with two out of nine
patients showing . ,
increased levels; four out of nine patients showing no change and three out of
nine patients
showing decreased levels.
V. The Effect Of LHRH Therapy On The Level Of Naive Cells Relative To Memory
Cells:
The major changes seen post-LHRH agonist treatment were within the T cell
population of the peripheral blood. In particular there was a selective
increase in the
proportion of naive (CD45RA+) CD4+ cells, with the ratio of naive (CD45RA+) to
memory
(CD45R0~'') in the CD4+ T cell subset increasing in six out of nine patients
(data not shown).
VI. Conclusion
Thus it can be concluded that_LHRH agonist treatment of an animal such as a
human
having an atrophied thymus can induce regeneration of the thymus. A general
improvement
has been shown in the status of blood T lymphocytes in these prostate cancer
patients who
have received sex-steroid ablation therapy. It is likely that such cells are
derived from the
thymus as no other source of mainstream (TCRa(3+CD8 a(3 chain) T cells has
been
described. Gastrointestinal tract T cells are predominantly TCR y8 or CD8 as
chain.
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EXAMPLE 18
REGENERATION OF THE PERIPHERAL IMMUNE CELL POOL FOLLOWING
HEMATOPOIETIC STEM CELL TRANSPLANTATION IN HUMANS
I. Allogeneic and Autolo~ous HSCT
This example relates to clinical trials undertaken with HSCT patients. To
assess the
clinical potential for restoring thymus and bone marrow function in humans,
prostate cancer
patients (>60 years) who routinely undergo sex-steroid ablation therapy based
on LHRH-
agonist (chemical castration) treatment have been analyzed. Patients were
examined at the
time of presentation and after 4-months of treatment by which time serum
testosterone
concentration was at castrate levels for all patients.
Materials and Methods:
Patients. Eighty-two Patients were all due to undergo high-dose therapy (HDT)
with
PBSCT for malignant disease or bone marrow failure (n = 22 for allogeneic
control patients, n
= 20 for allo LHRH-A treated patients, n = 20 for autologous controls and n =
20 .for
autologous LHRH-A treated patients). Test patients were given 3.6 mg
(effective for 4 weeks)
Zoladex (LHRH-A) 3-weeks prior to autologous or allogeneic stem cell
transplantation and
then monthly injections for 4-months. All patients were analyzed pre-
treatment, weeldy for 5-
weeks after transplantation and then monthly up to 12 months. Ethics approval
was obtained
from The Alfred Committee for Ethical Research on Humans (Trial Number
01/006).
FRCS Analysis of Whole Peripheral Blood. The appropriate antibody cocktail (20
Ol) was added to 200 D1 whole blood and incubated in the dark at room
temperature (RT) for
30min. RBC were lysed and remaining cells washed and resuspended in 1%PFA for
FACS
analysis. Samples were stained with antibodies to CD19-FTTC, CD4-FITC, CD8-
APC, CD27-
FITC, CD45RA-PE, CD45R0-CyChrome, CD62L-FITC and CD56-PE (all from Pharmingen,
San Diego, CA).
Ki67 Analysis. For detection of proliferating cells, samples were surface
stained with
CD27-FITC, CD45R0-CyChrome, and CD4- or CD8-APC (Pharmingen, San Diego, CA).
Following red cell lysis, samples were incubated for 20 min, RT, in the dark
in 500 ~ul of 1X
FACS permeabilizing solution (Becton-Dickinson, USA; 1X solution was made from
lOX
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stock in R.O.H20). Washed samples (2 ml FAGS buffer, 5 min., 600gmaX, RT) were
incubated with either anti-Ki67-PE or anti-Ki67-FITC (or the appropriate
isotype controls)
for 30 min. at RT, in the dark. Samples were then washed and resuspended in 1%
PFA for
analysis.
Preparation of PBMC. Purified lymphocytes were prepared for T-cell stimulation
assays and TREC analysis, by ficoll-hypaque separation and following
centrifugation, the
plasma layer was removed and stored at -20°C prior to analysis of sex
steroid levels. Cells
not used for T lymphocyte stimulation assays were resuspended in freezing
media and stored
in liquid nitrogen prior to TREC analysis.
T Lymphocyte Stimulation Assay. For mitogen stimulation, purified lymphocytes
were plated out in 96-well round-bottom plates at a concentration of 1 x 105
cells/well in 100
w1 of RPMI-FCS. Cells were incubated at 37°C, 5% C02 with PHA in doses
from 1-10
p,g/ml. For TCR-specific stimulation, cells were incubated for 48 hours on
plates previously
coated with purified anti-CD3 (1-10 ~.ghnl) and anti-CD28 (I0 q.g/ml).
Following plaque
formation (48-72 hours), 1 ~.Ci of 3H-thymidine was added to each well and
plates incubated
for a further 16-24 hours. Plates were harvested onto filter mats and
incorporation of 3H-
Thymidine was determined using liquid scintillation on a [3-counter (Packard-
Coulter, USA).
TREC Anal,
Cell Sorting. Frozen samples were rapidly thawed and stained with anti-CD4-
FITC
and anti-CD8-APC for 30 min on ice, washed (2 ml FACS buffer) and fixed with
3%
formalin in PBS (with agitation). Samples were incubated for a further 30
min., washed and
resuspended in 500 ~,l FACS- buffer for sorting. CD4~ and CD8+ cell
populations were sorted
on a MoFloO cell sorter (Cytomation Inc.).
DNA Isolation. Cells were sorted and resuspended in Proteinase K (PK)
digestion
buffer (2x 105 cells/20 ~,1 of a 0.8 mg/mL solution). . Samples were incubated
for 1 hour at
56°C followed by l0min at 95°C to inactivate the proteinase.
Real-Time PCR using Molecular Beacons. Real-time PCR for analysis of TREC
content in sorted cells was performed as described previously (Zhang et al.,
(1999) J. Exp.
Med. 190:725). The primers were sense, 5'-GGATGGAAAACACAGTGTGACATGG-3'
(SEQ ll~ N0:4) and antisense, 5'-CTGTCAACAAAGGTGATGCCACATCC-3' (SEQ ID
NO:S). One cycle of denaturation (95°C for lOmin) was performed,
followed by 45 cycles of
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amplification (94°C for 30 s, 60°C for 30 s, and 72°C for
30s). To normalize for cell
equivalents in the input DNA, a separate real-time PCR assay was used to
quantify the CCRS
coding sequence, which contains no pseudogenes.
Statistical Analysis. Statistical analysis was performed using Instat II
software. For
prostate cancer HSCT studies, a Mann-Whitney U-test was performed. For human
studies,
each patient acted as an internal control by comparing pre- and post-treatment
results and
were analyzed using paired student t-tests or wilcoxon signed rank tests.
Results:
Fig. 49 depicts analysis of natural killer (NK) cell recovery at various time
points (2-8
weeks) following HSCT in control patients. As shown in Figs. 49A-B,
respectively, a similar
trend was observed for both control allogeneic and autologous transplant
recipients. Tn
contrast, allogeneic patients who were given LHRH-A treatment 3 weeks prior to
HSC'I'
showed a significantly higher number of NKT (V ~ 24+V ~ 11+) cells from D 14-
5M post-
transplant (Fig. 49C; data is expressed as mean ~ 1 SEM of 6-20 patients;
*=p_<0.05). NKT
cells were analyzed based on their V 024+V D 11+ phenotype.
Fig. 50 depicts FACS analysis of NKT cell reconstitution at various time
points {day
14, 21, 28 and 35) following HSCT in control patients. An early recovery was
observed in
allogeneic patients, and was seen predominantly within the CD8+ population
early post- - -
transplant, which indicated extrathymic routes of regeneration. Also, CD4+NKT
cells were r
evident from 1 month past-transplant.
Fig. 51 depicts B cell reconstitution following HSCT at various time points (2-
12
months) following HSCT in control patients. As shown in Fig. 51B, B cell
regeneration
occurs relatively faster in autologous transplant patients as compared to that
of allogeneic
patients (Fig. 51A). However, a return to control values (shaded) was not
evident until at least
6 months post-transplant in both groups.
Fig. 52 depicts CD4+ reconstitution following HSCT at various time points (2-
12
months) following HSCT in control patients. While B cell numbers were
returning to control
values by 6 months post-transplant (see Figs. 48A-B), CD4+ T cell numbers were
severely
reduced, even at 12 months post-transplant, in both autologous (Fig. 52B) and
allogeneic
(Fig. 52A) recipients.
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Fig. 53 depicts CD8+ regeneration following HSCT at various time points (2-12
months) following HSCT in control patients. As shown in Fig. 53A-B, CD8+ T
cell
numbers regenerated quite rapidly post-transplant in both allogeneic and
autologous
recipients, respectively. However, as shown in Fig. 53C, the CD8+ T cells are
mainly of
extrathymic origin as indicated by the increase in TCRyB+ T CD8+ T cells,
CDBaa T cells,
and CD28~CD8+ T cells.
Fig. 54 depicts FACS analysis of proliferation in various populations of CD4+
and
CD8+ T cells before (Fig. 54A) and 28 days after (Fig. 54B) HSCT in control
patients using
the marker Ki-67. Cells were analyzed on the basis of naive, memory and
activated
l0 phenotypes using the markers CD45R0 and CD27. The majority of proliferation
occurred in
CD8+ T cell subset, which further indicated that these cells were
extrathymically derived and
that the predominance of proliferation occurred within peripheral T cell
subsets.
Fig. 55 depicts naive CD4+ T cell regeneration at various time points (2-12
months)
following HSCT in control patients and LHRH-A treated patients. Fig. 55A
depicts FACS
analysis of naive,CD4+ T cells (CD45RA+CD45R0-CD62L+), and shows a severe loss
of
these cells throughout the study. As shown in Figs. 55B-C, naive CD4+ T cell
began to
regenerate by 12 months post-HSCT in autologous transplant patients (Fig. 55C)
but were
still considerably lower than the control values in allogeneic patients (Fig.
55B). These
results indicated that the thymus was unable to restore adequate numbers of
naive T cells
post-transplant due to the age of the patients. In contrast, in patients that
were given LHRH-
A 3-weeks prior to allogeneic HSCT showed a significantly higher number of
naive CD4+ T
cells at both 9 & 12 months post-transplant compared to controls (p<_0.05 both
9 & 12 months
post-transplant compared to control (non-LHRH-A treated) (Fig. 55D). This
indicates
enhanced regeneration of the thymic-dependent T cell pathway with sex. steroid
ablation
therapy.
Fig. 56 depicts TREC levels at various time points (1-12 months) following
HSCT in
control patients. Analysis of TREC levels, which are only seen in recent
thymic errugrants
(RTE), emphasized the inability of the thymus to restore levels following
transplant in both
allogeneic (Fig. 52A) and autologous (Fig. 52B) patients. Again, this was due
to the age of
the patients, as well as the lack of thymic function due to thymic atrophy,
which has
considerable implications in the morbidity and mortality of these patients. n
contrast,
patients undergoing allogeneic peripheral blood stem cell transplantation
demonstrated a
significant increase in CD4+TREC+ cells/ml blood when treated with an LHRH-A
prior to
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allogeneic transplantation (p<_0.01 at 9 months post-transplant compared to
control (non-
LHRH-A treated). Allogeneic patients who were given LHRH-A treatment showed a
significantly higher number of CD4+TREC+ cells/ml blood at 9 months post-
transplant (Fig.
56C) compared to controls. Autologous LHRH-A treated patients also showed
significantly
higher levels at 12 months post-transplant (Fig. 56D). This indicates enhanced
regeneration
of the thymus with sex steroid ablation therapy. Data is expressed as mean ~ 1
SEM of 5-18
patients. *=p_0.01.
LHRH-A administration significantly increases NK but not B cell numbers in the
peripheral blood. Overall, no significant change in B cell numbers was
observed with
LHRH-A treatment (Fig. 47). However, a significant increase in NK cell numbers
was
observed with treatment (p_<0.01) (Fig. 47). Therefore, removal of sex steroid
results in
significantly increased numbers of T cells and NK cells.
A significant increase in the total lymphocyte, T cell (predominantly CD4+)
and NK
cells was observed (Figs. 45 and 47) consistent with previous studies of
patients treated with
LHRH-agonists (Garzetti et al., (1996) Obstet. Gyaecol. 88: 234-40; Oliver et
al., (1995)
Urol. hZt., 54:226-229; Umesaki et al., (1999) Gyreecol. Obstet. Ifzvest.
48:66-8). More
detailed analysis of the T cell compartment revealed a significant increase,
in the numbers of
naive CD4+ T cells and both naive and memory CD8+ T cells following LHRH-A
treatment
(Fig. 44).
To determine if the increase in naive T cells was through peripheral expansion
(as
seen for example with IL-7 administration (Snares et al., J. Imnzuhol. (1998)
161:5909-5917)
or as a direct result of thymic reactivation, analysis of cellular
proliferation (Ki-67 antigen+),
together with TREC levels was performed (Hazenberg et al., (2001) J. Mol. Med.
79:631-40).
No change in the level of proliferation was seen with agonist treatment in
naive, activated or
memory populations of both CD4+ (Fig. 48A) and CD8+ T cells (Fig. 48B)
(remaining at a
low 2-4%). This indicates that the treatment does not directly induce
proliferation of T cells,
and that the levels of TRECs would not be influenced by excessive
proliferation in the
periphery. This does not rule out the possibility of peripheral expansion at
earlier time-
points. However this would presumably only account for increased
activated/memory cell
levels. Direct evidence for an increase in thymic function and T cell export
was found
following analysis of TREC levels in 10 patients (Fig. 46B). Within both the
CD4+ and CD8+
T cell population, five out of ten patients showed an increase (>25% above
initial
presentation values) in absolute TREC levels (per ml of blood) by 4 months of
LHRH-A
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treatment. This was also reflected in a proportional increase (per 1x105
cells). This correlated
with six out of ten patients showing an overall increase in total TREC levels.
Only 1 patient
showed a decrease in total TRECs (about 30% decrease). Since TRECs are diluted
out with
mitosis (Zhang et al., (1999) J. Exp. Med. 190:725-732), which could occur
intrathymically
as part of normal T cell development or following export (Hazenberg et al.,
(2001) J. Mol.
Med. 79:631-40), the absolute TREC levels would represent very much an
underestimate of T
cell export. The marked increase in total TREC+ cells in the periphery
following treatment
with the agonist is thus fully consistent with regeneration of the thymus-
dependent T cell
pathway (Douek et al., (1998) Natut-e 396:690-695 (1998); Douek et al., (2001)
J. Immuytol.
167:6663-8; Hochberg et al., (2001) Blood 98:1116-21). Together, these data
demonstrate the
ability of sex steroid inhibition to improve thymic output in adult humans and
provides a
basis for restoring naive T cell numbers following severe T-cell depletion in
many clinical
conditions.
EXAMPLE 19
SEX STEROID ABLATION ENHANCES IMMUNE RECONSTITUTION
FOLLOWING HEMATOPOIETIC STEM CELL TRANSPLANTATION IN MICE
This experiment was done to test the hypothesis that sex steroid inhibition in
recipients of an allogeneic HSCT can improve their post-transplant immune
reconstitution.
Thus, these experiments aimed to establish whether sex steroid ablation
influenced
hematopoietic recovery following allogeneie HSCT. Fourteen days after HSCT, BM
and
thymic cell numbers were significantly increased in the castrated mice
compared to sham
controls. These remained elevated at day 28 at which time splenic cellularity
was also
increased in the castrates. In the thymus, T cell precursors and DC were
significantly after
HSCT and castration. BM precursors and developing B cells were also
significantly increased
after HSCT and castration. These central increases translated to a significant
increase in
donor-derived peripheral T and B cells after allogeneic HSCT. Every immune-
enhancing
strategy carries the risk of exacerbating the development of graft-versus-host
disease
(GVHD). Mice were castrated at the same time as GVHD induction in an
allogeneic setting.
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There was no significant difference in GVHD incidence or severity when
comparing
castrated and sham-castrated mice. Furthermore, GVT activity was not
diminished in the
absence of sex steroid. It has been previously shown that lymphoid recovery is
enhanced in
allo-HSCT recipients after IL-7 treatment. The combination of IL-7 treatment
and castration
appeared to have an additive effect in the thymus following HSCT. These
results indicate that
castration and the resulting ablation of sex steroids enhance hematopoietic
recovery
following allogeneic HSCT without increasing GVHD and maintaining GVT.
Materials and Methods:
Reagents. Antimurine CD 16/CD32 FcR block (2.462) and all following
fluorochrome-labeled antibodies against marine antigens were obtained from
Pharmingen
(San Diego, CA): Ly-9.1(30C7), CD3(145-2C11), CD4 (RM4-5), CD8(3.2(53-5.8), T-
cell
receptor-(3 (TCR-~3; H57-597), CD45R/B220 (RA3-6B2), CD43 (S7), IgM-FITC (R6-
60.2),
CDllb (M1/70), Ly-6G(Gr-1) (RB6-8C5), c-kit (2B8), Sca-1 (D7), CDllc (HL3) I-
Ak (11-
5.2), isotypic controls: rat IgG2a-k (R35-95), rat IgG2a-1 (B39-4), rat IgG2b-
(A95-1), rat
IgGl-k (R3-34), hamster IgG-groupl-k (A19-3), hamster IgG-group 2-1 (Ha4/8),
and 2.462
and Fcr ~ (FcR blocking). Streptavidin-FTTC, PercP -phycoerythrin (PE) also
were obtained
from Pharmingen (San Diego, CA). Recombinant human IL,-7 was provided by Dr
Michel
Mon.-e (Cytheris, Vanves, France).
To confirm that .the human recombinant IL-7 could stimulate marine cells,
thymidine
incorporation proliferation assays were performed with an IL-7-dependent
marine pre-B cell
line 2E8 and found that the human IL-7 used in the studies had a proliferative
effect on
marine cells that was equal to marine IL-7. Tissue culture medium consisted of
RPMI 1640
supplemented with 10% heat inactivated, fetal calf serum, 100 U/mL penicillin,
100 mg/rnL
streptomycin, and 2 mM L-glutamine (as well as 50 mM 2-mercaptoethanol for the
culture of
32Dp210 cells and proliferation assays).
Mice and HSCT. Male C57BL/6J (B6, H-2b), C3FeB6F1/J([B6 3 C3H]Fl; H-2b/k),
B lO.BR (H-2k), B6D2F1/J (H-2b/d), CBA/J (H-2k), Balb/c (H2-d), IL7-/- and KGF-
/- mice
were obtained from the Jackson Laboratory (Bar Harbor, ME) and used in
experiments when
they were between 8 and 12 weeks of age. KGF-l- and IL7-l- were used between 4
and 7
months of age. HSCT protocols were approved by the Memorial Sloan-Ketterzng
Cancer
Center Institutional Animal Care and Use Committee. The BM cells were removed
aseptically from femurs and tibias. Donor BM was depleted of T cells by
incubation with
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anti-Thy-1.2 for 30 minutes at 4°C followed by incubation with Low-TOX-
M rabbit
complement (Cedarlane Laboratories, Hornby, ON, Canada) for 1 hour at
37°C. Splenic T
cells (for GVHD analysis) were obtained by purification over a nylon wool
column followed
by red cell removal with ammonium chloride red cell lysis buffer. Cells (5x106
BM cells with
or without splenic T cells and leukemia cells) were resuspended in Dulbecco
modified
essential medium (Life Technologies, Grand Island, NY) and transplanted by
tail vein
infusion (0.25 mL total volume) into lethally irradiated recipients on day 0.
Prior to
transplantation, on day 0, recipients received 1300 cGy total body irradiation
(l3~Cs source)
as split dose with 3 hours between doses (to reduce gastrointestinal
toxicity). Mice were
housed in sterilized microisolator cages and received normal chow and
autoclaved
hyperchlorinated drinking water (pH 3.0).
Surgical Castration. Mice were anaesthetized and a small scrotal incision was
made
to reveal the testes. These were sutured and removed along with surrounding
fatty tissue. The
wound was closed using surgical staples. Sham-castration required the same
surgical
procedure, except for the removal of the testes. Castration was performed one
day prior to
BM transplant for both immune reconstitution and GVHD studies. .
Administration of IL-7. IL-7 were either given from days 0 to 13 or 21 to 27
intraperitoneally at 10 ~,glday for immune reconstitution studies. PBS was
injected into
control mice at the same time points.
Flow cytometric analysis. BM cells, splenocytes or thymocytes were washed in
FACS buffer (phosphate buffered saline (PBS)/2% bovine serum albumin
(BSA)/0.1% azide)
and 1-3 x 106 cells were incubated for 30 minutes at 4°C with CD16/CD32
FcR block. Cells
were then incubated for 30 minutes at 4°C with primary antibodies and
washed twice with
FACS buffer. Where necessary, cells were incubated with conjugated
Streptavidin for a
further 30 minutes at 4°C. The stained cells were resuspended in FAGS
buffer and analyzed
on a FACSCaliburTM flow cytometer (Becton Dickinson, San Jose, CA) with
CellQuestTM
software.
Proliferation assays. Splenocytes (4 x 105 cellslwell) from sham-castrated
(n=5) and
castrated (n=5) mice were incubated for 5 days with irradiated (2000 cGy)
BALB/C
splenocytes as stimulators (2 x 105 cells/well) in 96-well plates and
splenocytes (4 x 105
cells/well) were stimulated with ocCD3 ( 145-2c 11 ) and ocCD28 (37.51 ) (2.5
mg/mL as a final
concentration) for 4 days. Cultures were pulsed during the final 18 hours with
1 mCi/well
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[3H]-thymidine and DNA was harvested on a Harvester 96 (Packard). Stimulation
indices
(SI) were calculated as the ratio of stimulated cells (cpm) over unstimulated
cells (cpm).
SlCr release assays. Target cells were labelled with 100 mCi 5lCr at 2 x106
cells/mL for 2 hours at 37°C and 5% CO2. After 3 washes, labelled
targets were plated at
2.5x103cells/well in U-bottomed plates (Costar, Cambridge, MA). Splenocytes
cultured with
irradiated BALB/C splenocytes (1:2 ratio) for 5 days were added at various
effector-to-
target ratios in a final volume of 200 mL to 4 to 6 wells and incubated for 4
to 6 hours at
37°C and 5% CO2. Subsequently, 35 mL supernatant was removed from each
well and
counted in a gamma counter (Packard, Meriden, CT) to determine experimental
release.
Spontaneous release was obtained from wells receiving target cells and medium
only, and
total release was obtained from wells receiving 5% Triton X-100. Percent
cytotoxicity was
calculated by the following formula: percent toxicity = 100 X [(experimental
release -
spontaneous release)/(total release - spontaneous release)].
Detection of alloreactive T-cell clones with intracellular IFN-y staining
Briefly,
cells were incubated for 12 to 15 hours (for secondary allogeneic stimulation
with T cell-
depleted [TCD], irradiated stimulator cells) with Brefeldin A (10 mglmL),
harvested, washed,
stained with primacy (surface) fluorochrome (FTTC, PerCP, and APC)-conjugated
antibodies,
fixed, and permeabilized with the Cytofix/Cytoperm kit (Pharmingen), and
subsequently
stained with aIFN~y - PE. FAGS analysis was conducted by gating for the
designated
populations. Flow cytometer and software were used as mentioned below.
Delayed Type Hypersensitivity Assay. Sham-castrated and castrated mice were
sensitized day 42 after allo-BMT by tail vein injection with 200 ~,l of 0.01 %
sheep red blood
cells (Colorado Serum, Denver, CO) in PBS. Sensitized animals were challenged
at day 46 in
the right hind footpad with 501.x.1 of 20% sheep 12BC suspension while the
left hind footpad
received the same volume of 50 p,1 of PBS solution as a control. 24 and 48 hr
later foot~pad
swelling was measured with a dial-thickness gauge (Mitutoyo, Kanagawa, Japan).
The
magnitude of the response was determined by subtracting measurements of PBS-
injected left
footpads from the experimental right ones.
Assessment of GVHD. The severity of GVHD was assessed with a clinical GVHD
scoring system as first described by Cooke et al. ((1996) Blood 88:3230-9).
Briefly, ear-
tagged animals in coded cages were individually scored every week for 5
clinical parameters
(weight loss, posture, activity, fur, and skin) on a scale from 0 to 2. A
clinical GVHD index
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was generated by summation of the 5 criteria scores (0-10). Survival was
monitored daily.
Animals with scores of 5 or more were considered moribund and were humanely
killed.
Assessment of GVT - P815 (H-2d) mastocytoma induction and assessment of
mastocytomic death versus death from GVHD. B6D2F1/J recipients received 1 x
I03
P815 (H-2d) cells intravenously on day 0 of allogeneic HSCT (5 x 106 T cell
depleted (TCD)
BM cells and 5 x 105 T cells of C57/BL6 origin). Survival was monitored daily
and the
cause of death after HSCT was determined by necropsy as previously described.
Briefly,
death from leukemia was characterized by hepatosplenomegaly and the presence
of
mastocytoma cells in liver and spleen on microscopic examination, whereas
death from
GVHD was defined as the absence of hepatosplenomegaly and leukemic cells in
liver and
spleen, and the presence of clinical symptoms of GVHD as assessed by the
clinical GVHD
scoring system at the time of death.
Semi-Quantitative RT-PCR. Total cellular RNA from whole BM was reverse-
transcribed
using Superscript II reverse transcriptase (Life Technologies, Rockville,
USA). cDNA was
PCR-amplified for 35 cycles (94°C for 30 sees; 56°C for 30 sees;
72°C for 60 sees) with PCR
Master Mix (Promega, Madison, USA . HPRT: 5'CACAggACTAgAACACCT gC 3' and 5'
gCTggTgAAAAggACCTCT 3' TGF(31: 5' CTACTgCTTCAgCTC CACAg 3' and 5'
TgCACTTgCAggAgCgCAC 3 y and KGF: 5' gCCTTgTCACg ACCTgTTTC 3' and 5'
AgTTCACACTCgTAgCCgT T Tg 3'.
Enzymic digestion of IL7-/- Thymii. IL7-/- mice contain a large proportion of
CD45- thymic stroriial cells and each thymus was subjected to enzymic
digestion in 0.125'0
(w/v) collagenase/dispase (Roche Applied Sciences, Indianapolis, USA) with 0.1
% (w1v)
DNase, releasing most of the stromal and haematopoietic cells from the thymi
allowing for
the accurate calculation of thymic cellularity. Anti-CD45 was used to identify
CD45- stromal
cells.
Statistics. All values are expressed as mean~SEM. The Mantel-Cox log-rank test
was used for survival data and all other statistical analysis was performed
with the
nonparametric, unpaired Mann-Whitney U test. A P value of less than .05 was
considered
statistically significant.
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lZesults:
I. Castration increases BM, thynuc and splenic cellularity following
allogeneic
HSCT. Male CBA mice were castrated one day prior to allogeneic HSCT. Mice were
subjected to1300 cGy total body irradiation followed by 5 x 106 B lO.BR TCD BM
cells.
There were significantly more cells in the BM (16 x 106~ 1.4 x 106) and thymus
(55.4 x 106~
1.8 x 106) of castrated mice, compared to the sham castrated controls (9.5 x
106~ 3.0 x 105
and 25 x 106~ 2.6x106, respectively), as early as 14 days after HSCT (Figs.
29A-B). These
numbers remained significantly elevated in castrated mice 28 days after HSCT
(BM: 22 x
106~ 4.0 x 106 vs. 14 x106~ 2.2 x106; thymus: 72 x 106~ 5.9 x 106 vs. 45x106~
2.9x106).
Splenic cellularity in the castrated mice was also significantly elevated
above sham-castrated
spleen cell numbers at day 28 (253 x 106 ~ 28.4 x 106 vs. 126 x 106 ~ 13.9
x106) (Fig. 29C ).
The castrated mice had begun to approach pre-transplant cellularities by day
28. By 42 days
after HSCT there was no longer a significant difference between castrated and
sham-castrated
mice with respect to thymic and splenic cellularity. Since the sham-recipients
were young
mice they had active post-transplant lymphopoiesis but the time required to
generate normal
cellularity in the primary and secondary lymphoid tissues was markedly delayed
compared to
castrated recipients.
II. There are significantly more donor-derived HSCs in the BM of castrated
mice 28
days after HSCT. Several studies have shown that sex steroids inhibit the
proliferation
and/or differentiation of early hematopoietic precursors (Thurmond et al.,
(2000) Endocriraol.
141:2309-2318; Medina et al., (2001) Nat. Intmunol. 2:718; I~ouro et al.,
(2001) Blood
97:2708). Therefore, the impact of castration on the HSC numbers in the
allogeneic
transplant setting has been investigated. The number of donor-derived HSC was
very low in
both sham-castrated and castrated mice 14 days after allogeneic HSCT
(2.98x102~1.25x1.02
and 2.66x102~8.8x101 respectively) (Fig. 30A). However, by day +28 there are
significantly
more Ly9.1+ Lin- Sca-1+ c-kit+ donor-derived HSCs in the castrated mice
(4.8x103~1.1x103),
compared to the sham-castrated controls (1.1x103~4.1x102) (Fig. 30A).
III. Castration prior to allogeneic HSCT enhances donor-derived B cell
recovery. In
the analysis of B cell recovery three stages in B cell development was
distinguished: Pro-B
cells (CD45R+CD43+IgM-), pre-B cells (CD45R+CD43-IgM-) and immature B cells
(CD45R+CD43-IgM+). Fourteen days after allogeneic HSCT, there were
significantly more
pxe-B cells in the BM of castrated mice (5.5x106~1.7x106) compared to the sham-
castrated
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controls (2.08x106~5.0x104) (Fig. 30By:~ At 28 days there were, also
significantly more pre-B
cells (sham-cx: 3.1x106~3.7x105 c.f. cx: 6.6x106~6.6x105) and immature B cells
(sham-cx:
1.3x106~2.6x105 c.f. cx: 3.0x106~3.4x105) in the BM of castrated mice (Fig.
30B). The
increase in BM B cells and their precursors translated to a significant
increase in the number
of immature B cell in the spleens of castrated mice, 28 days after HSCT (sham-
cx:
64.9x106~6.4x106 cf. cx: 112.0x106~10.0x106) (Fig. 30C). These results are in
agreement
with previous studies that suggest that castration enhances B cell production
and export from
the BM.
IV. T cell reconstitution following allogeneic HSCT is enhanced by castration.
Thymocytes and peripheral cells were divided into developmental stages on the
basis of
expression of CD3, CD4 and CDB: Triple Negatives (TN) (CD3-CD4-CD8-), double
positive
(DP) (CD4+CD8+), single positive CD4 (SP CD4) CD3+CD4+CD8- and single positive
CD8
(SP CD8) CD3+CD4-CD8+ (Figs. 31A-D). As early as 14 days after allogeneic HSCT
there
are significantly more TN, DP, SP CD4 and SP CD8 thymocytes in castrated mice
compared
to sham castrated controls. 28 days after HSCT, DP and CD4 SP cell numbers
remain
significantly elevated in the castrated group. By day 42, all thymocyte
subsets are equivalent
in sham-castrated and castrated mice. Both host and donor-derived DC are
thought to play an
integral role in the avoidance of graft rejection (Morelli et al., (2001)
Senzdra. Inzrr~urcol.
13:323-335). Fourteen days after allogeneic HSCT, there are significantly more
host-derived
CD11ch1 DC in the thymii of castrated mice. Both host and donor-derived DC in
the thymus
were significantly increased in castrated mice 28 days after allogeneic HSCT
{Figs. 3IE-F).
The increase in thymocyte numbers in castrated mice translated to a
significant increase in
the number of donor-derived mature CD4+ and CD8+ T cells in the spleens of
castrated mice
compared to the sham-castrated controls at day 28 (Fig. 31G).
V. On a per cell basis, there is no significant difference between T cells
from sham-
castrated and castrated mice. In order to determine the functional potential
of peripheral T
cells in castrated mice after alto-HSCT, a series of in vitro assay were
performed. The
number of donor-derived T cells in castrated and sham-castrated controls 6
weeks after allo-
HSCT are represented in Fig. 32A. The proliferative capacity of the splenic T
cells was tested
in 2 ways: ocCD3/oiCD28 cross-linking (Fig. 32B) and in a 3'd party MLR (using
irradiated
BALB/C splenocytes as stimulators) (Fig. 32C). There is no significant
difference in the
proliferative capacity of peripheral T cells when comparing sham-castrated and
castrated
mice in either of these settings. Six weeks after alto-HSCT splenocytes were
cultured with
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irradiated BALB/C splenocytes (3rd party) for 5 days. Following 5 days of
allogeneic
stimulation the vast majority of cells in culture were CD8+ T cells. Half
these cells were used
in a CTL (SICr release) assay to determine the cytotoxicity of splenocytes
from sham-
castrated and castrated mice. Splenocytes were tested for their ability to
kill SICr loaded A20
(BALB/C B cell lymphoma tumour cell line) cells at different effector: target
ratios (Fig.
32D) . There was no significant difference between sham-castrated and
castrated mice with
respect to cytotoxicity. The other half of the cells cultured for 5 days were
restimulated
overnight with either 3rd party (BALB/C) or syngeneic (B lO.BR) irradiated
splenocytes and
Brefeldin A to determine IFN-y production. Fig. 32E shows IFNy production by
donor-
derived CD8+ splenic T cells following BALB/C primary stimulation and either
BALB/C or
B10.BR secondary stimulation (control). This is represented graphically in
Fig. 32F. There is
no significant difference in the proportion IFN-y producing donor-derived CD8+
when
comparing sham-castrated and castrated mice.
In order to assess immune function ira vivo a DTH assay was used whereby, 42
days
after castration and alto-BMT mice were sensitised with sRBCs. On day 46 days
they were
challenged and 24 and 48 hr later footpad swelling was determined. The DTH
response is
significantly enhanced 48 hrs after challenge when mice are castrated at the
time of allo-
HSCT compared to sham-castrated controls (Fig. 32G).
These functional assays demonstrate that the T cells in castrated recipients
are
comparable on a per cell basis with T cells from sham-castrated recipients and
are capable to
respond to novel antigens with intact proliferation, cytoxicity and cytokine
production.
However, the significantly more rapid T Bell reconstitution in castrated
recipients translates in
an enhanced DTM response even at 6 weeks after transplant.
VI. Castration prior to allogeneic-HSCT does not exacerbate GVHD and maintains
GVT activity. Both GVHD and GVT are mediated, primarily, by alloreactive donor-
derived
T cells, which are transferred with the allograft. Any treatment used to
enhance immune
reconstitution has the potential to exacerbate GVHD or, conversely, decrease
GVT activity.
To establish that castration does not have a stimulatory effect on
alloreactive T cells of donor
origin, GVHD was induced by the addition of allogeneic donor T cells to the
allograft. There
was no significant difference in morbidity or mortality due to GVHD when
comparing
castrated and sham-castrated mice (Fig. 33A). To assess the effects of
castration on GVT
activity the mastocytoma cell line P815 (H-2d) was injected into B6D2F1/J
recipients at the
time of transplant. Animals that died during the experiment were autopsied and
the cause of
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mortality (tumor vs. GVHD) was determined. Mortality due to mastocytoma
remained
unchanged following castration (six out of nine mice) when compared to sham-
castrated
controls (five out of eight mice). This suggests that castration does not
diminish GVT
response following HSCT (Fig. 33B).
VII. IL-7 and Castration have and additive effect following allogeneic HSCT.
It has
previously been shown that IL-7 treatment can increase the number of T and B
cells in
otherwise untreated animals and can also enhance lymphoid recovery following
cyclophosphamide treatment, irradiation, syngeneic and allogeneic HSCT
(Alpdogan et al.,
(2001) Blood 98:2256; Bolotin et al., (1996) Blood 88:1887; Faltynek et al.,
(1992) J. ~
Immunol. 149:1276; Morrissey et al., (1991) J. Immunol. 146:1547). IL-7 is
known to
increase T cell numbers through increased thymic activity as well as
peripheral expansion.
It was, therefore, decided to combine 1L-7 administration with castration in
recipients
of allogeneic HSCT. 14 days after treatment there are significantly more cells
in the thymi of
castrated mice and those given the combined treatment (castration and IL-7
administration).
At this early time point there is no difference seen between the PBS treated,
sham-castrated
controls and the IL-7 treated, sham-castrated mice. There is also no
significant difference
seen between the castrated group and those receiving the combined treatment
suggesting that
it is only the effects of castration acting 14 days after alto-HSCT, IL-7
treatment and
castration (Fig. 34A). At a later time point, 28 days after allo-HSCT, the
cellularity of the
thymi in both the castration alone group and the IL-7 alone group is
significantly higher that
the control group. The combination of IL-7 treatment and castration had an
additive effect on
thymic cellularity when analyzed 28 days after allogeneic HSCT (Figs. 34B).
VIII. Semi-Quantitative RT-PCR for IL-7, TGF-(31 and KGF reveals an increase
in
KGF and a decrease in TGF-(31 following alto-HSCT and castration. RT-PCR
analysis of
whole bone marrow cells revealed undetectable levels of IL-7 transcript in
both sham-
castrated and castrated mice as late as 6 weeks after alto-HSCT. When template
from control,
untransplanted mice was used IL-7 was detected (data not shown). TGF(31 and
KGF are
known to be key mediators of hematopoiesis. Using HPRT equibrated template
there appears
to be a decrease in TGF(3land an increase in KGF 2 weeks after castration and
allo-BMT
(Fig.34C).
IX. Changes that occur following castration were seen in KGF-~- mice but not
ILT~-
mice. In order to further study the possible mechanisms behind the enhanced
immune
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reconstitution following castration KGF-~- and IL7-~- mice (4-6 months old)
were castrated and
2 weeks later thymus, spleen and BM were analysed (TGF(31-~- mice die
prepubertally, Shull
et al. (1992) Natut-e 359:693). Thymic cellularity is significantly increased
when comparing
sham-castrated and castrated KGF-~- mice. Although no differences were seen in
the total
cellularity of the BM and spleen at this early time point, changes were seen
in the B cell
compartment of the BM, as seen previously in wildtype mice (Elks et al. (2001)
Irzt.
Immuzcol. 13:553; data not shown). Due to the fact that a large proportion of
cells in the thymi
of IL7-~- mice are CD45- stromal cells, enzymic digestion was used to obtain a
single cell
suspension when using these mice. By doing this many more cell are released
into suspension
which accounts for the slightly larger thymic cellularity seen in this
experiment compared to
previous literature (von Freeden-Jeffry et a1.(1995) J. Exp. Med. 181:1519).
No differences
were seen in the thymi (Fig. 34G), spleen (Fig. 34H) or BM (Fig. 34I) of IL7-~-
mice when
comparing castrated mice and sham-castrated controls. These finding suggest an
important
role for IL-7 in the enhanced immune reconstitution seen following castration
and allo-
HSCT.
Discussion
Recipients of an allogeneic HSCT experience a prolonged period of immune
deficiency, which is associated with life-threatening infections. With
increasing age of the
recipient, this infection risk increases, as does the time it takes for full
immunological
reconstitution. The period of immunodeficiency following HSCT can be greater
than one
year, and recent long-term studies demonstrated a decrease in TREC+CD4+ T
cells in older
HSCT patients compared to their donors (Storek et al., (2001) Blood 98:3505);
Lewin et al.,
(2002) Blood 100:2235). This suggests that thymic damage and the subsequent
decline in T
cell production may be more prolonged than once thought. The majority of post-
HSCT
infections are associated.with a lack of CD4+ peripheral T cells (Storek et
al., (1997) Am. J.
Hefzzatol. 54:131 ). Therefore, the increase in peripheral T cell number that
occurs following
castration may decrease the incidence of these infections leading to enhanced
overall survival
of transplant patients.
Many groups have focused their research on hematopoietic reconstitution
following
HSCT, and the most promising results have come with the use of haematopoietic
growth
factors and cytokines. G-CSF, for example, is used to mobilise donor stem
cells (Dreger et
al., (1993) Blood 81:1404). Noach et al., showed that pre-treatment with SCF
and IL-11 or
SCF and Flt-3 ligand resulted in enhanced donor cell engraftment ((2002) Blood
100:312).
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KGF appears to enhance engraftment and reconstitution in both syngeneic and
allogeneic
settings as well as ameliorating GVHD (Panoskaltsis-Mortari et al., (2000)
Blood 96:4350;
Krijanovski, et al., (1999) Blood 94:825). IL-7 enhances immune reconstitution
following
syngeneic HSCT (Bolotin et al., (1996) Blood 88:1887) and can enhance immune
reconstitution and maintain GVT activity without exacerbation of GVHD after
allogenic
HSCT (Alpdogan et al., (2001) Blood 98:2256).
Several studies have shown that sex steroid ablation, be it by surgical or
chemical
castration, of male mice increases both BM and splenic B cell numbers (Ellis
et a1.,(2001)
Int. Imr~zuhol. 13:553; Erben et al., (2001) Flor-f~2. Metab. Res. (2001)
33:491; Wilson et al.,
(1995) Blood 85:1535; Masuzawa et al., (1994) T. Cliff. Ifavest. 94:1090). The
increase in
peripheral B cell number is predominantly due to an increase in
B2201°CD24h' recent BM
emigrants (Ellis et al., (2001) Int. Irnmunol. 13:553). Olsen et al., have
demonstrated that
androgens enhance the production of TGF-[3 by stromal cells within the BM,
which in. turn
suppresses B cell development (J. Cli~c. luvest. (2001 ) 108:697). In
addition, neutralization
1.5 of TGF-(3 ih. vitro reverses B cell suppression by dihydrotestosterone.
TGF-~3 has also been
shown to down-regulate stromal IL-7 production and subsequently inhibit the
proliferation of
B cell progenitors (Tang et al., (1997) J. Immuyzol. 159:117). Therefore one
possible
explanation for the effects of castration/androgen ablation, in this instance,
following w
allogeneic HSCT, suppresses the production of TGF-(3, in turn enhancing B cell
development, explaining the increased B cell numbers in the BM and spleen of
castrated mice
compared to the sham-castrated controls.
The proliferation of hematopoietic stem cells is also regulated by TGF-(3.
Batard et
al., have demonstrated that physiological concentrations of TGF-(31 inhibit
the proliferation
and differentiation of HSCs fu vitro ((2000) J. Cell. Sci. 113:383-90).
Furthermore,
disruption of TGF-(3 signaling in HSCs (via the transient expression of a
mutant type II
receptor) enhances survival and proliferation of these cells (Fan et al.,
(2002) ,T. Im~iunol.
168:755-62). It is therefore possible that the increased number of HSCs seen
28 days after
allogeneic HSCT and castration may be due do a decrease in the production of
TGF-(3 by BM
stromal cells.
Both estrogen and androgen can effect the differentiation and proliferation of
HSCs
(Thurmond et al., Endocrihol., (2000) 141:2309-18; Medina et al., (2001) Nat.
Inaf~aunol.
2:718-24; Kouro et al., (2001) Blood 97:2708-15). Estrogen directly inhibits
the proliferation
and differentiation of HSC, as well as some lymphoid precursor subsets (Medina
et a1.,(2001)
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Nat. Immuhol. 2:718; I~ouro et al. (2001) Blood 97:2708). HSCs express
functional estrogen
receptors (ERs) and estrogen administration decreases the number of Liri c-
kit+Sca-1+ HSCs
(Thurmond et al., (2000) Efadocrinol. 141:2309; Kouro et al., Blood (2001)
97:2708). The
study conducted by Thurmond et al., suggests that the transition between c-
kit+Sca-1+
precursors and the more mature subsets (c-kit+Sca-1- and c-kit Sca-1-is
blocked when ERoc is
present in the hematopoietic cells of the BM (Endocrinol. (2000) 141:2309).
ERs are also
present on BM stromal cells (Girasole et al., (1992) J. Cliya. Invest. 89:883;
Smithson et al.,
(1995) J. Im~~uhol. 155:3409) suggesting that estrogen may also have an effect
on the
production of growth factors by the stroma, which in turn affects HSC
proliferation and/or
differentiation. Although most evidence suggests an indirect effect of
androgens on HSCs via
the BM stroma, the presence of functional androgen receptors on lymphoid
components of
the BM does not exclude a direct effect.
Olsen et al. have shown that it is the presence of a functional androgen
receptor on the
thymic epithelium but not the thymocytes that is essential for age-related
thymic involution
and the subsequent regeneration via sex steroid ablation Olsen et al., (2001)
Eudocrifaol.
142:1278).
Although the molecular mechanisms for thymic involution and/or regeneration
remain
unknown there are several potential candidates. Thymic IL-7 levels decline
with age
(Aspinall, et al., (2000) Uaccif2e 18:1629; Andrew et al., (2002) Exp.
Gerohtol. 37:455;
Ortman et al., (2002) lut. Immurcol. 14:813). It remains unclear as to whether
this is due to a
decrease in the number of cells that produce IL-7 or a decrease in the ability
of the existing
cells to produce the cytokine. However, IL-7 treatment of old mice can reverse
age-related
increases in thynuc apoptosis and enhance thymopoiesis (Andrew et al., (2001)
J. ImmuiZOl.
166:1524). Stem cell factor (SCF) and M-CSF mRNA expression is also decreased
in the
mouse thymus with age (Andrew et al., (2002) Exp. Gerofztol. 37:455). At the
intracellular
signaling level, E2A a transcription factor essential for the development of
DN thymocytes is
decreased, as is Foxnl (whn) a transcription factor present in and involved in
the proliferation
and differentiation of thymic epithelial cells (Ortman et al., (2002) Ifit.
Immunol. 14:813).
Sempowski et al., have monitored mRNA steady-state levels in ageing humans and
shown a
significant increase in Leukemia Inhibitory Factor (LIF), Oncostatin M, IL-6
and SCF mRNA
(J. hrzf~auriol. (2000) 164: 2180).
The above studies suggest that the response to castration is multifactorial.
The
experiments with castration of IL7-/- mice suggest that increased production
of 1L-7 is an
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important component of the castration effect. However, an additive effect on
thymic
cellularity was observed when recipients were treated with both high dose IL-7
and
castration, which would suggest that castration provides more thymopoietic
effects than
increased IL,-7 levels alone.
DC are the key mediators of negative selection in the thymus (Jenkinson et
al., (1985)
Trarcsplantat. 39:331; Matzinger et al., (1989) Nature 338:74-60) and in a
transient setting
have been implicated in inducing graft acceptance by presenting alloantigens
in the thymus
after transplantation, removing donor-specific T cells. Tomita et al. have
demonstrated that
donor-derived cells in the thymii of MHC class I mis-matched recipients
mediate deletion of
donor reactive cells (Tomita et al., (1994) J. Imnzuuol. 153:1087-1098). They
have also
shown that thymus-derived DC injected intravenously traffic to the host
thymus.
Furthermore, it has been shown that intrathymic injection of both host cells
pulsed with allo-
antigen, donor cells, or donor soluble peptides increases graft acceptance
(Gaa.~rovillo et al.,
(1999) Transplantatiofi 68:1827; Ali et al., (2000) Transplantation 69:221;
Garrovillo Qt al.,
(2001) Am. ,T. Tr~ansplaht. 1:129; Oluwole et al., (1993) Trahsplautataorc
56:1523-1527).
These studies show that castration significantly increased the number of host
and donor-
derived DC in the thymus following allogeneic HSCT. That is, sex steroid
ablation enhances
the differentiation andlor proliferation of thymic DC. Thus, castration, used
in conjunction
with hematopoietic stem cells and solid organ transplantation, increases graft
acceptance.
Conclusion:
The current study has revealed that blocking sex steroids has a profound
positive
effect on immune reconstitution following myeloablation and HSCT. HSCs, and B
and T
progenitors are markedly enhanced. This provides an important platform for
increasing the
efficiency of engraftment and post-transplant strategies that depend on an
intact
hematopoietic system, such as vaccination against tumor or microbial antigens
or gene
therapy targeting donor HSCT. The increase in DC in the thymus have importance
in
inducing and sustaining tolerance to allogeneic grafts. In addition, GVHD is
not exacerbated
and GVT activity is not diminished in castrated recipients. These results
demonstrate that
transient sex steroid ablation (using, e.g., LHRH analogs) are useful as a
prophylactic therapy
to enhance immune reconstitution.
EXAMPLE 20
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SEX STEROID ABLATION ENHANCES TCR-SPECIFIC STIMULATION
FOLLOWING HEMATOPOIETIC STEM CELL TRANSPLANTATION
In order to assess the functional nature of regenerated T cells, patients were
analyzed
for responsiveness to TCR specific stimulation.
Materials and Methods:
Patients. Test patients were given Zoladex (LHRH-A) 3-weeks prior to stem cell
transplantation and then monthly injections for 4-months. All patients were
analyzed pre-
treatment, weekly for 5-weeks after transplantation and monthly up to 12
months. Ethics
approval was obtained from The Alfred Committee for Ethical Research on Humans
(Trial
Number 011006).
Preparation of PBMC. Purified lymphocytes were used for T-cell stimulation.
assays and TREC analysis nd were prepared as above.
T Lymphocyte Stimulation Assay. Analysis of TCR specific stimulation was
performed using anti-CD3 and anti-CD28 cross-linking from 1-12 months post-
transplant,.
unless otherwise indicated. For TCR-specific stimulation, cells were incubated
for 48 hours
on plates previously coated with purified anti-CD3 (1-10 pg/ml) and anti-CD28
(10 ~g/mlj.
Following plaque formation (48-72 hours), 1 p,Ci of 3H-Thymidine was added to
each well
and plates incubated for a further 16-24 hours. Plates were harvested onto
titter mats ana
incorporation of 3H-thymidine was determined using liquid scintillation on a
(3-counter
(Packard-Coulter, USA).
w I. LHRH-A administration enhances responsiveness to TCR specific stimulation
following allogeneic stem cell transplantation. LHRH-A treated patients showed
enhanced
proliferative responses (assessed by 3H-thymidine incorporation) compared to
control
patients at all time-points except 6 and 9 months due to low patient numbers
analyzed at this
time; (Figs. 57A-B). In allogeneic transplant patients treated with a LHRH-A,
a significant
increase in responsiveness to anti-CD3/CD28 stimulation was observed at 4 and
5-months
post-transplant compared to control patients. While control patients showed an
enhanced
response at both 6 and 9 months post-transplant, LHRH-A treated patients
showed a greater
responsiveness at 12-months post-transplant. At 6 and 9 months post-transplant
control
patients had similar responsiveness to pre-treatment values. However at all
other time-points,
they were considerably lower. In contrast, LHRH-A treated patients had
equivalent
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responsiveness at all time-points except 6 months compared to pre-treatment.
LHRH-A
treated patients showed enhanced ~ proliferative responses (assessed by 3H-
Thymidine
incorporation) compared to control patients at l, 3 and 4 months post-
transplant. This
indicates a contribution of direct peripheral T cell effects, as new CD4+ T
cells are not
evident until at least 1-2 months post-transplant (Fig. 57A-B).
II. LHRH-A administration enhances responsiveness to TCR specific stimulation
following autologous stem cell transplantation. A similar response as that
seen in allograft
recipients was also observed with autograft recipients (Fig. 57C). Those
patients treated with
a LHRH-A demonstrating an enhanced proliferative response to TCR stimulation
at both 4
and 9 months post-transplant. LHRH-A treated patients showed enhanced
proliferative
responses (assessed by 3H-thymidine incorporation) compared to control
patients at all time-
points except 5 months. Restoration to pre-treatment values was observed by 12
months post-
transplant in both control and LHRH-A treated patients.
III. It.HRH-A administration enhances responsiveness to TCR specific
stimulation
following treatment for chronic cancer sufferers.
Patients with chronic haematological malignancies who were Immunosuppressed .
patients as determined by documented serious infection associated with: CD4 <
0.4 x 109/L;
or lymphoproliferative disorder (e.g., CLL, myeloma, lymphoma) and receiving
regular
prophylactic intravenous gammaglobulin (documented hypogarnmaglobulinemia); or
previous treatment with fludarabine, deoxycorfomycin and 2-CdA within 4 year ;
or prior
allogeneic or autologous stem cell transplant within 2 years were enrolled in
the study: Ethics
approval was obtained from The Alfred Committee for Ethical Research on Humans
(Trial
number 01/006). Patents were given LHRH-A as follows and results are presented
up to 6-
weeks post-LHRH-A administration.
d -1 (or before): consent signed and' pre-treatment investigations performed
d0: Zoladex administered (males 10.8mg; females 3.6mg)
d+28: females-Zoladex 3.6mg administered
d+56: females-Zoladex 3.6mg administered
d+84: females and males-Zoladex 3.6mg adnunistered
Analysis of TCR specific stimulation was performed using anti-CD3 and anti-
CD28
cross-linking from D7 post-administration. LHRH-A treated patients showed
enhanced
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proliferative responses (assessed by ~H-Thymidine incorporation) compared to
pre-treatment
levels in a "cyclical" fashion (Fig. 63). That is, on the day of injection, an
increase in T cell
proliferation was observed and this appeared to decrease slightly prior to the
subsequent
injection. This also indicates the probability of a direct influence of LHRH-A
on existing
peripheral T cells. This reflected the administration of the agonist with
monthly depot
injections. These results indicate an influence directly on peripheral T
cells. However, the
enhanced response seen at 12-months post-treatment reflect changes in thymic-
derived T
cells as well, since agonist administration was ceased from 4-months for all
patients.
Conclusion:
Since an increased responsiveness to TCR-specific stimulation was observed as
early
as D35 post-transplantation, this is due predominantly to pre-existing T
cells, since newly
derived T cells only just begin to exit the thymus at this stage. The
influence of newly
derived thymic-generated T cells is only observed after this time-point, so it
can be concluded
that the increased T cell function is due to an improvement in the pre-
exisiting T cells and not
T cells derived from the regenerated thymus.
EXAMPLE 21
SEX STEROID ABLATION ENHANCES MITOGENIC STIMULATION
FOLLOWING HEMATOPOIETIC STEM CELL TRANSPLANTATION
In order to assess the functional nature of regenerated T cells, patients were
analyzed
for responsiveness to mitogenic stimulation.
Materials and Methods:
Patients. Patients were those enrolled in Clinical Trial protocol No. 01/006
as above.
Prior to stem cell transplant, patients were given LHRH-A (3-weeks prior).
Patients who did
not receive the agonist were used as control patients.
Preparation of PBMC. Purified lymphocytes were used for T-cell stimulation
assays and TREC analysis and were prepared as above.
Mitogen Stimulation Assay. Analysis of mitogenic responsiveness was performed
using pokeweed mitogen (PWM) and tetanus toxoid (TT) from 1-12 months post-
transplant.
For mitogen stimulation, PBMC were plated out in 96-well round-bottom plates
at a
concentration of 1 x lOs/well in 100 g,1 of RPMI-FCS. Cells were incubated at
37°C, 5% COZ
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with TT (2LFAU/ml) or PWM (10 ug/ml) . Following plaque formation (48-72
hours), 1 ~,Ci
of 3H-thymidine was added to each well and plates incubated for a further 16-
24 hours.
Plates were harvested onto filter mats and incorporation of 3H-thymidine was
determined
using liquid scintillation on a (3-counter (Packard-Coulter, USA).
I. LHRH-A administration enhances responsiveness to mitogenic stimulation
following allogeneic stem cell transplantation.
Analysis of mitogen responsiveness showed that allogeneic patients undergoing
LHRH-A treatment had an increased responsiveness to PWM at all time-points
post-
transplant compared to controls (Fig. 58A). That is, patients treated with
LHRH-A prior to
stem cell transplantation showed an enhanced responsiveness to PWM stimulation
at all time-
points studied compared to control patients.
Similar results were evident following analysis of response to TT (Fig. 58B).
LHRH-
A treated patients had , enhanced responses at all time-points compared to
control patients
except at 12 months post-transplantation.
II. LHRH-A administration enhances responsiveness to mitogenic stimulation
following autologous stem cell transplantation. Patients treated with LHRH-A
prior to
stem cell transplantation showed an enhanced responsiveness to PWM stimulation
at the
majority of time-points studied compared to control patients (p<_0.001 at 3
months) (Fig.
59A). By 12-months post-transplantation, LHRH-A treated patients had restored
responsiveness to pre-treatment levels while control patients were still
considerably reduced.
Also, similar to allograft patients above (Fig. 58B), autograft patients also
showed an
increased response to TT when given LHRH-A prior to treatment Patients treated
with
LHRH-A prior to stem cell transplantation showed an enhanced responsiveness to
TT
stimulation at the majority of time-points studied compared to control
patients (Fig. 59B). By
12-months post-transplantation, LHRH-A treated patients had restored
responsiveness to pre-
treatment levels while control patients were still considerably reduced.
l~nnn~noinn~
Since an increased responsiveness to mitogenic stimulation was observed as
early as
D35 post-transplantation, this is due predominantly to pre-existing T cells
since newly-
derived T cells are only just beginning to exit the thymus at this stage. The
influence of
newly-derived thymic-generated T cells is observed only after this time-point.
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EXAMPLE 22
SEX STEROID ABLATION ENHANCES RATE OF ENGRAFTMENT IN
HEMATOPOIETIC STEM CELL TRANSPLANT PATIENTS
Materials and Methods:
Materials and methods used for these experiments are described above.
Additional
materials and methods are as follows:
Allogeneic and autologous patients (control or LHRH-A treated) were analyzed
for
total WBC and total granulocyte or neutrophil numbers following HSCT. Three
weeks prior
to-HSCT, patients were treated with LHRH-A. Patients who did not receive the
agonist were
used as control patients. Total white blood cell (WBC) counts, granulocyte (G)
and
neutrophil counts per ~,1 of blood were determined up to 35 days post
transplant. A sample of
whole peripheral blood was analyzed either using a Cell-Dyn 1200 automated
cell counter '
(Abbott) or hemocytometer counts done in duplicate. This allows calculation of
total whiie
blood cells, lymphocytes and granulocyte numbers following transplant.
Analysis of
engraftment was performed from D14-D35 post-transplant.
Results:
I. Autologous Stem Cell Transplant patients undergoing LHRH-A treatment prior
to transplant enhance rate of engraftment. Total white blood cell (WBC) counts
and
granulocyte (G) counts per ~,1 of blood were determined at days 14, 28, and 35
post
transplant. As shown in Figs. 60A-D, autologous patients who were given LHRH-A
treatment showed a significantly higher number of WBC at D14 post-transplant
compared to
controls (Figs. 60B) (p<_0.05), with 87% showing granulocyte engraftment
(>_500 cells/p,l
blood) compared to 45% of controls (p<_0.05) at this time point. Autologous
patients who
were given LHRH-A treatment also showed a significantly higher number of
neutrophils at
D10-12 post-transplant compared to controls (Fig. 60C; data is expressed as
mean~1SEM of
8-20 patients. *= p_<0.05). In addition, although not significant, autologous
patients had
higher lymphocyte counts throughout the time-points analyzed in LHRH-A treated
compared
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to control group (Fig. 60D). This indicates that~iI,HRH-A therapy
significantly increases
lymphocyte levels following stem cell transplantation.
II. Allogeneic Stem Cell Transplant patients undergoing LHRH-A treatment prior
to transplant enhance rate of engraftment. As shown in Figs. 61A, C and D,
allogeneic
patients who were given LHRH-A treatment showed a significantly higher number
of WBC
at D14 post-transplant compared to controls (Figs. 61A) (p<_0.05) with 64%
showing
granulocyte engraftment (>_500 cells/~l blood) compared to 44% of controls at
this time point.
In addition, allogeneic patients who were given LHRH-A treatment showed a
significantly
higher number of neutrophils at D9, 12 and 19 post-transplantation compared to
controls
(Fig. 61C; data is expressed as mean~1SEM of 8-20 patients. *= p<_0.05).
Additionally,
analysis of patients undergoing peripheral blood stem cell transplantation
demonstrated a
significant increase in lymphocyte counts when treated with an LHRH-A prior to
allogeneic
transplantation (p<0.05 at days 10, 12, 13 and 17-21 post-transplantation)
(Fig. 61D).
Conclusion:
In both the allogeneic and autologous transplant models, a significant
increase in
WBC and granulocyte numbers at D14 post-transplant was observed with LHRIi-A
treated
patients compared to controls (Figs. 60 and 61). This enhanced rate of
engraftment is cnicial
for the overall patient morbidity with neutropenia (<_200 neutrophils/ml
blood) indicative of
increased infection rates. As such, an early recovery of WBC and granulocyte
numbers
demonstrates a better survival rate for LHRH-A treated patients. Inhibition of
sex steroids
enhances engraftment and reconstitution prior to full thymic regeneration or
the release of
new T cells as a result of. full thymic regeneration.
EXAMPLE 23
SEX STEROID ABLATION INCREASES T CELL PROLIFERATIVE RESPONSES
WITHIN ONE WEEK
These studies were conducted to determine if sex steroid ablation was capable
of
enhancing proliferative responses as early as 3 to 7 days following castration
in mice.
Materials and Methods:
Eight week-old mice were castrated and analyzed for anti-CD3/anti-CD28
stimulated
T cell proliferative response 3 days (Figs. 62A, C, and E) and 7 days (Figs.
62B, D, and F)
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after surgery. Peripheral (cervical, axillary, brachial and inguinal) lymph
node (Figs. 62A
and B), mesenteric lymph node (Figs. 62C and D), and spleen cells (Figs. 62E
and F) were
stimulated with varying concentrations of anti-CD3 and co-stimulated with anti-
CD28 at a
constant concentration of 10 pg/ml for 48 hours. Cells were then pulsed with
tritiated
thymidine for 18 hours and proliferation was measured as 3H-T incorporation.
Control puce
were sham-castrated, n=4, ~p<0.05 (non-parametric, unpaired, Mann-Whitney
statistical test).
Results:
Sex steroid ablated mice show enhanced CD28/CD3-stimulated T cell
proliferation at
3 days (Figs. 62A,C, and E) and 7 days (Figs. 62 B,D, and F) post-castration.
T cells isolated
from the peripheral LN showed a significant increase in proliferative
responses at 3 days (10
~.g/ml anti CD-3) and 7 days (2.5 p.g/ml and 1.25 p,g/ml anti-CD3) post-
castration.
Additionally, T cells isolated from the mesenteric lymph nodes (Figs. 62C and
D) and
spleen (Figs. 62E and F) also showed a significant increase in anti-CD3-
stimulated
proliferation over sham-castrated mice at 3 days post-castration.
Conclusion:
As early as 3-7 days post castration (prior to new T cells migrating from the
thymus),
there is an increase in responsiveness of T cells to stimulation with anti-CD3
and CD28
cross-linking. These data indicate that sex steroid ablation has direct
effects on the
functionality of the peripheral T cell pool, prior to thymic reactivation.
To determine the extent of direct effects of LHRH-A on peripheral T cells in
human
patients, studies are conducted. Control patient T cells are incubated with
various loses of
LHRH-A and are analyzed at varying time-points (D3, D7 and D14) for the level
of
proliferation compared to control (media alone) samples. This allows the
determination of
whether the LHRH-A acts directly on the existing T cells by causing their
activation, as was
observed in the mouse model.
EXAMPLE 24
SEX STEROID ABLATION ENHANCES HAEMOPOIESIS
FOLLOWING CONGENIC HSCT
Results:
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I. Castration enhances engraftment in the BM, thymus, and spleen following
HSCT. Mice were castrated 1 day before congenic HSCT. 5x106 Ly5.1+ BM cells
were
injected intravenously into irradiated (800 rads) C57BL6 mice. The BM, spleen
and thymus
were analyzed by flow cytometry at various time points (2-6 weeks) post-
transplant (Fig. 35).
As shown in Fig. 358, two weeks after castration and HSCT, there are
significantly more
cells in the BM of castrated mice as compared to sham-castrated controls.
Similarly, as
shown in Fig. 35C, there is a significant increase in thymic cell number 2, 4
and 6 weeks
post-transplant as compared to sham castrated controls. As shown in Fig. 35C,
in the
periphery, splenic cell numbers are also significantly higher than controls 4
and 6 weeks post-
transplant in the castrated recipients.
II. Castration enhances engraftment of HSC in the BM following congenic HSCT.
Mice were castrated 1 day before congenic HSCT. 5x106 Ly5.1+ BM cells were
injected
intravenously into irradiated (800 rads) C57BL6 mice. The BM was analyzed for
lin-c-
kit+sca-1+ HSC by flow cytometry at two weeks post-transplant (Fig. 36). Two
weeks after
BMT transplantation and castration there are significantly more donor-derived
HSCs in the
BM of castrated mice compared to sham castrated controls.
III. Castration enhances engraftment of HSC in the BM following congenic HSCT
(2.5x106 cells). Mice were castrated 1 day before congenic HSCT. 2.5x106
Ly5.1+ BM cells
were injected intravenously into irradiated (800 rads) C57BL6 mice. The BM was
analyzed
for lin-c-kit+sca-1+ HSC by flow cytometry at two weeks post-transplant (Fig.
37A-B). Fig.
37A depicts percent of conunon lymphoid precursors in the BM. Fig. 37B depicts
the
number of common lymphoid precursors in the BM. Two weeks after BMT
transplantation
and castration there is a significantly increased proportion of donor-derived
HSCs in the BM
of castrated mice compared to sham castrated controls.
IV. Castration enhances engraftment of HSC in the BM following congenic HSCT
(5x106 cells). 5x106 Ly5.1+ BM cells were injected intravenously into
irradiated (800 rads)
C57BL6 mice. The BM was analyzed for lin-c-kit+sca-1+ HSC by flow cytometry at
two
weeks post-transplant (Fig. 37C-D). Fig. 80A depicts percent of common
lymphoid
precursors in the BM. Fig. 37D depicts the number of common lymphoid
precursors in the
BM. Two weeks after BMT transplantation and castration there is a
significantly increased
proportion of donor-derived HSCs in the BM of castrated mice compared to sham
castrated
controls.
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v. Castration enhances the rate of engraftment of donor-derived DC in the
thymus
following congenie HSCT. 5x106 LyS.l+ BM cells were injected intravenously
into
irradiated (800 rads) C571BL6 mice. Thymocytes were a~lalyzed by flow
cytometry at two
weeks post-transplant. Donor-derived DC were defined as CD45.1+CDllc+MHC class
II+
+or-
CDl 1b . Donor-derived CDllb+ and CDl 1b- DC are significantly increased in
the thymii
of castrated mice compared to sham-castrated controls 2 weeks after BMT (Fig.
38).
~JI. Castration enhances the rate of engraftment of donor-derived B cells in
the
spleen following congenic HSCT. 5x106 Ly5.1+ BM cells were injected
intravenously into
irradiated (800 rads) C57IBL6 mice. Splenocytes were analyzed by flow
cytometry at two
weeks post-transplant. There are significantly more B220~ B cells in the
spleens of castrated
mice, as compared the sham-castrated controls, 2 weeks after congenics BMT
(Fig. 39).
EXAMPLE 25
TREATMENT OF CANCER PATIENTS WITH G-CSF AND/OR GM-CSF
DECREASES THE INCIDENCE OF NEUTROPENIA FOLLOWING
CHEMOTHERAPY
This example illustrates the use of G-CSF and/or G.M-CSF' for the increase in
neutrophil levels and decrease of incidence of infection in patients receiving
chemotherapy
Materials anti Method s:
A randomized double blind, placebo controlled study is conducted in 100
patients
with small cell lung cancer.
G-CSF administration. Neupogen0 (Amgen, Thousand Oaks, CA) is administered
at a dosage of 4-8 ~,g/kg/day s.c. from days 4-17 following chemotherapy
according to
manufacturer's instructions.
GM-CSF administration. In a first study, patients receive a single SC dose of
6 mg
of Neulasta" (Amgen, Thousand Oaks, CA) on day 2 of each chemotherapy cycle or
Filgrastim0 (Amgen, Thousand Oaks, CA) at 5 p,g/kg/day SC beginning on day 2
of each
cycle according to manufacturer's instructions. In a second study, subjects
were randomized
to receive a single SC injection of Neulasta° at 100 ~.g/kg on day 2 or
Filgrastim0 at 5
~,g/kg/day SC beginning on day 2 of each cycle of chemotherapy.
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Patients. All patients had been diagnosed with small cell lung cancer and were
treated with standard cycles of cyclophosphamide, doxorubicin and etoposide.
GM-CSF
patients are diagnosed with metastatic breast cancer.
Results:
Treatment with G-CSF (Neupogen0) in this manner resulted in a clinically and
statistically significant decrease in the incidence of infection as measured
by febrile
neutropenia, infection rates, in-patient hospitalization and antibiotic use.
Numerous other
Phase I/II trials reported by this company (www.neupogen.com) report that the
use of G-CSF
resulted in measurable increases in neutrophils, thereby supporting the
clinical use of G-CSF
to treat cancer patients receiving immunosuppressive chemotherapy.
Similar results were seen using GM-CSF (Neulasta0) in a randomized, double-
blind
active control study (using Neupogen~), employing doxorubicin 60 mg/m2 and
docetaxol 75
mg/m2administered every 21 days for up to 4 cycles in the treatment of
metastatic breast
cancer (www.neulasta.com).
15' Duration of neutropenia was chosen as the primary endpoint (obviously with
FDA
approval). . Patients that did not receive Neulasta~ had a 100% incidence of
severe
Neutropenia with a mean duration of 5-7 days and a 30-40% incidence of febrile
Neutropenia.
In both studies the patients were administered Neulasta~ on Day 2 (cf.
Neupogen~
where the drug was administered on day 4).
Both studies (one was fixed dose, the other a weight-adjusted dose for
Neulasta0)
demonstrated no difference in either drug at the primary endpoint of mean days
of severe
Neutropenia. W both cases this being approximately 1.7 days (cf. 5-7 days with
no therapy).
The rates of febrile Neutropenia were comparable for both studies at approx.
10-20%.
EXAMPLE 26
TREATMENT OF CANCER PATIENTS WITH SEX STEROID ABLATION
THERAPY AND G-CSF (AND/OR GM-CSF) DECREASES THE INCIDENCE OF
NEUTROPENIA AND INFECTION RATES FOLLOWING CHEMOTHERAPY
In a randomized double blind study, 80 patients with small cell lung cancer
are
randomized into Groups 1-4 that receive G-CSF (Neupogen0) or receive G-CSF and
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Lupron~. All patients in all groups are monitored daily for the first 4 days
of the
chemotherapy cycle and thereafter every third day. All patients are monitored
for CBC,
neutrophil count, haematocrit and differential T cell analysis using
techniques well known to
those skilled in the art. Additionally, all patients are monitored twice daily
for temperature
and for any side effects.
Group #1 - G-CSF only
The first group consists of a group of patients (n=20) that receive G-CSF on
days 4-17
following standard chemotherapy with cyclophosphamide (1 g/m2/day),
doxorubicin (50
mg/m2/day) and etoposide (120 mg/m2/day X 3). All drugs are administered
intravenously
(LV.) in accordance with the administration and dosage instructions in the
relevant package
inserts. All patients receive this therapeutic regimen in 21 day cycles. In
this group the dose
of G-CSF is 5 p,g/kg/day delivered subcutaneously (S.C.) in accordance with
the dosage
instructions set out on the package insert for Neupogen~ (optional range of 0-
10 p,g/kglday).
Subsequent chemotherapy cycles are optionally given according to the duration
and severity
of ANC nadir. In accordance with conventional oncology techniques, treating
clinicians are
also optionally able to decrease the doses used if the ANC increased towards
10,000/mm3, at
which point G-CSF is discontinued.
Group #2 - G-CSF plus "high dose" Lupron~, 21 days pre-chemotherapy
The second group consists of a group of patients (n=20) that are given a "high
dose"
of Lupron~ (3 month sustained release product, dose of. 22.5 mg, S.C.) twenty-
one days
prior to chemotherapy and are also administered G-CSF (5 p.g/kg/day) (optional
range of 0-
10 pg/kg/day) in accordance with the aforementioned protocol from days 4-17
following
chemotherapy with cyclophosphamide (1 g/m2/day), doxorubicin (50 mg/m2/day)
and
etoposide ~ (120 mg/m2/day X 3). All patients receive this therapeutic regimen
in 21 day
cycles. Subsequent chemotherapy cycles are optionally given according to the
duration and
severity of ANC nadir. In accordance with conventional oncology techniques,
treating
clinicians are also optionally able to decrease the doses used if the ANC
increased towards
10,000/mma, at which point G-CSF is discontinued. All drugs are administered
in
accordance with the dosage and administration instructions set out in the
respective package
inserts.
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Group #3 - G-CSF plus "low dose" Lupron~ 21 days pre-chemotherapy
The third group consists of a group of patients (n=20) that are give a "low
dose" of
Lupron0 (3 month sustained release product, dose of 11.25 mg, S.C.) twenty-one
days prior
to chemotherapy and were adnunistered G-CSF (5 ~,g/kglday) from days 4-17
following
chemotherapy with cyclophosphamide (1 g/m2/day), doxorubicin (50 mg/m2/day)
and
etoposide (120 mg/mz/day X 3). All patients receive this therapeutic regimen
in 21 day
cycles. Subsequent chemotherapy cycles are optionally given according to the
duration and
severity of ANC nadir. In accordance with conventional oncology techniques,
treating
clinicians are also optionally able to decrease the doses used if the ANC
increased towards
10,000/mm3, at which point G-CSF is discontinued. The chemotherapy drugs, the
G-CSF
and Lupron0 are administered in accordance with the package inserts for those
drugs.
Group #4 - G-CSF plus "high dose' Lupron~ 14 days pre-chemotherapy
The fourth group consists of a group of patients (n=20) that are administered
a ".b.igh
dose" of Lupron~ (3 month sustained release product, dose of 22.5 mg, S.C.) 14
days prior to
the commencement of chemotherapy. These patients are administered G-CSF (5
~.g/kglda.y)
from days 4-17 following chemotherapy with cyclophosphamide (1 g/m2/day),
doxorubicin
(50 mg/m2/day) and etoposide (120 mg/m2/day X 3). All patients received this
therapeutic ,
regimen in 21 day cycles. Subsequent chemotherapy cycles are optionally given
according to ;:
the duration and severity of ANC nadir. In accordance with conventional
oncology
techniques, treating clinicians are also optionally able to decrease the doses
used if the ANC
increased towards 10,000/mm3, at which point G-CSF is discontinued. The
chemotherapy
drugs, the G-CSF and Lupron~ are administered in accordance with the package
inserts for
those drugs.
Results:
It is expected that treatment with both G-CSF and "high dose" Lupron~ (Group
2)
will result is a clinically and statistically significant reduction in the
incidence of infection--as
manifested by infection rates, antibiotic use, WBC counts, mean days of severe
neutropenia
(ANC <500/mm3) and febrile neutropenia)--as compared to the G-CSF treated
group (Group
1). It is also expected that treatment with G-CSF and the "low dose" Lupron0
(Group 3) will
produce results that are not statistically different from those patients
treated with the higher
Lupron~ dosage (Group 2). Finally, it is expected that the administration of
Lupron0 on day
14 pre-chemotherapy (Group 4) at the 3 month/sustained release, 22.5 mg S.C.
dosage, will
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produce data that are not statistically different from those patients in Group
2. There are
expected to be, however, a number of data points that indicate that
administration of
Lupron~ at an earlier stage of the treatment cycle may be of benefit to some
patients.
EXAMPLE 27
TREATMENT OF BONE MARROW TRANSPLANTATION PATIENTS WITH SEX
STEROID ABLATION THERAPY REDUCES WBC COUNTS. SEVERE
NEUTROPENIA, FEBRILE NEUTROPENIA, AND INFECTION RATES
In a randomized unblinded study 20 patients are treated with a GnRH analog
prior to
receiving a BMT. This patient group is to a control group (n=19) who also
undergo a BMT,
but will not receive a GnRH analog.
All patients receive an allogeneic graft from a matched donor in - accordance
with
accepted medical techniques (Lincz et al., (2001) Leuk. afzd Lyni~h. 40: 373).
All patients in the treated group received four (4) monthly doses of a GnRH
analog
(Lupron ° 7.5 mg, S.C.). Lupron~ is initially administered 21 days
prior to the BMT.
Prior to transplant, the BM from all patients is ablated using standard
ablating dnags
and techniques Segeren et al., (1999). Br. J. Haem. 105: 127-130.
Blood samples from each patient are drawn at days -21, ~-2, 0, 1, 2, 3, 7, 10,
14 and 21.
The blood samples are then evaluated for WBC, neutrophils, T cells (by ~FACS
analysis, as
described in detail above), granulocytes levels, and hematocrit, using the
methods described
in detail elsewhere herein, as well as methods well known to those skilled in
the art.
Additionally, all patients are monitored for infection rates (including viral
infection),
the use of antibiotics, febrile neutropenia, and mean days of severe
neutropenia (ANC<0.5 x
109/L). These analysis are performed as outlined above, with the patients
being followed up
at 1 month, 3 months, 6 months and 9 months from the date of transplant.
Results:
It is expected that up to day 21 from transplantation, treatment with GnRH
agonist
(Lupron0) will result in a clinically and statistically significant reduction
in WBC counts,
mean days of severe Neutropenia (ANC <500/mm3) and febrile neutropenia, as
compared to
the non-GnRH agonist treated group.
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It is also expected that, due to the small number of patients, the incidence
of viral and
other infection rates will not be statistically significant between the two
groups up to month
1. However, it is expected that after 3, 6 and 9 months from transplant, these
infection rates
will be statistically different between the 2 groups, wherein the GnRH agonist
treated patients
will be performing better (i. e., more easily managed and recover faster).
EXAMPLE 28
TREATMENT OF CANCER PATIENTS WITH SEX STEROID ABLATION
THERAPY INCREASES HAEMOPOIESIS AND NEUTROPHIL COUNTS
In a randomized unblinded study, male patients (n=30) with metastatic prostate
cancer
receive, in the normal course of their treatment, a GnRH agonist (Lupron
Depot~ injection
(7.5 mg s.c. monthly)). All patients also received an anti-androgen drug.
Cosudex~ may
also be given (5 mg/day or 50 mg/day orally) for 1 month.
All patients undergo a Full Blood Analysis (FBA) prior to the commencement of
therapy. FBA involves standard pathology analysis of lymphocytes, white blood
cells,
hematocrit and red blood cell content. In addition, analysis of immunological
parameters
including T cell stimulation, cytokine production, immune cell subsets and
TREC analysis
are performed.
Additionally, blood samples from. each patient are drawn at days -2, 0, 1, 2,
3, 7, 10,
14, 21, and 28, and monthly thereafter for 6 months. The.blood samples are
then .evaluated
for WBC, neutrophils, T cells (by FACS analysis, as described in detail
above), granulocytes
levels, arid hematocrit, using the methods described in detail elsewhere
herein, as well as
methods well known to those skilled in the art.
It is expected that the majority of patients (approximately 70%) will have an
increase
in haemopoiesis (as measured by T cell analysis) and neutrophil count, as
compared to the
baseline levels (days -2 and 0), within the first 14 days of GnRH agonist
therapy.
EXAMPLE 29
TREATMENT OF PATIENTS WITH SEX STEROID ABLATION THERAPY
INCREASES INFLUENZA VACCINATION EFFICACY
In a randomized unblinded study male patients (n=45) with metastatic prostate
cancer
received in the normal course of their treatment a GnRH agonist (Lupron DepotO
injection
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(7.5 mg s.c. monthly)). All patients also received an anti-androgen drug.
Cosudex~ may
also be given (5 mglday or 50 mglday orally) for 1 month.
All patients underwent a Full Blood Analysis (FBA) prior to the commencement
of
therapy.
The patients that receive the GnRH agonist and Cosudex~ are randomly assigned
into
one of three groups of 15 patients per group as follows:
Group #1
The first group consists of 15 patients that receives an influenza vaccination
(e.g.,
Fluarix0 (GlaxoSmithI~line, Australia), 0.5 ml, i.m.) at Day 0, according to
manufacturer's
instructions.
Group #2
The second group consists of 15 patients that receive an influenza vaccination
(e.g.,
Fluarix~ (GlaxoSmithKline, Australia), 0.5 ml, i.m.) at Day 21, according to
manufacturer's
instructions.
Group #3
The third group consists of 15 patients that receive an influenza vaccination
(e.g.,
Fluarix~ (GlaxoSmithKline, Australia), 0.5 ml, i.m.) at the 8 weeks, according
to
manufacturer's instructions
Group #4 - Control
The fourth group is a control group that consists of an additional group of 15
males of
similar age that do not have prostate disease and receive no medication. The
control group
receives an influenza vaccination (e.g., Fluarix0 (GlaxoSmithKline,
Australia), 0.5 ml, i.m.)
at Day 0, according to manufacturer's instructions.
All patients are monitored throughout (on days -2, 0, 2, 3, 5, 7, 14, 21, 28
and
monthly thereafter for a further 6 months) for WBC, neutrophils, T cells (by
FACS analysis,
as described in detail above), granulocytes levels, and hematocrit, using the
methods
described in detail elsewhere herein, as well as methods well known to those
skilled in the
art. At each time point, the patients are also monitored for the presence or
absence of
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Influenza (types A and B) virus, utilizing nasopharyngeal swabs and FLU OIA
(Thermo
BioStar).
The patients in each group are further monitored for the presence of
hemagglutination-inhibition antibody to H1N1 strains of Influenza at day -2,
as well as all
time points post-inoculation with the influenza vaccine using techniques well
known to those
in the art.
Results:
It is expected that no patients will report any infective episodes with
influenza in any
of Groups 1-3, as compared to the control patients in Group 4 that return a
positive swab to
influenza.
It is also expected that patients in all groups will have a higher
hemagglutination-
inhibition antibody titre than prior to GnRH agonist therapy. Patients in
Groups 2 ~uld 3 are
expected to produce higher antibody titres than those patients in Groups 1 and
4. However, it
is expected that all groups will have similar antibody titres from 12 weeks
onwards. .
Additionally, it is expected that the protection rates (percentage of subjects
with
hemagglutinin-inhibitors greater than 40 for H1N1 strain of influenza) will be
greatest in
Group 2 and least in Group 4.
EXAMPLE 30
LHRH-A TREATMENT EFFECTIVELY DEPLETES SERUM TESTOST~;RONE
Materials and Methods:
Detection of sex steroid levels in patient sera was performed using a lasI-
Testosterone
radioimmunoassay (RIA). Prior to the assay, all reagents, samples and controls
were brought
to room temperature. Control tubes had either buffer alone - non-specific
binding (NSB) tube
or 0 ng/ml testosterone standard (B~). Buffer alone, standards (0-10 ng/ml
testosterone) or
test samples were added to each tube, followed by sex binding globulin
inhibitor (SBGI) to
limit non-specific binding of the radio-labeled testosterone. The ~ 25I-
testosterone was added
to each tube followed by an anti-testosterone antibody (except for the NSB
tubes). Tubes
were then incubated at 37°C for 2 hours. Following this, a secondary
antibody was added to
all tubes which were vortexed and incubated for a further 60 rains. Tubes were
centrifuged
(1000gmaX) for 15 rains, supernatant removed and the precipitate counted on a
Packard Cobra
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auto-beta counter. Triplicate cpm results were averaged and a standard curve
constructed
using the formula for percent bound testosterone (B/Bo):
%B/Bo = Sample - NSB
Bo - NSB
Sample = average cpm of particular test sample
NSB = average cpm of non-specific binding tube
Bo = average cpm of 0 ng/ml standard (total binding tube)
The level of testosterone in each test sample was determined from the standard
curve.
Results:
LHRH-A administration to prostate cancer patients results in castrate levels
of
serum testosterone. In order to determine the efficacy of LHRH-A treatment,
serum
testosterone levels were analyzed for all patients before treatment and at 4
months of
treatment with LHRH-A. Analysis was performed using a radioimmunoassay (RIA)
wish
i2sl-Testosterone. The concentration of serum testosterone was within the
range of 1-3 ng/ml
testosterone (mean = 2.3 ng/ml) prior to hormonal treatment (Fig. 46A). At 4-
months of
treatment, patients had essentially no detectable serum testosterone
indicating successful
abrogation of sex steroid release.
LHRH-A administration does not affect the percent of lymphocyte subsets within
the peripheral blood. Following 4-months of treatment with LHRH-A, no changes
in the
proportion of any lymphocyte subset was observed compared to pre-treatment
values. These
values are all within normal ranges (data not shown). Peripheral blood
lymphocytes were
analyzed by FACS for proportions and cell numbers of T, B, and myeloid-derived
(NK and
macrophages) cells. No change in proportion of any cell subset was observed
following
LHRH-A administration. Furthermore, the proportions of all lymphocyte subsets
were within
normal ranges for this age group (Fig. 84C); Hannet et al., 1992; Xu et al.,
1993).
EXAMPLE 31
THYMIC REACTIVATION FOLLOWING TRANSIENT ABLATION OF SEX
STEROIDS WITH GOSERELIN ACETATE (ZOLADEXO) IN PATIENTS
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UNDERGOING AUTOLOGOUS OR ALLOGENEIC STEM CELL
TRANSPLANTATION
In this example, goserelin acetate (Zoladex~) is administered prior to
autologous or
allogeneic peripheral blood stem cell transplantation (PBSCT). The primary
endpoint is
thymic re-activation as measured by irz vitro assays. Patients will be
followed for six months
post-transplant. Twenty (10 allografts and 10 autografts) patients will be
entered into the
study. This example investigates the effect of inhibiting sex steroid
production at the level of
LHRH, using agonists thereof to desensitize the pituitary and hence prevent
release of LH
and FSH. In turn this causes a block in the gonadal production of androgens
and estrogen
which removes the inhibitory effects on thymic function. The groups examined
in this trial
are patients undergoing high-dose chemoradiotherapy (HDT) and PBSCT.
Goserelin acetate (Zoladex~) is a potent synthetic decapeptide analogue of
LHRH.
When given acutely, goserelin acetate will release LH from the pituitary
gland. However,
following chronic administration, goserelin acetate is a potent inhibitor of
gonadotrophin
production resulting in gonadal suppression and, consequently, sex organ
regression. In
animals and humans, following an initial stimulation of pituitary, LH
secretion and a transient
elevation in serum testosterone, chronic administration results in inhibition
of gonadotrophin
secretion. The result is a sustained suppression of pituitary LH occurring
within
approximately three weeks of initiation of therapy and a reduction in serum
testosterone
levels in males to a range normally seen in surgically castrated men. This
suppression is then
maintained as long as therapy is continued.
Patients are male or female, aged 18 yrs. or older that are due to undergo
high-dose
therapy (HDT) with PBSGT for malignant disease or BM failure. The I0.8 mg
implant
formulation (for men) of the Zoladex~ is dispersed in a cylindrical rod of
biodegr adable and
biocompatible polyglactins and is released continuously over 12 weeks
following
subcutaneous injection. The 3.6 mg implant (for women) is dispersed in a
cylindrical rod of a
biodegradable and biocompatible polyglactin and is released continuously over
28 days
following subcutaneous injection. The implants are commercially supplied in a
purpose-
designed applicator with 14-16 gauge needles.
Reduction of sex steroids in the blood to minimal values may take several
weeks.
Consequently, 21 days prior to PBSC infusion (day 0), patients are injected
with the sex
steroid ablation therapy in the form of LHRH agonist Zoladex0 (implant). For
males, 10.8
mg goserelin as a single dose (effective for 3 months) are administered on day
-21 with a
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further injection of 3.6 mg on day 63 (effective for 28 days). For females 3.6
mg (effective
for 28 days) are administered on day -21 and days 7, 35 and 63. This should be
effective in
reducing sex steroid levels sufficiently to reactivate the thymus (predicted 4
months post
PBSCT). Thus, in this example, only 4-6 months of treatment are administered.
Other doses
may be deemed acceptable as determined readily by those in the art.
PBPCs are infused on day 0. The reactivated thymus takes up the infused
precursor
cells and convert them into new T lymphocytes and epithelial thymic cells.
Maximum sex
steroid 'ablation' is at the time of PBSC infusion, and hence infused PBSC is
able to assist in
thymic reconstitution. It is expected that within 3-4 weeks after PBSCT the
first new T cells
will be present in the blood stream but the therapy will be maintained for 3
months post-
PBSCT to allow complete normalization of the immune system.
Thymic function is determined by assessment of T cell subsets by flow
cytometry, T
cell responses ifa vitro, and production of TRECs.
Prior to the start of the study, routine pre-HDT investigations are performed,
and
baseline FBE, electrolytes, LFTs documented. Other pre-treatment analyses
include serum (3-
HCG (women), thymus CT, bone density studies, protein electrophoresis and
immunoelectrophoresis, hormone studies: TFTs, FSH, LH, estrogen, progesterone,
-.
testosterone. Additionally, various baseline T cell assays will be performed.
Leukocytes will
be purified from 50 ml of blood and examined as follows:
(a) Flow Cytometry
Naive vs. memory T cells
CD27-FITC, CD45RA-PE, CD45R0-PerCP,CD4- or CD8-APC
CD27-FITC, CD45R0-PerCP, CD4/CD8-APC, Ki-67-PE
CD62L, CD45R0-PerCP, CD103, CD4/CD8-APC
T cell subsets
CD4-FITC, CD8-APC, a(3TCR-PE, yBTCR-B/S-PerCP
CD25-PE, CD69-CyChrome, CD4-FTTC, CD8-APC
CD28-CyChrome, a(3TCR-PE, CD4-FITC,CDB-APC
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B cells / myeloid cells
CD19-FITC, CD3-PerCP, CD56-PE, CD34-APC
CDllb-CyChrome, CDllc-PE, CD4-FITC, CD8-FITC
Cytokines
IL-4-PE, IFNg-APC, CD4-FITC, CD8
Other markers
CDlla, CD95, HLA-DR, CD2, CD5
All patients serve as internal controls because they are examined pre- and
post-
treatment. Staining specificity controls will include isotype controls with
FITC/PE/APC etc.
and blocking of FcR prior to staining.
(b) T cell function
Blood lymphocytes will be examined for their ability to respond to CD3 cross-
linking ifs
vitro.
(c) TREC analysis
Naive T cells will be isolated and probed for the presence of T cell receptor
excision
circles which are formed as a result of rearrangement of the TCR genes as
described above.
Their presence is a very strong indication of export from the thymus (being
the only source
of mainstream T cell production). Because cell division is associated with
thymic
development post-rearrangement of the TCR genes, TREC levels may be an
underestimate
of thymic migrants (about 10°l0 of actual levels).
EXAMPLE 32
TREATMENT OF A PATIENT WITH PERNICIOUS ANEMIA
A adult (e.g., 35 years old) human female patient is suffering from pernicious
anemia,
an autoimmune disease. Her CD34+ hematopoietic stem cells (HSC) are recruited
from her
blood following 3 days of G-CSF treatment (2 injections /day, for 3 days, 10
g/kg). Her HSC
can be purified from her blood using CD34. To collect the CD34+ cells,
peripheral blood of
the donor (i.e., the person who will be donating his/her organ or skin to the
recipient) is
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collected, and CD34+ cells isolated from the peripheral blood according to
standard methods.
One non-limiting method is to incubate the peripheral blood with an antibody
that
specifically binds to human CD34 (e.g., a murine monoclonal anti-human CD34+
antibody
conunercially available from Abcam Ltd., Cambridge, UK), secondarily stain the
cells with a
detectably labeled anti-murine antibody (e.g., a FITC-labeled goat anti-mouse
antibody), and
isolate the FITC-labeled CD34+ cells through fluorescent activated cell
sorting (FACS).
Because of the low number of CD34+ cells found in circulating peripheral
blood, multiple
collection and cell sorting may be required from the donor. The CD34+ may be
cryopreserved until enough are collected for use.
Because the antigen for pernicious anemia, the patient's collected HSC are
transfected
by any means to express the antigen (namely, the gastric proton pump). HSC can
be
transfected by using a variety of techniques including, without limitation,
electroporation,
viral vectors, laser-based pressure wave technology, lipid-fusion (see, e.g.,
the methods
described in Bonyhadi et al. 1997). In one example, her HSC are transfected
with the Cl
chain of the H/K-ATPase proton pump, using the MHC class II promoter for the
expression.
To stop the ongoing autoimmune disease, the patient will to undergo T cell
depletion
and/or other immune cell depletion. She will also undergo thynuc regeneration
to replace
these T cells and hence overcome the immunodeficiency state. To do this, she
will receive 4
one monthly injections of Lupron (7.5 mg) to deplete the sex steroids (by 3
weeks) thereby
allowing reactivation of her thymus. This will also allow uptake of the HSC
and to establish
central tolerance to the autoantigen in question. It is not clear why
autoimmune disease starts
but cross-reaction to a microorganism is a likely possibility; depleting all T
cells will thus
remove these cross-reactive cells. If the disease wasyinitiated by such cross-
reaction if may
not be necessary to transfect the HSC with the nominal autoantigen. Simply
depleting T cells
followed by thymic reactivation by disrupting sex steroid signaling may be
sufficient. One
standard procedure for removing T cells is as follows. The human patient
receives anti-T cell
antibodies in the form of a daily injection of 15 rng/kg of Atgam (xeno anti-
~T cell globulin,
Pharmacia Upjohn) for a period of 10 days in combination with an inhibitor of
T cell
activation, cyclosporin A, 3 mg/kg, as a continuous infusion for 3-4 weeks
followed by daily
tablets at 9mg/kg as needed. This treatment does not affect early T cell
development in the
patient's thymus, as the amount of antibody necessary to have such an effect
cannot be
delivered due to the size and configuration of the human thymus. The treatment
is
maintained for approximately 4-6 weeks to allow the loss of sex steroids
followed by the
reconstitution of the thymus. The prevention of T cell reactivity may also be
combined with
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inhibitors of second level signals such as interleukins, accessory molecules
(blocking, e.g.,
CD2S), signal transduction molecules or cell adhesion molecules to enhance the
T cell
ablation or other immune cell depletion.
EXAMPLE 33
TREATMENT OF A PATIENT WITH TYPE I DIABETES
A similar approach to that described in Example 32 is undertaken with a
patient with
Type I diabetes. The T cells will be removed by broad-based depletion methods
(see above),
thymic rejuvenation instigated by 4 month Lupron treatment and the patient's
immune system
recovery enhanced by injection of pre-collected autologous HSC transfected
with the pro-
insulin gene using the MHC class II promoter. The HSC will enter the thymus,
differentiate
into DC (and all thymocytes), and present pro-insulin to the developing T
cells. All those
potentially reactive to the pro-insulin will be killed by apoptosis, leaving a
repertoire free to
attack foreign infectious agents.
In the case that the autoimmune disease arose as a cross-reaction to an
infection or
simply "bad luck" it would be sufficient to use autologous HSC to help boost
the thymic
regrowth. If there is a genetic predisposition to the disease (family members
can often get
autoimrnune disease) the thymic recovery would be best performed with
allogeneic highly
purified HSC to prevent graft versus host reaction through passenger T cells.
Umbilical cord
blood is also a good source of HSC and there are generally no or very few
alloreactive T
cells. Although card blood does not have high levels of CD34+ HSC, they may be
sufficient
for establishment of a microchimera - even ~ 10% of the blood cells being
eventually (after 4-
6 weeks) could be sufficient to establish tolerance to the autoantigen with
sufficient
intrathymic dendritic cells.
EXAMPLE 34
TREATMENT OF A PATIENT SUFFERING FROM ALLERGIES
In the case of allergy, a similar principle as Examples 32 and 33 would be
undertaken.
The allergic patient would be depleted of T cells as above. In severe cases
where there is
exacerbation through IgE or IgG producing B cells (plasma cells) it may be
necessary to use
myeloablation as for chemotherapy. Alternatively, whole body irradiation may
be used (e.g.,
6 Gy). The entire immune system would be rejuvenated by the use of 3-4 month
GnRH and
injection intravenously of the HSC (allogenic or autologous as appropriate).
Allogeneic
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would be used in the case of genetic disposition to allergy but otherwise
mobilized
autologous HSC would be used.
EXAMPLE 35
EFFECTS OF CASTRATION ON NOD AND NZB MICE
Non-obese diabetic mice (NOD mice) are a very well characterized model for
type I
diabetes. Extensive research has confirmed that the pathology of this disease
is due to
abnormal T cell infiltration of the pancreas and autoimmune destruction of the
insulin-
producing islet cells. The structure of the thymus in these animals is
abnormal - there is
ectopic expression of medullary epithelial cells (identified by mAb MTS 10),
the presence of
large B cell follicles and thymocyte-rich areas which lack the epithelial
cells.
To examine the impact of sex steroids on these mice, 20 three week old female
NOD
mice were surgically ovariectimised and 20 were sham-operated. This stage was
chosen
because it is prior to disease onset. Blood glucose was monitored from 10
weeks of age. By
21 weeks of age, over 60% of the sham-castrated mice had developed diabetes
but <20% of
the castrated group had. There was insulitis (infiltration of the pancreas)
but no islet
destniction. After surgical castration, there was also a normalization of the
thymic defects
with well-defined cortex and medulla, loss of the B cell follicles, an
increase in CD25+
regulatory cells. The increased in regulatory T cells may be very important
because they
could alter the pathogenic cytokine profile of the emigrating thymocytes.
Hence castration
has a dramatic impact on the development and progression of diabetes in NOD
mice.
Sixteen (16) ovariectomized and sixteen (16) sham-operated NOD mice were
examined for 21 weeks for the development of diabetes (elevated blood glucose
levels; BGL)
and insulitis. At autopsy they were also examined for the presence of thynuc
structural
abnormalities. As shown in Fig. 45, whereas 60% of the sham-operated NOD mice
had
diabetes, fewer than 20% of the castrated group had diabetes. This clearly
shows a retarding
or even prevention of the diabetes.
As shown in Fig. 64, castrated NOD mice had a marked increase in total
thymocyte
number but no differences in total spleen cells. In the diabetic castrated
mice there was a
marked decrease in total thymocyte number, which may have pre-disposed these
mice to
disease and suggests that the diabetes trigger may have occurred before the
castration.
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There was a significant increase in all thymocyte subclasses (Fig. 66A) but
there was
no change in their proportions (data not shown). Interestingly there no change
in B cells
compared to sham-castrated mice (Fig. 66C) nor in the total T or B cells in
the spleen (Fig.
66B).
In parallel with the increase in total thymocytes post-castration, there was a
marked
increase in CD25+ regulatory cells (data not shown). There was no such change
in the
spleen.
The effect of castration was also examined on NZB mice, which are a model for
systemic lupus erythematosis (SLE). NZB mice have marked abnormalities in the
thymus
which are manifest before disease onset and are closely associated with
disease. These
defects include a poorly-defined cortex-medulla demarcation and abnormal
clusters of B cell
s (see Takeoka et a.l., (1999) Clin. Immurzol.. 90:388).
Mice were castrated or sham -castrated at 4-7 weeks of age and examined 4
weeks
later.
There was a marked increase in total thymocytes (Fig. 67A) and spleen cells
(Fig.
67B). There was also a marked increase in thymic regulatory cells (CD25+ and
NKT cells).
The cytokines from these mice maybe influencing the effector T cells and
modulating their
potential pathogenicity. By immunohistology, the castrated mice had a normal
thymic
architecture and a loss of the B cell follicles (data not shown).
EXAMPLE ~6
w EFFECT OF CASTRATION ON IMMUNIZATION yVITH
TUMOR-SPECIFIC ANTIGEN
Human Papillomavirus (HPV) infection causes genital herpes, which may lead to
cervical cancer in some women. In fact, over 90% of all cervical cancers
contain HPV
DNA. Papillomaviruses are double-stranded DNA viruses that infect skin and
mucosal
surfaces. More than 80 types of HPV have been identified to date. HPV16 is one
of the major
types associated with cervical cancers.
E7 is the major oncogenic protein associated with HPV16-induced cervical
cancer.
Expression of the E7 open reading frame with activated r-as has been shown by
other groups
to be sufficient to transform primary epithelial cells in culture to a
malignant phenotype (Lin
et al.(1996) Cancer Res. 56:21). Thus, E7 is an attractive tumor specific
antigen for use in
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immunotherapy and/or vaccination for cervical cancer and precursor lesions.
Indeed, other
groups have shown that mice immunized with an optimal dose of 50 ~.g/ml of an
E7-GST
fusion protein, with Quil A as adjuvant were protected against a subsequent
challenge with an
HPV 16E7-transfected tumor cell line (Fernando et al., (1999) Clarz. Exp.
Imzzzufzol. 115:397.
This experiment was undertaken to determine if castration of mice was able to
enhance the efficacy of vaccination (as a prophylactic vaccine) andlor
immunotherapy (as an
therapeutic vaccine) with a suboptimal dose of HPV 16E7.
Materials and Methods
Adult (>9 mon. old) C57BL/6 mice received a subcutaneous injection of E7-
positive
syngeneic E7+TC1 tumor cells derived from primary epithelial cells of C57BL16
mice
cotransformed with HPV-16 E6 and E7 and c-Ha-ras oncogenes. Five days later,
the mice
were surgically castrated as described above by a scrotal incision, revealing
the testes, Which
were tied with suture and then removed along with surrounding fatty tissue.
The wound was
then closed using surgical staples. Sham-castrated mice were prepared
following the above
procedure without removal of the testes and were used as negative controls.
Seven days
following tumor challenge, castrated or sham-castrated mice were injected with
a suboptimal
dose (5 ~.g/ml) of the E7GST fusion protein. Positive control mice received
the optimal 50
~,g/ml dose of the E7GST fusion protein. Twenty five days later, mice were
analyzed for
tumors (visually, and tumor mass), and T cell responsiveness (lFNy production
using
standard ELIspot methods and CTL lytic activity by SICr release assays using
HPV16E7-
pulsed target cells as described above).
Results
As shown in Figure 68, only 22% (4118) of castrated mice receiving the
suboptimal
dose of E7GST had tumors, as compared to 47% (9/19) tumor occurrence in the
sham-
castrated suboptimal dose controls. Notably, the proportion of protected
castrated mice
receiving the suboptimal dose E7GST vaccine (78%) was comparable to the
proportion of
protected positive control mice, which received a 10-fold higher (optimal)
dose of the vaccine
(3/4, 75%). All of the castrated mice that did not receive the vaccine had
tumors.
As shown in Figure 69, castrated mice receiving the suboptimal E7GST dose had
a
significantly higher number of cells in the spleen as compared to all other
groups (Fig. 69A)
(p > 0.01). However, the overall number of activated (CD25+) CD4+ (Fig. 69B)
and CD8k
(Fig. 50C) T cell in the spleens of the castrated, suboptimal E7GST group
remained the same
as compared to all the other groups tested.
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As shown in Figure 70A, nonspecific production of IFNy in the splenocytes of
castrated mice receiving the suboptimal GST-E7 dose was comparable to the
positive control
mice receiving a 10-fold higher (optimal) dose of the vaccine. Sham-castrated
mice receiving
the suboptimal dose vaccine had a moderate level of IFNy production, whereas
the negative
control animals had little to no nonspecific splenocyte production of IFNy
This data
parallels that seen with respect to tumor incidence (see Figure 68), and
indicates that the
production of IFNy by splenocytes is directly proportional to tumor incidence
in these
animals. This is not surprising given that Thl cell mediated responses are
primarily
responsible for tumor protection.
As shown in Figure 70B, the level of E7-specifc production follows the same
trends
as discussed above with respect to nonspecific production of IFNy. While the
level of E7-
specific IFNy production in castrated mice receiving the suboptimal GST-E7
dose was
slightly lower than that of the positive control mice receiving a 10-fold
higher (optimal) dose
of the vaccine, it was still higher than the levels observed in the sham-
castrated mice
receiving the suboptimal dose. Unce again, the negative control animals had
little to no E7-
specific splenocyte production of IFNy.
Finally, the mice were analyzed for E7-specific CTL killing of target cells
infected .
with HPV16E7. As shown in Figure 71A, castrated rriice receiving the
suboptimal E7GST
dose had comparable levels of E7-specific CTL as compared to the positive
control. mice
receiving a 10-fold higher (optimal) dose of the vaccine. Sham-castrated mice
receiving the
suboptimal dose vaccine had a moderate level of E7-specific CTL responses,
whereas the
negative control animals had little to no E7-specific CTL. This data parallels
that seen with
respect to tumor incidence (see Figure 49) and lFNyproduction (see Figure 70).
Figure 11B
shows that E7-specifc CTL activity is inversely proportional to tumor mass.
That is, mice
having the highest levels of CTL activity also had no tumors; whereas the few
mice that did
have tumors, were shown to have low E7-specifc CTL lytic activity.
Conclusion
These experiments show that castration can potentially increase vaccine
efficacy in
patients. Equivalent IFNy production, CTL activity, and protection from tumor
challenge is
achieved in noncastrated mice receiving a 10-fold higher (optimal) dose of an
HPV16-E7
vaccine as compared to castrated mice receiving a suboptimal dose of the
vaccine.
A similar experiment could be undertaken to determine the efficacy of
castration plus
E7 vaccination in the context of an immunotherapeutic vaccination. In this
instance, mice
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would be castrated as indicated above. Mice are injected with TCl cells at 6
weeks post-
castration, and immunized with a GSTE7 vaccination 1 week later. Mice are then
analyzed
for tumors (visually, and tumor mass), and T cell responsiveness (IFNy
production using
standard ELIspot methods and CTL lytic activity by S~Cr release assays using
HPV16E7-
pulsed target cells as described above). It is expected that mice receiving
this therapeutic
vaccination protocol will have reduced tumor mass as compared to sham
castrated controls.
Additionally, it is expected that castrated mice will need a lower dose of
vaccine than their
sham castrated counterparts.
EXAMPLE 37
ALTERNATIVE VIRAL VACCINATION STRATEGIES
The methods of the present invention can be used to improve the efficacy of a
variety
of art-recognized viral vaccines by prior, subsequent, or concurrent
administration of an
inhibitor of sex steroid signaling, such as a GnIRIi analog (or other method
of castration). In
addition to the examples provided above, non-limiting examples of viral
vaccines are as
follows.
I. Hepatitis B Viral Vaccine.
An example of a viral vaccine and a recombinant DNA vaccine are those
developed
for Hepatitis B by Glaxo Smith Kline (Engerix B~), and Merck Sharpe and Dohme
(HS
VaxII~), respectively. The Engerix B~ vaccine preparation is a 20 ~.g per 1 mL
dose
administered according to manufacturer's instructions. The HB Vax II~ vaccine
preparation
is a 1 mL 10 ~,g/mL dose administered according to manufacturer's
instructions. A number of
pediatric formulations are also available for these and other vaccines. These
vaccines may or
may not contain preservatives such as Thiomersal. (Australian Immunization
Handbook, gtn
edition). Vaccine doses are typically in the range of 0.5mL to 1mL
administered by i.m.
injection. The usual course of vaccination may vary but usually consists of a
single, primary
immunization followed by at least one booster immunization at intervals of
approximately
one or more months.
II. Hepatitis A Viral Vaccine.
Examples of an inactivated viral vaccine are those developed for the treatment
of
Hepatitis A by Aventis Pasteur (Avaxim0), Glaxo Smith Kline (Havrix 1440~ and
Havrix
Junior0) and others. In the case of Avaxim0, each 0.5 mL dose contains 160
ELISA units of
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hepatitis A (GBM strain) viral antigens. In the case of Havrix 14400, each
0.5mL dose
contains 1440 ELISA units of hepatitis A virus (HM 175 strain). Vaccine doses
of such
monovalent vaccines are in the range of 0.5rnL to 1 mL by IM injection. The
usual course of
vaccination may vary but usually consists of three vaccinations at six month
intervals.
III. Hepatitis A and Hepatitis B Multivalent Vaccine.
Additionally, polyvalent formulations may be used, which contain more than one
viral
antigen. For instance, Glaxo Smith Kline's TwinrixG contains 720 ELISA units
of Hepatitis
A viral antigens and 20~ug of recombinant DNA hepatitis B surface antigen
protein, and are
administered by i.m. injection at 0, 3, and 6 months. Another polyvalent
vaccine is Aventis
Pasteur's Vivaxim~. Each 1 mI, dose contains 160 ELISA units of inactivated
Hepatitis A
virus antigens and 25~g purified typhoid capsular polysaccharide. Supplied in
a dual
chamber syringe this polyvalent vaccine is administered i.m. in two or three
doses.
IV. Cytomegalovirus Vaccine.
Vical, Inc. has developed a immunotherpautic DNA-based vaccine against CMV.
The vaccine is administered in three doses of either 1 or 5 mgs, as provided
in the
manufacturer's instructions. The DNA plasmid encodes CMV phosphoprotein 65
(pp65) and
glycoprotein B (gB), and the vaccine is formulated with a poloxamer.
V. EBV 'Vaccine.
Medlmmune, GlaxoSmithhline, and Henogen have co-developed a soluble
recombinant subunit vaccine against EBV. Live recombinant vaccinia vectors
have been
used to express EBV gp220/350, and have been showmto confer protection in
primates and
EBV-negative infants. Additionally, clinical trials of an EBNA-3A peptide have
been
conducted in Australia. (for a review of this virus vaccine and others, see,
e.g., THE JORDAN
REPORT 2000: ACCELERATED DEVELOPMENT OF VACCINES, published by the Division of
Microbiology and Infectious Diseases, National Institute of Allergy and
Infectious Diseases,
National Institutes of Health (available at
http://www.maid.nih.~publications/pdf/jordan.pdf -last visited Apr. 2004))
EXAMPLE 38
ALTERNATIVE CANCER VACCINATION STRATEGIES
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The methods of the present invention can be used to improve the efficacy of a
variety
of art-recognized cancer vaccines by prior, subsequent, or concurrent
administration of an
inhibitor of sex steroid signaling, such as a GnRH analog (or other method of
castration). In
addition to the examples provided above, non-limiting examples of cancer
vaccines are as
follows.
I. Melanoma Vaccine.
The methods of the present invention can be used to improve the efficacy of a
melanoma vaccine by prior, subsequent, or concurrent administration of an
inhibitor of sex
steroid signaling, such as a GnRH analog (or other method of castration). An
example of
such a melanoma vaccine is the autologous mela~ioma cellular vaccine developed
by AVAX
corporation (Philadelphia, PA). Metastatic tumor are excised, maintained at 4
°C, and
delivered to the laboratory within 48 h of excision. Tumor cells are extracted
by enzymatic
dissociation with collagenase and DNAse, aliquotted, frozen in a controlled
rate freezer, and
stored in liquid nitrogen in a medium containing human albumin and 10%
dimethylsulfoxide
1.5 until needed. On the day that a patient is to be treated, an aliquot of
cells are thawed, washed,
and irradiated to 2500 cGy. The tumor cells are then washed, incubated for 30
min. with
initrofluorobenzene (DNFB), and then washed with saline. (Miller et al. (1976)
J. Irv~n~.unol.
117:1519. After washing, the cells are counted, suspended in 0.2 rnl Hanks
solution with
human albumin, and maintained at 4°C until administered.
Vaccine doses are in the range of 0.5-25.0 x 106 cells. Just prior to
injection, 0.1 ml
of BCG (Tice, Organon Teknika, Durham, N.C.) may be added to the vaccine. The
dose of
BCG may be progressively attenuated to produce a local reaction consisting of
an
inflammatory papule without ulceration. The mixture is injected intradermally
into one or
more sites, e.g." the upper arm. Mufti-dose vaccination is conducted over a
variable period
(e.g., monthly for 12 months or weekly for 6-12 weeks) and may include the
administt~ation
of low dose (300 mg/M2) cyclophosphamide, a cytotoxic drug that paradoxically
augments
cell-mediated immunity when administered at the proper time in relation to
immunization.
II. Lung Cancer Vaccine.
The methods of the present invention can also be used to improve the efficacy
of a
lung cancer vaccine by prior, subsequent, or concurrent administration of an
inhibitor of sex
steroid signaling, such as a GnRH analog (or other method of castration). An
example of a
such a lung cancer vaccine is the DNA vaccine for non-small cell lung cancer
that by Corixa
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Corporation. The vaccine is a recombinant DNA vaccine that is administered
with a
recombinant adenovirus adjuvant. A Biojector~ device (Bioject, Tualatin, OR)
is used for
vaccine administration and is a reusable needle-free injector powered by a
compressed COZ
cartridge.
. Other non-limiting lung vaccines that may be used with the methods of the
invention
include L523S for non-small cell lung cancer (Wang et al. (2003) Br. J. Cancer
88:887) and
BEC-2, GM2, Globo H, fucosyl GM1, and polysialic acid for small cell lung
cancers (Krug
(2004) Sera. Orecol. 31:112).
III. Prostate Cancer Vaccine.
The methods of the present invention can also be used to improve the efficacy
of a
prostate cancer vaccine by prior, subsequent, or concurrent administration of
an inhibitor of
sex steroid signaling, such as a GnRH analog (or other method of castration).
One non-
limiting example of such a vaccine is Provenge0 (Dendreon Corp.), which is a
vaccine
against androgen independent prostate cancer. This vaccines results in the
generation of a T
cell immune response to the prostate cancer associated antigen prostatic acid
phosphatase
(PAP). DC precursors are obtained from the patient by leukopheresis and
isolated from other
white blood cells using a cell separation device. These cells are then co-
cultured with a
recombinant PAP fused delivery cassette for about 36 hours to allow the DC to
mature. The
mature DC are then used in the vaccine. The Provenge vaccine is delivered as
three 30-
minute intravenous infusions at two week intervals. Other prostate cancer
vaccine candidates
for use in the invention are known in the art (see, e.g., Shaffer and Scher
(2003) Laracet
Otacol. 4:407).
IV. Colorectal Cancer Vaccine.
The methods of the present invention can also be used to improve the efficacy
of a
colorectal cancer vaccine by prior, subsequent, or concurrent administration
of an inhibitor of
sex steroid signaling, such as a GnRH analog (or other method of castration).
One non-
limiting example of such a vaccine is TrovaxT"" (Oxford BioMedica). This
vaccine is a solid
tumor associated antigen, 5T4, delivered by modified vaccinia virus Ankara
(MVA) via
intramuscular injection. It is delivered at weeks 0, 4 and 8, coincident with
chemotherapy.
Other cytotoxic T-lymphocyte precursor-oriented peptide vaccines for
colorectal carcinoma
patients are known in the art (see, e.g., Sato et al. (2004) Br. J. Cancer
90:1334.
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V. Ovarian Cancer Vaccine.
The methods of the present invention can also be used to improve the efficacy
of a
ovarian cancer vaccine by prior, subsequent, or concurrent administration of
an inhibitor of
sex steroid signaling, such as a GnRH analog (or other method of castration).
One non-
limiting example of such a vaccine is M-FP (CancerVac Pty. Ltd.) for the
treatment of
metastatic ovarian cancer. It is an autogenic cellular vaccine, whereby DC are
injected
subcutaneously. A recombinant version of MUCl is fused to mannan to promote
antigen
uptake by the patients purified dendritic precursor cells. Activated DC
presenting MUC1
peptides are then injected s.c. into the patient.
EXAMPLE 39
CASTRATTON EFFECT ON BM AND SPLEEN IN THYMECTOMIZED
MICE
These preliminary experiments were performed to determine if castration
effects were
apparent in thymectomized patients (mice). The results indicated that
disruption of sex
steroid signaling has direct or indirect effects on the immune system (e.g.,
immune cells in
the BM and thymus), irrespective of the presence of a regenerated thymus.
Materials and Methods
Mice were castrated and th.ymectomized using routine methods known in the art.
Mice were divided into the following groups: untreated ~(i.e., naive,
"untreated"), sham.
castrated ("sham-cx"), and castrated ("cx"), and each of those three groups
was then
thymectomized ("tx") or sham thymectoized ("shtx") for a total of six groups
analysed. Each
of the six groups was analysed as 2 weeks and 4 weeks following myeloablation
and BMT'
(see methods above).
Results
I. Thymectomy does not impact the effect of sex steroid inhibition on the
BM.
As shown in Fig. 72A, at 4 weeks post-BMT, Tx/Cx mice had an increase in the
number of BM common lymphoid progenitors (CLPs), which is comparable to the
ShTx/Cx
mice.
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As shown in Fig. 72B, at 4 weeks post-BMT, TxlCx mice have an increase in the
total
number of B cells in the BM, which is comparable to the ShTx/Cx mice. The
Tx/Cx mice
and the ShTx/Cx mice also have increased numbers of B cells in the BM, as
compared to the
either the ShamCx/Tx or ShamCx/ShTx controls.
As shown in Fig. 72C, at 4 weeks post-BMT, TxlCx mice also have an increase in
the
total number of immature B cells in the BM, which is comparable to the ShTx/Cx
mice. The
Tx/Cx mice and the ShTx/Cx mice also have increased numbers of immature B
cells in the
BM, as compared to the either the ShamCx/Tx or ShamCx/ShTx controls.
Thus, the results in Figs. 72A-C support the conclusion that the effect of
castration on
increasing the number and functionality of cells in the BM, including
increasing engraftment,
does not require a reactivated thymus, and is instead due to direct effects on
the BM and other
cells of the immune system.
II. Thymectomy does not impact the effect of sex steroid inhibition on the
spleen.
As shown in Fig. 72D, at 4 weeks post-BMT, Tx/Cx mice also appear to have an
increase in the total number of cells in the spleen, which is comparable to
the ShTx/Cx mice.
The Ta/Cx mice and the ShTx/Cx mice also have increased total numbers of
splenocytes, as
compared to the either the ShamCx/Tx or ShamCxlShTx controls.
As down in Fig. 72E, at 4 weeks post-BMT, Tx/Cx mice also appear to have an
increase in the total number of B cells in the spleen, which is comparable to
the ShTx/Cx
mice. The Tx/Cx mice and the ShTx/Cx mice also have increased numbers of B
cells in the
spleen, as compared to the either the ShamCx/Tx or ShamCx/ShTx controls.
Thus, the results in Figs. 72D-E support the conclusion that the effect of
castration on
increasing the number and functionality of immune cells in the spleen,
including enhanced
reconstitution, does not require a reactivated thymus, and is instead due to
direct effects on
the BM and other cells of the immune system.
All publications mentioned in the above specification are herein incorporated
by
reference. Various modifications and variations of the described methods and
system of the
invention will be apparent to those skilled in the art without departing from
the scope and
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spirit of the invention. Although the invention has been described in
connection with specific
preferred embodiments, it should be understood that the invention as claimed
should not be
unduly limited to such specific embodiments. Indeed, various modifications of
the described
modes for carrying out the invention which are apparent to those skilled in
biology or related
fields are intended to be within the scope of the following claims.
Those skilled in the art will. recognize, or be able to ascertain, using no
more than
routine experimentation, numerous equivalents to the specific embodiments
described
specifically herein. Such equivalents are intended to be encompassed in the
scope of the
following claims.
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