Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR THE INDUCTION OF PROFESSIONAL AND CYTOKINE-PRODUCING
REGULATORY T CELLS
This application claims the benefit of the filing date of U.S.S.N. 60/342,655,
filed December 21, 2001
and U.S.S.N. 60/374,102, filed April 19, 2002.
FIELD OF THE INVENTION
The field of the invention is generally related to methods used for the
induction of T cells with
suppressive activity. More specifically, the methods are used to generate
professional regulatory T
cells and cytokine-producing T cells with enhanced suppressive activity.
BACKGROUND
A number of immune disorders can be characterized by the failure of the immune
system to
distinguish self from non-self. For example, autoimmune diseases are caused by
the failure of the
immune system to distinguish self from non-self. In these diseases, the immune
system reacts
against self tissues and this response ultimately causes inflammation and
tissue injury. Autoimmune
diseases can be classified into two basic categories: antibody-mediated
diseases such as systemic
lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic
anemia,
thrombocytopenia purpura, Grave's disease, Sjogren's disease and
dermatomyositis; and cell-
mediated diseases such as Hashimoto's disease, polymyositis, disease
inflammatory bowel disease,
multiple sclerosis, diabetes mellitus, rheumatoid arthritis, and scleroderma.
Alternatively, the ability of the immune system to recognize and respond to
foreign antigens is
undesirable in some situations. For example, the rejection of solid organ
transplants, i.e., graft
rejection, occurs when the immune system of the recipient recognizes foreign
histocompatibility
antigens. Likewise, transplantation of hematopoietic stem cells from an
unrelated (or allogeneic)
donor can trigger a deadly response called graft versus host disease (GVHD)
because the donor stem
cell preparations generally contain T lymphocytes. GVHD results when the donor
T lymphocytes
recognize histocompatibility antigens of the recipient as foreign and respond
by causing multi-organ
dysfunction and destruction.
Methods for alleviating the symptoms of autoimmune disorders and for
preventing graft rejection
typically involve the use of steroids with potent anti-inflammatory and
immunosuppressive action,
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such as prednisone. Other strong immunosuppressive drugs that can be used
include azathioprine,
cyclosporin, and cyclophosphamide. All of these drugs have undesirable side
effects due to the
global reduction of the immune system.
A more desirable strategy would be to identify methods that do not have
undesirable side effects.
Methods for "resetting" the immune system, by generating regulatory T cells
(also referred to as
suppressor T cells) are described in U.S. Patent Nos. 6,228,359, and
6,358,506, 6,557,765 and
U.S.S.N.s. 09/653,924 and 09/833,526, all of which are incorporated herein by
reference in their
entirety. These methods are directed towards restoring normal regulatory cell
function in an affected
individual.
Accordingly, it is an object of the present invention to provide methods for
treating peripheral lymphoid
tissue for the generation of regulatory T cells that can be used to treat
autoimmune disorders, as well
prevent immune responses resulting in graft rejection and graft versus host
disease.
SUMMARY OF THE INVENTION
In accordance with the objects outlined above, the present invention provides
compositions and
methods that can be used to generate regulatory T cells in a sample of ex vivo
peripheral blood
mononuclear cells (PBMCs). The regulatory cells so generated may be
professional CD4+ CD25+
regulatory cells or cytokine-producing regulatory cells. Preferably, both the
number and suppressive
activity of the regulatory cells are increased.
The regulatory compositions may comprise a number of components, including
cytokines, T cell
activators, T cell stimulators, non T accessory cells, and neutralizing anti-
cytokine antibodies. These
components may be added in any number of combinations including one or more
compounds from
the same class of compounds, i.e., two or more cytokines, may be mixed
together. The composition
also may contain compounds from different classes of compounds, such as a one
or more cytokine, T
cell activator, etc.
In an additional aspect, the present inventions provides methods for
inhibiting aberrant or undesirable
immune responses comprising administering the regulatory T cells generated
using the regulatory
compositions described herein.
In a further aspect, the present invention provides kits for the practice of
the methods of the invention,
i.e., the incubation of cells with the regulatory compositions.
ERIEF DESCRIPTION OF THE FIGURES
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Figures 1A-C depict some preferred embodiments for generating regulatory T
cells beginning with a
population of T cells (1) comprising mostly conventional T cells (2) and
virgin professional regulatory T
cells (3). Targeting virgin CD4+ CD25+ cells with a regulatory composition
results in a treated T cell
population (6) comprising > 50% activated professional regulatory CD4+ CD25+ T
cells with
enhanced suppressive activity (4). These cells may be the progeny of the
virgin regulatory cells or
other T cells that have been induced to develop potent contact-dependent
suppressive activity (Figure
1 A).
Figure 1 B depicts another preferred embodiment comprising mixing activated
professional regulatory
T cells (4) with conventional T cells (2) and stimulating with a T cell
activator. In this embodiment, the
professional regulatory cells induce other T cells to become cytokine-
producing T cells (5) by a
phenomenon called T cell tolerance.
In another embodiment, treatment of conventional T cells (2) depleted of CD4+
CD25+ with a
regulatory composition induces these cells to become activated cytokine
producing regulatory T cells
(5) with enhanced suppressive activity (Figure 1C).
Figures 2A and B illustrate that TGF-(3 co-stimulation markedly increases the
percentage and
absolute numbers of total CD4+ CD25+ and CD4+ CD25- cells. Does dependent
effects of TGF-(3
are shown. The cytokines produced by T cells co-stimulated by TGF- also
increase CD8+ cells.
Figure 3 depicts that the suppressive effects of TGF-(3 may be overcome by
neutralizing IL-2 with a
monoclonal antibody. The dose-dependent co-stimulatory properties of TGF- ~i
were abolished with
small amounts of anti-IL-2. These doses did not affect T cell activation
without TGF-Vii. Larger
amounts of anti-IL-2 completely inhibited IL-2 activity, which in turn led to
suppressive effects on TGF-
Vii.
Figures 4A-F show that the combination of IL-2 and TGF- (3 stimulates
professional CD4+ CD25+
cells and increases their suppressive activity. Using alloantigens as the T
cell activator, this is
demonstrated for the small "virgin" CD25+ subset isolated from the naive
fraction of CD4+ CD45RA+
cells (Figures 4A and B). Suppression was assessed by the inhibiting the
generation of cytotoxic T
lymphocyte (CTL) activity against the lymphoblasts of the stimulator using the
well established
chromium release assay with three effector to target cell ratios. Most CD4+
CD25+ cells are
contained in the previously activated, or memory CD45R0+ subset. Studies
showing the suppressive
activity of these regulatory cells are shown in Figure 4C and 4D. The
suppressive effects of memory
CD4+ CD25+ cells alloactivated for 5 days with IL-2 (10 U/ml) ~ TGF-beta 1 (1
ng/ml) on other T cells
is shown. The mixed lymphocyte was repeated with the same donors and the
activated CD4+ CD25+
cells were added to autologous responder T cells in a 1:10 ratio. The
responder cells were labeled
with carboxyfluorescein and the percentage and absolute numbers of cycling
CD8+ cells was
assessed 6 days after activation. The clear bar indicates cycling CD8+ cells
without added activated
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CD4+ CD25+ cells. The gray bar indicates the effect of CD4+ CD25+ cells
activated with IL-2, and
the black bar the effect of cells activated with IL-2 and TGF-Vii. The
suppressive activity is not
decreased by anti-TGF-Vii. Using inhibition of the generation of alto-CTL
activity to assess
suppressive activity, the combination of IL-2 and TGF-~i increases the
cytokine-dependent
suppressive activity of conventional naive CD4+ CD45RA+ CD25- cells and of
previously activated or
memory CD4+ CD45RO+ CD25- cells (Figures 4E and F). Anti- TGF-(3 abolishes the
suppressive
effects of the cells by increasing CTL activity to levels observed with
control CD4+ cells. The
suppressive activity induced by the superantigen, staphylococcal enterotoxin
B, in the presence of
TGF-~i is also abolished by anti-TGF-(3. In this example, conventional, CD4+
CD25- cells are induced
to become regulatory cells.
Figures 5A-D confirm the critical role of the CD25+ subset in mediating the co-
stimulatory effects of
TGF-[3. Naive CD4+ CD45R0- cells were prepared and these were further
fractionated into CD25+
and CD25- cells by cell sorting. These cells were activated in the allogeneic
mixed lymphocyte
reaction with IL-2 (10U/ml) ~ TGF-beta 1 (1 ng/ml). Figure 5A shows that the
TGF-(3 mediated 2 fold
increase in cell number was abolished by removal of the <1 % CD25+ cells. The
addition of CD25+
cells to CD25- cells in a 1:10 ratio restored this effect. Figure 5B and C
show a similar experiment
where TGF-~i induced enhancement of cell numbers was only modest, but the
phenotype of the cells
was markedly altered. TGF-(3 markedly increased the number of cells expressing
both CD25 and
CTLA-4. This effect was lost when the CD25+ subset was removed. The addition
of CD25+ cells to
CD25- cells in a ratio of 1:10 restored the effect. Figure 5D shows that when
the cells were
restimulated without TGF-a, those preparations containing CD25+ cells markedly
increased in
comparison with those where the CD25+ subset had been removed.
Figure 6 shows an experiment suggesting that TGF-~i conditioned, activated
CD4+ CD25+ cells can
induce conventional CD4+ CD25- cells to develop suppressive activity. The
experimental design is
similar to that described in Figure 5. Naive CD4+ cells, purified CD25+ cells,
CD25 depleted cells and
a mixture of CD25+ and GD25- cells that had been alloactivated with and
without TGF-~i were added
to fresh T cells in a 1:10 ratio and alloactivated with the same allogeneic
stimulator cells. The
percentage of CD8+ CD25+ cells after 6 days of culture was determined. The
horizontal line shows
the percentage of CD8+ CD25+ cells without added CD4+ cells. Marked
suppression by CD25+ cells,
but not by CD25- cells is shown. When CD25+ cells were mixed with CD25- cells
in a ratio of 1:10
and alloactivated with TGF-(3, their suppressive activity was equivalent to a
similar number of purified
CD4+ CD25+ cells. This experiment also illustrates that the suppressive-
inducing property of IL-2 and
TGF-~i is not exclusively limited to the CD25+ subset of CD4+ cells. IL-2 and
TGF-(i treatment of
naive CD4+ CD45RA+ cells that had been depleted of CD25+ cells also induced
suppressive activity,
The suppression was not abolished with anti-TGF-Vii.
Figures 7A and B illustrate another experiment where IL-2 and TGF-~i induce
CD4+ CD45RA+ CD25-
cells to become suppressor cells. Various CD4+ CD45RA+ subsets were activated
~ TGF-~i and
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tested for suppressive activity by inhibition of the generation of CTL
activity. In this experiment, the
suppressive activity of total CD4+ CD45RA+ cells was identical to the CD4+
CD45RA+ CD25- subset
(Figure 7A). Anti-TGF-(3 did not reverse this suppressive activity (Figure
7B).
Figures 8A-J demonstrate that IL-2 and TGF-~i enhance the growth and
suppressive activity of the
previously activated or memory fraction of CD4+ CD45R0+ CD25+ cells after the
endogenous
suppressive effects are overcome. The endogenous negative feedback effects of
CD4+ CD45R0+
CD25+ cells on suppressor cell induction was shown by alloactivating total
CD4+ cells with TGF-~i to
generate regulatory cells. Inhibition of the generation of CTL activity was
used to assess suppression.
TGF-(3 show that the suppressive activity developed by total CD4+ cells was
significantly less than
when the CD45R0 subset, or the CD25+ fraction was depleted (Figures 8A and B).
Figure 8C shows
that the removal of the CD25+ suppressor cells correspondingly increased the
number of CD4+ cells
following alloactivation. The horizontal line indicates the starting number of
CD4+ CD45R0+ cells.
Because most CD4+ CD25+ cells are contained in the CD45R0 fraction, the TGF-(3
consistently
decreased the number of T cells recovered following the mixed lymphocyte
reaction. Figures 8D-F
show that IL-2 can overcome the inhibitory effects of TGF-Vii. CD4+ CD45R0
cells were prepared and
some were depleted of CD25+ cells. The two populations were stimulated with
allogeneic cells with
IL-2 and TGF-~i as described above. The total number and those express CD25
and CTLA-4 after 6
days was measured. In comparison with IL-2 alone, the combination of IL-2 and
TGF-~i modestly, but
significantly enhanced the total number (Figure 8D), the number of CD25+ cells
(Figure 8E), and the
number of CTLA-4+ cells (Figure 8F). By contrast, TGF-(3 had inhibitory
effects on CD4+ CD45R0+
CD25- cells. These studies suggested that the co-stimulatory effects of IL-2
and TGF- specifically
targeted the CD4+ CD45R0+ CD25 subset. Figure 8G illustrates that the
combination of IL-2 and
TGF-(3 enhances the growth of CD4+ CD45R0+ CD25+ cells. The horizontal line
indicates the
starting population of cells. While the numbers of these CD25+ T cells
decreased by 50% in medium,
with IL-2, and with TGF-Vii, they increased by 33% when cultured with IL-2 and
TGF-(i. Figure 8H
shows TGF- ~i -dependent up regulation of CD122. CD25+ were targeted and
depletion of CD25+
abolished this effect. Figures 81 and J show that even CD25+ depleted naive
CD4+ could be induced
to develop suppressive activity independent of TGF- Vii. The addition of IL-2
to TGF- ~i enabled a
CD4+ CD45RA+ CD25- cells to develop potent suppressive activity that could not
be abolished by
anti-TGF- Vii.
Figures 9A-D illustrate the co-stimulatory effects of TGF-~i on the expression
of surface markers
which are characteristically displayed by professional CD4+ CD25+ regulatory T
cells. Besides CD25,
these include CTLA-4 and CD62L. The total numbers of CD25+ cells, CTLA-4+
cells and CD62L cells
were increased in cultures containing TGF-~ (Figures 9A, B, C). In addition,
TGF-~i enhanced
expression of CD122, the beta chain of the IL-2 receptor (Figure 9D). This
receptor binds IL-2 and IL-
15 and is involved in signal transduction. The CD4+ CD25+ subset was the
specific target of TGF-~i
as depletion of this subset abolished the marked enhancement of receptor
expression.
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Figure 10 illustrates that blocking the beta chain of the IL-2 receptor with
anti-122 may abolish the
suppressive activity of CD25+ cells. T cells were stimulated with allogeneic
cells and CTL activity was
assessed. Partial depletion of CD25+ cells with immunomagnetic beads enhanced
CTL activity,
suggesting that total suppressor cell activity was diminished. Blocking IL-2
signaling through the IL-
2Rbeta chain with various doses of anti-CD122 enhanced CTL activity even
greater. This effect was
due to blocking the activity of T cells with lower amounts of cell-surface
CD25, and inhibiting
suppressor cells that had lost expression of CD25.
Figures 11A and B illustrate that in addition to the combination of IL-2 and
TGF-Vii, IL-15 and TGF-~i
has co-stimulatory effects on CD4+ cells. CD4+ cells were stimulated with
allogeneic non-T cells ~
TGF-(3, and expression of CD122 and CD25 CTLA-4 double positive cells were
quantified by flow
cytometry. Adding IL-15 to TGF-(3 enhanced expression of these markers as well
as the combination
of IL-2 and TGF-(3.
DETAILED DESCRIPTION
The present invention is directed to methods of generating regulatory T cells
ex vivo. The methods
involve isolating naive T cells and treating them with a regulatory
composition. Treatment with a
regulatory composition increases the numbers and suppressive activity of the
generated regulatory T
cells. This enhanced suppressive activity is thought to be mediated through
the induction of a cell
surface receptor critical for T cell proliferation and differentiation.
Moreover, the regulatory T cells generated by the methods described herein
induce other T cell
populations to develop regulatory cells with enhanced suppressive activity.
This ability to induce
suppressive activity may occur through a phenomenon called infectious
tolerance (see Waldmann H.,
et al, (1993) Science, 259: 974-7). Regulatory T cells prevent self-reactive T
cells from becoming
activated and causing immune pathology. Regulatory T cells also prevent
microbial antigens from
inducing immune-mediated tissue injury (see Zheng, et al., (2002) J. Immunol.,
169: 4183-4189).
Several regulatory T cell subsets have been identified (see Zheng, et al.,
(2002) J. Immunol., 169:
4183-4189). For example, in the thymus, a subset of CD4+ T cells (CD4+ CD25+)
constitutively
express the alpha chain of the IL-2 receptor complex (CD25). These CD4+ cells
have a broad range
of suppressive activities that include prevention of autoimmunity and graft
rejection, control of
homeostatic lymphocyte proliferation, and regulation of germinal center
formation in lymph nodes.
These cells suppress by a contact-dependent, TGF-~i independent, mechanism and
are referred to as
"professional" regulatory T cells.
Other regulatory cells, called Th3 or Th3-like cells, can be either CD4+ or
CD8+ T cells that produce
immunosuppressive levels of TGF-(3. These cells are produced in peripheral
lymphoid tissue, such as
mucosal lymphoid tissues in response to oral or intranasal immunization. Th3
cells have a protective
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role in several experimental autoimmune diseases, including experimental
allergic encephalitis and
diabetes mellitus.
When a naive CD4+ cell population comprising primarily conventional T cells
(CD4+ CD25-) and a
few virgin CD4+ CD25+ professional regulatory T cells is treated with a
regulatory composition, some
of the virgin professional regulatory T cells are stimulated to proliferate,
resulting in the generation of
activated professional regulatory cells with enhanced suppressive activity
(see Figure 1A). As
illustrated in Figure 1A, naive (CD45RA+RO-) CD4+ cells are generally 99%
conventional T cells
(CD4+ CD25-). However, when activated with a regulatory composition, the
percentage of CD4+
CD25+ cells is markedly increased, such that these cells now comprise greater
than 50% of the total
20
treated CD4+ cell population. The CD4+ CD25+ population comprises the progeny
of the virgin CD4+
CD25+ cells and other T cells that were activated by the virgin CD4+ CD25+
cells to become
regulatory cells. This phenomenon is dependent upon the virgin CD4+ CD25+
regulatory cell subset
as it can be abrogated by removing this subset (Yamagiwa et al, (2001 ) J.
Immuno. 166: 7282-89).
When activated professional regulatory T cells are mixed with conventional T
cells, they generate a
population of cytokine producing regulatory T cells when treated with a T cell
activation (Figure 1 B).
Significantly, repeated treatment with a regulatory composition comprising
cytokines is not needed to
generate this population of cytokine producing regulatory T cells.
Cytokine-producing regulatory T cells may also be generated from conventional
T cells depleted of
professional CD25+ T cells by treating the conventional T cells with a
regulatory composition (see
Figure 1 C).
Thus, by targeting a small number of virgin CD4+ CD25+ cells in T cell
preparations with a regulatory
composition results in (1) a marked expansion of professional regulatory T
cells with enhanced
suppressive activity; and (2) induction of conventional T cells to become
cytokine producing cells.
The proportions of professional and cytokine-producing cells that are
generated depends upon the
composition of the T cell population that is treated, the composition of the
regulatory composition and
whether non T accessory cells are added.
Administration of one or more of the regulatory T cell subsets generated by
one of above approaches
can be used to inhibit undesirable immune responses in an individual. For
example, in individuals
with antibody-mediated autoimmune disorders, the present invention restores
the capacity of
peripheral blood T cells to down regulate antibody production and restores
cell mediated immune
responses. In patients with cell-mediated disorders, the present invention
generates regulatory T
cells that suppress cytotoxic T cell activity in other T cells. In patients
receiving a solid organ
transplant , the present invention prevents the recipient's T cells from
destroying the donor organ. In
patients receiving a stem cell transplant, the present invention prevents the
donor stem cells from
destroying the recipient's cells and tissues.
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Accordingly, the present invention is drawn to methods of generating
regulatory T cells that comprise
isolating T cells from peripheral blood mononuclear cells(PBMC) and treating
those cells with a
regulatory composition comprising at least one compound that induces the
generation of regulatory T
cells with suppressive activity.
Peripheral T lymphocytes
In mature human immune systems, peripheral T cells, both CD4+ and CD8+, can be
separated into
two major subsets, naive versus memory-effector, by a number of correlated
functional and
phenotypic features, including: (a) activation requirements; (b) effector
function (e.g., cytokine
synthesis); (c) homing behavior; (d) adhesion function; and, (e) cell surface
phenotype. The putative
naive subset, which predominates in immature (i.e., neonatal) immune systems
and resembles the
most mature thymocytes demonstrates the following features: (a) little or no
response to recall
antigens; (b) little or no ability to produce effector cylines such as
inferferon-y; (c) high costimulatory
requirements for TCR-mediated activation; (D) inefficient maturation into MHC-
restricted cytotoxic T
cells; (e) efficient in vivo localization in secondary lymphoid tissues but
not tertiary sites; (f) relatively
low susceptibility to apoptosis and, corresponding to these functional
characteristics, (g) a
predominance of the high molecular weight, low activity RA isoforms of the
CD45 protein tyrosine
phosphatase; (h) uniform low expression of many general adhesion molecules,
such as
CD11a/CD18(LFA-1), CD54 (ICAM-1), CD2, CD58 (LFA-3), and CD44, the apoptosis-
triggering
molecule CD95/FAX, and the tertiary skin-selective homing receptor CLA; (i)
uniform high expression
of the peripheral lymph node homing receptor L-selectin and the costimulatory
molecule CD27; and Q)
uniform moderate expression of the Peyer's patch homing receptor a4~i7
integrin (Picker and
Siegelman, (1999) "Lymphoid Tissues and Organs" in W. E. Paul, ed.,
Fundamental Immunology, 4tn
ed., chapter 14, pp 479-531 ).
In contrast, the memory-effector subset, which contains the vast majority of
cells capable of
responding to recall antigens and can be generated in vitro from the
aforementioned naive subset
following appropriate activation, predominantly expresses the low molecular
weight, high activity, RO
CD45 isoform and shows efficient effector function (e.g., production of
effector cytokines, cytotoxicity),
increased CD95/FAS expression, and increased susceptibility to apoptosis, high
levels of
CD11a/CD18 and the other general adhesion molecules listed above, and
heterogenous expression
of the L-selectin, a4(37 integrin, and CD27 (Picker and Siegelman, (1999)
"Lymphoid Tissues and
Organs" in W. E. Paul, ed., Fundamental Immunology, 4'n ed., chapter 14, pp
479-531). Although the
specific markers of memory-effector T-cell differentiation apply largely to
humans, analogous naive-
memory T-cell dichotomy exists in animals as well.
Thus, the methods of the present invention begin with the isolation of T
cells. As described below, the
methods provide for the isolation of naive and memory-effector T cells. By
"naive T cells" herein is
meant T cells that express the CD45RA+/RO-, are undifferentiated because they
have not been
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exposed to an immunizing antigen and have the characteristic features of
"naive T cells" discussed
herein. By " memory-effector T cell" herein is meant T cells that express
CD45RA-/RO+, are
differentiated because they have been exposed to an immunizing antigen and
have the characteristic
features described above for "memory-effector" T cells.
Isolation of T Cells
Peripheral blood mononuclear cells (PBMC) are taken from heparinized venous
blood of an individual
using standard techniques (see Zheng, et al., (2002) J. Immunol., 169: 4183-
4189). By "peripheral
blood mononuclear cells" or "PBMC" herein is meant lymphocytes (including T-
cells, B-cells, NK cells,
etc.) and monocytes. Preferably, only PBMC are taken, either leaving or
returning red blood cells to
the patient. This is done as is known in the art, for example using
leukophoresis techniques. In
general, a 5 to 7 liter leukopheresis step is done, which essentially removes
PBMC from a patient,
returning the remaining blood components. Collection of the cell sample is
preferably done in the
presence of an anticoagulant such as heparin, as is known in the art.
20
In general, the sample comprising the PBMC can be pretreated in a wide variety
of ways. Generally,
once collected, the cells can be additionally concentrated, if this was not
done simultaneously with
collection or to further purify and/or concentrate the cells. The cells may be
washed, counted, and
resuspended in buffer.
The PBMCs are generally concentrated for treatment, using standard techniques
in the art. In a
preferred embodiment, the leukopheresis collection step results a concentrated
sample of PBMCs, in
a sterile leukopak, that may contain reagents or doses of the regulatory
composition, as is more fully
outlined below. Generally, an additional concentration/purification step is
done, such as Ficoll-
Hypaque density gradient centrifugation as is known in the art.
Separation or concentration procedures include but are not limited to magnetic
separation, using
antibody-coated magnetic beads, affinity chromatography, cytotoxic agents,
either joined to a
monoclonal antibody or used with complement, "panning", which uses a
monoclonal antibody
attached to a solid matrix. Antibodies attached to solid matrices, such as
magnetic beads, agarose
40
beads, polystyrene beads, follow fiber membranes and plastic surfaces, allow
for direct separation.
Cells bound by antibody can be removed or concentration by physically
separating the solid support
from the cell suspension. The exact conditions and procedure depend on factors
specific to the
system employed. The selection of appropriate conditions is well within the
skill in the art.
Antibodies may be conjugated to biotin, which then can be removed with avidin
or streptavidin bound
to a support, or fluorochromes, which can be used with a fluorescence
activated cell sorter (FAGS), to
enable cell separation. Any technique may be employed as long as it is not
detrimental to the viability
of the desired cells.
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In a preferred embodiment, the PBMC are separated in a automated, closed
system. One such
example is the Nexell Isolex 300i Magnetic Cell Selection System. Generally, a
closed system is
preferable to maintain sterility and to insure standardization of the
methodology used for cell
separation, activation and development of suppressor cell function.
In a preferred embodiment, the PBMC are washed to remove serum proteins and
soluble blood
components, such as autoantibodies, inhibitors, etc., using techniques well
known in the art.
Generally, this involves addition of physiological media or buffer, followed
by centrifugation. This may
be repeated as necessary. The PBMC can be resuspended in physiological media,
preferably AIM-V
serum free medium (Life Technologies) (since serum contains significant
amounts of inhibitors of
TGF-Vii) although buffers such as Hanks balanced salt solution (HBBS) or
physiological buffered saline
(PBS) can also be used.
In a preferred embodiment, peripheral blood lymphocytes (PBL) are prepared by
adding PBMC to a
continuous Percoll density gradient and the high density fraction collected.
In some embodiments, the
PBMC are concentrated and washed as described above prior to the isolation of
the PBL. T cells are
prepared by immediate rosetting with 2-aminoethylisothiouronium bromide-
treated SRBC. T cells are
further purified from rosetting cells by staining with antibodies (Abs) to
CD16, CD74, and CD11 b and
deleting reactive cells using immunomagnetic beads. The percentage of CD3+
cells in this fraction is
usually greater than 96% (see Zheng, et al., (2002) J. Immunol., 169: 4183-
4189).
In a preferred embodiment, CD8+ cells are prepared by negative selection (see
Zheng, et al., (2002)
J. Immunol., 169: 4183-4189).
Ina preferred embodiment, CD4+ cells are prepared from T cells that are
stained with Abs to CD8 by
negative selection using immunomagnetic beads. The purity of CD4+ cells is
usually greater that
95%. CD25-depleted T cells are prepared from CD4+ T cells by cell sorting.
Before sorting, the
CD4+ CD25+ population was approximately 3-5% among total CD4+ T cells. After
sorting, the CD4+
CD25+ population was less than 0.3% (see Zheng, et al., (2002) J. Immunol.,
169: 4183-4189).
In a preferred embodiment, the CD4+ cells are further purified to include only
undifferentiated, naive
CD4+ T cells. This is done by depleting the CD4+ cells of CD45R0+ cells using
monoclonal
antibodies.
NK T cells may be isolated by standard techniques known to those of skill in
the art (see for example,
Gray, et al. (1998) J. Immunol., 160: 2248, incorporated herein by reference
in its entirety).
If desired, B cells can be obtained from nonrosetting PBMC treated with 5 mM ~-
leucine methyl ester
(LME) for depletion of monocytes and NK cells. The cells so obtained ar then
stained with Abs to
CD3, CD16, and CD11b and depleted of reactive cells by immunomagnetic beads.
the resulting
CA 02469800 2004-06-09
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population is greater than 90% CD20+ and less than 0.5% CD3+ (see Zheng, et
al., (2002) J.
Immunol., 169: 4183-4189).
In some embodiments, T cell subsets, e.g., CD8+ or CD4+ T cells are not
isolated from the PBMC
until after treatment with a regulatory composition. In these embodiments, the
T cells are isolated
following treatment with a regulatory composition and one or more of the
treated T cell subsets are
returned to a patient with an immune disorder.
As will be appreciated by those of skill in the art, there are a number of
other ways to isolate T cells, in
addition to the preferred embodiments provided above.
Once purified or concentrated the cells may be aliquoted and frozen,
preferably, in liquid nitrogen or
used immediately as described below. Frozen cells may be thawed and used as
needed.
Cryoprotective agents, which can be used, include but are not limited to
dimethyl sulfoxide (DMSO)
(Lovelock, J. E. and Bishop, M. W. H., 1959, Nature 183:1394-1395; Ashwood-
Smith, M. J., 1961,
Nature 190:1204-1205), hetastarch, glycerol, polyvinylpyrrolidine (Rinfret, A.
P., 1960, Ann. N.Y.
Acad. Sci. 85:576), polyethylene glycol (Sloviter, H. A. and Ravdin, R. G.,
1962, Nature 196:548),
albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-
mannitol (Rowe, A. W., et al., 1962,
Fed. Proc. 21:157), D-sorbitol, i-inositol, D-lactose, choline chloride
(Bender, M. A., et al., 1960, J.
Appl. Physiol. 15:520), amino acids (Phan The Tran and Bender, M. A., 1960,
Exp. Cell Res. 20:651 ),
methanol, acetamide, glycerol monoacetate (Lovelock, J. E., 1954, Biochem. J.
56:265), and
inorganic salts (Phan The Tran and Bender, M. A., 1960, Proc. Soc. Exp. Biol.
Med. 104:388; Phan
The Tran and Bender, M. A., 1961, in Radiobiology Proceedings of the Third
Australian Conference
on Radiobiology, Ilbery, P. L. T., ed., Butterworth, London, p. 59 ).
Typically, the cells may be stored
in 10% DMSO, 50% serum, and 40% RPMI 1640 medium. Methods of cell separation
and purification
are found in U.S. Patent No. 5,888,499, which is expressly incorporated by
reference.
Generation of Regulatory T cells
Once isolated, the T cells may be treated with a regulatory composition to
generate activated
regulatory T cells with enhanced suppressive activity. By "regulatory T cells"
herein is meant CD8+ or
CD4+ T cell subsets that develop the ability to prevent cytotoxic T cell
activity in other T cells, inhibit
antibody production, suppress delayed type hypersensitivity responses, inhibit
monocyte, dendritic
cell or B cell function as antigen presenting cells, etc. As discussed above,
several regulatory T cell
subsets exist. These T cell subsets can be broadly divided into two
categories: (1 ) professional
regulatory T cells; and (2) cytokine-producing regulatory cells.
By "professional regulatory T cells" herein is meant a subset of CD4+ T cells
that constitutively
express the alpha chain of the IL-2 receptor complex, CD25. Thus, professional
regulatory T cells are
CD4+ CD25+. These cells exhibit a broad range of suppressive activities
including suppressing
activation of other T cells, down regulating antibody production, and
inhibiting cytotoxic T cell activity.
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These suppressive activities require the professional regulatory T cell to
directly bind to other cells
(i.e., contact-dependent) and deliver one or more inhibitory signals.
Cytokines, such as interleukin 10
or TGF-~3 s are not required for the induction of these suppressive activities
(i.e, cytokine-
independent); thus the activity is not abolished by the addition of
neutralizing monoclonal antibodies to
these cytokines.
Professional regulatory T cells can be further classified as virgin or
activated professional regulatory
cells. By "virgin professional regulatory T cells" herein is meant
professional regulatory T cells that
have not been treated with the regulatory compositions described herein. By
"activated professional
regulatory T cells" herein is meant professional regulatory T cells that have
been treated with the
regulatory compositions described herein, and as a result of the treatment
exhibit enhanced
suppressive activity. Activated professional regulatory cells are derived from
CD4+ cells.
By "cytokine producing regulatory T cells" herein is meant NK T cells, CD4+ or
CD8+ Th3 or Th3-like
cells that produce immunosuppressive~levels of TGF-Vii. As these cells do not
constitutively express
the alpha chain of the IL-2 receptor complex, they are CD4+ CD25- cells. While
cytokine producing
regulatory T cells can also suppress autoimmunity, they function principally
as general feedback
regulators of Th1 and Th2 cells. Activated cytokine producing regulatory T
cells can be generated
from CD4+ cells, CD8+ cells, or NK T cells that have been treated with a
regulatory composition.
In a preferred embodiment, treatment of PBMC or isolated T cell subsets
increases both the number
of regulatory T cells and their suppressive activity. As illustrated in Figure
1, the number of virgin
professional regulatory in a T cell population is less than 1 % of the total.
Following treatment with a
regulatory composition, the percent of professional regulatory T cells in the
population is increased
from less than 1 % to greater than 10%. Preferably, the percent of
professional regulatory T cells in
the population is increased to greater than 25%. More preferably, the percent
of professional
regulatory T cells in the population is increased to greater than 50%. More
preferably, the percent of
professional regulatory T cells in the population is increased to greater than
70%.
"Suppressive activity" herein refers to ability of a regulatory T cells to
inhibit the activation of other
lymphocytes, including T cells and B cells, monocytes, and dendritic cells. By
"enhanced"
suppressive activity" herein is meant regulatory T cells that have been
activated in the presence of a
regulatory composition comprising TGF-Vii. These regulatory T cells are able
to inhibit the activity of
other immune cells in fewer numbers than non-conditioned regulatory cells. The
suppressive activity
of professional regulatory T cells can be determined using an allogeneic mixed
lymphocyte reaction
as is known in the art and described in several of the Figures. For example,
professional regulatory
CD4+ CD25+ T cells isolated from lymphoid tissues have suppressive activity
when added to other
cells at 1:1 to 1:4 ratios (Shevach, E.M. (2000) Regulatory T cells in
autoimmmunity. Annu. Rev.
Immunol.,18, 423-449). By contrast, 1:10 to less than 1:100 activated CD4+
CD25+ cells generated
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in the presence of a regulatory composition comprising TGF-~i exert strong
suppressive effects
(Yamagiwa et al, (2001) J. Immuno. 166: 7282-89).
Once isolated, the cells are treated with a regulatory composition. By
"treated" herein is meant that
the cells are incubated with the regulatory composition for a time period
sufficient to develop
regulatory T cell activity. The incubation will generally be under
physiological temperature.
By "regulatory composition" herein is meant a composition that can induce T
cells to suppress
undesirable immune responses. By "undesirable immune responses" herein is
meant immune
disorders characterized by the failure of the immune system to distinguish
self from non-self or to
respond to foreign antigens, or immune responses to transplanted tissues.
Thus, undesirable
immune responses include, but are not limited to, inhibition of T cell
activation, inhibition of
spontaneous antibody and autoantibody production, or cytotoxicity, or both.
Regulatory compositions may comprise a number of components, including: (1)
cytokines; (2)
stimulator cells (i.e., irradiated T cell-depleted mononuclear cells (see
U.S.S.N. 09/833,526); (3) T cell
activators; (4) non T accessory cells; and (5) anti-cytokine neutralizing
monoclonal antibodies.
The concentration of the regulatory composition will vary depending on the
identity of the compounds
included in the composition, but will generally be at physiologic
concentration, i.e. the concentration
required to give the desired effect, i.e. an enhancement of specific types of
regulatory cells.
Generally, regulatory compositions include cytokines. Suitable cytokines
include, but are not limited
to, IL-2, IL-4, IL-5, IL-15, TGF-p and TNF-a. Preferred cytokines include IL-
2, IL-15 and TGF-(3.
In a preferred embodiment, TFG-p is a component the regulatory composition. By
"transforming
growth factor -p" or "TGF-~~~ herein is meant any one of the family of the TGF-
ps, including the three
isoforms TGF-p1, TGF-p2, and TGF-p3; see Massague, J. (1980), J. Ann. Rev.
Ce118io16:597.
Lymphocytes and monocytes produce the t31 isoform of this cytokine (Kehrl,
J.H. et al. (1991), Inf J
Cell Cloning 9: 438-450). The TFG-a can be any form of TFG-p that is active on
the mammalian
cells being treated. In humans, recombinant TFG-p is currently preferred. In
general, the
concentration of TGF-p used ranges from about 2 picograms/ml of cell
suspension to about 10
nanograms, with from about 10 pg to about 4 ng being preferred, and from about
100 pg to about 2 ng
being especially preferred, and 1 ng/ml being ideal.
In a preferred embodiment, IL-2 is used in the regulatory composition. The IL-
2 can be any form of IL-
2 that is active on the mammalian cells being treated. In humans, recombinant
IL-2 is currently
preferred. In general, the concentration of IL-2 used ranges from about 1
Unitlml of cell suspension to
about 100 U/ml, with from about 5 U/ml to about 25 Uiml being preferred, and
with 10 Ulml being
especially preferred. In a preferred embodiment, IL-2 is not used alone.
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In a preferred embodiment, IL-15 is used in the regulatory composition. The IL-
15 can be any form of
IL-15 that is active on the mammalian cells being treated. In humans,
recombinant IL-15 is currently
preferred. In general, the concentration of IL-15 used ranges from about 1
Unit/ml of cell suspension
to about 100 U/ml, with from about 5 U/ml to about 25 U/ml being preferred,
and with 10 U/ml being
especially preferred. In a preferred embodiment, IL-15 is not used alone.
In a preferred embodiment, the regulatory composition comprises T cell
activators. Suitable T cell
activators include soluble antigens, peptide fragments of antigens,
alloantigens, anti-CD2, anti-CD3,
anti-CD28, LFA-3, and mitogens. As will be appreciated by those of skill in
the art, anti-CD3, soluble
antigens, and peptide fragments of antigens are T cell receptor (TCR)
activators.
CD2 is a cell surface glycoprotein expressed by T lymphocytes. By "CD2
activator" herein is meant
compound that will initiate the CD2 signaling pathway. A preferred CD2
activator comprises anti-CD2
antibodies (OKT11, American Type Culture Collection, Rockville MD and GT2,
Huets, et al., (1986) J.
Immunol. 137:1420). In addition, a combination of anti-CD2 antibodies can be
used, including the
CD2 ligand LFA-3, in the regulatory composition. In general, the concentration
of CD2 activator used
will be sufficient to induce the production of TGF-p. The concentration of
anti-CD2 antibodies used
ranges from about 1 ng/ml to about 10 uglml, with from about 10 nglml to about
100 ng/ml being
especially preferred.
In some embodiments it is desirable to use a mitogen to activate the cells;
that is, many resting phase
cells do not contain large amounts of cytokine receptors. The use of a mitogen
such as Concanavalin
A (ConA) or staphylococcus enterotoxin B (SEB) can allow the stimulation of
the cells to produce
cytokine receptors, which in turn makes the methods of the invention more
effective. When a mitogen
is used, it is generally used as is known in the art, at concentrations
ranging from 1 ug/ml to about 10
ug/ml is used. In addition, it may be desirable to wash the cells with
components to remove the
mitogen, such as a-methyl mannoside, as is known in the art.
In a preferred embodiment, non T accessory cells or an equivalent surrogate
are used in the
regulatory composition. Non T accessory cells that may be included in the
regulatory composition are
B cells, macrophages, monocytes, and dendritic cells.
In a preferred embodiment, anti-cytokine neutralizing monoclonal antibodies
are used in the
regulatory composition. Suitable anti-cytokine neutralizing monoclonal
antibodies include anti-TGF-Vii.
In some embodiments, such as the generation of regulatory T cells for use in
graft rejection or Gi/HD,
stimulator cells (e.g. irradiated T cell depleted cells) are included in the
regulatory composition (see
U.S.S.N.s. 09/653,924 and 09/833,526).
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Accordingly, a regulatory composition comprising at least one of the above
components may be used
to generate activated regulatory T cells. The regulatory compositions may
contain more than one
compound from the same class of compounds, i.e., two or more cytokines, may be
mixed together.
The composition also may contain compounds from different classes of
compounds, such as a
cytokine and a T cell activator, etc. Thus, regulatory compositions
containing: (1) one cytokine; (2)
two or more cytokines; (3) at least one cytokine, and a T cell activator; (4)
at least one cytokine, a T
cell activator, and a stimulator; (5) at least one cytokine, a T cell
activator, and non T accessory cells;
and (6) at least one cytokine, a T cell activator, and an anti-cytokine
antibody, (7) at least one cytokine
and at least one T-cell activator with or without non T accessory cells; and
(8) at least one cytokine, at
least one T-cell activator, at least one anti-cytokine antibody, with or
without non T accessory cells;
and (9) at least one cytokine, at least one T-cell activator, at least one
stimulator, at least one anti-
cytokine antibody, with or without non T accessory cells, may be used to
generate activated
regulatory T cells. As will be appreciated by those of skill in the art, the
above list of combinations is
not meant to be exhaustive, but is provided as examples for the possible
combinations of components
that may be included in the regulatory compositions of the present invention.
As will be appreciated by those of skill in the art, the combination that is
used will depend on whether
T cell proliferation, T cell differentiation, or both is the desired outcome.
Moreover, the number of
cytokine-producing regulatory cells to professional regulatory cells that are
generated by a given
treatment will vary depending on the percentage of CD4+ CD25+ cells in the
starting population, the
nature of the T cell activator and signals provided by non-T accessory cells.
In a preferred embodiment, IL-2 and TGF-(3 are used together to generate
activated regulatory T cells
with enhanced suppressive activity. As will be appreciated by those of skill
in the art, both
professional regulatory T cells and cytokine-producing T cells are produced
using this regulatory
composition. Moreover, professional regulatory T cells and cytokine-producing
T cells may be
generated from various T cell subsets, including CD4+, CD8+, naive CD4+ cells,
etc.
In a preferred embodiment, IL-15 and TGF-(3 are used together to generate
activated regulatory T
cells with enhanced suppressive activity. As will be appreciated by those of
skill in the art, both
professional regulatory T cells and cytokine-producing T cells are produced
using this regulatory
composition. Moreover, professional regulatory T cells and cytokine-producing
T cells may be
generated from various T cell subsets, including CD4+, CD8+, naive CD4+ cells,
etc.
In a preferred embodiment, IL-2, TGF-Vii, and a CD2 activator, are used to
generate activated
regulatory T cells with enhanced suppressive activity. As will be appreciated
by those of skill in the
art, both professional regulatory T cells and cytokine-producing T cells are
produced using this
regulatory composition. Moreover, professional regulatory T cells and cytokine-
producing T cells may
be generated from various T cell subsets, including CD4+, CD8+, naive CD4+
cells, etc. ~ther
preferred combinations include IL-15, TGF-(3, and a CD2 activator.
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In a preferred embodiment, IL-2, IL-15, and TGF-~i are used to generate
activated regulatory T cells
with enhanced suppressive activity.
In a preferred embodiment, IL-2, TGF-(3, and an anti-cytokine antibody are
used to generate activated
regulatory T cells with enhanced suppressive activity. Other preferred
combinations include IL-15,
TGF-Vii, and an anti-cytokine .
In a preferred embodiment, IL-2, TGF-Vii, and a TCR activator are used to
generate T cells with
suppressive activity. Other preferred combinations include IL-2, TGF-Vii, a
TCR activator, and non T
accessory cells; IL-15, TGF-Vii, and a TCR activator; IL-15, TGF-~3, a TCR
activator, and non T
accessory cells.
As will be appreciated by those of skill in the art, repeated stimulation of
the T cells with our without a
regulatory composition in secondary cultures may be necessary to develop
maximal suppressive
activity.
In a preferred embodiment, T cell activators are used to generate cytokine
producing regulatory T
cells. In this embodiment, the T cell activators are used in combination with
conventional T cells and
activated professional regulatory T cells to generate activated cytokine
producing regulatory T cells.
Once the cells have been treated, they may be evaluated for suppressive
activity and suitability for
transplantation into a patient. For example, a sample may be removed for:
sterility testing; gram
staining, microbiological studies; LAL studies; mycoplasma studies; flow
cytometry to identify cell
types; functional studies, etc. These and other lymphocyte studies may be done
before and after
treatment. A preferred analysis is to label a test or target population of
cells that are capable of
eliciting an immune response in the treated T cells, incubate the treated T
cells with the labeled
population, and determine cell survival as a measure of suppressive activity
(see Figures). Assays,
such as those described in Example 1 and in the brief description of the
Figures may be used to
determine suppressive activity.
Assays such as those described in U. S. Patent No. 6,358,506, incorporated
herein by reference in its
entirety, can also be used to identify professional regulatory T cells. The
addition of neutralizing
antibodies and IL-10 to a population of treated cells can be used to identify
cytokine-producing T cells
(see Example 1 ).
lJses for Regulatory T cells
Once generated, T cells with suppressive activity may be administered to
alleviate an immune
response in a patient. By "immune response" herein is meant host responses to
foreign or self
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antigens. Preferably, T cells with suppressive activity are used to prevent
aberrant immune response
or undesirable immune responses to foreign antigens. By "aberrant immune
responses" herein is
meant the failure of the immune system to distinguish self from non-self or
the failure to respond to
foreign antigens. In other words, aberrant immune responses are
inappropriately regulated immune
responses that lead to patient symptoms. By "inappropriately regulated" herein
is meant
inappropriately induced, inappropriately suppressed and/or non-responsiveness.
Aberrant immune
responses include, but are not limited to, tissue injury and inflammation
caused by the production of
antibodies to an organism's own tissue, impaired production of IL-2, TNF-a and
IFN-y and tissue
damage caused by cytotoxic or non-cytotoxic mechanisms of action. By
"undesirable immune
responses" herein is the responses to foreign antigens observed in transplant
patients. Thus,
undesirable immune responses include responses associated with GVHD and graft
rejection.
By "patient" herein is meant a mammalian subject to be treated, with human
patients being preferred.
In some cases, the methods of the invention find use in experimental animals,
in veterinary
applications, and in the development of animal models for disease, including,
but not limited to,
rodents including mice, rats, and hamsters; and primates.
In a preferred embodiment, the present invention inhibits aberrant immune
responses. In patients
with antibody-mediated autoimmune disorders, the present invention restores
the capacity of
peripheral blood T cells to down regulate antibody production and restores
cell mediated immune
responses by treating them with an regulatory composition ex vivo. In patients
with cell-mediated
disorders, the present invention generates regulatory T cells which suppress
cytotoxic T cell activity in
other T cells.
Accordingly, in a preferred embodiment, the present invention provides methods
of treating antibody-
mediated autoimmune disorders in a patient. By "antibody-mediated autoimmune
diseases" herein is
meant a disease in which individuals develop antibodies to constituents of
their own cells or tissues.
Antibody-mediated autoimmune diseases include, but are not limited to,
systemic lupus
erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia,
thrombocytopenia
purpura, Grave's disease, dermatomyositis and Sjogren's disease. The preferred
autoimmune
disease for treatment using the methods of the invention is SLE.
In addition, patients with antibody-mediated disorders frequently have defects
in cell-mediated
immune responses. By "defects in cell mediated immune response" herein is
meant impaired host
defense against infection. Impaired host defense against infection includes,
but is not limited to,
impaired delayed hypersensitivity, impaired T cell cytotoxicity and impaired
production of TGF-Vii.
Other defects, include, but are not limited to, increased production of IL-10
and decreased production
of IL-2, TNF-a and IFN-y. Using the methods of the present invention, purified
T cells are stimulated
to increase production of IL-2, TNF-a and IFN-y and decrease production of IL-
10. T cells which can
be stimulated using the current methods include, but are not limited to, CD4+
and CD8+.
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In one embodiment, antibody-mediated disorders are not treated.
In a preferred embodiment, the present invention provides methods of treating
cell-mediated
autoimmune disorders in a patient. By "cell-mediated autoimmune diseases"
herein is meant a
disease in which the cells of an individual are activated or stimulated to
become cytotoxic and attack
their own cells or tissues. Alternatively, the autoimmune cells of the
individual may stimulate other
cells to cause tissue damage by cytotoxic or non-cytotoxic mechanisms of
action. Cell-mediated
autoimmune diseases include, but are not limited to, Hashimoto's disease,
polymyositis, disease
inflammatory bowel disease, multiple sclerosis, diabetes mellitus, rheumatoid
arthritis, and
scleroderma.
By "treating" an autoimmune disorder herein is meant that at least one symptom
of the autoimmune
disorder is ameliorated by the methods outlined herein. This may be evaluated
in a number of ways,
including both objective and subjective factors on the part of the patient.
For example, immunological
manifestations of disease can be evaluated; for example, the level of
spontaneous antibody and
autoantibody production, particularly IgG production in the case of SLE, is
reduced. Total antibody
levels may be measured, or autoantibodies, including, but not limited to, anti-
double-stranded DNA
(ds DNA) antibodies, anti-nucleoprotein antibodies, anti-Sm, anti-Rho, and
anti-La. Cytotoxic activity
can be evaluated as outlined herein. Physical symptoms may be altered, such as
the disappearance
or reduction in a rash in SLE. Renal function tests may be performed to
determine alterations;
laboratory evidence of tissue damage relating to inflammation may be
evaluated. Decreased levels of
circulating immune complexes and levels of serum complement are further
evidence of improvement.
In the ease of SLE, a lessening of anemia may be seen. The ability to decrease
a patient's otherwise
required drugs such as immunosuppressives can also be an indication of
successful treatment. Other
evaluations of successful treatment will be apparent to those of skill in the
art of the particular
autoimmune disease.
In a preferred embodiment, the quantity or quality, i.e. type, of antibody
production, may be evaluated.
Thus, for example, total levels of antibody may be evaluated, or levels of
specific types of antibodies,
for example, IgA, IgG, IgM, anti-DNA autoantibodies, anti-nucleoprotein (NP)
antibodies, etc. may be
evaluated. Regulatory T cells may also be assessed for their ability to
suppress T cell activation or to
prevent T cell cytotoxicity against specific target cells in vitro (see U.S.
Patent No. 6,358,506,
incorporated herein by reference in its entirety).
After the treatment, the cells are transplanted or reintroduced back into the
patient. This is generally
done as is known in the art, and usually comprises injecting or introducing
the treated cells back into
the patient, via intravenous administration, as will be appreciated by those
in the art. For example,
the cells may be placed in a 50 ml Fenwall infusion bag by injection using
sterile syringes or other
sterile transfer mechanisms. The cells can then be immediately infused via IV
administration over a
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period of time, such as 15 minutes, into a free flow IV line into the patient.
In some embodiments,
additional reagents such as buffers or salts may be added as well.
After reintroducing the cells into the patient, the effect of the treatment
may be evaluated, if desired,
as is generally outlined above. Thus, evaluating immunological manifestations
of the disease may be
done; for example the titers of total antibody or of specific immunoglobulins,
renal function tests,
tissue damage evaluation, etc. may be done. Tests of T cells function such as
T cell numbers,
phenotype, activation state and ability to respond to antigens and/or mitogens
also may be done.
The treatment may be repeated as needed or required. For example, the
treatment may be done
once a week for a period of weeks, or multiple times a week for a period of
time, for example 3-5
times over a two week period. Generally, the amelioration of the autoimmu ne
disease symptoms
persists for some period of time, preferably at least months. Over time, the
patient may experience a
relapse of symptoms, at which point the treatments may be repeated.
i4its
In a preferred embodiment, the invention further provides kits for the
practice of the methods of the
invention, i.e., the incubation of cells with the regulatory compositions. The
kit may have a number of
components. For example, the kit may comprise a cell treatment container that
is adapted to receive
cells from a patient with an antibody-mediated or cell-mediated autoimmune
disorder. The container
should be sterile. In some embodiments, the cell treatment container is used
for collection of the
cells, for example it is adaptable to be hooked up to a leukophoresis machine
using an inlet port. In
other embodiments, a separate cell collection container may be used.
In a preferred embodiment, the kit may also be adapted for use in a automated
closed system to
purify specific T cell subsets and expand them for transfer back to the
patient.
The form and composition of the cell treatment container may vary, as will be
appreciated by those in
the art. Generally the container may be in a number of different forms,
including a flexible bag, similar
to an IV bag, or a rigid container similar to a cell culture vessel. It may be
configured to allow stirring.
Generally, the composition of the container will be any suitable, biologically
inert material, such as
glass or plastic, including polypropylene, polyethylene, etc. The cell
treatment container may have
one or more inlet or outlet ports, for the introduction or removal of cells,
reagents, regulatory
compositions, etc. For example, the container may comprise a sampling port for
the removal of a
fraction of the cells for analysis prior to reintroduction into the patient.
Similarly, the container may
comprise an exit port to allow introduction of the cells into the patient; for
example, the container may
comprise an adapter for attachment to an IV setup.
The kit further comprises at least one dose of an regulatory composition.
"Dose" in this context
means an amount of the regulatory composition such as cytokines, that is
sufficient to cause an
effect. In some cases, multiple doses may be included. In one embodiment, the
dose may be added
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to the cell treatment container using a port; alternatively, in a preferred
embodiment, the dose is
already present in the cell treatment container. In a preferred embodiment,
the dose is in a lyophilized
form for stability, that can be reconstituted using the cell media, or other
reagents.
In some embodiments, the kit may additionally comprise at least one reagent,
including buffers, salts,
media, proteins, drugs, etc. For example, mitogens, monoclonal antibodies and
treated magnetic
beads for cell separation can be included.
In some embodiments, the kit comprise written instructions for using the kit.
The following examples serve to more fully describe the manner of using the
above described
invention, as well as to set forth the best modes contemplated for carrying
out various aspects of the
invention. It is understood that these examples in no way serve to limit the
true scope of this
invention, but rather are presented for illustrative purposes. All references
cited herein are
incorporated by reference in their entirety.
EXAMPLES
Example 1
Effects of TGF-(3 co-stimulation on CD4+ and CD8+ T cells
Effect on Growth of CD4+ and CD8+ cells
As shown in Figure 1, co-stimulation by TGF-~i markedly increases the
percentage and absolute
numbers of total CD4+ CD25+ and CD4+ CD25- cells. However, the increase in
CD4+ CD25- cells
was dependent upon the CD4+ CD25+ subset, as depletion of the CD4+ CD25+
subset abolished the
growth promoting effects of TGF-Vii. A similar, but smaller effect was
observed with CD8+ cells.
In these experiments, CD4+ or CD8+ cells were depleted of CD25+ cells by
staining with anti-CD25.
Stained cells were removed using immunomagnetic beads. Total T cell subsets
and CD25 depleted T
cell subsets were mixed with allogeneic irradiated non-T cells and cultured
for 6 days with grade d
amounts of TGF-Vii. At the conclusion of the culture period, the total number
of each subset and those
that expressed CD25 was determined.
Effect on the Expansion of CD4+ expressing different cell surtace markers
Figures 2A and 2B illustrate the expression of cell surface markers on CD4+
subsets after stimulation
by TGF-~i in an allogeneic mixed lymphocyte reaction. Total CD4+ cells, CD4+
cells depleted of
CD25, and naive CD45RA+ CD45R0- cells were studied. A dose dependent effect of
TGF-~i on the
expression CD25 on total CD4+ cells and naive cells was observed. Depletion of
CD25 in the starting
population abolished this effect. Thus, TGF-Vii, appears to expand the CD4+
CD25+ subset of CD4+
cells.
CA 02469800 2004-06-09
WO 03/059264 PCT/US02/41288
A similar TGF-~i dose-dependent effect was observed in the expression of CD62L
(L selectin) on
CD4+ subsets. This result is consistent with the results of others showing
that CD62L is expressed
by professional CD$+ CD25+ cells. Co-stimulatory effects of TGF-(3 were also
in CD4+ CD25- cells.
In addition, TGF-~i also increased the expression of CTLA-4 and CD122, the ~i
chain of the IL-2
receptor.
Effect on Suppressive Activity of CD4+ Subsets
Figures 3A-D depict the effect of TGF-(3 in inducing suppressive activity by
various CD4+ T cell
subsets. In these experiments, purified CD4+ T cell subsets were obtained by
cell sorting and
conditioned with TGF-[3 (1 ng/ml) in an alto mixed lymphocyte reaction (MLR)
as described above.
The purified CD4+ subsets were thene tested for their ability to inhibit the
generation of T cell
cytotoxicity. Figures 3A and B show that purified CD4+ CD25+ T cells have
significant suppressive
acitivty and that this activity is significantly increased, i.e., enhanced, by
conditioning with TGF-Vii.
Figures 3C and 3D show the TGF-(3 has similar effects, i.e., a marked increase
in the suppressive
activity, on other T cell subsets: CD45RA+ CD45R0- CD25-, and CD45RA- CD45R0+
subsets of
CD4+ cells. Addition of IL-2 is not required for this enhancement of
suppressive activity.
The addition of neutralizing monoclonal antibodies and IL-10 blocked the
suppressive activity of these
cells, suggesting that al least some of the observed suppressive activity is
cytokine-dependent.
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