Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND CO~OSITIONS FOR THE SELECTIVE EXPANSION OF GAMMA/DELTA T-CELLS
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to methods for expanding gamma-delta (y8) T
cells in hematolymphoid cell populations for the purpose of generating cell
populations
enriched for y8 T cells. Such enriched populations can be used in a variety of
applications, including therapeutic uses such as treatment of cancer and
infectious
diseases, promotion of wound healing and enhancement of bone man ow
engraftment,
as well as for functional and structural studies of ys T cells and their
interactions with
other components of the immune system.
Background Art
The subset of T cells known as y8 T cells is normally present in
hematolymphoid cell populations as a very small fraction. Although the role of
y8 T
cells is not fully understood, data from correlative clinical studies in
humans and
experimental data from murine models suggest that y8 T cells may serve to
facilitate
hematopoietic stem cell (HSC) engraftment in the setting of allogeneic bone
marrow
transplant (BMT). Current models postulate that yS T cells may facilitate
engraftment
across major histocompatibility complex {MHC) burners by eliminating or
suppressing
the function of host-derived cellular elements capable of rejecting donor HSC.
There is
also evidence to suggest that ys T cells can play a beneficial role in the
control of
infectious disease, in inhibiting tumor growth and in promoting wound healing.
In
order to employ y8 T cells at a clinical scale, it would be necessary to
isolate or expand
y8 T cells to a significant degree, given their relative infrequency in
peripheral blood
and other hematological tissues. Isolating y8 T cells from fresh peripheral
blood (PB)
or bone marrow (BM) is impractical and prohibitively expensive. Attempts to
expand
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y8 T cells ex vivo using a variety of standard mitogenic stimuli, including
anti-CD3
antibodies or anti-TCR y8 antibodies have yielded y8 T cells which are less
functional
in vivo (26). The reason for such decreased function may be related to
observations
that, in humans, y8 T cells are extremely sensitive to TCR/CD3 engagement
(especially
S in the presence of IL-2) and undergo apoptosis upon receiving mitogenic
stimuli
through the TCR (27). In support of this finding, studies have shown that
human y8 T
cell clones readily undergo apoptosis when stimulated simultaneously by anti-
CD3/TCR MAb plus exogenous IL-2 (28,29). This mechanism of activation-induced
cell death (AICD) has not previously been overcome, thereby significantly
limiting the
usefulness of the y8 T cells expanded as previously described.
Furthermore, enrichment of resting yS T cells without cytokine and/or mitogen
activation, by isolating this cell fraction from a population of
hematolymphoid cells and
pooling the isolated y8 T cells from several populations to increase their
number has
1 S not proven useful for obtaining y8 T cells in the amounts needed for
clinical use.
Specifically, y8 T cells have a very limited life span (about 1-2 weeks) and
by the time
a sufficient number of y8 T cells could be isolated and pooled from primary
cultures,
most of the cells will have died or would be very near death. Such cells would
not be
useful for administration to a subject for the various clinical applications
described
herein because the y8 T cells would not survive long enough in the subject to
facilitate
engraftment, inhibit an infectious process, inhibit tumor growth or promote
wound
healing. Thus, what is needed is a method of expanding y8 T cells which can
survive
both ex vivo for a period of time sufficient to produce sufficiently large
numbers of y8
T cells for clinical use and in vivo for a period of time sufficient to impart
their
intended clinical effect.
The present invention overcomes previous shortcomings in the art by providing
methods for increasing the percentage of y8 T cells, which can survive for
prolonged
periods, in a population of hematolymphoid cells and for administering
hematolymphoid cell populations which are enriched for these y8 T cells to
subjects to
treat cancer, treat infections, promote wound healing and enhance transplant
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engraftment. The present invention also provides populations of hematolymphoid
cells
having increased percentages of y8 T cells which can survive for prolonged
periods.
SUMMARY OF THE INVENTION
The present invention provides a method of increasing the percentage of
gamma-delta T cells in a population of hematolymphoid cells and which gamma-
delta
T cells can survive for a prolonged period, comprising: a) contacting a
population of
hematolymphoid cells with interleukin 12 and a ligand of CD2 which induces
responsiveness to interleukin 12; and b) contacting the cells of step (a) with
an antibody
to CD3 and interleukin-2.
Also provided is a method of screening a ligand of CD2 for the ability to
induce
responsiveness to interleukin 12 comprising: a) contacting a population of
1 S hematolymphoid cells with the ligand and interleukin 12; b) contacting the
cells of step
(a) with an antibody to CD3 and interleukin 2; c) maintaining the cells of
step (b) in
culture for at least seven days; and d) determining the percentage of viable
gamma delta
T cells in the population of cells of step (c), whereby greater than 10%
viable gamma
delta T cells identifies a ligand of CD2 having the ability to induce
responsiveness to
interleukin-12.
Further provided is a population of hematolymphoid cells having greater than
10% gamma delta T cells and which gamma-delta T cells can survive for a
prolonged
period.
The present invention also provides a method of treating cancer, treating an
infection, promoting wound healing and enhancing bone marrow engraftment in a
subject comprising administering to the subject an effective amount of the
cells of this
mvent~on.
Various other objectives and advantages of the present invention will become
apparent from the following detailed description.
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DETAILED DESCRIPTION OF THE INVENTION
As used herein, "a" or "an" can mean multiples. For example, "a cell" can
mean at least one cell or more than one cell.
The present invention is based on the surprising discovery that the percentage
of
y8 T cells in a population of hematolymphoid cells can be increased to
percentages
never before achieved by the administration to the cell population of a
particular
combination of cytokines and mitogenic stimuli in a specific order. In
particular, this
invention provides the discovery that y8 T cells can be expanded in a
population of
hematolymphoid cells by first administering IL-12 and a ligand of CD2,
followed by
administering IL-2 and T cell mitogenic stimulus. A further unexpected
discovery is
that the expanded y8 T cells are capable of surviving for a prolonged period
of time,
making them very useful as source populations for studying the functional and
1 S structural aspects of y8 T cells, for identifying additional cytokines
and/or other
substances which activate y8 T cells and for analyzing their interactions with
other
immune components, as well as for developing clinical uses for these cells.
Thus, the present invention provides a method of increasing the percentage of
gamma-delta T cells in a population of hematolymphoid cells and which gamma-
delta
T cells can survive for a prolonged period, comprising: a) contacting a
population of
hematolymphoid cells with interleukin 12 and a ligand of CD2 which induces
responsiveness to interleukin 12; and b) contacting the cells of step (a) with
a T cell
mitogen and interleukin-2. In addition, the method of this invention can
further
comprise the step of contacting the cells of step (a) with interferon-y.
That the percentage of y8 T cells in the population of hematolymphoid cells
has
increased as a result of this method can be determined according to standard
methods
well known in the art and as described herein for determining percentages of
various
cell types in a mixed population of cells. For example, the percentage of y8 T
cells can
be measured in a population of hematolymphoid cells by fluorescence activated
cell
sorting (FACS) as described in the Examples herein.
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As used herein, y8 T cells "which can survive for a prolonged period" means
y8 T cells which can survive under the culture conditions described herein for
a period
of time which is greater than the period of time during which hematolymphoid
cells in
primary culture normally survive. For example, a prolonged period of time for
survival
of y8 T cells means survival of y8 T cells for greater than three weeks and
more
preferably, for greater than six weeks and most preferably, for greater than
eight weeks,
under the culture conditions described herein. It is contemplated that the y8
T cells of
this invention could be kept alive and functional for the methods described
herein
indefinitely upon subsequent restimulation of the y8 T cells according to the
methods
described herein.
Also as used herein, "a ligand of CD2 which induces responsiveness to
interleukin 12" means any natural or synthetic molecule, including antibodies
to CD2,
that, upon interaction with CD2 itself, results in the generation of the
cellular and
molecular events of signal transduction, thus increasing a cell's
responsiveness to IL-
12. By responsiveness to IL-12 is meant that the cells which bind the ligand
of CD2
are enhanced in their ability to bind and/or respond to IL-12. A ligand of CD2
which
can be used in the methods of this invention can include, but is not limited
to, an
antibody or antibody fragment which specifically binds CD2, CD58, a natural or
synthetic homologue of CD58, a receptor-binding fragment of CD58, CD48, a
natural
or synthetic homologue of CD48 and a receptor-binding fragment of CD48, any of
which induce responsiveness to IL-12. A ligand of CD2 can be screened for the
ability
to induce responsiveness to interleukin-12 according to the methods described
herein.
For example, the ligand of CD2 can be the monoclonal antibody S5.2 (mouse
IgG2a,
Becton Dickinson).
It is also appreciated by one of skill in the art that IL-12 and IL-2 can
include
fragments of IL-12 or IL-2, respectively, which retain the binding and signal
transducing activity of an entire IL-12 or IL-2 molecule.
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The T cell mitogen of this invention can be any substance, now known or later
identified to have a mitogenic effect on T cells. For example, the T cell
mitogen of this
invention can be, but is not Limited to, an antibody to CD3, pokeweed mitogen,
ionomycin, phorbol myristate acetate (PMA), a superantigen (e.g., substances,
such as
bacterial products, which activate the T cell receptor nonspecifically by
binding to less
polymorphic domains) and any other T cell mitogen now known or later
identified to be
a T cell mitogen.
Thus, the present invention further provides a method of screening a ligand of
CD2 for the ability to induce responsiveness to interleukin 12 comprising: a)
contacting
a population of hematolymphoid cells with the ligand and interleukin 12; b)
contacting
the cells of step (a) with an antibody to CD3 and interleukin 2; c)
maintaining the cells
of step (b) in culture for at least seven days; and d) determining the
percentage of viable
gamma delta T cells in the population of cells of step (c), whereby greater
than 10%
viable gamma delta T cells identifies a ligand of CD2 having the ability to
induce
responsiveness to interleukin-12. The screening method of this invention can
further
comprise the step of contacting the cells of step (a) with interferon gamma.
The
percentage of viable y8 T cells can be determined according to the methods
provided in
the Examples herein and as are well known in the art. For example, the y8 T
cells can
be selectively separated from other cell types by FACS, as described herein
and the
viability of the y8 T cells can be determined by trypan blue staining, as also
described
herein.
As used herein, "hematolymphoid cells" means any cells derived froze bone
marrow precursor cells, which comprise myeloid cells, erythroid cells,
lymphoid cells,
platelets and the like, as is well known in the art. The hematolymphoid cells
which can
be used in the methods of this invention can be bone marrow cells, peripheral
blood
mononuclear cells and/or cord blood cells, as well as cells from any tissue in
which y8
T cells and/or their precursors can be found (e.g., skin, intestinal
epithelium, thymus,
liver, spleen, fetal tissues such as fetal liver and fetal thymus).
Furthermore, the
hematolymphoid cells of this method can be from any animal which produces y8 T
cells, which can be any mammal and in a preferred embodiment, is a human.
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Furthermore, the hematolymphoid cells used in the methods of this invention
can be cells which are removed from a subject and returned to the subject
(autologous).
Additionally, the hematolymphoid cells can be removed from a donor and
administered
to a recipient of the same species, that is a genetically different
(allogeneic); removed
from a donor and administered to a recipient that is genetically identical
(syngeneic),
and/or removed from a donor and administered to a recipient of a different
species
(xenogeneic).
The subject, donor and/or recipient of the present invention can be diagnosed
with cancer (e.g, leukemia, lymphoma and solid tumors) tissue injury (e.g.,
due to
trauma, burns, graft-versus-host disease or autoimmune destructive processes)
and/or
an infection by a pathogen (e.g., human immunodeficiency virus or other
pathogenic
virus, pathogenic bacteria, parasites, mycoplasma, pathogenic fungi, etc.).
In addition, the hematolymphoid cells used in the methods of this invention
can
be removed from a donor and administered to a transplant recipient. The
transplant
recipient can be diagnosed with cancer, tissue injury and/or an infection by a
pathogen.
Thus, the cells of this invention can be used to enhance engraftment of the
recipient's
transplanted tissue and/or treat the recipient's cancer, infection and/or
tissue injury.
The population of hematolymphoid cells in which the percentage of y8 T cells
is increased by the methods described herein can be further enriched for y8 T
cells to
yield a population of hematolymphoid cells having any percentage of y8 T
cells, up to
100%'y8 T cells. Such enriched cell populations can be administered, in a
pharmaceutically acceptable Garner, to a subject for a variety of therapeutic
treatments,
as described herein. Thus, the method of increasing the percentage of y8 T
cells in a
population of hematolymphoid cells as described herein can also comprise the
step of
further increasing the percentage of y8 T cells in the population of
hematolymphoid
cells of step (c) of the above-described method by procedures well known in
the art and
as described herein, such as selective separation of 'y8 T cells by
fluorescence activated
cell sorting (FACS), affinity column chromatography, immunomagnetic
separation,
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density gradient centrifugation and cellular panning, as well as any other
method for
selectively separating cell subpopulations, as would be well known to an
artisan. In a
preferred embodiment, the y8 T cells are selectively separated by FACS with
negative
staining methods, as described in the Example section herein, to avoid
stimulation of
the y8 T cells.
The present invention also provides a population of hematolymphoid cells
having greater than 10% y8 T cells and which y8 T cells s can survive for a
prolonged
period, as defined herein. The population of hematolymphoid cells of this
invention
can have greater than 20%y8 T cells, greater than 30%y8 T cells, greater than
40% y8
T cells, greater than 50%y8 T cells, greater than 60% y8 T cells, greater than
70% y8
T cells, greater than 80% y8 T cells and greater than 90% y8 T cells and can
have any
percentage of y8 T cells up to 100% 'y8 T cells.
The hematolymphoid cells having the percentages of y8 T cells listed above can
be bone marrow cells, peripheral blood mononuclear cells and/or cord blood
cells, as
well as cells from any tissue in which y8 T cells and/or their precursors can
be found,
as described herein. Thus, typically the other cell types included in the
population of
hematolyrnphoid cells can include but are not limited to, red blood cells,
platelets,
white blood cells (e.g., neutrophils, eosinophils, basophils, monocytes,
lymphocytes),
tissue cells (e.g, macrophages, epithelial cells, endothelial cells, dendritic
cells, mast
cells) and the like as would be well known to an artisan. Furthermore, the
hematolymphoid cells of this invention can be from any animal which produces
y8 T
cells, which can be any mammal and in a preferred embodiment is a human.
As described above, the cells of this invention can be administered to a
subject
to treat various disorders as well as to enhance transplant engraftment. Thus,
the
present invention provides a method of treating cancer in a subject comprising
administering to a subject diagnosed with cancer an effective amount of the
cells of this
invention. The cancer of this invention can be any malignant blood disorder,
acute
and/or chronic leukemia, multiple myeloma, lymphoma, solid tumors (e.g.,
breast
cancer, pancreatic cancer). In addition, the cells of this invention can be
administered
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to a subject to treat aplastic anemia, bone marrow failure and any other
hematological
disorder and/or immunological disorder which can be treated by administration
of the
cells of this invention.
Further provided is a method of treating an infection in a subject comprising
administering to a subject diagnosed with an infection an effective amount of
the cells
of this invention.
A method of promoting wound healing in a subject is also provided, comprising
administering to a subject having a wound an effective amount of the cells of
this
invention.
In addition, a method of enhancing bone marrow engraftment in a bone marrow
recipient is provided, comprising administering to the recipient an effective
amount of
the cells of this invention.
The hematolymphoid cells of this invention can be removed from a subject or
donor and treated according to the methods described herein to increase the
percentage
of'y8 T cells in the hematolymphoid cell population and administered to the
same
subject or to a recipient by ex vivo methods for removing cells, maintaining
the cells in
culture and re-administering them, as well known in the art. Standard methods
are
known for removal of cells from a subject or donor (e.g., phlebotomy,
apheresis) and
infusion of cells into a subject or recipient.
It is also contemplated that the percentage of yb T cells can be increased in
a
population of hematolymphoid cells in vivo. For in vivo methods, the ligand of
CD2,
the T cell mitogen, IL-I2 and IL-2 can be administered to the subject in a
pharmaceutically acceptable carrier. For example, an anti-CD2 antibody which
induces
responsiveness to IL-12 (e.g., MAb 55.2) can be administered intravenously to
a
subject in a dosage range of 0.01 to 10 mg/kg body weight; IL-12 can be
administered
intravenously to a subject in a dosage range of 100 to 1000 nanograms/kg of
body
weight (41); an anti-CD3 antibody (e.g., OKT3) can be administered
intravenously to a
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subject in a dosage range of 1-10 mg (manufacturer's package insert and
Physician's
Desk Reference, latest edition); and IL-2 can be administered intravenously to
a subject
in a range of 200,000 to 800,000 international units (IU) (manufacturer's
package insert
and Physician's Desk Reference, latest edition). The exact amount of these
substances
5 can vary from subject to subject, depending on the species, age, weight and
general
condition of the subject, the severity of the disorder being treated, the
particular
substance being used, its mode of administration and the like. Thus, it is not
possible
to specify an exact amount for every substance in every subject. However, an
appropriate amount can be determined by one of ordinary skill in the art using
only
10 routine experimentation given the teachings herein (see, e.g., Remington's
Pharmaceutical Sciences).
As an example, on Day 0, 0.1 mg/kg of anti-CD2 and S00 nanogram/kg of IL-
12 is administered in a pharmaceutically acceptable Garner as an intravenous
infusion
to the subject. On Day 1, 600,000 IU of IL-2 is administered in a
pharmaceutically
acceptable carrier as a 15 minute intravenous infusion and 5 mg of anti-CD3 as
a single
dose is administered in a pharmaceutically acceptable carrier, intravenously
as a rapid
injection. To determine the efficacy of administration of these substances,
the
peripheral blood of the subject can be analyzed by FACS before and after
administration to determine if the percentage of y8 T cells has increased. The
time
intervals for such measurement of the peripheral blood can be hours, days,
weeks
and/or months after administration of the substances listed herein. Other
clinical
parameters which can be monitored to determined the efficacy of administration
of
these substances can include measurement of 1) disease progression or
response, 2) rate
of graft failure, 3) graft-versus-host disease assessment, 4) time to
engraftment after
bone marrow transplant, 5) rate of infectious complications and/or 6)
objective
parameters of epithelial mucosal tissue injury, as would be known to one of
skill in the
art.
Thus, the present invention further provides a method for 1 ) treating cancer
in a
subject; 2) treating an infection in a subject; 3) promoting wound healing in
a subject;
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and/or 4) enhancing engraftment of a transplant in a subject by increasing the
percentage of y8 T cells in the subject comprising:
a) administering to the subject on day 0 an effective amount of a ligand of
CD2
which induces responsiveness to IL-12, in a pharmaceutically acceptable
carrier and an
effective amount of IL-12; and
b) administering to the subject on day 1 an effective amount of a T cell
mitogen
in a pharmaceutically acceptable carrier and an effective amount of IL-2 in a
pharmaceutically acceptable carrier, whereby the administration of the
substances of
steps (a) and (b) increases the percentage of y8 T cells in the subject.
As described above, the cells and/or substances administered to a subject in
vivo
can be in a pharmaceutically acceptable carrier. By "pharmaceutically
acceptable" is
meant a material that is not biologically or otherwise undesirable, i.e., the
material may
be administered to a subject, along with the cells or substances, without
causing any
undesirable biological effects or interacting in a deleterious manner with any
of the
other components of the pharmaceutical composition in which it is contained.
The
carrier would naturally be selected to minimize any degradation of the active
ingredient
and to minimize any adverse side effects in the subject, as would be well
known to one
of skill in the art.
The cells of the present invention and the substances which increase the
percentage of y8 T cells in vivo are typically administered parenterally and
are most
typically administered by intravenous injection, although other parenteral
routes of
administration, such as intramuscular, intradermal, subcutaneous,
intraperitoneal
administration, etc., is also contemplated. Injectables can be prepared in
conventional
forms, either as liquid solutions or suspensions, solid forms suitable for
solution of
suspension in liquid prior to injection, or as emulsions. A more recently
revised
approach for parenteral administration involves use of a slow release or
sustained
release system, such that a constant level of dosage is maintained. See, e.g.,
U.S.
Patent No. 3,710,795, which is incorporated by reference herein.
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The present invention is more particularly described in the following examples
which are intended as illustrative only since numerous modifcations and
variations
therein will be apparent to those skilled in the art.
EXAMPLES
EXAMPLE I
Isolation of PBMC, adherent cell-depleted PBMC and CDl4+ monocytes.
PBMC, replete with monocytes, were isolated from healthy, human volunteers by
Ficoll gradient centrifugation of peripheral blood anticoagulated with
heparin. PBMC
were depleted of monocytes by removal of plastic-adherent cells, as previously
described (15). CD14+ monocytes were purified directly from PBMC by sorting
FITC-
CD14 (Leu-M3)+cells, as described below.
1 S Generation and maintenance of cell cultures. Cultures of PBMC or
monocyte-depleted PBMC were initiated at a cell density of 1 x 10~ cells/mL in
24-well
flat-bottom tissue culture trays (Costar, Cambridge, MA) and were maintained
in 5
C02 at 37° C in complete medium consisting of RPMI-1640 (Applied
Scientific, San
Francisco, CA), 10% autologous human plasma, 2 mM L-glutamine, 100 U/mL
penicillin, 100 U/mL streptomycin and 50 pM 2-ME (GIBCO, Grand Island, NY). On
the day of culture initiation, human rIFN-y (Boehringer Mannheim,
Indianapolis, IN)
was added at a concentration of 1,000 U/mL. 24 hours later, cultures were
stimulated
by the addition of 10 ng/mL anti-CD3 MAb (Muromonab-CD3, Orthoclone OKT3,
Orthobiotec, Raritan, NJ) and 300 U/mL rIL-2 (Boehringer Mannheim,
Indianapolis,
IN).
Commercially prepared recombinant IL-12 (rIL-12) (Genetics Institute,
Cambridge, MA) was used at a final concentration of 10 U/mL and was added as a
single dose to appropriate cultures on the day of initiation. A neutralizing
poiyclonal
anti-IL-12 antibody, a neutralizing monoclonal anti-IL-12 antibody, or an
irrelevant
isotype control antibody (R&D Systems, Minneapolis, MN) were used at
concentrations of 20 ~g/mL and were added as a single dose to cultures on the
day of
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initiation. Cell density in all cultures was maintained at 1 to 2 x 10~
cells/mL, with the
addition of fresh media and transferred to larger tissue culture flasks as
required. Fresh
complete medium with 300 U/mL IL-2 was added to cultures every 5 days.
Analysis and cell sorting by flow cytometry. Cells were stained using FITC- or
PE-directly conjugated MAbs recognizing CD3, CD 14, CD 19, CD20 or CD56
(Becton-
Dickinson, San Jose, CA). Directly-conjugated isotype-matched irrelevant
antibodies
served as controls. Cells were stained for 30 min at 4° C in staining
buffer consisting
of Hank's buffered saline solution (HBSS, Mediatech, Herndon, VA) containing
2%
autologous human plasma. Excess Ab was removed by dilution with 10 volumes of
staining buffer, followed by centrifugation at SOOx g. Stained cells were
immediately
analyzed using a FACSCAN flow cytometer (BDIS, San Jose, CA), or sterile-
sorted
using a FACSVANTAGE cell sorter (BDIS, San Jose, CA). List-mode data were
acquired using forward- and side-scatter gates appropriate for viable lymphoid
cells or
monocytes. Data analysis was performed using CellQuestTM or LYSIS-II software
(BDIS, San Jose, CA).
Detection of IL-12 by ELISA. Monocytes, B lymphocytes, T lymphocytes and
NK cells were isolated directly from fresh PBMC by sorting the respective CD
19
CD14+ CD14 CD19+, CD3+CD56-and CD3 CD56+cell populations. Equivalent
numbers (1 x 105) of each sorted population were cultured separately as
described
above, with or without the addition of IFN-'y on the day of culture
initiation.
Supernatants were harvested after 72 hours, centrifuged to remove cellular
debris and
stored at -20° C. Detection of IL-12 in culture supernatants was
performed using an
enzyme-linked immunoassay following the manufacturer's instructions (IL-12
QuantikineTM Assay, R&D Systems, Minneapolis, MN).
Upon phenotypic analysis of cultures established as described above, it became
apparent that inclusion of one-anti-CD2 mAb (clone S5.2, mouse IgG2a, Becton
Dickinson) resulted in over-representation of y8 T cells in short-term
cultures as
detected by FAGS. At 14 days, cultures established as above (with or without
anti-
CD2 MAb S5.2) were analyzed for percentage of T cells using FITC-conjugated
MAb
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to 'y8 TCR, gating on propidium iodide (PI) negative populations of lymphoid
cells.
Cultures established without addition of IL-12, to which a single dose of anti-
CD2
MAb 55.2 was added at 10 mcg/mL, were found to contain a significant
percentage of
y8 T cells (24%). In contrast, isotype control antibody for 55.2 (mouse IgG2a,
Becton
Dickinson) resulted in no increase in percentage of'y8 T cells.
Anti-CD2 MAb-induced y8 T cell proliferation was augmented by the addition
of exogenous rIL-12. Ifboth anti-CD2 MAb and rIL-12 (10 U/mL) were included at
the initiation of cultures, an even greater percentage of y8 T cells was
detected after 14
days in culture (32%). Addition of IL -12 with only the isotype control for
anti-CD2
resulted in a minimal increase of y8 T cell percentage. If anti-CD2 MAb was
added to
culture but neutralizing MAb to IL-12 was also added at the initiation of
culture, y8 T
cell proliferation was completely inhibited, indicating that the CD2-mediated
y8 T cell
proliferation is dependent upon the presence of endogenous IL-12 in these
cultures.
Endogenous IL-12 exerts an important effect upon y8 T cell expansion in this
culture system. Monocytes are an important cellular source of IL-12 and
interferon-
gamma (IFN~y) stimulates the production of IL-12 by monocytes. Though IFNy
plays a
role in the stimulation of IL-12 production in these conditions, it is also
possible that
IFNy induces the expression of other soluble factors or surface structures
which may
also contribute to the observed y8 T cells expansion.
An extensive literature exists describing the role of various anti-CD2 mAbs to
engage various epitopes of CD2 and the resulting proliferation of various T
cells
subsets, including ~y8 T cells (11,13,15). In addition to 55.2, several other
anti-CD2
MAbs (and their corresponding isotype controls) were assessed for the ability
to induce
y8 T cell expansion under the culture conditions described, now incorporating
the
addition of exogenous IL-12 at the time of initiation (day 0: IFNy, 1000 u/mL
and Il-
12, 10 U/mL; day 1: OKT-3, 10 ng/mL and IL-2, 300 U/mL). Antibodies to be
tested
were added to cultures at the time of initiation (day 0) at equivalent
concentrations (5
mcg/mL). These antibodies included 6F 10.3 (mouse IgG 1, Immunotech); 3 9C 1.
5 (rat
IgG2a, Immunotech) and LT-2 (mouse IgG2b, Serotech). None of these reagents
tested
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was comparable to S5.2 in ability to significantly induce y8 T cell expansion.
These
data suggest that anti-CD2 MAb 55.2 engages a particular CD2 epitope not
engaged by
the other CD2 MAbs, resulting in the observed effect, or alternatively, S5.2
is more
effectively cross-linked by FcR-bearing cells present in the heterogeneous
PBMC
5 cultures, resulting in more efficient signaling via CD2. Anti-CD2 MAb 55.2
is a
commercially available preparation, available in a low endotoxin azide-free
form.
Cultures of fresh PBMC were initiated as described, receiving IFNy on day O
and OKT3 and IL-2 on day 1. In addition, on day 0, IL-12 (10 U/mL) or PBS was
10 added as was an amount of MAb 55.2 (0, 1, 5 and 10 mcg/mL). After 14 days
in
culture, absolute cell numbers of both y8 T cells and a~3 T cells were
calculated from
total cell numbers present in cultures and the percentage of cells in culture
of the
corresponding phenotype as determined by FACS. Data were expressed as fold
expansion of T cells over day 0 cell numbers for a(3 T cells or y8T cells.
Data from
15 these experiments demonstrated that addition of exogenous IL-12 without the
addition
of anti-CD2 MAb 55.2 resulted in only a marginal augmentation of y8 T cells
(expansion 40 fold to 60 fold), which was minimal compared to the expansion
elicited
by engaging CD2 in the presence of IL-12 (over 200 fold). Also, addition of
this
combination appears to have a more pronounced effect on y8 T cells as compared
to a~i
T cells.
These studies show that engagement of CD2 is critical for expansion of yS T
cells from these cultures. This expansion requires the presence of IL-12,
whether
provided exogenously or produced endogenously. Cultures of PBMC were
stimulated
with IFNy on day O, followed by OKT3 and IL-2 on day 1. In cultures to which
neither IL-12 nor anti-CD2 MAb S5.2 were added, y8 T cells expanded only 38
fold.
In identical cultures receiving anti-D2 MAb S5.2 (S mcg/mL) on day 0, yS T
cells were
expanded 83 fold, far less than the 229 fold expansion observed if both MAb
55.2 and
IL-12 (10 U/mL) were included. Thus, endogenously produced IL-12, while less
pronounced in its ability to stimulate y8 T cell expansion (presumably related
to its less
than pharmacological concentration present in culture) is still clearly
important, as
indicated by loss of'y8 T cell expansion on adding neutralizing monoclonal
anti-human
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16
IL-12 antibody (25 mcg/mL, R&D Systems). Isotype control for anti-human IL-12
antibody had no effect.
Assessment of y8 T cell expansion in purified sorted populations:
Measurements of proliferation. Data derived in clones showed that some anti-
CD2
MAbS can preferentially stimulate proliferation of y8 T over a(3 T cells. The
proliferative capacities of y8 T cells and a(3 T cells sorted to high purity
from identical
cultures of fresh PBMC stimulated as described herein, were compared.
[3H]thymidine
incorporation and cell survival (as determined by cell counting) were examined
in order
to determine if y8 T cells had a proliferative advantage over a(3 T cells, or
possibly
surviving better, thus accounting for their eventual over-representation in
cultures.
Sorting of al3 T cells and y8 T cells by negative selection. In order to avoid
undesirable stimulation which might occur if y8 T cells were sorted using
either anti-
TCR~yB or anti-CD3 antibodies, y8 T cells were isolated by negative sorting.
Thus, y8
T cells were identified and sorted on the basis of being TCRa(3- and CDS+.
(Greater
than 96% of CD3+ cells also stained for CDS). All cells sorted were taken from
the PI-
gate to assure viability. Briefly, y8 T cells were stained using FITC- or PE-
directly
conjugated MAbS recognizing CDS and TCRa(3 (Becton-Dickinson, San Jose, CA).
Directly-conjugated isotype-matched irrelevant antibodies served as controls.
Cells
were stained for 30 min at 4° C in staining buffer consisting of Hank's
buffered saline
solution containing 3% autologous human plasma. Excess Ab was removed by
dilution
with 10 volumes of staining buffer, followed by centrifugation at SOOx g.
Stained cells
were immediately sorted using a FACSVANTAGE cell sorter (BDIS, San Jose, CA)
equipped with a high-speed (Turbo-Sort ) sort option. Data analysis was
performed
using CellQuestTM software (BDIS, San Jose, CA). In a similar manner, a[i T
cells
were sorted as TCRyB- and CDS+.
ys T cells sorted from 3 week old short-term cultures (initiated with mAb
55.2) proliferate to a greater degree than a(3 T cells. Cultures of human PBMC
were
initiated as described above, receiving on day 0: IFN-y, IL-12 and anti-CD2
mAb 55.2.
OKT3 and IL-2 were added on day 1. Cultures were maintained at a cell density
of 1-2
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17
x lOG cells/mL for 3 weeks with the addition of fresh medium and IL-2 (100
U/mL) as
needed. On day 21, highly pure y8 T cell and a~i T cell populations were
sorted from
these initial cultures by negative selection as described above and were
replated at
equivalent cell density (5,000 cells/well) in 96-well micro-titer trays,
receiving either
IL-2 at 100 U/mL or nothing. After 24 hours, sorted cells were labeled and
harvested
as above. In these cultures, y8 T cells proliferated to a greater extent in
response to IL-
2 when compared to a~3 T cells arising from identical cultures, in contrast to
the above
data obtained in fresh PBMC cultures.
Because y8 T cells have been reported to be highly sensitive to TCR/CD3
engagement (especially in the presence of IL-2) and undergo apoptosis upon
receiving
mitogenic stimuli through the TCR (10), an interpretation of these data is
that y8 T
cells found in these longer-term cultures represent the outgrowth of a subset
of cells
which upon engagement of CD2 were imparted a survival advantage, i.e., became
resistant to apoptosis or activation-induced cell death (AICD) caused by
engagement of
TCR/CD3.
Total cell numbers. To investigate whether y8 T cells present in longer-term
cultures represented the out-growth of a population of cells more resistant to
apoptosis
(either through inherent or acquired means), the following experiment was
performed.
PBMC were stimulated as described with IFN-y, IL-12 and MAb S5.2 on day 0,
followed by OKT3 and IL-2 on day 1. Cultures were maintained at a cell density
of 1-2
x 10~ cells/mL for 3 weeks with the addition of fresh media and IL-2 (100
U/mL) as
needed. On day 21, highly pure y8 T cell populations and a(3T cell populations
were
sorted from these initial cultures by negative selection as described above.
150,000
highly purified cells of each sorted population were placed separately in 2
mLs of
complete RPMI containing 100 U/mL IL-2. Cells were stimulated with nothing
(indicated as "no addition"); IL-12 alone; plastic-immobilized anti-CD2 MAb
S5.2
alone; or both IL-12 and plastic-immobilized anti-CD2 MAb 55.2. Cells were
kept at
37°C in S% COz for one week at which point they were counted and scored
as live
using trypan blue, which was confirmed with acridine orange assessment using a
fluorescence microscope. These data demonstrated that, whereas a(3 T cells
survived
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18
very poorly and responded to no signals, in contrast, greater numbers of y8 T
cells
survived that were stimulated with nothing. These data also demonstrated that
engagement of CD2, but only in the presence of IL-12, imparted an even greater
survival advantage on y8 T cells, whereas IL-12 alone or MAb 55.2 alone did
not.
In conjunction with the data obtained from the [3H]thymidine incorporation
studies described herein, these findings confirm that some 'y8 T cells persist
in 55.2-
initiated cultures, surviving to a greater extent than a[3 T cells. These data
also have
important in vivo immunotherapeutic implications. If a subset of y8 T cells is
selected
to survive by an initial encounter in vitro with a "death-rescue signal" (CD2
engagement in the presence of IL-12) and if the "death rescue signal" can
again impart
a survival advantage at a later time, then engagement of the natural ligand
for CD2
(CD58/LFA-3) in the presence of IL-12 in vivo can also serve to preserve cell
viability
and/or function.
Human T cells are thought to undergo apoptosis upon engagement of their
TCR/CD3 complex. This process has been termed activation-induced cell death
(AICD) and likely plays an important role in regulating T cell proliferative
responses to
mitogenic stimuli both in vitro and in vivo. The mechanisms by which AICD
occurs are
not entirely understood, although it has been shown that in mature, previously
stimulated T cells, initiation of programmed cell death (apoptosis) likely
involves the
transduction of death-initiating signals generated by engagement of the Fas
(CD95/Apo-1) antigen present on T cells and that apoptosis triggered by
TCR/CD3
signaling is not restricted to CD4+CD8+ immature thymocytes or transformed
leukemic T cell lines but can also occur in IL-2-dependent normal y8 T cells
(10,19-
21 ). It has also been shown that Fas/CD95 engagement and subsequent
mobilization of
intracellular Caz+are causally related to apoptosis occurnng in human y8 T
cells clones
(22). In light of these observations, studies were conducted to determine if
CD2
engagement in the presence of IL-12 provides a "protective" signal to a subset
of y8 T
cells which rescues them from AICD caused by mitogenic OKT3 and IL-2 and to
determine if these "AICD-protected" cells are the y8 T cells which eventually
come to
be "over-represented" in 55.2-stimulated cultures. Studies were also conducted
to
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19
prospectively identify which y8 T cells might be receiving protective signals
from CD2
engagement in the presence of IL-12.
Flow c tometry. Initial observations were made using bulk T cell cultures and
not T cell clones or T cell lines. Therefore, the issue of apoptosis was
examined using
a flow cytometric approach. Flow cytometry using a four-color dual laser
configuration
(FACS Calibur flow cytometer or FACS Vantage cell sorter, Becton Dickinson)
allows
a discrete population of cells (such as 'y8 T cells) to first be defined by
surface
phenotype from within a heterogeneous population of cells. Simultaneously,
biologically relevant processes related to activation, proliferation, cytokine
production
or apoptosis can be examined, provided the proper reagents and methods are
employed.
Annexin V conjugated to FITC has been used to detect apoptosis in a variety of
cell
types. Annexin V binds with high affinity to phosphatidylserine which is
normally
confined to the inner plasma membrane leaflet of live, non-apoptotic cells.
1 S Phosphatidylserine externalization is an early and widespread event
associated with
apoptosis in a variety of human cell types, regardless of the initiating
stimulus. Thus, in
combination with fluorescent-conjugated antibodies, Annexin V-FITC can be used
to
examine apoptosis in a phenotypically defined subpopulation of cells in
heterogeneous
cell cultures. Furthermore, by utilizing propidium iodide (PI) as an indicator
of cell
viability, viable cells (Annexin-/PI-) can be distinguished both from
apoptotic cells
(Annexin+/Ph) and from necrotic cells (Annexin+/PIT).
1. 'y8 T cells at rest are more prone to apoptosis than a~i T cells. T cell
subsets from fresh PBMC isolated from several different individuals were first
characterized for their "baseline" apoptotic tendencies. y8- and a~i T cells
were first
identified and gated on the basis of surface staining with directly conjugated
antibodies
recognizing CD3 and TCR~yB. Gated populations of y8- and a~i T cell
populations
were then analyzed with respect to Annexin-FITC and PI. Statistics were
expressed as
the percentage of cells appearing in the corresponding dot-plot quadrants.
Viable,
apoptotic and necrotic regions were defined using the Jurkat T cell line
treated with the
anti-Fas (CD95/Apo-I) monoclonal antibody CH-11 (Kamiya Biomedical) or isotype
control IgM.
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These data revealed that, whereas 89% of a(3 T cells were found to be "viable"
(Annexiri /PI-), only 59% of y8 T cells were found to be "viable", that is are
not
actively undergoing apoptosis, as measured by Annexin V-FITC binding. These
cells
were all isolated, processed and analyzed in the exact same manner, minimizing
the
5 possibility that an artifact associated with preparation alone accounts for
these findings.
Furthermore, experiments were performed using various combinations of Annexin
V-
FITC and -PE as well as anti-CD3, anti-TCRyB and anti-TCRa(3 reagents
conjugated
to alternate fluorophores to address the possibility that compensation
artifacts alone
account for these findings. Results of several separate experiments were found
to be
10 comparable.
2. Immediate effects of mitogenic and "protective" signals on a~3-and y8 T
cell
apoptosis: Measurement of AICD within discrete subpopulations of T cells in
bulk cultures after 18 hours.
"Rescue" and "Death" si nals. The above data suggest a slightly greater
susceptibility to apoptosis in resting y8 T cells as compared to a(3 T cells.
Studies
were also conducted to determine whether y8 T cells display a greater
susceptibility to
apoptosis upon receiving mitogenic (AICD) signals and whether CD2 engagement
in
the presence of IL-12 could serve as a "rescue signal" from AICD caused in y8
T cells.
For the purposes of this study, the following combination was employed as the
"standard" method of generating 55.2-stimulated ~r8 T cells. On day 0, fresh
PBMC in
complete RPMI containing 10% autologous human plasma at a density of 1 x 106
cell/mL received: IFN-y (1,000 U/mL); IL-12 (20 U/mL) and anti-CD2 MAb S5.2 (5
mcg/mL). On day 1 cultures received: OKT3 (10 ng/mL) and IL-2 (300 U/mL).
Furthermore, the day 0 stimuli were designated as "rescue" signals and day 1
stimuli (a
standard mitogenic combination) were designated as the "death" signals.
PBMC were prepared fresh from peripheral blood by Ficoll density
centrifugation. Cultures were initiated receiving either day 1 "death" signals
alone; day
0 "rescue" signals alone or both day 0 + day 1 signals, i.e., "standard"
conditions. At
18 hours after receiving the day 1 signals (mitogen), cultures were analyzed
for
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21
apoptosis by four-color flow cytometry as described above. y8- and a(3 T cell
populations from within each PBMC culture were first identified and gated in
two
colors (CD3-APC and TCRyB-PE). Statistics were expressed as the percentage of
cells
appearing in the corresponding dot-plot quadrants: viable (Annexin-/PI-),
apoptotic
S (Annexin+/PI-).
These results demonstrated that y8 T cells were highly sensitive to AICD in
response to day 1 ("death") signals alone. As stated previously, close to 60 %
of resting
y8 T cells were viable (Annexin /Ph). However, after receiving mitogenic
stimuli, this
percentage falls to S%. In contrast, a(3 T cells in the same cultures
demonstrated no
such sensitivity to these mitogenic conditions, with close to 90% of these
cells
remaining viable.
Furthermore, whereas only S% of 'y8 T cells were found to be viable after
receiving day 1 "death" signals alone, close to 20% of y8 T cells were found
to be
viable, if, in addition to receiving the same day 1 "death" signals, they
received day 0
"rescue" signals. The greatest proportion of viable 'y8 T cells was found in
cultures
initiated with "rescue" signals alone, i.e., those receiving no mitogenic
stimulation via
OKT3 and IL-2. However, in these cultures, significant T cell expansion
(either a(3 or
y8) did not occur even after two weeks, as assessed by gross cell numbers.
Apoptosis
induced in a~i T cells under the same conditions was negligibly affected by
either
"death" or "rescue" signals. In this regard, a(3 T cells serve as a control,
indicating that
it is the y8 T cell compartment which is most affected by these manipulations.
These finding taken together, demonstrate that CD2 engagement in the presence
of IL
12 can provide a "protective" signal to a subset of y8 T cells which
eventually arises as
the dominant population in these cultures.
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3. Long-term effects of mitogenic and "protective" signals on a~3-and y8 T
cell
apoptosis: Measurement of AICD within discrete subpopulations of T cells in
bulk cultures after 7 days.
S Similar experiments described above were performed, but cultures were
maintained for longer periods, to determine if CD2 engagement in the presence
of IL12
provides a "protective" signal which allows for an apoptosis resistant
population of y8
T cells to dominate the culture. PBMC were prepared fresh from peripheral
blood by
Ficoll density centrifugation. Cultures were initiated, receiving either day 1
"death"
signals alone or both day 0 + day 1 signals, i.e., "standard" conditions.
Cultures
receiving no mitogenic stimulation via OKT3 and IL-2 ("rescue" signals alone)
failed
to proliferate. One week after receiving the day 1 signals (mitogen), cultures
were
analyzed for apoptosis by four-color flow cytometry as described above.
In cultures initiated with both rescue signals and mitogenic stimuli, over 80%
y8 T cells were found to be viable after one week, a significantly larger
percentage than
that found at rest (less than 60 %). In contrast, in cultures stimulated
without the rescue
signals and receiving only the mitogenic signals, only approximately SO% of y8
T cells
were found to be viable. These data appear to indicate that CD2 engagement in
the
presence of IL-12 is indeed providing a "protective" signal to a small subset
of y8 T
cells, these cells possibly becoming the dominant population in cultures by
virtue of an
acquired resistance to AICD. These cells would be expected to be less
susceptible to
apoptosis, consistent with the Annexin/PI data from 18 h cultures, as
described above.
The cell cultures described herein which have been treated by standard
conditions and which have been shown to contain up to 80% viable y8 T cells
after one
week, have been maintained in culture for a time period exceeding eight weeks.
These
cultures can be pezpetuated indefinitely by cell passage and addition of fresh
medium as
needed and restimulation of the cells by administering the substances
described herein
for standard conditions at regular intervals, as can be determined by routine
methods.
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23
EXAMPLE II
Isolation of PBMC and adherent cell-depleted PBMC. Peripheral blood was
obtained by phlebotomy of normal, healthy human volunteers. All samples were
collected into tubes anticoagulated with heparin. PBMC were isolated by Ficoll
gradient centrifugation of whole blood. PBMC were depleted of monocytes by
removal of plastic-adherent cells, as previously described ( 1 S). Monocyte
depletion
was confirmed by FACS prior to use in cultures.
Generation and maintenance of cell cultures. Cultures of PBMC were initiated
at a cell density of 1 x 106 cells/mL in 24-well flat-bottom tissue culture
trays (Costar,
Cambridge, MA) and were maintained in S % CO~ at 37° C in complete
medium
consisting of RPMI-1640 (Mediatech, Herdnon, VA), 10% fetal bovine serum
(HyClone, Logan, UT), 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL
streptomycin and 50 pM 2-ME (GIBCO, Grand Island, NY). On the day of culture
initiation (day 0), human rIFN-y (1,000 U/mL, Boehringer Mannheim,
Indianapolis,
IN); human rIL-12 (10 U/mL, R&D Systems, Minneapolis, MN) and mouse anti-
human CD2 MAb clone 55.2 (1-10 ~glmL, mouse IgG2a, Camfolio, Becton Dickinson,
San Jose, CA) were added. 24 hours later (day 1 ), cultures were stimulated by
the
addition of 10 ng/mL anti-CD3 MAb OKT3 (mouse IgG2a, Orthobiotec, Raritan, NJ)
and 300 U/mL rIL-2 (Boehringer Mannheim, Indianapolis, IN). Where indicated,
neutralizing monoclonal anti-human IL-12 antibody, or an irrelevant isotype
control
antibody (R&D Systems, Minneapolis, MN) were added as a single dose at a final
concentration of 25 wg/mL on the day of culture initiation. Neutralizing
monoclonal
anti-human CD58 MAb clone L306 (mouse IgG2a, Camfolio, Becton Dickinson, San
Jose, CA) or IgG2a isotype control antibody were added to cultures where
indicated, at
a final concentration of S Ng/mL. In all cultures, cell density was maintained
at 1 to 2 x
106 cells/mL, with the addition of fresh media and transfer to larger tissue
culture flasks
as required. Fresh, complete medium with 10 U/mL IL-2 was added to cultures
every 7
days.
Immobilization of stimulatory antibodies. When used in an immobilized form,
MAbs OKT3 and anti-human CD2 MAb clone S5.2 were bound to plastic tissue
culture
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24
plates as previously described (30). Briefly, MAbs at a concentration of 10
~g/mL in
PBS were placed in 24 well flat-bottom tissue culture trays (Costar), making
certain to
coat the entire surface of each well. After incubating at 37° C for 2
hours or at 4°C
overnight, wells were washed 3 times with ice cold PBS followed by gentle
suction
aspiration of well contents.
~jHJthymidine proliferation assay. Highly pure y8 and a~i T cells were plated
at
equivalent cell numbers (5,000 cells/well) in 96-well micro-titer trays,
receiving either
IL-2 at 100 U/mL or PBS only. After 24 hours, sorted cells were incubated with
1 ~Ci
[3H]thymidine and after an additional 18 hours, cells were harvested onto
glass fiber
filters using a standard cell harvester (PHD Cell Harvester, Cambridge, MA);
radioactivity was measured using a liquid scintillation counter. All samples
were
assayed in triplicate with data presented as mean CPM.
Surface staining and purification of cells by FACS. Cultured or fresh cells
were
surface stained using FITC-, PE-, or allophycocyanin (APC)-directly conjugated
MAbs
recognizing CD3, CDS, TCR-~ys or TCR-a~i (Becton Dickinson, San Jose, CA).
Directly-conjugated isotype-matched irrelevant antibodies served as controls.
Cells
were stained for 30 min at 4° C in staining buffer consisting of Hank's
buffered saline
solution (HBSS, Mediatech, Herndon, VA) containing 2% FBS. Excess Ab was
removed by dilution with 10 volumes staining buffer followed by centrifugation
at 500
X G. Stained cells were immediately analyzed using a FACSCalibur flow
cytometer
(BDIS, San Jose, CA). For sterile sorting, stained cells were immediately
sorted using
a FACS Vantage cell sorter (BDIS, San Jose, CA) equipped with a high-speed
Turbo
Sort option. For analysis and sorting, propidium iodide (PI) uptake was used
to
exclude non-viable cells during acquisition. Data analysis was performed using
CellQuest software (BDIS, San Jose, CA).
Four color flow cytometrv utilizing annexin V FITC and PI to measure
apoptosis in a,13 and ya T cell subsets. Cultured PBMC were first
simultaneously
surface stained (1 x 10' total cells in 100 ~L) using anti-CD3-APC and anti-
TCR-y8-
PE MAbs, as described above. Cells were subsequently washed twice with cold
PBS
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and then washed twice again with 1X annexin binding buffer provided by
manufacturer
(Apoptosis Detection Kit, R&D Systems). Cells were then resuspended in 100 ~L
of
binding buffer to which the appropriate volume of annexin V-FITC and PI were
added,
as determined by titration. Cells were incubated for 15 minutes at room
temperature in
S the dark after which time 300 ~l 1X annexin binding buffer was added.
Without
washing, cells were immediately analyzed utilizing a dual laser, four color
FACSCalibur flow cytometer (BDIS, San Jose, CA). At this point, cells were
kept on
ice to prevent the capping and internalization of surface-bound MAbs. Prior to
acquisition of data, calibration and compensation of all fluorescence
detectors (FL1 x
I O FL2; FL2 x FL3 and FL3 x FL4) was performed using cells stained with
individual
positive and negative control reagents in the presence or absence of annexin V-
FITC
and/or PI. Agonistic mouse anti-human CD95/Fas MAb CH11 (mouse IgM, Kamiya
Biomedical, Seattle, WA) or mouse IgM isotype control antibody were used as
positive
and negative controls respectively to define apoptotic (annexin+/pI-), viable
(annexin-
15 /PI-) and necrotic (annexin+/pI+) quadrants within acquisition dot-plots.
S'Cr release cytotoxicity assay. Melanoma cell lines SK-MEL-3, SK-MEL-5
and SK-MEL-28 (Dr. B. McAlpine, Emory University, Atlanta, GA) were utilized
as
targets for cytotoxicity assays. 1 x 106 cells were labeled with 100 ~Ci
Na25'Cr04
20 (sodium chromate, aqueous; Amersham Pharmacia Biotech, Piscataway, NJ) for
1 hour
at 37° C and subsequently washed in RPMI containing 10% FBS. Cells were
allowed
to incubate an additional 30 minutes at 37 ° C followed by an
additional wash, then
plated (2 x 10'/well) in 96 well V-bottom microtitre trays. Highly purified
a(3 or y8 T
cells in varying numbers (final ratios, 40:1 to 0.5:1 ) were added to target
cells in a final
25 volume of 150 ~L. Trays were briefly centrifuged then incubated for 4 hours
at 37° C,
after which 50 ~L of supernatant was removed to determine 5'Cr release in CPM
as
measured by gamma counter. Percent specific target cell lysis was calculated
as
[(experimental release - spontaneous release) / (maximum release - spontaneous
release)] X 100. Maximum and spontaneous release were respectively determined
by
adding either 0.1% Triton X-100 or culture medium alone to labeled target
cells in the
absence of effector cells. Data are presented as the mean of triplicate
samples of a
representative experiment.
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26
Mitogenic stimulation of PBMC in the presence of anti-CD2 MAb S5.2 results
in a large expansion of y8 T cells. Culture of PBMC in the presence of
mitogenic anti-
CD3 MAb OKT3 and IL-2 is a well known method for the induction of in vitro T
cell
proliferation, particularly a~i T cells (31-33). IL-12-dependent expansion of
human
S CD56+ a~i T cells arising in OKT3/IL-2 -stimulated PBMC cultures,
particularly if
these cultures were first primed with IFN-y 24 hours prior to stimulation with
mitogens
has been described (2,3). It has since been determined that within these
cultures,
monocytes serve as an important cellular source of both endogenous IL-12 and a
contact-dependent factor in the form of CD58/LFA-3, both of which are critical
for the
in vitro expansion of these CD56+ a~3 T cells.
In the process of examining which surface antigens played a role in CD56+ a~3
T
cell expansion, monoclonal antibodies against various surface structures such
as
adhesion molecules or co-stimulatory receptors were separately included in
PBMC
cultures first primed with IFN-y, then stimulated 24 hours later with
mitogenic OKT3
and IL-2. Shown in Figure 1, inclusion of one particular mouse anti-human CD2
MAb
(55.2, IgG2a), but not its isotype control, resulted in a large increase in
the percentage
of y8 T cells in cultures 7 to 10 days after initiation. Several other anti-
human CD2
MAbs (and their corresponding isotype controls) were assessed for their
ability to cause
y8 T cell expansion under identical culture conditions. Neither antibody 6F
10.3 (mouse
IgG~), antibody 39C1.5 (rat IgG2a) nor antibody LT-2 (mouse IgG2b, not shown)
were
able to significantly induce y8 T cell expansion as compared to MAb 55.2.
yaT cell expansion induced by anti-CD2 MAb S5.2 in mitogen-stimulated
PBMC cultures requires the presence of IL-12. Mitogen-stimulated PBMC cultures
were initiated as described above, primed first with IFN-y then stimulated 24
hours later
with mitogenic OKT3 and IL-2. After 7 to 10 days, cultures were analyzed by
FACS
for the percentage of Y8 T cells. Whereas anti-CD2 MAb S5.2 (but not its
isotype
control) can induce y8 T cell expansion in cultures to which no exogenous IL-
12 is
added, the addition of exogenous IL-12 to identical cultures containing MAb
S5.2
results in a further increase in the percentage of y8 T cells (24% to 32%).
Importantly,
IL-12 alone cannot significantly induce y8 T cell expansion in mitogen-
stimulated
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27
PBMC cultures not containing MAb 55.2 since addition of IL-12 to cultures
containing
only the isotype control for MAb 55.2 results in a minimal increase in y8 T
cell
percentage.
Most importantly, if a neutralizing MAb to human IL-12 (but not its isotype
control) is added to mitogen-stimulated PBMC cultures initiated in the
presence of
MAb 55.2, y8 T cell proliferation is almost completely inhibited. These
findings
indicate that the S5.2-mediated y8 T cell expansion is dependent upon the
presence of
endogenous IL-12; however, these results also indicate that the addition of
exogenous
IL-12 in the presence of S5.2 can further augment y8 T cell expansion in these
cultures.
S5.2-mediated, IL-12-dependent increase in ya T cell percentage occurring in
mitogen-stimulated PBMC cultures is a conseguence of a preferential increase
in the
absolute cell numbers of y8 T cells. By determining the fold expansion
(absolute cell
numbers) of both y8 and a~i T cells in mitogen-stimulated PBMC cultures, it
was
determined that the increase in the percentage of y8 T cells induced by the
addition of
S5.2 and exogenous IL-12 was not occurring at the expense of ap T cell
expansion.
Cultures of fresh PBMC were initiated as described receiving IFN-y initially
(day 0),
followed 24 hrs later by mitogenic stimulation with OKT3 and IL-2 (day 1).On
day 0,
either IL-12 (10 U/mL) or PBS was added to cultures. Likewise, anti-CD2 MAb
55.2
was added at the indicated concentration (~g/mL). After 14 days, absolute
numbers of
both ap and y8 T cells present in cultures were determined by multiplying the
total cell
number present in culture by the percentage of a(3 and y8 T cells as measured
by FACS.
Data are presented as fold expansion over starting a~i and y8 T cell number.
In agreement with the findings above, addition of IL-12 to mitogen-stimulated
PBMC in the absence of MAb S5.2 is not sufficient to induce a significant
increase in
the expansion of y8 T cells. Second, whereas the fold expansion of y8 T cells
is greatly
increased by the addition of both MAb 55.2 and exogenous IL-12 (40 fold
expansion to
over 230 fold expansion), no corresponding increase in the fold expansion of
a~i T cells
is noted. Also, the addition of MAb 55.2 and IL-12 does not inhibit ap T cell
expansion
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28
at 55.2 concentrations which promote y8 T cell expansion, indicating that a
true
preferential expansion of ya T cells is occurring in response to these
stimuli. Both ap
and y8 T cell expansion appear to be inhibited at 55.2 concentration at 10
~g/mL (or
higher). The reasons for this remain unclear.
Anti-CD2 MAb 55.2 induces ya T cell expansion via an agonistic and not a
blocking interaction with CD2. The existence of "accessory" or "alternate" CD2
signaling pathways triggered by MAbs to CD2 which function exclusively in y8 T
cells
have previously been suggested by several investigators (28,34). While the
majority of
anti-CD2 MAbs capable of delivering a proliferative signal to either ap or y8
T cells
appear to do so only if combined with a second anti-CD2 MAb recognizing a
separate
CD2 epitope (28,34,35), single epitope-binding anti-CD2 MAbs have been
reported
which appear to stimulate only y8 T cells (28,34). In a similar manner, it is
proposed
that anti-CD2 MAb S5.2 functions to induce y8 T cell expansion in
heterogeneous
PBMC cultures. The following experiments were performed to show that MAb 55.2
functions in an agonistic and not a blocking capacity, thereby initiating
rather than
inhibiting CD2 signal transduction events leading to the observed IL-12-
dependent y8 T
cell expansion.
In mice and humans, both CD58 (LFA-3) and CD48 have each been shown to
serve as ligands for CD2; in humans, however, only CD58 has been shown to
interact
with CD2 on T cells in a functionally significant manner (20, 36-38). Thus, if
anti-CD2
MAb S5.2 were inducing y8 T cell expansion by blocking an inhibitory
interaction
between CD2 and CD58, then the effect of a neutralizing anti-CD58 MAb would be
the
same, i.e., enhancement of y8 T cell expansion. This was shown not to be the
case.
Cultures of fresh PBMC were initiated as described receiving IFN-y initially
(day 0)
and OKT3 and IL-2 the next day (day 1 ). On day 0, either anti-CDZ MAb 55.2
(mouse
IgG2a), mouse anti-human-CD58 MAb L066.4 (mouse IgG2a), or mouse IgG2a isotype
control were added separately to identical cultures. After 14 days, cultures
were
analyzed by FACS to determine the percentage of y8 T cells present. Under
these
conditions, whereas addition of anti-CD2 MAb S5.2 resulted in the expansion of
y8 T
cells, addition of a blocking MAb to CD58 did not cause the same. These data
indicate
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29
that MAb S5.2 is not causing y8 T cell expansion by disrupting a putative
inhibitory
CD2-CD58 interaction.
To further demonstrate that MAb 55.2 is acting in an agonistic rather than an
inhibitory manner, the capacity of both soluble and immobilized MAb 55.2 to
induce y8
T cell expansion within mitogen stimulated PBMC cultures was compared.
Antibodies
which bind to specific cell surface receptors usually cannot trigger signal
transduction
events, unless either immobilized or cross-linked. As CD 14+ cells (monocytes)
present
in the mitogen-stimulated cultures express FcR capable of cross-linking MAb
S5.2
(mouse IgG2a), the following experiments were performed using PBMC first
rigorously
depleted of monocytes, as described herein.
Utilizing monocyte-depleted PBMC, cultures were initiated as described above,
stimulated on day 0 with IFN-y, IL-12 and either soluble or plastic-
immobilized MAb
55.2, followed 24 hrs later by mitogenic stimulation with IL-2 and plastic-
immobilized
OKT3. After 21 days, cultures were analyzed by FACS to determine the
percentage of
y8 T cells present. The results of these experiments showed that y8 T cells
can be
induced to expand significantly in mitogen-stimulated cultures by immobilized
but not
soluble anti-human CD2 MAb S5.2. Immobilized or soluble IgG2a (isotype control
for
MAb 55.2) similarly had minimal effect on yS T cell expansion. Cultures
maintained
for longer periods (up to 35 days) similarly display a preferential expansion
of y8 T
cells induced by immobilized but not soluble MAb 55.2.
Enhanced y8 T cell expansion induced by MAb S5.2 does not occur simply as a
consequence of increased y6 T cell proliferation. Data derived in clones
clearly show
that some anti-CD2 MAbs can preferentially induce proliferation of y8 T cells
compared to «~3 T cells (34,35). If the increased y8 T cell expansion observed
in S5.2-
treated cultures was occurring simply on the basis of a preferential
proliferation induced
by MAb S5.2, then it is likely that y8 T cells isolated from these cultures
would
incorporate [3H]thymidine to a greater degree than «p T cells isolated from
identical
cultures.
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Cultures of human PBMC were initiated as described above receiving on day 0,
IFN-y, IL-12 and anti-CD2 MAb 55.2. OKT3 and IL-2 were added 24 hours later
(day
1). After 24 hours (day 2), both ap and y8 T cells were sorted to high purity
from these
cultures, then plated at equivalent densities (5,000 cells/well) in 96-well
micro-titer
5 trays and were either stimulated with IL-2 at 100 U/mL or left unstimulated
(PBS).
After 24 hours (day 3), [3H]thymidine was added to cultures and 18 hours
later, cells
were harvested onto glass fiber filters. Data were evaluated as mean CPM of
triplicate
samples. These data indicate that y8 T cells isolated from 55.2-stimulated
cultures do
not appear to be proliferating to a greater degree than ap T cells isolated
from identical
10 cultures. This indicates that the over-representation of y8 T cells
observed in longer-
term 55.2-treated cultures is not only a consequence of an initial
preferential
proliferation induced by the anti-CDZ MAb.
IL-12-dependent MAb SS.2-mediated signaling through CD2 protects yaT cells
1 S from activation-induced cell death. Janssen et al. have shown that y8 T
cells are
exquisitely sensitive to TCR/CD3 engagement (especially in the presence of IL-
2) and
undergo apoptosis upon receiving mitogenic stimuli through the TCR (27). Thus,
one
possible interpretation of these findings is that CD2 engagement by MAb 55.2
in the
presence of IL-12 provides a signal to a subset of y8 T cells which protects
them from
20 activation-induced cell death (AICD) caused by mitogenic OKT3 and IL-2. It
is then
these apoptosis-resistant y8 T cells which eventually come to be represented
in larger
numbers in 55.2-stimulated cultures.
Annexin V binds with high affinity to phosphotidylserine (PS) which is
25 normally confined to the inner plasma membrane leaflet of live, non-
apoptotic cells;
appearance of PS on the outer plasma membrane leaflet is an early and
widespread
event associated with apoptosis in a variety of human cell types, regardless
of the
initiating stimulus. These findings have been exploited to allow the
examination of
apoptosis by flow cytometric means utilizing annexin V conjugated to FITC
(39,40).
30 Thus, annexin V-FITC in combination with directly conjugated antibodies can
be used,
especially in a properly configured four color flow cytometer, to detect
apoptosis
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31
occurring in phenotypically defined subpopulations of cells within
heterogeneous cell
cultures.
In order to demonstrate that CD2 engagement by MAb 55.2 in the presence of
IL-12 protects y8 T cells from AICD, the following experiment was performed.
By
convention, day 0 stimuli (IFN-y, IL-12 and anti-CD2 MAb 55.2) are
designated.as
putative "protective" signals. PBMC cultures were initiated as described
above. Those
receiving day 0 rescue signals were defined as protected; those receiving no
day 0
signals (PBS only) were defined as unprotected. All cultures received OKT3 and
IL-2
24 hours later (day 1 ).
At various intervals after receiving the day 1 mitogenic signals, y8 and a~i T
cell
populations within protected and unprotected cultures were simultaneously
analyzed for
apoptosis using four color flow cytometry. a~ and y8 T cell populations were
first
delineated by electronically gating on the corresponding a~ and y8 T cells
defined by
anti-CD3-APC and anti-TCR-y8-PE MAbs. Apoptosis occurnng in ap and y8 T cell
populations was then determined examining the uptake of Annexin V-FITC and PI
in
the respective gated events.
The results of these experiments show the exquisite sensitivity to AICD
demonstrated by unprotected y8 T cells compared to protected y8 T cells. In
the
absence of protective day 0 signals, mitogen stimulation induces apoptosis in
a large
majority of ~yb T cell with only approximately 1/3 or 36% of y8 T cells
remaining viable
(Annexin-/PI-) after 2 days. In contrast, apoptosis occurs to a far lesser
extent in y8 T
cells first protected with day 0 signals (IFN-g, IL-12 and anti-CD2 MAb 55.2).
Fully
2/3 or 67% are found to be viable despite receiving the identical mitogenic
stimulation.
These results also show that apoptosis occurring in ap T cells in response to
mitogenic
stimulation is negligible under these conditions. In this regard, a~3 T cells
both serve as
a control and support the argument that it is the y8 T cell compartment in
cultures which
is most affected by these manipulations.
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32
Confirmatory experiments were next performed utilizing highly purified y8 and
ap T cells obtained by high-speed cell sorting to exclude the likelihood that
these
findings resulted from staining or compensation artifact introduced as a
consequence of
complex simultaneous multi-color analysis. Using highly purified a~3 and y8 T
cell
preparations, apoptosis was measured employing Annexin V-FITC and PI staining
alone without the need for additional surface staining. PBMC cultures were
initiated on
day 0 as described above. All cultures received OKT3 and IL-2 24 hours later
(day I ).
After 48 hours, viable y8 and a~3 T cells were sorted from both protected and
unprotected cultures; all samples immediately upon sorting were routinely >98%
pure
and >96 % viable, as determined by PI exclusion. Sorted populations were
plated at
equivalent cell densities and after 24 to 36 hours, apoptosis in each
population was
determined by two color FACS employing annexin V-FITC and PI only. In
agreement
with the findings above obtained in unsorted populations analyzed in four
colors,
purified a(i T cells from both mitogen-stimulated protected and unprotected
cultures
1 S were found to be viable (Annexin-/PI-) to an equivalent degree (routinely
at least 94%,
not shown). More importantly, and in complete agreement with the four color
analysis
of unsorted y8 T cells, unprotected compared to protected y8 T cells were
confirmed to
be far more sensitive to apoptosis induced by mitogen stimulation (54% viable
versus
80% viable).
Restimulation with IL-2 is a potent inducer of apoptosis in unprotected but
not
protected ya T cells. Cultures, both unprotected and protected, can be
maintained for up
to 6 to 8 weeks with weekly replenishment of tissue culture media including
recombinant human IL-2 (10-100 U/mL); during this period, cultures are not
routinely
restimulated with other mitogens or growth factors. Despite the significant
apoptosis
induced initially in unprotected y8 T cells by day I mitogenic stimulation
(OKT3 and
IL-2), surviving y8 T cells found in these cultures at the end of the first
week (prior to
refeeding) are found to be relatively viable, though still less viable than
those found in
protected cultures. It should be noted, however, that even at 7 days, a
significant
difference already exists in the percentage and absolute number of y8 T cells
found in
protected versus unprotected cultures: Whereas protected cultures are
comprised of up
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33
to 2S to 30% y8 T cells, unprotected cultures almost never contain more than 7
to 10%
y8 T cells at this point.
The effect of adding IL-2 to both protected and unprotected y8 T cells was
also
S demonstrated. On day 7, IL-2 (100 U/mL) was added to equivalent numbers of
cells
from both protected and unprotected PBMC cultures. After overnight incubation,
apoptosis in a~ and y8 T cell populations was determined (day 8) by measuring
the
uptake of Annexin V-FITC and PI in the respective gated populations. Agonistic
mouse anti-human CD9S/Fas MAb CH11 (mouse IgM) or mouse IgM isotype control
were included in identical cultures as controls. These data indicate that
while the
addition of IL-2 can induce apoptosis in both protected and unprotected y8 T
cells, this
effect is by far more pronounced in unprotected y8 T cells where the addition
of IL-2
markedly decreases the percentage of viable y8 T cells (37% viable on day 8).
In
contrast the addition of IL-2 only modestly decreases viability of y8 T cells
in protected
1 S cultures (73% viable on day 8).
Functional properties ofprotected y8T cells: Cytotoxicity measured against
melanoma cell lines. Recognition that MAb SS.2-mediated IL-12-dependent
signals
can protect human y8 T cells from mitogen-induced apoptosis provides the
biological
basis for developing the practical means to generate large numbers of human y8
T cells
The following experiment is provided to demonstrate that protected y8 T cells
possess
properties distinct from ap T cells arising from within the same cultures.
Cultures of human PBMC were initiated as described above receiving on day 0,
2S IFN-y, IL-12 and anti-CD2 MAb SS.2. OKT3 and IL-2 were added 24 hours later
(day
1). After 14 days, purified populations of y8 and ap T cells were isolated
from cultures
by high speed cell sorter. To avoid inadvertent activation of T cells via
engagement of
the T cell receptor (CD3), sorting was performed using an anti-CDS-PE MAb. For
the
same reason, y8 T cells were sorted as ap TCR , CDS+ cells. Likewise, a~i T
cells were
sorted as yb TCR , CDS+ cells. The cytotoxic activity of these highly purified
y8 and ap
T cells was then tested against 5'Cr-labeled human melanoma cell lines SK-MEL-
3, SK-
MEL-S and SK-MEL-28 at various effector to target ratios. Data were evaluated
as
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34
percent specific lysis. For each of the three tumor cell lines, the percent
specific lysis
by the 'y8 T cells exceeded that of the a~i T cells assayed. These data
indicate that
apoptosis-resistant y8 T cells can mediate antitumor cytotoxicity against
human
melanoma cell lines in vitro to a significantly greater degree than a~i T
cells derived
from identical cultures.
EXAMPLE III
Clinical applications of the administration of the y8 T cells of this
invention
to a subject and of the administration of the substances described herein for
in
vivo expansion of y8 T cells in a subject.
1) Either y8 T cells expanded ex vivo according to the methods of this
invention
or the substances described herein for increasing the percentage of y8 T cells
in vivo
can be administered to a subject diagnosed with a malignancy, according to the
dosage
regimens described herein, with the intent of exploiting the anti-neoplastic
(anti-tumor
or anti-leukemic effect) activity of these cells. Efficacy of this treatment
would be
determined by assessing a response of the malignancy as measured by tumor
regression
or failure of tumor progression.
2) The y8 T cells expanded ex vivo according to the methods described herein
can be administered as an adjuvant to an allogeneic bone marrow transplant in
a clinical
setting where graft failure would be a likely complication (such as where the
donor is an
HLA-mismatched sibling or a matched unrelated donor). Ex vivo expanded donor
or
recipient y8 T cells can be administered prior to, in conjunction with or
following the
administration to the recipient of a donor bone marrow stem cell product which
is first
depleted of all T cells (which is done in an attempt to minimize the
likelihood of graft-
versus-host disease). The intent of including ex vivo expanded y8 T cells with
the
transplanted cells in the recipient would be to facilitate engraftment of
donor-derived
hematopoietic stem cells in the transplant recipient. Efficacy of this
treatment can be
determined in a clinical setting by measuring a number of clinical indices as
would be
well known to the clinician, such as time to full engraftment, or decreased
incidence of
graft failure. Alternatively, the substances described herein can be
administered to the
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bone marrow transplant recipient according to the dosage regimens described
herein for
increasing the percentage of the recipient's y8 T cells in vivo to impart the
same
beneficial effects as described herein for the direct administration of ex
vivo expanded
y8 T cells. Efficacy of treatment by this in vivo stimulation method would
also be
5 assessed according to the same parameters as described herein for the
administration of
ex vivo expanded y8 T cells.
3) The ex vivo expanded y8 T cells of this invention can be administered as an
adjuvant to standard therapy (which can include, but is not limited to,
antibiotics), to a
10 subject diagnosed with an infectious process, including, but not limited
to, infection
with viral pathogens (such as HIV), bacterial pathogens or other infectious
agents.
Efficacy would be measured by assessing the subject's ability to effectively
clear
infectious organisms according to standard protocols well known in the art.
Alternatively, the substances described herein can be administered to the
subject to treat
15 an infectious process according to the dosage regimens described herein for
increasing
the percentage of the recipient's 'y8 T cells in vivo to impart the same
beneficial effects
as described herein for the direct administration of y8 T cells. Efficacy of
treatment by
this in vivo stimulation method would also be assessed according to the same
parameters as described herein for the administration of y8 T cells.
4) The ex vivo expanded y8 T cells of this invention can be administered as an
adjuvant to subjects diagnosed with a tissue injury, which can include but is
not limited
to, tissue trauma, burns, graft-versus-host disease or autoimmune destructive
processes.
'y8 T cells appear to contribute to wound healing by the elaboration of a
number of
important factors including, but not limited to, keratinocyte growth factor
(KGF), also
known as FGF-7. Alternatively, the substances described herein can be
administered to
the subject to treat a tissue injury according to the dosage regimens
described herein for
increasing the percentage of the recipient's y8 T cells in vivo to impart the
same
beneficial effects as described herein for the direct administration of 'y8 T
cells.
Efficacy of treatment by this in vivo stimulation method would also be
assessed
according to the same parameters as described herein for the administration of
y8 T
cells.
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36
In the protocols described above in which y8 T cells are administered to a
subject, y8 T cells are first expanded ex vivo according to the methods
described herein,
typically from either an autologous or allogeneic source and delivered
intravenously at a
S dose effective in mediating a biologically significant process, as
determined according
to the methods described herein. For example, the amount of cells administered
to a
subject can be in the range of 1 X 105 to 1 X 108 y8 T cells/kg body weight.
Although the present process has been described with reference to specific
details of certain embodiments thereof, it is not intended that such details
should be
regarded as limitations upon the scope of the invention except as and to the
extent that
they are included in the accompanying claims.
Throughout this application, various publications are referenced. The
disclosures of these publications in their entireties are hereby incorporated
by reference
into this application in order to more fully describe the state of the art to
which this
invention pertains.
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