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IMMUNOBIOLOGY
CD2-mediated IL-12–dependent signals render human ␥␦-T cells resistant
to mitogen-induced apoptosis, permitting the large-scale ex vivo expansion
of functionally distinct lymphocytes: implications for the
development of adoptive immunotherapy strategies
Richard D. Lopez, Shan Xu, Ben Guo, Robert S. Negrin, and Edmund K. Waller
The ability of human ␥␦-T cells to mediate
a number of in vitro functions, including
innate antitumor and antiviral activity, suggests these cells can be exploited in
selected examples of adoptive immunotherapy. To date, however, studies to examine such issues on a clinical scale
have not been possible, owing in large
measure to the difficulty of obtaining sufficient numbers of viable human ␥␦-T
cells given their relative infrequency in
readily available tissues. Standard methods used to expand human T cells often
use a combination of mitogens, such as
anti–T-cell receptor antibody OKT3 and
interleukin (IL)-2. These stimuli, though
promoting the expansion of ␣␤-T cells,
usually do not promote the efficient
expansion of ␥␦-T cells. CD2-mediated,
IL-12–dependent signals that result in the
selective expansion of human ␥␦-T cells
from cultures of mitogen-stimulated human peripheral blood mononuclear cells
are identified. It is first established that
human ␥␦-T cells are exquisitely sensitive to apoptosis induced by T-cell mitogens OKT3 and IL-2. Next it is shown that
the CD2-mediated IL-12–dependent signals, which lead to the expansion of ␥␦-T
cells, do so by selectively protecting sub-
sets of human ␥␦-T cells from mitogeninduced apoptosis. Finally, it is demonstrated that apoptosis-resistant ␥␦-T cells
are capable of mediating significant antitumor cytotoxicity against a panel of human-derived tumor cell lines in vitro. Both
the biologic and the practical implications of induced resistance to apoptosis
in ␥␦-T cells are considered and discussed because these findings may play
a role in the development of new forms of
adoptive cellular immunotherapy. (Blood.
2000;96:3827-3837)
© 2000 by The American Society of Hematology
Introduction
Human T lymphocytes recognize and respond to antigens via a
clonally expressed T-cell receptor (TCR). Whereas most mature T
cells express an ␣␤-TCR heterodimer, a few express an alternative
␥␦-TCR heterodimer.1-5 Although the physiologic role of human
␥␦-T cells remains unclear, evidence continues to accumulate to
suggest that ␥␦-T cells are involved in a number of important
physiologic and disease-related processes. For example, both
murine and human ␥␦-T cells have been shown6-10 to exhibit major
histocompatibility complex (MHC)-unrestricted cytotoxicity against
some tumors, in vitro and in vivo. In addition, ␥␦-T cells have been
shown11-13 to exert antiviral activity against a number of human
pathogens, including the human immunodeficiency virus. It has also
been proposed14 that ␥␦-T cells may play a role in wound healing or
tissue repair through the elaboration of a number of growth factors,
including keratinocyte growth factor. Recently, in both human clinical
studies and experimental animal models of allogeneic bone marrow
transplantation, it has been recognized that donor-derived ␥␦-T cells
may serve as facilitating cells, promoting the engraftment of donor
hematopoietic stem cells across varying degrees of MHC disparity.15,16
However, the exploitation of ␥␦-T cells for specific therapeutic
ends remains largely unrealized, largely because of the extreme
difficulty of obtaining sufficient numbers of viable ␥␦-T cells given
their relative infrequency in peripheral blood (PB) or other readily
available tissues. Simply isolating ␥␦-T cells from fresh PB or bone
marrow is likely to prove impractical. Expanding ␥␦-T cells ex
vivo using a variety of mitogenic stimuli, including anti-CD3 or
anti-TCR␥␦ antibodies, is an attractive alternative means by which
to obtain sufficient numbers of these cells. However, for reasons
that are not entirely clear, human ␥␦-T cells, when compared to
␣␤-T cells, appear to undergo apoptosis or activation-induced cell
death more readily upon TCR/CD3 engagement, especially in the
presence of IL-2.17 Human ␥␦–T-cell clones have also been
shown18,19 to readily undergo apoptosis when stimulated simultaneously by anti-TCR monoclonal antibody (mAb) plus exogenous
IL-2, leading some to propose that the induction of programmed
cell death upon repeated mitogenic stimulation might serve as a
regulatory mechanism whereby excessive in vivo ␥␦–T-cell proliferation is prevented. In any event, the fact that ␥␦-T cells may
simply die upon ex vivo expansion may represent a serious obstacle
to developing approaches to incorporate ␥␦-T cells into any form of
adoptive cellular immunotherapy.
Here, we identify a CD2-mediated, IL-12–dependent signal that
results in the selective expansion of human ␥␦-T cells in mitogenstimulated human peripheral blood mononuclear cell (PBMC)
cultures. Using 4-color flow cytometry integrating surface staining
with annexin V and propidium iodide (PI) uptake, we first confirm
From the Division of Bone Marrow Transplantation, Stanford University School
of Medicine, Stanford, CA; the Division of Hematology/Oncology, Bone Marrow
Transplant-Leukemia Program, Emory University School of Medicine, Atlanta,
GA; and the Bone Marrow Transplantation Program, University of Alabama at
Birmingham, AL.
Reprints: Richard D. Lopez, BMT Program, THT-541, University of Alabama at
Birmingham, 1900 University Boulevard, Birmingham AL 35294; e-mail:
[email protected].
Submitted March 30, 1999; accepted August 1, 2000.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by a grant from the Robert Wood Johnson Foundation.
© 2000 by The American Society of Hematology
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
3827
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3828
LOPEZ et al
in PBMC cultures the findings of others that human ␥␦-T cells are
indeed exquisitely sensitive to apoptosis induced by T-cell mitogens. Using these same methods, we then establish that the
CD2-mediated, IL-12–dependent signals that lead to the observed
expansion of ␥␦-T cells do so by selectively protecting a subset of
human ␥␦-T cells from programmed cell death induced by
mitogenic stimulation, in particular IL-2. Finally, we demonstrate
that highly purified apoptosis-resistant human ␥␦-T cells can
mediate antitumor activity against a variety of human tumor cell
lines in vitro. Both the biologic and the practical implications of
these findings are considered and discussed.
Materials and methods
PBMC and adherent cell-depleted PBMC
PBMC were isolated by Ficoll gradient centrifugation of whole blood
obtained from healthy human volunteers. Where indicated, PBMC were
depleted of monocytes by the removal of plastic-adherent cells, as
described.20
Generation and maintenance of cell cultures
Cultures were initiated at a cell density of 1 ⫻ 106 cells/mL in 24-well
flat-bottom tissue culture trays (Costar, Cambridge, MA) and maintained in
5% CO2 at 37°C in RPMI-1640 with 10% fetal bovine serum (HyClone,
Logan, UT), 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 U/mL
streptomycin, and 50 ␮mol/L 2-ME. On the day of culture initiation (day 0),
human recombinant interferon (rIFN)-␥ (1000 U/mL; Boehringer Mannheim, Indianapolis, IN); human rIL-12 (10 U/mL; R&D Systems, Minneapolis, MN), and mouse antihuman CD2 mAb clone S5.2 (1-10 ␮g/mL, mouse
IgG2a; Becton Dickinson, San Jose, CA) were added. Twenty-four hours
later (day 1), cultures were stimulated with 10 ng/mL anti-CD3 mAb OKT3
(mouse IgG2a; Orthobiotec, Raritan, NJ) and 300 U/mL human rIL-2
(Boehringer Mannheim). Where indicated, neutralizing monoclonal antihuman IL-12 antibody or an irrelevant isotype control antibody (R&D
Systems) was added as a single dose at a final concentration of 25 ␮g/mL.
Neutralizing monoclonal antihuman CD58 mAb clone L306 (mouse IgG2a;
Becton Dickinson) or IgG2a isotype control antibody were added to cultures
where indicated (5 ␮g/mL). Fresh medium with 10 U/mL IL-2 was added
every 7 days. Where indicated, mAb OKT3 and mAb S5.2 were bound to
plastic tissue culture plates as described.21
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
pended in 100 ␮L binding buffer. After the addition of annexin V-FITC and
PI, cells were incubated for 15 minutes at room temperature in the dark. At
this point, cells were kept on ice to prevent the capping and internalization
of surface-bound mAbs. Calibration and compensation of all fluorescence
detectors was performed using cells stained with individual positive and
negative control reagents in the presence or absence of annexin V-FITC,
PI, or both.
Induction of apoptosis in tumor target cells
by cocultured human lymphocytes
Tumor target cells (2.5 ⫻ 104 in 500 ␮L complete RPMI) were cultured
alone or cocultured with either human ␣␤- or ␥␦-T cells at varying
effector–target (E:T) ratios (1:1 to 20:1) in sterile 5-mL round-bottom
polystyrene tubes (Falcon, BD Labware, Franklin Lakes, NJ). Cells were
incubated for 4 hours at 37°C in 5% CO2, after which they were washed
with PBS and resuspended in 100 ␮L annexin binding buffer to which
annexin V-FITC was added. After 15 minutes at room temperature in the
dark, cocultured cells were gently vortexed to disrupt any tumor–
lymphocyte aggregates, and they immediately were analyzed by flow
cytometry. Voltages in the forward-scatter (FSC) and side-scatter (SSC)
detectors were set to permit the discrimination of tumor cells (high FSC and
high SSC) from lymphocytes (low FSC and low SSC) on the basis of light
scatter. Electronically gated tumor cells were subsequently analyzed for the
binding of annexin V-FITC using the FL1 detector.
Measurement of target cell viability on coculture with human
lymphocyte for longer periods at lower E:T ratios
We modified a previously described method to distinguish living from dead
cells.22 Briefly, target cells (2 ⫻ 103 in 100 ␮L complete RPMI) were
cultured alone or cocultured with either human ␣␤-or ␥␦-T cells at various
E:T ratios in 96-well flat-bottom tissue culture trays (Corning Glassware,
Corning, NY). Cells were incubated for 18 hours at 37°C in 5% CO2, after
which 100 ␮L of a solution containing 10 ␮g/mL ethidium bromide and 3
␮g/mL acridine orange (Sigma, St Louis, MO) was added. Cells were
immediately viewed using an inverted fluorescence microscope illuminated
with a 300-W xenon light source (Intracellular Imaging, Cincinnati, OH).
By using a blue filter set configured to excite for fluorescein (470 ⫾ 20 nm
excitation filter, 500 nm dichroic/beamsplitter filter, 515 nm emitter
filter; Chroma Technology, Brattleboro, VT), live cells were observed to
fluoresce green (acridine orange) whereas dead cells fluoresced orange
(ethidium bromide).
[3H]-thymidine proliferation assay
Proliferation assays were performed using standard methods as described in
figure text. Assays were performed in triplicate with data presented as mean
counts per minute (cpm) (⫾ SD).
Surface staining and purification of cells by FACS
Cells were stained using fluorescein isothiocyanate (FITC)-, phycoerythrin
(PE)-, or allophycocyanin (APC)-directly conjugated mAbs recognizing
CD3, CD5, TCR-␥␦, or TCR-␣␤ or directly conjugated isotype-matched
irrelevant control antibodies (Becton Dickinson). Cells were stained for 30
minutes at 4°C in Hank’s buffered saline solution (Mediatech, Herndon,
VA) containing 2% fetal bovine serum (FBS) and immediately analyzed
using a FACScalibur flow cytometer or sorted using a FACS Vantage cell
sorter (Becton Dickinson). PI uptake was used to exclude nonviable cells.
Data analysis was performed using CellQuest software (Becton Dickinson).
Four-color flow cytometry using annexin V-FITC and PI to
measure apoptosis in ␣␤- and ␥␦-T cells
Cells were first surface stained (1 ⫻ 105 total cells in 100 ␮L) using
anti-CD3–APC and anti-TCR-␥␦–PE mAbs. Cells were washed twice with
cold phosphate-buffered saline (PBS), washed twice again with annexin
binding buffer (Apoptosis Detection Kit; R&D Systems), and then resus-
Chromium Cr 51 release cytotoxicity assay
Human cervical carcinoma cell line HeLa (ATCC); human melanoma cell
lines SK-MEL-3, SK-MEL-5, and SK-MEL-28 (ATCC and kindly provided by Dr B. McAlpine, Emory University, Atlanta, GA); human
T-lymphoblastoid cell line CCRF-HSB-2 (ATCC); human peripheral blood
lymphoblast cell line NC-37 (ATCC); human myeloid leukemia cell line
K-562 (ATCC), human ovarian cancer cell line SK-OV-3 (ATCC), and
human B-cell lymphoma line OCI-Ly823 were used as targets for chromium
release assays. Target cells were labeled with 100 ␮Ci Na2 51CrO4
(Amersham Pharmacia Biotech, Piscataway, NJ) from 2 hours to overnight
at 37° C, after which cells were washed, trypsinized, and resuspended in
RPMI containing 10% FBS. Cells were then plated (2 ⫻ 103/well) in
96-well V-bottom microtiter trays. Purified ␣␤- or ␥␦-T cells in varying
numbers were added to target cells in a final volume of 150 ␮L. Trays were
briefly centrifuged and then incubated for 4 hours at 37° C, after which
50 ␮L supernatant was removed to determine 51Cr release in cpm.
Percentage specific target cell lysis was calculated as [(experimental
release⫺spontaneous release)/(maximum release⫺spontaneous release)]⫻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 (⫾ SD) of triplicate samples.
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BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
Results
Mitogenic stimulation of PBMC in the presence of anti-CD2
mAb S5.2 results in a large expansion of ␥␦-T cells
Previously, we described the expansion of human CD56⫹ ␣␤-T
cells arising in OKT3/IL-2-stimulated PBMC cultures, particularly
if these cultures were first primed with IFN-␥ 24 hours before
stimulation with mitogens.24,25 In the process of examining the role
of various surface antigens involved in CD56⫹ ␣␤–T-cell expansion, the inclusion of one particular mouse antihuman CD2 mAb
(S5.2, IgG2a), but not its isotype control, resulted in a large increase
in the percentage (Figure 1A) and absolute number of ␥␦-T cells
INDUCED RESISTANCE TO APOPTOSIS IN ␥␦-T CELLS
3829
Table 1. Expansion of mitogen-stimulated human ␥␦-T cells is augmented by
anti-CD2 mAb S5.2 in the presence of recombinant human IL-12
Fold expansion ␥␦-T cells†
Culture condition*
IFN-␥
Anti-CD2
IL-12
Mean ⫾ SD (n ⫽ 5)
P
⫹
IgG2a
PBS
17.7 ⫾ 11.4
⫹
IgG2a
⫹
20.9 ⫾ 10.5
NS
⫹
PBS
⫹
23.0 ⫾ 13.1
NS
⫹
0.1 ␮g/mL
⫹
41.9 ⫾ 20.3
.08
⫹
1 ␮g/mL
⫹
72.2 ⫾ 60.8
.10
⫹
5 ␮g/mL
⫹
67.1 ⫾ 33.7
⬍.02
PBMC were obtained from 5 healthy donors; short-term cultures of PBMC from
each donor were initiated, maintained, and analyzed for the fold expansion of ␥␦-T
cells after equivalent culture durations.
*On day 0, IFN-␥ (1000 U/mL), anti-CD2 mAb S5.2 (indicated concentration,
isotype control IgG2a, or PBS) and IL-12 (10 U/mL or PBS) were added to PBMC
cultures (1 ⫻ 106 cells/mL). Twenty-four hours later (day 1), all cultures were
stimulated with OKT3 (10 ng/mL) and IL-2 (300 U/mL).
†Data are means (⫾SD) from separate experiments performed using PBMC
derived from 5 persons. P values are between indicated condition and control
(IFN-␥ ⫹ IgG2a ⫹ PBS), derived using a standard Student t test.
(Figure 1B). Table 1 shows the results of experiments performed
using PBMC obtained from several additional healthy donors. Data
are presented as the mean fold expansion of these cultures (⫾ SD,
n ⫽ 5) determined after equivalent-length short-term cultures.
Expansion of ␥␦-T cell induced by anti-CD2 mAb S5.2 requires
the presence of IL-12 and occurs as a consequence
of an increase in ␥␦–T-cell absolute numbers
The importance of IL-12 in the mAb S5.2-mediated expansion of
␥␦-T cell is shown in Figure 2, where the greatest percentage and
absolute numbers of ␥␦-T cells are found in cultures initiated in the
presence of anti-CD2 mAb S5.2 and exogenous IL-12. Importantly,
if a neutralizing mAb to human IL-12 (but not its isotype control,
not shown) is added to cultures initiated in the presence of mAb
S5.2, both the percentage of ␥␦-T cells (Figure 2A, lower
histogram) and the absolute number of ␥␦-T cells (Figure 2B, right
column, anti–IL-12) are significantly diminished. Furthermore, as
indicated in Figure 2, panel C, ␥␦–T-cell expansion induced by the
addition of S5.2 and exogenous IL-12 does not occur as a
consequence of the inhibition of ␣␤–T-cell growth or expansion. In
addition, from these data we conclude that 5 ␮g/mL is the optimum
mAb S5.2 concentration to promote ␥␦–T-cell expansion because
in most instances, at higher concentrations (⬎10 ␮g/mL), culture
growth is often globally inhibited in the presence of either the
anti-CD2 mAb S5.2 or the corresponding isotype control IgG2a
antibody (not shown). We attribute this finding to possibly the
nonspecific inhibitory effects of the low concentration of sodium
azide present in the mAb S5.2 preparation.
Figure 1. Anti-CD2 mAb S5.2 induces ␥␦–T-cell expansion from mitogenstimulated PBMC cultures. Cultures of human PBMC were initiated (day 0) by
pre-incubating with IFN-␥ (1000 U/mL) and then 24 hours later (day 1) with the
addition of both OKT3 (10 ng/mL) and IL-2 (300 U/mL). The indicated anti-CD2 mAb
or its corresponding isotype control antibody (5 ␮g/mL) was added on day 0. After 7 to
10 days, cultures were analyzed by FACS. Viable T-lymphocytes were first identified
by gating on the CD3-PE⫹ and PI⫺ populations. (A) Percentage of T-lymphocytes
staining with an anti-␥␦-TCR–FITC mAb is shown in each histogram. Results are
representative of experiments performed using materials obtained from at least 5
different persons. (B) Numbers of ␥␦-T cells found in cultures initiated in the presence
of the indicated anti-CD2 mAbs or isotype controls (P ⬍ .02 between mAb S5.2 and
IgG2a control). Other antihuman CD2 mAbs tested—6F10.3 (mouse IgG1), 39C1.5
(rat IgG2a), and LT-2 (mouse IgG2b, not shown)—do not induce ␥␦–T-cell expansion.
Data represent absolute numbers of ␥␦-T cells (mean ⫾ SD) determined in
quadruplicate; experiments were performed at least 3 separate times from samples
obtained from different persons.
Anti-CD2 mAb S5.2 induces ␥␦–T-cell expansion by an
agonistic and not a blocking interaction with CD2
The existence of accessory or alternative CD2 signaling pathways
triggered by mAbs to CD2, which function exclusively in ␥␦-T
cells, has previously been suggested by several investigators.18,26
Although most anti-CD2 mAbs capable of delivering proliferative
signals to either ␣␤- or ␥␦-T cells appear to do so only if combined
with a second anti-CD2 mAb recognizing a separate CD2 epitope,
single epitope-binding anti-CD2 mAbs have been reported that
appear to preferentially stimulate ␥␦-T cells.18,26,27 We performed
the following experiments to show that mAb S5.2 functions in an
agonistic and not a blocking capacity, thereby initiating rather than
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3830
LOPEZ et al
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
Figure 2. Expansion of ␥␦-T cell induced by anti-CD2 mAb S5.2 requires the presence of IL-12 and occurs as a consequence of an increase in ␥␦–T-cell absolute
numbers. Mitogen-stimulated PBMC cultures were initiated as described above. All cultures were primed with IFN-␥ on the day of culture initiation (day 0) in the presence of
anti-CD2 mAb S5.2 (or IgG2a isotype control, not shown). In addition, IL-12, PBS control, or antihuman IL-12 mAb (or isotype control for anti–IL-12 mAb, not shown) was
included in these cultures. Twenty-four hours later (day 1), all cultures were stimulated with mitogenic OKT3 and IL-2. After 7 days, both the percentage and the absolute
number of ␥␦-T cells were determined in cultures. Viable T cells were first identified by gating on the CD3-PE⫹ and PI⫺ populations. (A) Percentage of T cells staining with an
anti-␥␦-TCR–FITC mAb is shown in each histogram. (B) Absolute number of ␥␦-T cells found in indicated culture conditions (mean ⫾ SD) determined in quadruplicate. These
results are representative of experiments performed using materials obtained from at least 8 different persons. (C) Mitogen-stimulated PBMC cultures were initiated as
described above (day 0, IFN-␥; day 1, OKT3 and IL-2). On day 0, either IL-12 (10 U/mL) or PBS (⫺) was added to cultures. Likewise, anti-CD2 mAb S5.2 (or IgG2a isotype
control, not shown) was added at the indicated concentration (␮g/mL). After 14 days, absolute numbers of both ␣␤-T cells and ␥␦-T cells in cultures were determined by
multiplying the total cell number in culture by the percentage of ␣␤- and ␥␦-T cells, as measured by FACS. Data are presented as fold expansion (mean ⫾ SD) over starting
numbers of ␣␤-T cells (open bars) and ␥␦-T cells (solid bars), determined in triplicate. Results are representative of experiments performed using materials obtained from at
least 8 different persons.
inhibiting CD2 signaling events that contribute to IL-12–dependent
␥␦–T-cell expansion.
In mice and humans, both CD58 (LFA-3) and CD48 have 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.28-31 We reasoned, therefore, that if
anti-CD2 mAb S5.2 were inducing ␥␦–T-cell expansion by blocking interactions between CD2 on ␥␦-T cells and CD58 expressed
on other cells in culture, then the effect of a neutralizing anti-CD58
mAb would be the same—the enhancement of ␥␦–T-cell expansion. This is clearly not the case, as is shown in Figure 3, panel A.
To further demonstrate that mAb S5.2 is acting in an agonistic
rather than an inhibitory manner, we next compared the capacity of
both soluble and immobilized mAb S5.2 to induce ␥␦–T-cell
expansion. Antibodies that bind to specific cell surface receptors
usually cannot trigger signaling through these receptors unless
immobilized or cross-linked. Consistent with this, previous reports18,26 have shown that the single anti-CD2 mAbs known to
induce proliferative responses in ␥␦–T-cell clones appear to do so
only if immobilized or if accessory cells that can cross-link the
mAb through Fc receptors (FcR) are present. Because CD14⫹ cells
(monocytes) present in our mitogen-stimulated cultures express
FcR capable of cross-linking mAb S5.2 (mouse IgG2a), we
performed the following experiments using PBMC first depleted of
monocytes (⬍0.1% CD14⫹ cells; not shown). Figure 3, panel B
shows that in mitogen-stimulated cultures, ␥␦-T cells can be
induced to expand significantly by immobilized, but not soluble,
mAb S5.2.
IL-12–dependent mAb S5.2-mediated signaling through CD2
protects ␥␦-T cells from activation-induced cell death
Especially in the presence of IL-2, ␥␦-T cells rapidly undergo
apoptosis after receiving mitogenic stimuli through the TCR.17
Thus, one possible interpretation of our findings is that CD2
engagement by mAb S5.2 in the presence of IL-12 provides a
signal to a subset of ␥␦-T cells that renders them resistant to
activation-induced cell death caused by mitogenic OKT3 and IL-2.
Annexin V binds with high affinity to phosphatidylserine (PS),
which is normally confined to the inner plasma membrane leaflet of
nonapoptotic cells; the appearance of PS on the outer plasma
membrane leaflet is an early event associated with apoptosis. These
findings have been exploited to allow the examination of apoptosis
by flow cytometric means.32,33 Thus, annexin V-FITC, in combination with directly conjugated antibodies, can be used to detect
apoptosis occurring in phenotypically defined subpopulations of
cells within heterogeneous cell cultures.
To demonstrate that CD2 engagement by mAb S5.2 in the
presence of IL-12 protects ␥␦-T cells from activation-induced cell
death, we performed the following experiment. By convention, we
designated day 0 stimuli (IFN-␥, IL-12, and anti-CD2 mAb S5.2)
as protective signals. PBMC cultures were initiated as described
above. Those receiving day 0 stimuli were defined as protected;
those not receiving day 0 stimuli (PBS only) were defined as
unprotected. All cultures received OKT3 and IL-2 on day 1. After
receiving the day 1 mitogenic signals, ␥␦- and ␣␤–T-cell populations within protected and unprotected cultures were assessed for
apoptosis using 4-color flow cytometry 3 days after mitogen
stimulation. As shown in Figure 4, in the absence of protective day
0 signals, mitogen stimulation induces apoptosis in most ␥␦-T
cells. In contrast, apoptosis occurs to a far lesser extent in ␥␦-T
cells receiving day 0 protective signals. These results also show
that apoptosis occurring in ␣␤-T cells in response to mitogenic
stimulation is negligible under either of these conditions. In this
regard, ␣␤-T cells serve as a control and support in part our
argument that it is the ␥␦–T-cell compartment in cultures that is
most affected by our manipulations.
To demonstrate that the combination of CD2-mediated signals
and IL-12 signaling promotes the expansion of apoptosis-resistant
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BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
INDUCED RESISTANCE TO APOPTOSIS IN ␥␦-T CELLS
3831
contrast, a greater percentage of apoptotic ␥␦-T cells (annexin⫹/
PI⫺) and a significantly lower expansion of viable ␥␦-T cells are
noted in cultures to which no mAb S5.2 was added (Figure 5A-C).
Interestingly, as shown in Figure 5, panel D, in cultures to which
only mAb S5.2 has been added (no exogenous IL-12 or IFN-␥), it
appears that though a significant proportion of ␥␦-T cells in these
cultures remains viable, a significantly reduced expansion of viable
␥␦-T cell occurs. We interpret this to indicate that whereas
engagement of CD2 with mAb S5.2 may generate a critical signal
that induces resistance to apoptosis in mitogen-stimulated ␥␦-T
cells, these signals in the absence of IL-12 are not sufficient to
induce a significant expansion of apoptosis-resistant ␥␦-T cells.
Thus, in conjunction with CD2-mediated signals, IL-12 appears to
act synergistically to induce the greatest degree of expansion of
apoptosis-resistant ␥␦-T cells. It is especially important to emphasize that day 0 signals alone (IFN-␥, IL-12, and anti-CD2 mAb
S5.2), without day 1 signals (OKT3 and IL-2), cause no significant
␣␤- or ␥␦–T-cell proliferation (not shown).
Late, but not early, enhanced ␥␦–T-cell proliferation
characterizes mAb S5.2 and IL-12–induced ␥␦–T-cell expansion
Figure 3. Anti-CD2 mAb S5.2 induces ␥␦–T-cell expansion through an agonistic
and not a blocking interaction with CD2. (A) Anti-CD2 mAb S5.2 does not induce
␥␦–T-cell expansion by disrupting a CD2–CD58 interaction. Mitogen-stimulated
PBMC cultures were initiated as described above, now with the inclusion of IL-12 (day
0, IFN-␥, IL-12; day 1, OKT3 and IL-2). On day 0, either PBS (⫺), mAb S5.2 (mouse
IgG2a), antihuman-CD58 mAb L066.4 (mouse IgG2a), or mouse IgG2a isotype control
was added separately to identical cultures. After 14 days, cultures were analyzed
using FACS. Viable T cells were first identified by gating on the CD3-PE⫹ and PI⫺
populations. Percentage of T cells staining with an anti-␥␦-TCR–FITC mAb is shown
in each histogram. Results are representative of experiments performed using
materials obtained from at least 3 different persons. (B) Immobilized, but not soluble,
anti-CD2 mAb S5.2 can induce ␥␦–T-cell expansion in mitogen-stimulated, monocytedepleted PBMC cultures. Monocyte-depleted PBMC cultures were initiated as
described above, stimulated on day 0 with IFN-␥, IL-12, and either soluble or
plastic-immobilized mAb S5.2. Twenty-four hours later (day 1), cultures were
mitogenically stimulated with IL-2 and plastic-immobilized OKT3. After 21 days,
cultures were analyzed using FACS; the percentage CD3-APC⫹/␥␦-TCR-FITC⫹ cells
in each dot plot was indicated. Immobilized or soluble IgG2a (isotype control for mAb
S5.2) had a minimal effect on ␥␦–T-cell expansion (not shown). Results are
representative of experiments performed using materials obtained from at least 3
different persons.
␥␦-T cells, the following experiment was performed. Separate
PBMC cultures were prepared (Figure 5A-E) receiving on day 0 as
indicated, IFN-␥, IL-12, or anti-CD2 mAb S5.2. After 24 hours
(day 1), all cultures received mitogenic stimulation with OKT3 and
IL-2; after 21 days, ␥␦-T cells in each culture were analyzed for
apoptosis. The percentages of viable and apoptotic cells in each dot
plot are indicated in the corresponding quadrants and in the mean
fold expansion (⫾SD) of apoptosis-resistant ␥␦-T cells in these
cultures. As shown in Figure 5, panel E, the smallest percentage of
apoptotic ␥␦-T cells and the greatest fold expansion of nonapoptotic ␥␦-T cells is found in cultures that received protective signals,
including both exogenous IL-12 and anti-CD2 mAb S5.2. In
We have postulated that signaling through CD2 in the presence of
IL-12 can protect ␥␦-T cells from mitogen-induced apoptosis.
Alternatively, these signals might be leading to enhanced ␥␦–T-cell
expansion by simply providing an early proliferative advantage to
␥␦-T cells compared with ␣␤-T cells. The following experiments
were performed to show that this is not the case. By measuring
[3H]-thymidine incorporation, we compared proliferative capacities of ␥␦- and ␣␤-T cells at both early time points (culture
initiation, Figure 6A) and late time points (3 week cultures, Figure
6B). These data show that ␥␦-T cells isolated early from protected
cultures do not proliferate to a greater degree than ␣␤-T cells
isolated from identical cultures. This is in contrast to ␥␦-T cells
isolated later from protected cultures, which clearly manifest
enhanced proliferative capacities compared to ␣␤-T cells. These
data do not support a model where overrepresentation of ␥␦-T cells
in longer-term S5.2-treated cultures occurs as a consequence of an
early ␥␦–T-cell proliferative advantage.
IL-2 is a potent inducer of apoptosis in unprotected
but not protected ␥␦-T cells
Despite the significant early mitogen-induced apoptosis occurring
in unprotected ␥␦-T cells, after 7 days, surviving ␥␦-T cells are
present in these cultures, though to a lesser extent than in protected
cultures (Figure 7). Nonetheless, 1 day after the subsequent
addition of IL-2 (day 8), a significantly greater percentage of
unprotected but not protected ␥␦-T cells are induced to undergo
apoptosis. This indicates that compared to unprotected ␥␦-T cells,
protected ␥␦-T cells remain relatively resistant to IL-2–induced
apoptosis. Furthermore, the ability of agonistic mouse antihuman
CD95/Fas mAb CH11 to induce apoptosis in both protected and
unprotected ␥␦-T cells suggests that the greater resistance to
apoptosis of unprotected ␥␦-T cells does not result from a simple
loss of CD95/Fas expression and is supported by the findings that
CD95/Fas expression determined by FACS does not differ between
protected and unprotected ␥␦-T cells (data not shown).
In vitro antitumor activity of apoptosis-resistant ␥␦-T cells
measured against tumor cell lines
We next examined whether apoptosis-resistant ␥␦-T cells exert
measurable antitumor activity against human tumor cells in vitro.
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LOPEZ et al
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
Figure 4. CD2-mediated IL-12–dependent signals render human ␥␦-T cells resistant to mitogen-induced
apoptosis: analysis by 4-color flow cytometry. PBMC
cultures were initiated as described above. Those receiving day 0 signals (IFN-␥, IL-12, and anti-CD2 mAb S5.2)
by convention were defined as protected. Those receiving
no anti-CD2 mAb S5.2 or IL-12 on day 0 (PBS only) were
defined as unprotected. All cultures received OKT3 and
IL-2 24 hours later (day 1 mitogenic signals). Both ␣␤- and
␥␦–T-cell populations were first delineated by electronic
gating on the corresponding ␣␤- and ␥␦-T cells defined by
anti-CD3-APC and anti-TCR-␥␦–PE mAbs. Apoptosis occurring in ␣␤- and ␥␦–T-cell populations was then determined examining the uptake of annexin V-FITC and PI in
the respective gated events. Cells incubated with antihuman CD95/Fas mAb CH11 (mouse IgM,) or mouse IgM
isotype control antibody were used as positive and negative controls, respectively, to define apoptotic, viable, and
necrotic quadrants within dot plots. Percentages of ␣␤- or
␥␦-T cells appearing in the corresponding dot plot quadrants are indicated: viable (annexin⫺/PI⫺), apoptotic (annexin⫹/PI⫺), and necrotic (annexin⫹/PI⫹). Results shown
are representative of experiments performed using materials obtained from at least 4 different persons.
We explored this question using 3 distinct methods. In all
experiments, apoptosis-resistant ␥␦-T cells and control ␣␤-T cells
were expanded and isolated simultaneously from a given individual. In virtually all instances, control ␣␤-T cells derived from
either protected (day 0 plus day 1 signals) or unprotected cultures
(day 1 mitogenic stimulation alone) were indistinguishable with
regard to antitumor activity (not shown). Thus, ␣␤-T cells derived
from protected or unprotected cultures were used interchangeably
as controls for MHC-restricted alloreactivity.
We first examined the antitumor activity of apoptosis-resistant
␥␦-T cells against a number of human tumor cell lines using a
standard 4-hour 51Cr-release assay. Labeled tumor cells were
Figure 5. Both CD2-mediated signals and IL-12 signaling contribute to the
expansion of apoptosis-resistant ␥␦-T cells. On day 0, separate PBMC cultures
were initiated. Where indicated (⫹), IFN-␥ (1000 U/mL), IL-12 (10 U/mL), or anti-CD2
mAb S5.2 (5 ␮g/mL) was added to cultures with PBS (⫺) added as a control. After 24
hours, all cultures received mitogenic stimulation with OKT3 and IL-2 (day 1).
Cultures were maintained and expanded as described, and, after 21 days, ␥␦-T cells
in each culture (first gated as CD3-APC⫹, TCR-␥␦-PE⫹) were analyzed for apoptosis
using 4-color FACS, as described above. The percentages of viable (annexin⫺/PI⫺)
and apoptotic (annexin⫹/PI⫺) ␥␦-T cells in each dot plot are indicated in the
corresponding quadrants. The absolute number of viable ␥␦-T cells (annexin⫺/PI⫺)
found in each culture was determined with data expressed as the mean fold
expansion of viable ␥␦-T cells (⫾ SD), determined in triplicate. Results shown are
representative of experiments performed using materials obtained from at least 3
different persons.
Figure 6. [3H]-thymidine incorporation in sorted, highly purified ␣␤- and ␥␦-T
cells: late, but not early, enhanced ␥␦–T-cell proliferation induced by mAb S5.2.
Protected PBMC cultures were initiated as described with all cultures receiving IFN-␥,
IL-12, and mAb S5.2 on day 0 and OKT3 and IL-2 on day 1. (A) Early time points. After
24 hours (day 2), ␣␤- and ␥␦-T cells were sorted to high purity using FACS (greater
than 98% pure and greater than 96% viable; not shown). Then they were plated at
equivalent densities (5000 cells/well) in 96-well microtiter trays and were either
stimulated with IL-2 at 100 U/mL or left unstimulated (PBS; indicated as no IL-2). After
an additional 24 hours, [3H]-thymidine was added to cultures; 18 hours later, cells
were harvested onto glass fiber filters. (B) Late time points. As above, but after 3
weeks, ␣␤- and ␥␦-T cells were sorted to high purity from cultures initiated in parallel;
these cells were then assessed for proliferative capacity as described. Data are
presented as mean cpm (⫾ SD) of triplicate determinations. Results are representative of experiments performed using materials obtained from at least 2 different persons.
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BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
INDUCED RESISTANCE TO APOPTOSIS IN ␥␦-T CELLS
3833
Figure 7. IL-2 is a potent inducer of apoptosis in unprotected but not protected
␥␦-T cells. Unprotected and protected PBMC cultures were initiated on day 0, as
described above. All cultures received OKT3 and IL-2 after 24 hours (day 1 mitogenic
signals). On day 7, ␥␦-T cells in both unprotected and protected cultures were
analyzed for apoptosis, as measured by the uptake of annexin V-FITC and PI (upper
dot plots, day 7). The percentages of viable ␥␦-T cells (annexin⫺/PI⫺) and apoptotic
␥␦-T cells (annexin⫹/PI⫺) in each dot plot are indicated in the corresponding
quadrants. Subsequently, IL-2 (100 U/mL) was added to equivalent numbers of cells
from both protected and unprotected PBMC cultures. After overnight incubation,
apoptosis in ␥␦-T cells was once again determined (middle dot plots, day 8, IL-2).
Agonistic mouse antihuman CD95/Fas mAb CH11 (mouse IgM) was included in
identical cultures as a positive control (lower dot plots, day 8, CH-11). Day 8 cultures
(protected and unprotected) to which PBS alone or to which mouse IgM isotype
control for CH11 was added were essentially unchanged with respect to apoptosis
when compared to day 7 cultures (not shown). Addition of IL-2 had a minimal effect on
apoptosis detected in ␣␤-T cells within either protected or unprotected cultures (not
shown). Results are representative of experiments performed using materials
obtained from at least 2 different persons.
incubated with apoptosis-resistant ␥␦-T cells or control ␣␤-T cells
derived from a given person. Specific tumor lysis was measured,
and results obtained from 2 separate persons are shown in Figure 8,
panel A and panel B, respectively. Table 2 compiles the results of
additional experiments performed using apoptosis-resistant ␥␦-T
cells and control ␣␤-T cells derived from other healthy donors and
tested against the indicated tumor cell lines. To allow comparison
of multiple experiments, these data are presented as percentagespecific tumor lysis at an E:T ratio of 20:1.
Although 51Cr-release assays remain an established means by
which to measure the in vitro cytotoxic activity of effector
lymphocytes, we performed the following experiment to demonstrate that we could also measure the specific induction of apoptosis
in sensitive target cells on coculture with effector ␥␦-T cells. HeLa
cells were initially chosen for further experimentation. HeLa cells
were cultured alone or cocultured for 4 hours with apoptosisresistant ␥␦-T cells or control ␣␤-T cells at varying E:T ratios (1:1
to 20:1). As shown in Figure 9, panel A, T-lymphocytes alone (left
plot) and HeLa target cells alone (center plot) have characteristic
light scatter properties that allow both cell populations to be
distinguished, even when mixed together in coculture (right plot).
By gating only on HeLa cells, it was then possible to analyze HeLa
target cells for their uptake of annexin V-FITC; this served as a
measure of the ability of cocultured ␥␦- or ␣␤-T cells to induce
Figure 8. Antitumor activity of apoptosis-resistant ␥␦-T cells demonstrated
against human tumor cell lines. Purified ␥␦- and ␣␤-T cells used as effector cells
were sorted from 21-day cultures and were routinely enriched from cultures to 97% or
greater pure and 98% or greater viable (not shown). To avoid the activation of T cells
by the engagement of TCR, ␥␦-T cells were sorted as ␣␤-TCR⫺, CD5⫹ cells.
Similarly, ␣␤-T cells were sorted as ␥␦-TCR⫺, CD5⫹ cells. After sorting, all lymphocytes used as effector cells were cultured overnight in complete RPMI containing 10
U/mL IL-2 and were routinely found to be 95% or greater viable (not shown).
51Cr-labeled tumor cell targets (SK-MEL-5, SK-OV-3, NC-37, HeLa, and K-562) were
incubated at the indicated E:T ratios with apoptosis-resistant ␥␦-T cells (filled circles)
or control ␣␤-T cells (open circles) derived from 2 separate persons (column A and
column B, respectively). After a 4-hour incubation at 37°C, supernatants were
removed to determine 51Cr release in cpm. Data are presented as the mean
percentage specific target lysis (⫾ SD) of triplicate determinations.
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LOPEZ et al
Table 2. Percentage specific lysis (51Cr release) of human tumor cell targets
by apoptosis-resistant ␥␦-T cells and control ␣␤-T cells
derived from healthy donors
Effector lymphocytes added
and resultant specific lysis‡
Tumor target*
Lymphocyte source,
age (y)/gender†
␥␦-T cells
SK-MEL-3
38/M
19.8 ⫾ 0.9
3.5 ⫾ 0.8
20/F
16.2 ⫾ 2.9
4.8 ⫾ 1.9
␣␤-T cells
28/F
16.6 ⫾ 1.9
4.1 ⫾ 2.0
SK-MEL-5
38/M
14.1 ⫾ 0.6
2.9 ⫾ 0.9
28/F
26.2 ⫾ 5.4
5.3 ⫾ 2.2
SK-MEL-28
38/M
2.8 ⫾ 0.7
0.5 ⫾ 1.3
20/F
1.4 ⫾ 1.2
2.1 ⫾ 0.4
HeLa
41/F
75.3 ⫾ 2.0
9.1 ⫾ 0.4
47/F
65.7 ⫾ 1.0
8.5 ⫾ 1.2
OCI-Ly8
35/M
60.1 ⫾ 2.0
45.2 ⫾ 4.7
HSB-2
30/M
23.0 ⫾ 0.4
ND
K562
47/F
3.5 ⫾ 0.2
1.6 ⫾ 0.2
Comparison of cytotoxicity at E:T ratios of 20:1.
*Human tumor cell lines are described in “Materials and methods.”
†Highly pure (⬎97%, not shown) apoptosis-resistant ␥␦- and control ␣␤-T
lymphocytes were isolated by FACS from 21-day cultures of PBMC derived from
healthy donors, as described in “Materials and methods.” Age and sex of PBMC
donors are indicated.
‡Tumor target cells were labeled with 51Cr and cocultured with effector lymphocytes, as described in “Materials and methods.” Either apoptosis-resistant ␥␦-T cells
or control ␣␤-T cells derived from the indicated donors were added at various E:T
ratios (0.5:1 to 40:1). Percentage specific tumor lysis (mean ⫾ SD of triplicate
determinations) at an E:T ratio of 20:1 shown for comparison.
ND, not done.
apoptosis (Figure 9B). These representative data demonstrate that
apoptosis-resistant ␥␦-T cells can induce a significantly greater
degree of apoptosis in HeLa cells when compared to control ␣␤-T
cells. Similar results were obtained using other target cells such as
human melanoma cell lines (not shown).
Finally, we examined the ability of apoptosis-resistant ␥␦-T
cells to kill tumor targets using acridine orange and ethidium
bromide uptake as a means to distinguish live from dead target
cells. As shown in Figure 9, panel C, at an E:T ratio as low as 1:1,
Figure 9. Co-culture of tumor cells with apoptosis-resistant ␥␦-T cells: detection of tumor cell death. (A) T lymphocytes alone (left plot) and HeLa target cells
alone (center plot) have characteristic light scatter properties that allow each cell
population to be distinguished, even when mixed together in coculture (right plot). (B)
HeLa cells were cocultured for 4 hours with apoptosis-resistant ␥␦-T cells (TCR-␥␦) or
control ␣␤-T cells (TCR-␣␤) at the indicated E:T ratios (0:1 to 20:1). Cocultured cells
were then analyzed using FACS. Gating on the appropriate cell population (high
forward scatter and high side scatter), uptake of annexin V-FITC by HeLa cells was
determined and was taken as a measure of the ability of cocultured ␣␤- or ␥␦-T cells
to induce apoptosis. Light microscopy and FACS using anti-CD3 mAbs were used to
confirm that no tumor–lymphocyte aggregates remained after vortexing samples (not
shown). Data are presented as histograms, and the percentage of HeLa cells staining
with annexin V-FITC (and thus apoptotic) is indicated. These data are representative
of experiments performed at least 3 times on materials obtained from 3 separate
persons. (C) Measurement of HeLa target cell viability after coculture with apoptosisresistant ␥␦-T cells or control ␣␤-T cells for longer periods at lower E:T ratios. Target
HeLa cells were cultured alone or were cocultured with either human ␣␤- or ␥␦-T cells
at a 1:1 E:T ratio for 18 hours. On the addition of ethidium bromide and acridine
orange, cells were immediately viewed under fluorescence. As viewed using a 20⫻
objective lens, tumor cells were readily distinguished from effector lymphocytes by
size alone, permitting the enumeration of live (green) and dead (orange) tumor cells
in each well. The percentage of tumor cells remaining viable was thus derived by
dividing the number of green tumor cells by the number of green plus orange tumor
cells ([green]/[green ⫹ orange]) in each well. Quantitations were performed in
quadruplicate, with data presented as the mean viable tumor cells remaining per
high-power field ⫾ SD. Parallel determinations using trypan blue and a standard
inverted microscope were also made and were in agreement with the results of these
studies (not shown). These data are representative of experiments performed at least
3 times on materials obtained from 4 separate persons.
HeLa tumor cell viability was significantly decreased after 18
hours of coculture with apoptosis-resistant ␥␦-T cells but not with
control ␣␤-T cells. Although in agreement with the 51Cr-release
and annexin data above, these experiments may be of additional
biologic and even clinical significance given that this degree of
tumor cell killing was induced at a significantly lower E:T ratio
(1:1). Higher E:T ratios (ranging from 5:1 to 20:1, not shown)
resulted in a similar induction of target cell death with the addition
of ␥␦-T cells but not control ␣␤-T cells.
Discussion
The existence of alternative CD2 signaling pathways that function
predominantly, if not exclusively, in ␥␦- but not ␣␤-T cells has
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BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
been established by the important work of others.18,19,34 These
pathways were largely revealed by the recognition that certain
anti-CD2 mAbs could generate signals in cloned T cells, resulting
in proliferation as measured by standard means, such as [3H]thymidine incorporation. Although our current work is in agreement with these fundamental findings, we are able to extend these
findings in several important biologic and possibly clinically
relevant ways: our recognition that anti-CD2 mAb S5.2 can
generate IL-12–dependent signals that protect human ␥␦-T cells
from mitogen-induced apoptosis now provides us with the biologic
basis for obtaining large numbers of viable and, as we show,
functional human ␥␦-T cells.
The relation between CD2 signaling, IL-12, and acquired
resistance to apoptosis in ␥␦-T cells is likely complex, but several
observations may help elucidate the mechanisms underlying our
observation, beginning with signaling through CD2 itself. In most
studies, the capacity of anti-CD2 mAbs to signal through CD2 is
almost entirely assessed in terms of proliferation.26,27,35,36 However,
engagement of CD2 is not always associated with transduction of
proliferative signals. Breitmeyer and Faustman37 have shown that
the rosetting of human T cells by sheep red blood cells (which
express a functional homologue of human CD58/LFA3, the natural
ligand for CD2) results in the paralysis of TCR/CD3-mediated
signal transduction and activation. These studies demonstrated that
both calcium mobilization and proliferative responses to subsequent mitogenic anti-TCR antibodies were blocked for up to 48
hours after CD2 engagement. Similarly, in a more recent report,
Miller et al38 showed that interaction between CD58/LFA-3 and
CD2 can lead to T-cell unresponsiveness to antigenic or mitogenic
stimuli in vitro. Conversely, it has been reported that the ability of
certain anti-CD2 mAbs to stimulate ␥␦–T-cell clones was significantly diminished by the co-engagement of CD3, suggesting a
possible antagonistic interaction between ␥␦-T cell CD2 and CD3
signaling pathways.26
Reasoning along these lines, we propose that ␥␦–T-cell expansion may occur in our system, not simply as a consequence of
CD2-mediated proliferative signals per se but rather as a consequence of postreceptor CD2-mediated disruption or moderation of
signals that could otherwise induce apoptosis in already apoptosisprone ␥␦-T cells. In such a model, it is particularly important to
emphasize that mAb S5.2 functions in an agonistic rather than in a
blocking capacity, initiating rather than inhibiting CD2 signaling
events (Figures 3).
In any event, it is important to note that CD2 signaling in the
absence of IL-12 is insufficient to lead to the expansion of
apoptosis-resistant ␥␦-T cells in mitogen-stimulated PBMC cultures, as is established by the neutralization experiments shown in
Figure 2, panels A and B. This suggests that CD2 signaling is
necessary, but not sufficient, for the expansion of apoptosisresistant ␥␦-T cells. Conversely, it is evident that in the absence of
CD2 signaling, IL-12 can neither enhance ␥␦–T-cell expansion
(Figure 2C, Table 1) nor inhibit apoptosis (Figure 5C) in mitogenstimulated ␥␦-T cells. Taken together, these data suggest that, in
our system, ␥␦-T cells do not optimally respond to IL-12 without
first receiving signals through CD2. Such an interpretation would
be in keeping with the findings of Gollub et al,29,30 who have
previously shown that responsiveness to IL-12 in activated T cells
was indeed regulated and dependent on signals delivered through
CD2. Thus, the requirement for CD2 signaling in our system might
be explained on the basis that these signals simply enhance
responsiveness to IL-12 in ␥␦-T cells, leading to apoptosis
resistance. This would be in part supported by data that show
INDUCED RESISTANCE TO APOPTOSIS IN ␥␦-T CELLS
3835
IL-12–receptor (IL-12R) is found to be up-regulated on protected but
not on unprotected ␥␦-T cells (unpublished data, manuscript in
preparation).
It is unclear how IL-12 might inhibit apoptosis in mitogenstimulated ␥␦-T cells, though the observations of Perussia et al39
may provide an important clue. In one study, it was shown that
whereas IL-12 always acts synergistically with IL-2 in inducing
␣␤–T-cell proliferation, in contrast, IL-12 could significantly
inhibit IL-2–induced proliferation in resting ␥␦-T cells.39 Thus, it is
conceivable that during the first critical 24 hours (day 0) of our
protected cultures, responsiveness to IL-12 is first established
through CD2-mediated signals delivered by mAb S5.2. Subsequent
strong mitogenic signals delivered on day 1, in particular, IL-2,
would then be less able to induce apoptosis in ␥␦-T cells. Thus,
␥␦-T cells spared this initial IL-2–mediated apoptosis would be
those observed to expand in our cultures. That resistance to
apoptosis in ␥␦-T cells might be a consequence of blunted
responsiveness to IL-2 is supported by the findings shown in Figure
7, where clearly a differential susceptibility to IL-2–induced
apoptosis is noted between protected and unprotected ␥␦-T cells.
This is consistent with our observation that protected ␥␦-T cells fail
to up-regulate the expression of CD25/IL-2R␣ when compared to
unprotected ␥␦-T cells (unpublished data, manuscript in preparation) and is further supported by our findings that ␥␦-T cells
isolated early from protected cultures appear to proliferate to a
lesser extent in response to IL-2 (Figure 6).
Although the principal tenet of our model is that ␥␦-T cells in
protected cultures preferentially expand as a consequence of
acquired resistance to apoptosis, it is important to appreciate that
␥␦-T cells can and do expand in unprotected cultures as well, albeit
to a lesser extent (Figures 1B, 2C, 5, Table 1). This must be taken
into account, particularly when the magnitude of ␥␦–T-cell expansion is compared between various culture conditions, especially
after longer periods of expansion (Figure 5). In view of this, it must
be emphasized that in protected cultures, not all ␥␦-T cells are
resistant to apoptosis; conversely, in unprotected cultures, not all
␥␦-T cells are apoptotic (Figures 4, 5, 7). This suggests that
protective signals might simply alter the proportion of apoptosisresistant and -sensitive ␥␦-T cells present at a given time in culture.
Hence, if a significant fraction of ␥␦-T cells in protected cultures
acquire resistance to apoptosis early, even if only transiently, this in
itself could account for the greater ␥␦–T-cell expansion observed at
later time points. This is supported by the observation that in both
protected and unprotected cultures, the proportions of viable and
apoptotic ␥␦-T cells change in a predictable manner over time: at
very early time points (up to culture day 1), protected and
unprotected cultures contain comparable total numbers and similar
proportions of viable and apoptotic ␥␦-T cells (not shown).
However, by culture day 2 to day 4, though total ␥␦–T-cell numbers
still remain comparable (not shown), protected cultures are routinely found to contain a greater proportion of apoptosis-resistant
␥␦-T cells (Figure 4). Eventually, as protected and unprotected
cultures enter exponential growth phases (usually simultaneously
between day 6 and day 10; not shown), protected cultures—already
containing a larger proportion of viable ␥␦-T cells (Figure 7)—
invariably proceed to surpass unprotected cultures with regard to
expansion of viable ␥␦-T cells (Figure 1). This survival advantage
of ␥␦-T cells in protected cultures is further accentuated after
restimulation with IL-2 (Figure 7) and is ultimately reflected in the
larger total numbers of viable ␥␦-T cells found in protected cultures
at even later time points up to 45 days and beyond (not shown).
Although we do not propose that protective signals render ␥␦-T
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LOPEZ et al
cells permanently resistant to apoptosis, it is interesting to note that
by the third week in culture, compared to unprotected cultures,
protected cultures still contain a larger proportion of apoptosisresistant ␥␦-T cells, though the differences in the magnitude of
␥␦-T cell–expansion is often no longer as great (Figure 5).
With regard to antitumor activity, the data provided in Figures 8
and 9 and in Table 2 establish that apoptosis-resistant ␥␦-T cells,
expanded and isolated from a number of persons, do indeed possess
the ability to kill a variety of human tumors in vitro as measured by
several methods. Although the mechanism of ␥␦–T-cell tumor
recognition is not addressed here, several points are noteworthy.
First, in virtually all instances in which killing is observed,
apoptosis-resistant ␥␦-T cells were found to kill tumor targets to a
significantly greater degree than ␣␤-T cells on a cell-for-cell basis.
These data suggest that ␥␦-T cells recognize target cells by
mechanisms distinct from those used by ␣␤-T cells, as is known.1,2,4
Second, it is interesting to note that a number of tumor cell lines of
epithelial origin were found to be relatively sensitive to killing by
␥␦-T cells. This is especially intriguing given the recent findings
that ␥␦-T cells expressing particular V␦ TCR can recognize an
MHC class I-related molecule frequently expressed on tumor cells
of epithelial origin.40-42 Third, that the prototypic natural killer
(NK)–sensitive target K562 is found to be relatively resistant to
␥␦-T-cell–mediated killing might also suggest the involvement of
mechanisms distinct from NK or lymphokine-activated killer
cell–mediated killing. These cytotoxicity data, though not exhaustive—especially with respect to the number of donors tested or the
number of tumor targets assessed—nevertheless serve to underscore the practical significance of our findings, namely that an
otherwise rare subset of human lymphocytes can now readily be
expanded, isolated, and subjected to more rigorous study. This is
particularly relevant as one considers the potential for using such
cells in various forms of adoptive immunotherapy.
Whether ␥␦-T cells have therapeutically exploitable biologic
properties such as antiviral, antitumor, or hematopoietic stem cell
graft-facilitating effects, remains to be determined. Although far
larger numbers of apoptosis-resistant ␥␦-T cells would be required
to design adoptive cellular therapy clinical experiments, it should
be emphasized that usually only 2 mL PBMC (1 ⫻ 106 cells/mL),
derived from 3 to 5 mL fresh blood, is used as starting material to
generate cultures larger than 50 to 100 ⫻ 106 T cells containing
40% to 60% ␥␦-T cells after 2 to 3 weeks or longer. Thus, with
proper culture optimization, many more than 1 ⫻ 109 viable ␥␦-T
cells could readily be obtained after ex vivo expansion using, as
starting materials, safely procurable volumes of fresh autologous or
allogeneic peripheral blood. Such ␥␦-T-cell–enriched products
could readily be subjected to positive or negative selection
techniques (immunomagnetic columns, high-speed FACS, panning, and so on) to obtain a product essentially “pure” in terms of
␥␦–T-cell content and, thus, ideal for examining clinical questions,
something that was until now not practically possible.
Acknowledgment
We thank Christopher Ferrigno for his technical contributions to
this work.
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2000 96: 3827-3837
CD2-mediated IL-12−dependent signals render human γδ-T cells
resistant to mitogen-induced apoptosis, permitting the large-scale ex
vivo expansion of functionally distinct lymphocytes: implications for the
development of adoptive immunotherapy strategies
Richard D. Lopez, Shan Xu, Ben Guo, Robert S. Negrin and Edmund K. Waller
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