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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3315-3322
Interleukin-12 Inhibits Graft-Versus-Host Disease Through an
Fas-Mediated Mechanism Associated With Alterations in Donor T-Cell
Activation and Expansion
By
Bimalangshu R. Dey,
Yong-Guang Yang,
Gregory L. Szot,
Denise A. Pearson, and
Megan Sykes
From the Bone Marrow Transplantation Section, Transplantation Biology
Research Center, Surgical Service, Massachusetts General
Hospital/Harvard Medical School, Boston, MA.
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ABSTRACT |
We have recently made the paradoxical observation that a single
injection of recombinant murine interleukin-12 (IL-12) on the day of
bone marrow transplantation (BMT) inhibits graft-versus-host disease
(GVHD) in lethally irradiated mice receiving fully major histocompatability complex (MHC)-mismatched bone marrow
and spleen cells. We have now examined the mechanism of this effect of
IL-12 on acute GVHD. By day 4 post-BMT, IL-12-treated mice showed
marked reductions in splenic donor CD4+ and
CD8+ T cells compared with GVHD controls. Expression of
the early activation markers IL-2R alpha chain (CD25) and CD69 on
splenic donor CD4+ cells was considerably higher at early
time points (36 and 72 hours post-BMT) in IL-12-treated mice compared
with GVHD controls. However, the later, GVHD-associated increase in
CD25 and very late antigen-4 (VLA-4) expression on donor T cells was
greatly depressed in IL-12-protected mice compared with GVHD controls. The marked GVHD-associated expansion of host-reactive T helper cells by
day 4 was also completely inhibited in the IL-12-treated group.
Expression of Fas was increased on donor CD4 cells of IL-12-treated mice compared with those of controls on days 3 through 7 post-BMT. Furthermore, the ability of IL-12 to protect against GVHD was at least
partially dependent on the ability of donor cells to express functional
Fas molecules. We conclude that IL-12 treatment at the time of BMT
markedly perturbs the activation of alloreactive donor
CD4+ T cells that play a critical role in the
pathogenesis of acute GVHD. We hypothesize that these perturbations
culminate in Fas-dependent apoptosis of donor T cells, thus impeding
their expansion and their GVHD-promoting activity.
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INTRODUCTION |
DESPITE MAJOR ADVANCES in
chemotherapeutics, immunosuppressive therapy, and supportive care, the
full therapeutic potential of bone marrow transplantation (BMT) for the
treatment of leukemia has not yet been realized. Graft-versus-host
disease (GVHD), which arises from an attack of donor T cells against
alloantigens of the recipient,1 is a continued threat to
the successful outcome of allogeneic BMT, especially with the
increasing use of unrelated donors. With modern immunosuppressive drugs
for GVHD prophylaxis, significant GVHD still occurs in 30% to 40% of
HLA-matched sibling transplants.2 The problems of GVHD and
failure of engraftment are magnified in the presence of HLA
mismatches,3,4 so that extensively HLA-mismatched BMT
cannot be routinely performed. Removing T lymphocytes from the marrow
graft (T-cell depletion [TCD]) effectively prevents GVHD, but at the
expense of an increase in marrow graft rejection and leukemic
relapse.5,6 Immunosuppressive drugs used for GVHD
prophylaxis may also increase the rates of leukemic relapse by
suppressing this T-cell reactivity.2 Therefore, an ability
to separate the beneficial engraftment-promoting and antileukemic
effects of donor T cells while diminishing their ability to induce GVHD
might allow HLA-mismatched BMT to be performed and lead to reduced
leukemic relapse rates.
We have recently made the paradoxical observation that a single
injection of recombinant interleukin-12 (IL-12) inhibits acute GVHD in
a fully major histocompatability complex (MHC) plus multiple minor
antigen-mismatched murine BMT model.7 Administration of a
single injection of IL-12 on the day of BMT led to marked inhibition of
acute GVHD mortality in lethally irradiated H-2b mice
receiving fully MHC plus multiple minor antigen-mismatched allogeneic
bone marrow and spleen cells. A small additional protective effect was
observed when T-cell-depleted host-type bone marrow cells (BMC) were
added to the inoculum, even though those host-type BMC were rapidly
eliminated, so that full donor chimerism was observed by about 7 days
post-BMT.7 The protective effect of IL-12 against GVHD was
surprising because this cytokine is known to induce Th1 differentiation
and cytotoxic T-lymphocyte function, and both Th1 immune responses and
cytotoxic T lymphocytes (CTL) have been implicated in the
pathogenesis of acute GVHD, including the fully mismatched model in
which the protective effect of IL-12 was originally
discovered.7 Moreover, the ability of IL-12 to inhibit GVHD
is dependent on the Th1 cytokine IFN- .8 Initial studies
addressing the mechanism of this effect suggested that the early
expansion of donor T cells that occurs in the first week
post-BMT9 was inhibited by IL-12.7 We have now
attempted to further delineate the mechanisms of this paradoxical
inhibitory effect of IL-12 on acute GVHD by examining the effect of
IL-12 on the upregulation of donor T-cell activation markers and on the
expansion of host-reactive T cells. The results suggest that IL-12
markedly perturbs the activation and early expansion of host-reactive
donor T cells, at least in part through a Fas-dependent mechanism.
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MATERIALS AND METHODS |
Mice.
Specific pathogen-free female C57BL/6 (B6, H-2b,
Kb Ib Db) and A/J
(H-2a, Kk Ik Dd) mice
were purchased from the Frederick Cancer Research Facility (NCI,
Frederick, MD). Female MRL/MpJ+ +/+ control (designated MRL)
and lpr/lpr MRL/MpJ-lpr (designated LPR) mice (both
H-2k) were purchased from the Jackson Laboratory, Bar
Harbor, ME. Animals were housed in sterilized
microisolator cages and received autoclaved feed and autoclaved,
acidified drinking water.
Preparation of BMC, spleen cells, and T-cell-depleted BMC.
BMC and spleen cell suspensions were prepared as
described.10 Host-type BMC were depleted of T cells with
anti-CD4 monoclonal antibody (MoAb) (GK1.5 in ascites, 1:5,000
dilution),11 and anti-CD8 MoAb (2.43 in ascites, 1:1,500
dilution)12 plus low toxicity rabbit complement (1:14
dilution), as described previously.13
Total body irradiation and BMT.
Recipient mice were lethally irradiated (9.75 to 10.25 Gy,
137Cs source, 1.1 Gy/min) and reconstituted within 4 to 8 hours with a single 1-mL intravenous inoculum containing 15 to 17 ×106 A/J spleen cells plus 10 × 106
A/J BMC with 5 × 106 TCD B6 BMC (allogeneic group),
or with 5 × 106 TCD B6 BMC only (syngeneic group). In
other experiments, irradiated B6 mice received TCD B6 BMC plus either
MRL control or Fas-deficient LPR BMC (10 × 106) and
spleen cells (10 × 106). To avoid bias from
cage-related effects, animals were randomized before and after BMT as
described.14 Survival was followed for 60 to 100 days. All
experiments used for cell or cytokine analysis in this report included
at least five control mice in each group that were followed for
survival and in which the protective effect of IL-12 was documented and
was similar in magnitude to that we have reported.7
IL-12 administration.
In earlier experiments, 4,900 IU of murine recombinant IL-12 (kindly
provided by Genetics Institute, Cambridge, MA), with specific activity
of 4.9 to 5.5 × 106 IU/mg, was injected
intraperitoneally into recipient mice approximately 1 hour before BMT.
In later experiments, the dose was reduced to 2,400 U, because other
experiments to be reported elsewhere indicated that similar
protection was afforded by either dose of IL-12 (M.S., unpublished
data, January 1996).
MoAbs and flow cytometry.
Spleen cells or cell suspensions from inguinal and mesenteric lymph
nodes were analyzed by two- or three-color flow cytometry (FCM) on a
FACScan (Becton Dickinson, Mountain View, CA). To block nonspecific
FcR binding of labeled antibodies, 10 µL of undiluted culture
supernatant of 2.4G2 (rat antimouse FcR MoAb)15 was added to the first incubation. To determine the percentage of donor-
and host-type T cells in spleens, cells were stained with fluorescein
isothiocyanate (FITC)-conjugated rat antimouse CD4 (PharMingen, San
Diego, CA) and anti-CD8 (Caltag, San Francisco, CA) MoAbs for 30 minutes at 4°C, washed, incubated with biotinylated anti-H-2Dd MoAb 34-2-1216 for 30 minutes at
4°C, washed again, incubated for 10 minutes with
phycoerythrin-streptavidin (PEA), then washed again. To study the
expression of activation markers on T cells, spleen cells were stained
with FITC-conjugated anti-CD25 or anti-VLA-4 MoAbs for 30 minutes at
4°C, washed, incubated with biotin-conjugated anti-CD4 or anti-CD8
MoAbs for 30 minutes at 4°C, washed again, incubated for 10 minutes
with PEA, then washed again. A control tube contained nonreactive mouse
IgG2a MoAb HOPC-FITC plus 34-2-12-biotin-PEA. Dead cells
were excluded during FCM analysis by gating out low forward
scatter/high propidium iodide-retaining cells. For three-color determination of the percentage of donor CD4 cells expressing CD25,
CD69, and VLA-4, spleen cells were stained with FITC-conjugated anti-CD25, anti-CD69, or anti-VLA-4 MoAbs, plus PE-conjugated anti-CD4
MoAb, washed, incubated with biotin-conjugated anti-H-2Kb
MoAb 5F1 (recipient class I antibody), washed again, and incubated with
CyChrome-Streptavidin for 10 minutes and then washed twice. To
determine the expression of Fas on donor CD4 T cells, cells were
stained with 5F1-FITC plus PE-conjugated anti-CD4 MoAb, washed, incubated with biotinylated anti-Fas MoAb (PharMingen), washed, incubated with CyChrome-Streptavidin, then washed twice. Five to ten
thousand 5F1-negative (ie, donor), CD4-positive cells were collected
for analysis of Fas expression.
Limiting dilution analysis.
For measurement of activated helper cell (Th) frequencies, varying
numbers of responder cells were incubated with 6 × 105, 30 Gy irradiated stimulator cells in 96-well
round-bottomed plates. Twenty-four wells each containing 30,000, 10,000, 3,333, 1,111, 370, and 123 responder cells were prepared. After
24 hours of incubation, 100 µL of supernatant was obtained from each
well and transferred to parallel plates containing 8,000 IL-2/IL-4-dependent CTLL cells per well. These were
incubated in the supernatants for 24 hours, and 1 µCi of
3H-thymidine was then added to each well. After an
additional 18 hours, cells were harvested with a Tomtec harvester
(Wallac, Gaithersburg, MD) and 3H-thymidine uptake was
counted on a Betaplate  counter (Pharmacia LKB,
Gaithersburg, MD). Wells were considered positive if
3H-thymidine uptake was three standard deviations greater
than the mean 3H-thymidine uptake in 24 wells containing
supernatants from stimulator cells alone. The Poisson distribution was
used to determine the frequency of antigen-responsive Th that
recognized each stimulator strain, and statistical analysis was
performed by  2 minimization analysis as described by
Taswell.17
Analysis of serum cytokine levels.
Animals were selected randomly from each group and were killed by
exsanguination under anesthesia. Their blood was clotted on ice for 10 or 15 minutes, and serum was separated by centrifugation at 4°C and
was stored at  80°C. Serum tumor necrosis factor (TNF)- levels were measured in duplicate wells using the Cytoscreen Mouse TNF- enzyme-linked immunosorbent assay (ELISA) kit (Biosource International, CA). The minimum detectable concentration of TNF- was
less than 3 pg/mL.
Histopathologic evaluation.
These evaluations were performed blindly on hematoxylin and
eosin-stained liver and lung tissue sections by a hematopathologist.
Statistical analysis.
Two-tailed Student's t-tests for comparison of means were
performed for evaluation of the effects of IL-12 on cell populations and cytokine levels between groups at individual time points. Survival
data were analyzed using the Kaplan-Meier method of life table
analysis, and statistical analysis was performed with the Mantel-Haenzsen test or the log rank test. A P value of less
than .05 was considered to be significant.
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RESULTS |
Effect of IL-12 on donor T-cell numbers.
We have previously shown marked reductions in donor CD4+
and CD8+ T cells in the spleens of IL-12-protected
compared with GVHD control mice on day 4 post-BMT, with a reversal of
this difference by day 7.7 We confirmed these results in
three additional experiments and extended the analysis to include day
5. Overall, T-cell numbers in the spleens of GVHD control mice
increased to a peak level on day 5, then declined between days 5 and 7. In spleens of the IL-12-treated group, donor CD4+ and
CD8+ cells were markedly decreased compared with GVHD
controls on day 4 (P = .04), increased by day 5 to a peak that
was lower than that in GVHD controls, but remained relatively constant
between days 5 and 7 (Fig 1A). Because
mortality in GVHD controls was considerable after day 7, no further
comparisons could be made at later time points.
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| Fig 1.
Effect of IL-12 treatment on donor T-cell expansion and
activation. Lethally irradiated B6 mice received A/J BMC and spleen cells plus TCD host-type (B6) BMC with or without IL-12, 4,900 IU,
administered on day 0. (A) Mean number of CD4+ and
CD8+ T cells in spleens of GVHD control and
IL-12-protected mice according to donor ( ;) versus host ( )
origin, as determined by separate staining with anti-CD4 versus
anti-Dd and anti-CD8 versus anti-Dd. Mean
values obtained from six mice per group at each time point are shown.
(B) Altered expression of CD25 on donor T cells in IL-12-protected
mice. Spleen cells were analyzed by two-color FCM to determine
percentages of CD4+ and CD8+ T cells
expressing CD25 on days 4, 5, and 7 after BMT. The mean of the products
of the percentages of CD25+ CD4 or CD8 cells and the
spleen cell yield for GVHD control ( ) and IL-12-protected mice
() are shown (n = 3 mice per group per time point). Black bars
() represent splenic T cells from normal A/J mice. Similar results
were obtained in two additional experiments. Because additional stains
(anti-Dd v anti-CD25) showed that all
CD25-expressing spleen cells were of donor origin in these animals, it
can be inferred that all CD25+ T cells shown in this
figure are of donor origin. (C) Mean number of VLA-4+ CD4
and CD8 cells in spleens of GVHD control () and IL-12-protected mice () on days 4, 5, and 7 post-BMT. The average of the product of
the percentage of VLA-4+ CD4 or CD8 cells and the spleen
cell yield (n = 3 mice per group per time point) is shown.
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We hypothesized that the markedly reduced numbers of donor T cells in
the spleens of IL-12-treated mice compared with GVHD controls on day 4 post-BMT might be due either to (1) redistribution of allogeneic T
cells into GVHD target organs such as liver and lung, or (2) a
significant inhibitory effect of IL-12 on the activation and expansion
of donor T cells. To address the possibility of redistribution,
histopathologic findings (tissue sections stained with
hematoxylin-eosin) were compared for lungs and livers of GVHD control
and IL-12-treated mice on day 4 post-BMT (n = 2 from each group).
Livers and lungs did not show significant cellular infiltrations in
either group on day 4 (data not shown), making tissue redistribution an
unlikely explanation for the marked reductions in donor T cells in
spleens of IL-12-treated mice at this time.
Effect of IL-12 on donor T-cell activation markers.
To address the hypothesis that IL-12 inhibits activation of donor T
cells, we compared the expression of activation-associated markers on
donor T cells in spleens of GVHD control and IL-12-protected mice.
Results of two-color analyses are summarized in Fig 1. The absolute
numbers of CD4+ and CD8+ cells expressing CD25
on day 4 after BMT were markedly decreased in IL-12-treated mice
compared with GVHD controls (Fig 1B). These striking differences in
CD25 expression on donor CD4 T cells on day 4 were confirmed directly
using three-color FCM in three of three experiments (total, n = 9 per
group). Although by day 5 the number of CD25+,
CD4+, CD25+, and CD8+ T cells had
increased in the spleens of IL-12-protected mice, their numbers
remained lower than in GVHD controls. The number of CD25+
donor T cells in both groups declined to baseline by day 7. The CD25+ T cells shown in Fig 1B were all of donor origin
because there were no CD25-expressing host cells detected by FCM in
either group (not shown).
The distribution and kinetics of VLA-4 expression were quite distinct
from those of CD25. Very few VLA-4-expressing cells were detected in
spleens of IL-12-protected mice on day 4, and half of these were of
host origin, whereas GVHD controls contained significant numbers of
VLA-4+ cells that were almost all of donor origin (not
shown). These included substantial numbers of both CD4+ and
CD8+ donor T cells (Fig 1C). By day 5, essentially all
CD4+ and CD8+ cells and VLA-4+
cells in spleens of both groups were entirely of donor origin (Fig 1A
and data not shown). Therefore, the VLA-4+ CD4+
and VLA-4+ CD8+ cells shown in Fig 1C are
mainly of donor origin, except for the IL-12-treated group on day 4, when some of the very few VLA-4+ CD4+ cells
present (Fig 1C, left half) might be of host origin. A marked reduction
in VLA-4 expression on donor T cells was observed on day 4 post-BMT in
IL-12-protected mice. Direct confirmation of this result for donor CD4
cells on day 4 was obtained using three-color FCM (total, n = 9 per
group; data not shown). VLA-4 expression among donor T cells later
increased in IL-12-protected mice to become similar to that of GVHD
controls on days 5 and 7 (Fig 1C).
Premature activation of donor CD4 cells by IL-12.
We hypothesized that IL-12 might activate donor T cells earlier than in
uninhibited GVHD, in which marked donor T-cell activation is not
evident in the spleen before day 4. To address this hypothesis, we
performed three-color FCM on spleen cells to examine CD25 and CD69
expression on gated donor CD4+ spleen cells at 36 hours and
on days 3 and 4 post-BMT. As is shown in
Fig 2A, CD25 expression on donor
CD4+ cells was considerably higher (bottom row, left and
middle) at 36 hours and on day 3 post-BMT in IL-12-treated mice
compared with GVHD controls (middle row, left and middle). However, on day 4 the pattern reversed, and CD25 expression was markedly increased on donor CD4+ cells of GVHD controls (middle row, right),
but not in IL-12-protected animals (bottom right), consistent with
results presented above.
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| Fig 2.
Increased expression of CD25 and CD69 on donor
CD4+ T cells at 36 hours and on day 3 in spleens of
IL-12-protected mice. Lethally irradiated B6 mice received A/J BMC and
spleen cells plus TCD B6 BMC with or without IL-12 on day 0. Spleen
cells were analyzed by three-color FCM to determine percentages of
CD4+ Dd+ donor cells expressing CD25 (A)
and CD69 (B). Similar results were obtained from six mice per group at
each time point in two independent experiments.
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Expression of the early activation marker CD69 on donor
CD4+ cells was also markedly higher in IL-12-treated mice
(Fig 2B, bottom row, left and middle) compared with GVHD controls
(middle row, left and middle) at 3 days and was also slightly higher at 36 hours post-BMT. Similar results were observed in a repeat
experiment. Therefore, expression of CD69 and CD25 on donor
CD4+ cells increased in IL-12-protected mice between 36 and 72 hours, then decreased by day 4, when CD25 expression increased
markedly in GVHD controls.
Effect of IL-12 on donor Th activity.
To investigate the possibility that IL-12 inhibits the function of
host-reactive donor Th cells, we used limiting dilution analysis (LDA)
to quantify these cells in spleens of GVHD control and IL-12-protected
mice. This approach is quantitative and allows dilution of cells with
suppressive activity,18 which we have found to inhibit
T-cell responses in bulk culture at early time points (data not shown).
To obtain the best estimate of activated Th present at the time of
animal sacrifice, spleen cells from BMT mice were cultured with
stimulator cells for only 24 hours before supernatants were obtained,
when their ability to induce proliferation of the IL-2- and
IL-4-responsive cell line CTLL was assessed. Results of Th LDA are
presented in Fig 3, which shows that the
total numbers of host-reactive (B6-reactive) Th per spleen were
markedly higher in spleens of GVHD control mice than in unprimed A/J
mice at day 4. However, the host-reactive Th frequencies and total
number of host-reactive Th cells were markedly diminished in
IL-12-treated mice compared with GVHD controls (P = .006), and
were even lower than those detected for naive A/J mice (Fig 3A). Donor
and host T cells were quantified by two-color FCM so that the frequency
of host-reactive cells could be expressed as a fraction of the donor T
cells present in spleens. Among the reduced numbers of donor
CD4+ cells in spleens of IL-12-treated animals on day 4 post-BMT, a markedly lower fraction responded to host antigens compared with CD4+ cells in GVHD control mice (greater than 1 in 25 CD4 cells were host-reactive in GVHD controls v approximately 1 in 1,000 CD4 cells in IL-12-protected mice). Spleens of recipients of
TCD B6 (syngeneic) marrow alone (with or without IL-12 treatment)
contained undetectably low numbers of host-reactive Th, similar to
those of normal B6 mice (data not shown).
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| Fig 3.
Reduced host-reactive Th frequencies in spleens of
IL-12-protected mice. Lethally irradiated B6 mice received A/J BMC and spleen cells plus TCD B6 BMC with or without IL-12 on day 0. B6-reactive and A/J-reactive (specificity control) Th frequencies per
spleen were determined by limiting dilution analysis on days 4 and 7 after BMT. The total numbers of B6 (host)-reactive (A) and A/J (donor)-reactive (B) Th per spleen, as determined by the product of the
Th frequency and the total number of spleen cells obtained from each
individual animal, are shown. ( ) GVHD control mice; ( )
IL-12-protected mice; (*) normal A/J mice; () normal B6 mice. Results of three experiments are combined.
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By day 7, total numbers of host-reactive Th and the fraction of donor
CD4+ cells reacting to host antigens had increased compared
with day 4 in IL-12-treated mice. In the same period, total numbers of host-reactive Th (Fig 3A) and the fraction of donor CD4+
cells responding to host antigens decreased markedly in spleens of GVHD
control mice. However, both the absolute number of Th per spleen (Fig
3A) and the fraction of CD4+ cells reacting to host
antigens (approximately 1 in 125 CD4 cells for GVHD controls v
1 in 250 to 500 in IL-12-protected mice) remained slightly higher in
the GVHD control group than in the IL-12-protected group on day 7.
As is shown in Fig 3B, the frequency of donor (A/J)-reactive Th cells
was undetectably low in spleens of both GVHD and IL-12-protected mice
on day 4 and 7. Thus, the high level of Th reactivity shown in Fig 3A
was specific for host antigens.
Fas is upregulated on the surface of splenic donor CD4 cells in
IL-12-treated mice.
Using three-color FCM analysis, we compared the expression of Fas on
gated splenic donor CD4 cells at 36 hours and on days 3, 4, 5, and 7 post-BMT from GVHD control and IL-12-treated mice. As is shown in Fig 4, at 36 hours after
BMT, Fas was expressed at low, but detectable, levels on donor CD4
cells in both groups. By contrast, on days 3 (Fig 4), 4 (not shown),
and 5 (Fig 4), Fas was upregulated on donor CD4 cells in
IL-12-protected mice to a greater extent than in the GVHD control
group. By day 7, Fas expression was increased above normal levels (not
shown) on donor CD4 cells of both the GVHD control and the
IL-12-treated group, but remained highest on those of IL-12-treated
mice (Fig 4).
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| Fig 4.
Upregulation of Fas on donor CD4 cells in
IL-12-protected mice. Lethally irradiated B6 mice received A/J BMC and
spleen cells plus TCD B6 BMC without or with IL-12 on day 0. At 36 hours and on days 3, 5, and 7 post-BMT, spleen cells were analyzed by
three-color FCM to examine the expression of Fas on 5F1-negative, ie,
donor CD4+ cells. Solid lines represent the Fas density
on donor CD4 cells recovered from spleens of GVHD mice and dotted lines
represent Fas expression on splenic donor CD4 cells recovered from
IL-12-protected mice. Similar results were obtained from six mice per
group at each time point in three independent experiments.
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Role of donor Fas expression in IL-12-mediated GVHD protection.
To determine the functional significance of the increase in expression
of Fas by donor CD4 T cells in IL-12-protected compared with GVHD
control mice, we evaluated the ability of IL-12 to inhibit GVHD when
Fas-deficient H-2k LPR donors were used. For comparison, we
used Fas-competent H-2k MRL mice as donors to B6
recipients. As is shown in Fig 5A, marked and highly significant (P < .001) prolongation of survival
was induced by IL-12 treatment in recipients of wild-type MRL BMC and
spleen cells. Although IL-12 treatment led to slight prolongation of
survival in recipients of Fas-mutant LPR marrow and spleen cells in the
same experiments (Fig 5B), this prolongation did not achieve
statistical significance (P = .6 comparing IL-12-treated and
control recipients of LPR marrow). A somewhat greater degree of IL-12
protection was detectable when a higher dose of LPR spleen cells was
administered (median survival time [MST], 7 days in controls
v 37.5 days in IL-12-treated recipients), but this protection did not achieve statistical significance (P = .2) or approach the level of protection observed with wild-type donors in the same
experiment. Thus, IL-12-mediated GVHD protection is at least partially
dependent on the ability of donor cells to express functional Fas
molecules, suggesting a role for Fas-mediated apoptosis of donor T
cells in this phenomenon.
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| Fig 5.
Requirement for donor Fas expression for maximal
IL-12-induced GVHD protection. B6 mice received lethal irradiation
followed by reconstitution with TCD B6 marrow, along with either (A)
wild-type MRL or (B) with Fas-mutant LPR BMC and spleen cells
(107). Results of two experiments, both of which produced
similar results, are combined (n = 11 to 12 mice per group). ( ),
GVHD controls; (---), IL-12-treated.
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DISCUSSION |
A single injection of IL-12 can significantly inhibit acute GVHD in a
fully MHC and multiple minor antigen-mismatched, A/J B10 murine BMT model.7 IL-12 significantly alters the kinetics of donor T-cell activation and expansion in the first week after BMT.
By 4 days after BMT, the numbers of allogeneic CD4 and CD8 cells in the
spleens of GVHD control mice are approximately two to three times
greater than the number administered, increase further on day 5, then
decline to approximately one third to one fourth of this level by day 7 post-BMT. This decline in splenic donor T cells could be due to their
emigration to GVHD target tissues in this period. Consistent with this
possibility, we have observed marked lymphocytic infiltration of the
livers and lungs of GVHD control mice on day 7 post-BMT (M.S., P. Nguyen, unpublished data, March 1996). The decline may
also reflect apoptotic cell death that can follow T-cell
activation,19-22 ie, activation-inducted cell death (AICD).
The expression of the activation-associated markers CD25 and VLA-4 on
donor T cells follows a generally similar kinetic pattern as the
expansion of donor CD4 cells (Fig 1), as does the level of IFN- in
the serum.7 All of these parameters peak at 4 to 5 days
post-BMT, suggesting that this is the time at which the full extent of
T-cell activation has occurred. These patterns are altered by IL-12
treatment in parallel with the observed protection from GVHD. A marked
reduction in the number of donor T cells is detected in spleens of
IL-12-treated mice compared with GVHD controls on day 47;
by day 5, the number of donor T cells increases several-fold, but still
remains significantly lower than that in GVHD controls (Fig 1). Between
day 5 and day 7 post-BMT, the total numbers of donor T cells remain
relatively constant in spleens of IL-12-protected mice, whereas they
decrease markedly in spleens of GVHD controls. Most strikingly, IL-12
markedly attenuates the powerful host-specific Th response that is
evident in spleens of GVHD mice on day 4. Although total donor T-cell
numbers are reduced in IL-12-treated mice at this time point, the
fraction of these donor CD4 cells that respond to host antigens is
specifically and markedly diminished. In contrast, the frequencies of
host-reactive CTL precursors, which are less markedly
expanded than host-reactive Th in spleens of GVHD mice, are unaffected
by IL-12 treatment (data not shown). Thus, IL-12-induced inhibition of
activation, expansion, and cytokine production by donor CD4 cells by
day 4 post-BMT may play a critical role in IL-12-induced protection
against acute GVHD. This significant inhibitory effect of IL-12 on
donor Th activity has also been observed in a haploidentical,
haplotype-mismatched strain combination (CBD2F1B6D2F1), in
which even more potent GVHD protection is induced by IL-12 than in the
A/J to B6 strain combination used here.23 The increasing
numbers of donor CD4 and CD8 cells in the spleens of IL-12-treated
mice between days 4 and 7, along with increasing numbers of
host-reactive donor Th in the same period and the delayed increase in
VLA-4 expression in this group, suggest that a delayed pattern of
expansion of allogeneic T cells can result in a delay in the onset of
GVHD.
Rather than reduced expansion, an alternative explanation for the early
reduction in numbers of donor splenic T cells in IL-12-protected mice
is that they redistribute to other tissues. However, histopathologic evaluation of livers and lungs and flow cytometric analysis of lymph
node cells on day 4 post-BMT (data not shown) did not support this
hypothesis. Thus, we favor the possibility that IL-12 blunts and delays
donor T-cell proliferative responses to host antigens.
Although IL-12 can enhance T-cell proliferation,24,25
recent studies showed that high doses26,27 of IL-12
administered to mice can lead to depletion of splenic CD4 and CD8 cells
and can significantly inhibit virus-induced CD8+ T-cell
expansion and CTL activation. These changes were accompanied by
induction of TNF- production.27 Our
analyses of sera have not shown differences between GVHD control and
IL-12-protected mice in TNF- levels (data not shown), suggesting
that different mechanisms may prevail in each model. Furthermore, in
our studies, the reduction in donor CD4 cells in spleens of
IL-12-treated animals was much more striking than that reported in the
above studies.26,27
In parallel with the markedly reduced level of host-reactive Th
expansion, IL-12 treatment in our GVHD model leads to inhibition of
GVHD-associated increases in serum IFN- levels, and in T-cell expression of the activation markers CD25 and VLA-4. The inhibition of
IFN- levels probably reflects the ability of IL-12 to inhibit early
donor CD4+ T-cell expansion and activation, because the
majority of IFN- appears to be produced by donor CD4+ T
cells in GVHD controls on day 49 (M.S. et al, unpublished
data, August 1995). Dallman et al28 have
shown decreased expression of IL-2R (CD25) and chain by graft-infiltrating leukocytes from animals rendered tolerant to an
allogeneic kidney graft. These cells showed a very poor proliferative response to IL-2, and this altered regulation of the IL-2 pathway may
have resulted in tolerance.28 Together, our results show that IL-12 alters the pattern of donor T-cell activation, but does not
completely abrogate this process, thus permitting delayed VLA-4
upregulation (Fig 1).
We observed a significant increase in Fas expression on donor CD4 cells
in IL-12-protected compared with control mice on days 3 through 7. Most importantly, the data presented in Fig 5 indicate that
IL-12-induced GVHD protection is at least partially dependent on the
expression of functional Fas molecules by the donor. The role played by
IFN- in this early Fas-mediated apoptosis is currently under
investigation. IL-12 is a potent inducer of IFN- production by both
natural killer (NK) cells and T cells.29-33
In our GVHD model, IL-12 treatment has a biphasic effect on serum
IFN- levels, causing an early increase on day 2 and 3, followed by
the later inhibition described above.7 IFN- has been
reported to induce T-cell apoptosis by upregulating Fas
expression.34 Furthermore, IFN- can upregulate Fas
expression on tumor cells and increase their sensitivity to
FasL-dependent killing.35 Despite the fact that IFN- has
been implicated in GVHD pathophysiology, exogenous IFN- has recently
been shown to be capable of inhibiting GVHD.36 Importantly,
the early increase in serum IFN- production induced by IL-12
treatment plays a critical role in GVHD protection in our
model.8 Together, our results lead to the hypothesis that early IFN- production induced by IL-12 treatment upregulates donor
CD4 cell Fas expression and sensitivity to FasL-mediated killing, that
these cells undergo apoptosis by a Fas-dependent pathway, and that the
graft-versus-host response, which is CD4-dependent in this strain
combination,37 is largely aborted. Consistent with this
possibility, preliminary studies have shown that IL-12 increases the
number of donor T cells undergoing apoptosis in the first few days
after BMT. The source of Fas ligand responsible for this early
Fas-mediated apoptosis of donor T cells is unknown. Fas ligand can be
expressed by activated CD8 CTL and Th1 CD4 clones,38,39 and
it has recently been reported that in vivo IL-12 treatment induces a
CD3+CD4 CD8B220+
T-cell population that is capable of Fas ligand-mediated cytolysis of
tumor cells.35
In addition to Fas, CD25 and CD69 expression was also increased on
donor CD4 cells at 36 and 72 hours post-BMT in spleens of
IL-12-protected mice compared with GVHD controls. In vitro studies
have shown that IL-12 provides a second signal to Th1 clones to induce
IL-2R chain expression and proliferation.40 It is
possible that the early exposure to IL-12 leads to premature activation
of donor CD4+ T cells, as is suggested by their higher
expression of these activation markers at early time points. Such
premature activation might make these cells susceptible to AICD before
they have the opportunity to expand. Indeed, IL-2 can promote T-cell
death in response to activation,41 and this AICD is
dependent on the expression of the high-affinity IL-2 receptor that
requires the -chain, CD25.41-44 Thus, we hypothesize
that IL-12-induced premature activation of donor T cells makes
host-reactive donor T cells susceptible to AICD around day 2 to 3 post-BMT. Whether this AICD is mediated directly through the
upregulated CD25 molecules is currently unclear. However, signaling
through CD25 has been shown to be critical for Fas-mediated apoptosis
of T cells activated in vitro.45
Importantly, graft-versus-leukemia effects are preserved
in IL-12-protected mice, and, like IL-12-mediated GVHD protection, this GVL effect is largely IFN--dependent.8 Thus, the
particular effects of IL-12 on donor T-cell activation and expansion
appear to be highly beneficial in that they inhibit GVHD, while
preserving the graft-versus-leukemia effects of donor T cells, both of
which are mediated, at least in part, by IFN-.
In conclusion, IL-12-induced protection against acute GVHD in mice is
associated with evidence of premature activation of donor T cells,
followed by inhibition of the GVHD-associated activation and expansion
of donor T-helper cells that recognize host antigens. These changes may
reflect early induction by IL-12 of Fas-mediated apoptosis of
host-reactive donor T cells through an IFN--dependent mechanism.
Experiments now in progress should help to elucidate the pathway(s) by
which this occurs.
| |
FOOTNOTES |
Submitted September 10, 1997;
accepted December 16, 1997.
Supported by National Institutes of Health (Bethesda, MD) Grant No.
CA64912 and American Cancer Society (Atlanta, GA) Grant No.
RPG-95-071-03-CIM.
Address reprint requests to Megan Sykes, MD, Bone Marrow
Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital, MGH East, Bldg 149-5102, 13th St,
Boston, MA 02129.
The publication costs of this article were defrayed in part by
page charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs Henry J. Winn and Michael Seiden for helpful review of the
manuscript, Guiling Zhao for outstanding animal husbandry and technical
assistance, and Diane Plemenos for expert assistance with the
manuscript.
| |
REFERENCES |
1.
Gale RP:
Graft-versus-host disease.
Immunol Rev
88:193,
1985[Medline]
[Order article via Infotrieve]
2.
Storb R,
Deeg HJ,
Pepe M,
Appelbaum F,
Anasetti C,
Beatty P,
Bensinger W,
Berenson R,
Buckner CD,
Clift R,
Doney K,
Longton G,
Hansen J,
Hill R,
Loughran TJ,
Martin P,
Singer J,
Sanders J,
Stewart P,
Sullivan K,
Witherspoon R,
Thomas ED:
Methotrexate and cyclosporine versus cyclosporine alone for prophylaxis of graft-versus-host disease in patients given HLA-identical marrow grafts for leukemia: Long-term follow-up of a controlled trial.
Blood
73:1729,
1989[Abstract/Free Full Text]
3.
Clift RA,
Storb R:
Histoincompatible bone marrow transplants in humans.
Ann Rev Immunol
5:43,
1987[Medline]
[Order article via Infotrieve]
4.
Anasetti C,
Amos D,
Beatty PG,
Appelbaum FR,
Bensinger W,
Buckner CD,
Clift R,
Doney C,
Martin PJ,
Mickelson E,
Nisperos B,
O'Quigley J,
Ramberg R,
Sanders JE,
Stewart P,
Storb R,
Sullivan KM,
Witherspoon RP,
Thomas ED,
Hansen JA:
Effect of HLA compatibility on engraftment of bone marrow transplants in patients with leukemia or lymphoma.
N Engl J Med
320:197,
1989[Abstract]
5.
Martin PJ,
Hansen JA,
Buckner CD,
Sanders JE,
Deeg HJ,
Stewart P,
Appelbaum FR,
Clift R,
Fefer A,
Witherspoon RP,
Kennedy MS,
Sullivan KM,
Flournoy N,
Storb R,
Thomas ED:
Effects of in vitro depletion of T cells in HLA-identical allogeneic marrow grafts.
Blood
66:664,
1985[Abstract/Free Full Text]
6.
Kernan NA,
Flomenberg N,
Dupont B,
O'Reilly RJ:
Graft rejection in recipients of T-cell-depleted HLA-nonidentical marrow transplants for leukemia.
Transplantation
43:842,
1987[Medline]
[Order article via Infotrieve]
7.
Sykes M,
Szot GL,
Nguyen PL,
Pearson DA:
Interleukin-12 inhibits murine graft-vs-host disease.
Blood
86:2429,
1995[Abstract/Free Full Text]
8.
Yang Y-G,
Sergio JJ,
Pearson DA,
Szot GL,
Shimizu A,
Sykes M:
Interleukin-12 preserves the graft-vs-leukemia effect of allogeneic CD8 T cells while inhibiting CD4-dependent graft-vs-host disease in mice.
Blood
90:4651,
1997[Abstract/Free Full Text]
9.
Wang M-G,
Szebeni J,
Pearson DA,
Szot GL,
Sykes M:
Inhibition of graft-vs-host disease (GVHD) by IL-2 treatment is associated with altered cytokine production by expanded GVH-reactive CD4+ helper cells.
Transplantation
60:481,
1995[Medline]
[Order article via Infotrieve]
10.
Sykes M,
Romick ML,
Sachs DH:
Interleukin 2 prevents graft-vs-host disease while preserving the graft-vs-leukemia effect of allogeneic T cells.
Proc Natl Acad Sci USA
87:5633,
1990[Abstract/Free Full Text]
11.
Dialynas DP,
Quan ZS,
Wall KA,
Pierres A,
Quintans J,
Loken MR,
Pierres M,
Fitch FW:
Characterization of murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: Similarity of L3T4 to human Leu3/T4 molecule.
J Immunol
131:2445,
1983[Abstract]
12.
Sarmiento M,
Glasebrook AL,
Fitch FW:
IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt2 antigen block T cell-mediated cytolysis in the absence of complement.
J Immunol
125:2665,
1980[Abstract]
13.
Sykes M,
Abraham VS,
Harty MW,
Pearson DA:
IL-2 reduces graft-vs-host disease and preserves a graft-vs-leukemia effect by selectively inhibiting CD4+ T cell activity.
J Immunol
150:197,
1993[Abstract]
14.
Sykes M,
Romick ML,
Hoyles KA,
Sachs DH:
In vivo administration of interleukin 2 plus T cell-depleted syngeneic marrow prevents graft-versus-host disease mortality and permits alloengraftment.
J Exp Med
171:645,
1990[Abstract/Free Full Text]
15.
Unkeless JC:
Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.
J Exp Med
150:580,
1979[Abstract/Free Full Text]
16.
Ozato K,
Mayer NM,
Sachs DH:
Monoclonal antibodies to mouse major histocompatibility complex antigens IV. A series of hybridoma clones producing anti-H-2d antibodies and an examination of expression of H-2d antigens on the surface of these cells.
Transplantation
34:113,
1982[Medline]
[Order article via Infotrieve]
17.
Taswell C:
Limiting dilution assays for the determination of immunocompetent cell frequencies.
J Immunol
126:1614,
1981[Abstract]
18.
Simon MM,
Eichmann K:
Limiting dilution analysis of alloreactive T helper cells: Precursor frequencies similar to that of alloreactive cytotoxic T cells.
Immunobiology
164:78,
1983[Medline]
[Order article via Infotrieve]
19.
Webb SR,
Hutchinson J,
Hayden K,
Sprent J:
Expansion/deletion of mature T cells exposed to endogenous superantigens in vivo.
J Immunol
152:586,
1994[Abstract]
20.
Sprent J,
Webb S:
Intrathymic and extrathymic clonal deletion of T cells.
Curr Opin Immunol
7:196,
1995[Medline]
[Order article via Infotrieve]
21.
Kabelitz D,
Pohl T,
Pechold K:
Activation-induced cell death (apoptosis) of mature peripheral T lymphocytes.
Immunol Today
14:338,
1993[Medline]
[Order article via Infotrieve]
22.
Russell JH:
Activation-induced death of mature T cells in the regulation of immune responses.
Curr Opin Immunol
7:382,
1995[Medline]
[Order article via Infotrieve]
23.
Yang Y-G,
Dey B,
Sergio JJ,
Sykes M:
IL-12 prevents severe acute GVHD and GVHD-associated immune dysfunction in a full MHC haplotype-mismatched murine bone marrow transplantation model.
Transplantation
64:1343,
1997[Medline]
[Order article via Infotrieve]
24.
Mehrotra PT,
Wu D,
Crim JA,
Mostowki HS,
Siegel JP:
Effects of IL-12 on the generation of cytotoxic activity in human CD8+ T lymphocytes.
J Immunol
151:2444,
1993[Abstract]
25.
Gately MK,
Desai BB,
Wolitzky AG,
Quinn PM,
Dwyer CM,
Podlaski FJ,
Familletti PC,
Sinigaglia F,
Chizonnite R,
Gubler U,
Stern AS:
Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor).
J Immunol
147:874,
1991[Abstract]
26.
Orange JS,
Wolf SF,
Biron CA:
Effects of IL-12 on the response and susceptibility to experimental viral infections.
J Immunol
152:1253,
1994[Abstract]
27.
Orange JS,
Salazar-Mather TP,
Opal SM,
Spencer RL,
Miller AH,
McEwen BS,
Biron CA:
Mechanism of interleukin 12-mediated toxicities during experimental viral infections: Role of tumor necrosis factor and glucocortioids.
J Exp Med
181:901,
1995[Abstract/Free Full Text]
28.
Dallman MJ,
Shiho O,
Page TH,
Wood KJ,
Morris PJ:
Peripheral tolerance to alloantigen results from altered regulation of the interleukin 2 pathway.
J Exp Med
173:79,
1991[Abstract/Free Full Text]
29.
Chan SH,
Perussia B,
Gupta JW,
Kobayashi M,
Pospisil M,
Young HA,
Wolf SF,
Young D,
Clark SC,
Trinchieri G:
Induction of interferon gamma production by natural killer cell stimulatory factor: Characterization of the responder cells and synergy with other inducers.
J Exp Med
173:869,
1991[Abstract/Free Full Text]
30.
Nastala CL,
Edington HD,
McKinney TG,
Tahara H,
Nalesnik MA,
Brunda MJ,
Getely MK,
Wolf SF,
Schreiber RD,
Storkus WJ,
Lotze MT:
Recombinant IL-12 administration induces tumor regression in association with IFN-gamma production.
J Immunol
153:1697,
1994[Abstract]
31.
Chan SH,
Kobayashi M,
Santoli D,
Perussia B,
Trinchieri G:
Mechanisms of IFN-gamma induction by natural killer cell stimulatory factor (NKSF/IL-12). Role of transcription and mRNA stability in the synergistic interaction between NKSF and IL-2.
J Immunol
148:92,
1992[Abstract]
32.
Macatonia SE,
Hsieh C-S,
Murphy KM,
O'Garra A:
Dendritic cells and macrophages are required for Th1 development of CD4+ T cells from ab-TCR transgenic mice: IL-12 can substitute for macrophages to stimulate IFN-gamma production.
Int Immunol
5:1119,
1993[Abstract/Free Full Text]
33.
D'Andrea A,
Aste-Amezaga M,
Valiante NM,
Ma X,
Kubin M,
Trinchieri G:
Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells.
J Exp Med
178:1041,
1993[Abstract/Free Full Text]
34.
Oyaizu N,
McCloskey TW,
Than S,
Hu R,
Kalyanaraman VS,
Pahwa S:
Cross-linking of CD4 molecules upregulates Fas antigen expression in lymphocytes by inducing IFN-gamma and TNF-alpha secretion.
Blood
84:2622,
1994[Abstract/Free Full Text]
35.
Tsutsui T,
Mu J,
Ogawa M,
Yu W-G,
Suda T,
Nagaga S,
Saji F,
Murata Y,
Fujuwara H,
Hamaoka T:
Administration of IL-12 induces a CD3+CD4-CD8-B220+ lymphoid population capable of eliciting cytolysis against Fas-positive tumor cells.
J Immunol
159:2599,
1997[Abstract]
36.
Brok HPM,
Heidt PJ,
Van der Meide PH,
Zurcher C,
Vossen JM:
Interferon-gamma prevents graft-versus-host disease after allogeneic bone marrow transplantation in mice.
J Immunol
151:6451,
1993[Abstract]
37.
Sykes M,
Harty MW,
Szot GL,
Pearson DA:
Interleukin-2 inhibits graft-versus-host disease-promoting activity of CD4+ cells while preserving CD4- and CD8-mediated graft-versus-leukemia effects.
Blood
83:2560,
1994[Abstract/Free Full Text]
38.
Ju S-T,
Cui H,
Panka DJ,
Ettinger R,
Marshak-Rothstein A:
Participation of target Fas protein in apoptosis pathway induced by CD4+ Th1 and CD8+ cytotoxic T cells.
Proc Natl Acad Sci USA
91:4185,
1994[Abstract/Free Full Text]
39.
Hanabuchi S,
Koyanagi M,
Kawasaki A,
Shinohara N,
Matsuzawa A,
Nishimura Y,
Kobayashi Y,
Yonehara S,
Yagita H,
Okumura K:
Fas and its ligand in a general mechanism of T cell-mediated cytotoxicity.
Proc Natl Acad Sci USA
91:4930,
1994[Abstract/Free Full Text]
40.
Yanagida T,
Kato T,
Igarashi O,
Inoue T,
Nariuchi H:
Second signal activity of IL-12 on the proliferation and IL-2R expression of T helper cell-1 clone.
J Immunol
152:4919,
1994[Abstract]
41.
Lenardo MJ:
Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis.
Nature
353:858,
1991[Medline]
[Order article via Infotrieve]
42.
Kneitz B,
Herrmann T,
Yonehara S,
Schimpl A:
Normal clonal expansion but impaired Fas-mediated cell death and anergy induction in IL-2-deficient mice.
Eur J Immunol
25:2572,
1995[Medline]
[Order article via Infotrieve]
43.
Smith KA:
Interleukin-2: Inception, impact, and implications.
Science
240:1169,
1988[Abstract/Free Full Text]
44.
Willerford D,
Chen J,
Ferry JA,
Davidson L,
Alt F:
IL-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment.
Immunity
3:521,
1995[Medline]
[Order article via Infotrieve]
45.
Van Parijs L,
Biuckians A,
Ibragimov A,
Alt FW,
Willerford DM,
Abbas AK:
Functional responses and apoptosis of CD25 (IL-2Ra)-deficient T cells expressing a transgenic antigen receptor.
J Immunol
158:3738,
1997[Abstract]
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R. L. Truitt, B. D. Johnson, C. Hanke, S. Talib, and J. E. Hearst
Photochemical Treatment with S-59 Psoralen and Ultraviolet A Light to Control the Fate of Naive or Primed T Lymphocytes In Vivo After Allogeneic Bone Marrow Transplantation
J. Immunol.,
November 1, 1999;
163(9):
5145 - 5156.
[Abstract]
[Full Text]
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G. A. Martins, L. Q. Vieira, F. Q. Cunha, and J. S. Silva
Gamma Interferon Modulates CD95 (Fas) and CD95 Ligand (Fas-L) Expression and Nitric Oxide-Induced Apoptosis during the Acute Phase of Trypanosoma cruzi Infection: a Possible Role in Immune Response Control
Infect. Immun.,
August 1, 1999;
67(8):
3864 - 3871.
[Abstract]
[Full Text]
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Abstract
Introduction
Abstract