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The Carlos and Marguerite Mason Transplantation Research Center and Department of Surgery, Emory University School of Medicine, Atlanta, GA 30322; and
Department of Surgery, Seoul National University College of Medicine, Seoul, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing cells in response to allogeneic stimulators as
well as proliferation in an in vivo graft-vs-host disease model. We
find that additional blockade of either CD25 or IL-2 significantly
extends allograft survival beyond that in mice receiving costimulation
blockade alone. This correlates with diminished frequencies of
IFN--producing allospecific T cells and reduced CTL activity.
Anti-CD25 therapy also synergizes with CD40/CD28 blockade in
suppressing proliferative responses. Interestingly, depletion of
CD4+ T cells, but not CD8+ cells, prevents
prolongation in allograft survival, suggesting an IL-2-independent role
for regulation in extended survival. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous studies indicate that CD4+ T cells play a central role in the development of alloresponses, and that costimulatory signals play an important role in determining the character of the CD4+ T cell response (10, 11, 12). It therefore seemed logical that a differential effect of costimulation blockade on these cells might account for strain differences in the alloresponse. However, we and others recently observed that CD8+ T cells can mount alloimmune responses in the absence of the CD40 and CD28 costimulatory pathways and in the absence of CD4+ T cell help (8, 9, 13, 14, 15, 16). From these studies we concluded that alternative pathways might provide the necessary signals for activation of alloreactive CD8+ cells. Here, we study the role of the IL-2 pathway in the ability of CD8+ T cells to mediate costimulation blockade-resistant rejection.
Until recently the IL-2 pathway was considered a necessary component of
adaptive immune responses to transplanted tissue. Blockade of either
the cytokine or the IL-2R prolongs heterotopic heart allograft survival
in rats and mice (17, 18, 19, 20). Recently, growing doubt has
mounted regarding the necessity of its role, given the evidence that
absence of IL-2 expression in knockout models and that
specific intragraft inhibition of IL-2 mRNA fails to delayallograft
rejection (21, 22, 23, 24). Further, the finding that
cytokinesIL-2, -4, -7, -9, -13, and -15 use a common signaling
domain (c) supports the idea that these cytokines are
partially interchangeable in immune responses, with the loss of a
single type having only a minor impact on overall immune function
(25). Despite this evidence, Abs directed against the
-chain of IL-2R (CD25) have shown clinical efficacy when used in
combination with standard immunosuppressive therapies
(26, 27, 28). For this reason we hypothesized that the use of
Abs directed against CD25 may prove efficacious in combination with
costimulation blockade.
In the current study we demonstrate that treatment with blocking Abs to
CD25 or with IL-2-neutralizing Abs synergizes with
costimulation blockade in prolonging skin allograft survival
in C57BL/6 mice. Enhanced graft survival was associated with a
significant decrease in the frequency of T cells producing IFN- and
a decreased cytotoxic response to allogeneic stimulator cells.
We further demonstrate that the observed prolongation of
allograft survival is dependent on the presence of
CD4+ cells, but not CD8+
cells. Finally, we show that in vivo proliferation is significantly
attenuated when using costimulation blockade or anti-CD25, and that
this attenuation is further enhanced when using these treatments
together.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult male BALB/c (H-2d) and C57BL/6 (H-2b) mice, 6–8 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals received humane care in accordance with Emory University institutional animal care and use committee protocols and the Principles of Laboratory Animal Care (29).
Treatment protocols
Skin graft recipients were treated with 500 µg each of hamster
anti-mouse CD40L Ab (MR1) and human CTLA4-Ig administered i.p. on
the day of transplantation (day 0) and on postoperative days 2, 4, and
6. Additional experimental groups were treated with 250 µg
anti-CD25 mAb (PC61) or 250 µg rat IgG administered i.p. on days
0, 2, 4, and 6 or 100 µg rat anti-mouse CD8 (53-6.72) or rat
anti-mouse CD4 (GK1.5) administered i.p. on days -2, -1, and 0
and weekly thereafter until the time of rejection. Depletion of CD4 and
CD8 cells was confirmed as >99% by flow cytometry of peripheral blood
leukocytes. Refer to Table I
for a
summary of the treatment protocols as they appear in Figs. 1 and 2.
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Analysis of splenocytes and peripheral blood was conducted using fluorochrome-conjugated Abs (rat IgG2a-APC, rat IgG2b-APC, anti-CD4-APC, anti-CD8-APC, PharMingen, San Diego, CA). Flow cytometry was performed using a FACSCalibur, and data were analyzed using CellQuest software (both BD Immunocytometry Systems, San Jose, CA).
Skin grafting
Full-thickness skin grafts (
1 cm2) were
transplanted on the dorsal thorax of recipient mice and secured with a
plastic adhesive bandage for 7 days. Graft survival was then followed
by daily visual inspection. Rejection was defined as the complete loss
of viable epidermal graft tissue. Statistical analyses were performed
using a Mann-Whitney U test.
IFN- ELISPOT assays
Allospecific T cell responses were measured by IFN- ELISPOT
assay using splenocytes obtained from skin-grafted C57BL/6 mice. Rat
anti-mouse IFN- (clone R4-6A2; BD PharMingen), the capture Ab,
was incubated at 4 µg/ml in PBS (100 µl/well) at 4°C overnight in
ester-cellulose-bottom plates (Millipore, Bedford, MA).
Following PBS wash, 1 x 106, 3 x
105, 1 x 105, 3
x 104, 1 x 104,
3 x 103, 1 x
103, and 3 x 102
dilutions of splenocytes were added to the plate in duplicate.
Stimulators were irradiated (2000 rad) donor (BALB/c) splenocytes added
at a 1/1 stimulator to effector ratio. Effector cells were incubated
for 14–16 h at 37°C with or without stimulators. After the culture
period cells were removed by washing the plate in PBS-Tween (0.05%).
Biotinylated anti-mouse IFN- (clone XMG1.2; BD PharMingen) was
added at 4 µg/ml (100 µl/well), and the plates were incubated for
2–3 h at 4°C. Unbound Ab was then removed, and HRP-avidin D (Sigma,
St. Louis, MO) was added. Spots were developed with the substrate
3-amino-9-ethyl-carbazole (Sigma) with 0.015%
H2O2. Each spot represents
an IFN--secreting cell, and the frequency of these cells was
determined by dividing the number of spots counted in each well by the
total number of cells plated at that dilution. Naive splenocytes
produce IFN- at a frequency of 
3/105 cells
with or without stimulation. Statistical analysis was performed using
two-tailed Student’s t test.
Cytotoxicity assay
BALB/c CL.7 cells were used as targets. Target cells were
suspended in saline and 5% FCS (1 x
107/ml) with 750 µCi 51Cr
(sp. act., 470 mCi/mg; NEN Life Science Products, Boston, MA) for 90
min at 37°C. Target cells were washed three times and plated in
96-well round-bottom plates at 1 x 104
targets/well in R10 medium (RPMI 1640 supplemented with penicillin,
streptomycin, 2-ME, and 10% FCS (HyClone, Logan UT)). Effectors were
prepared by harvesting splenocytes and lysing RBC. Effectors were
plated at the appropriate ratios in quadruplicate. Four wells were set
aside for total lysis by addition of 2% Triton-X to the targets, and
four wells were set aside for spontaneous lysis by the addition of R-10
without effector cells. After 5 h, the supernatant was harvested
and analyzed by gamma counting. The percent specific lysis was
determined using the following formula: 100 x
(cpmunknown -
cpmspontaneous)/(cpmtotal
- cpmspontaneous).
CFSE staining
Nylon wool-passed C57BL/6 splenocytes were incubated in 10 µM CFSE (Molecular Probes, Eugene, OR). After 10 min the staining was halted by the addition of cold RPMI. Irradiated (18 Gy) BALB/c mice then received 3 x 107 CFSE-labeled cells i.v. by penile vein injection. Recipient mice were treated on the day of transfer (day 0) and on day 2 with CTLA4-Ig and anti-CD40L, anti-CD25 alone, rat IgG alone, or CTLA-4Ig and anti-CD40L with the addition of anti-CD25 or rat IgG.
Determination of precursor frequencies
Precursor frequency was determined as previously described (30). In short, peaks were labeled with the number of times the cells had divided (n). A T cell that divides n times generates 2n daughter cells. Therefore, to obtain the number of precursors for each individual peak, the number of cells in a peak, n, is divided by 2n. For allogeneic transfers, the original number of precursors resulting in each of peaks 4–8 was determined. These resulting values were added together for peaks 4–8 and divided by the total number of initial precursors, which yielded a precursor frequency for divisions 4–8. These divisions were chosen based on the observation that syngeneic transferred cells do not proceed detectably beyond three divisions. Statistical analysis was performed using Student’s two-tailed t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that CD8+ T cells mediate
costimulation blockade-resistant rejection led us to postulate that
these cells use CD28/CD40-independent costimulatory pathways. IL-2 has
long been considered a central factor in the function of T cell
responses, but experiments in IL-2 knockout mice have failed to
demonstrate a primary role for the cytokine in allograft survival
(21, 22). Further, Dai et al. (31) showed
that IL-2-/- mice are refractory to
costimulation blockade in the setting of vascularized heart allografts.
However, since the observations in IL-2-/-
animals may be due to abnormal lymphocyte development in the absence of
IL-2, weconsidered the possibility that interruption of the IL-2
pathway in normal animals might inhibit the generation of
CD8+ T cell-dependent costimulation
blockade-resistant rejection responses. We therefore used either
anti-IL-2 Ab (S4B6) or anti-CD25 Ab (PC61) to target the IL-2
pathway during concomitant treatment with CTLA4-Ig and anti-CD40L.
Animals treated with anti-CD25 rejected skin grafts (mean survival
time (MST), 13 days) with the same kinetics as those receiving isotype
control (rat IgG; MST, 15 days; Fig. 1
A). As in our previous
experiments, B6 recipients treated with costimulation blockade alone
had a modest prolongation of BALB/c skin allograft survival (MST, 34
days; Fig. 1A). In contrast, allograft survival in
recipients treated with costimulation blockade in addition to
anti-CD25 Ab was dramatically prolonged (MST, 76 days) compared
with that in recipients treated with costimulation blockade plus
isotype control (MST, 32 days; p < 0.008; Fig. 1). To
distinguish between the mechanisms of inhibition of the IL-2 pathway or
targeted depletion of CD25+ cells, we treated
animals with costimulation blockade and Ab directed against IL-2
cytokine. This treatment resulted in a similar prolongation in
allograft survival (MST, 61 days; p < 0.004 vs CB) as
treatment with CB and anti-CD25.
Extended allograft survival mediated by anti-CD25 is dependent on CD4+ T cells
We next sought to determine whether the effects were duplicated in
the absence of specific T cell populations. The treatment protocols
were repeated in animals depleted of either CD4+
or CD8+ cells by mAb (GK1.5 or 53-6.72,
respectively). Depletions were confirmed as >99% by flow cytometry of
peripheral blood leukocytes. As expected, animals depleted of
CD8+ cells treated with anti-CD25 rejected
their allografts promptly (MST, 11 days; Fig. 2A). Also as
expected, animals depleted of CD8+ cells and
treated with both costimulation blockade and anti-CD25 showed
significant prolongation of allograft survival (MST, 59 days; Fig. 2A). This is consistent with previous findings demonstrating
prolonged allograft survival in animals depleted of
CD8+ cells and treated with anti-CD40L and
CTLA4-Ig alone (8).
Animals depleted of CD4+ cells showed relatively
rapid rejection when treated with only anti-CD25, and surprisingly,
no enhancement of allograft survival over costimulation blockade alone
when treated with anti-CD25 and costimulation blockade together
(Fig. 2B). These results clearly demonstrate that a simple
model in which costimulation blockade impacts
CD4+ T cells and anti-CD25 treatment impacts
CD8+ is not sufficient to explain the mechanism
of these effects.
T cells from grafted animals treated with CTLA4-Ig, anti-CD40L,
and anti-CD25 show diminished production of IFN- to allogeneic
stimulator cells
To explore the effects of anti-CD25 mAbs on the generation of
breakthrough T cell responses in mice receiving CTLA4-Ig and
anti-CD40L, IFN- ELISPOT assays were performed 20 and 35 days
post-transplant. We compared the precursor frequency of T cells
producing IFN- in response to BALB/c stimulator cells between
animals receiving various treatment regimens (Figs. 3 and 4).
Animals receiving costimulation blockade alone demonstrate a
breakthrough IFN- response on day 20, which persists on day 35.
Conversely, animals receiving additional anti-CD25 showed no
breakthrough IFN- response at these time points
(p < 0.05 vs CB alone on day 20;
p < 0.01 on day 35). Interestingly, animals depleted
of CD4+ T cells and receiving both costimulation
blockade and anti-CD25 showed no response on day 20, but
demonstrated a small breakthrough response on day 35 (Fig. 4B; p < 0.0005 vs naive). This response
occurred before the initiation of skin graft rejection in these
animals.
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A direct ex vivo CTL assay was performed by measuring the release
of 51Cr from a BALB/c fibroblast cell line in the
presence of splenocytes derived from engrafted animals receiving the
indicated treatments (Fig. 3B). By day 20 costimulation
blockade-treated animals demonstrated effective killing of allogeneic
targets. Animals receiving additional anti-CD25 were
indistinguishable from naive animals in their ability to lyse targets
(p < 0.01 vs CB alone). Along with the results
of ELISPOT assays, these findings demonstrate that addition of
anti-CD25 to costimulation blockade abrogates the costimulation
blockade-resistant phenotype by inhibiting the generation of
allospecific T cells (i.e., cytotoxicity and IFN- secretion). Like
prolonged allograft survival, inhibition of effector function is
dependent on the presence of CD4+ T cells.
Costimulation blockade using anti-CD40L and CTLA4-Ig acts synergistically with anti-CD25 to inhibit both CD4+ and CD8+ T cell proliferation in a graft-vs-host disease model
Next, we studied the effects of anti-CD25 Ab on the expansion of CD4+ and CD8+ T cells in vivo in a graft-vs-host disease model using the fluorescent dye CFSE (32). Cells labeled with CFSE lose half their fluorescence each time they divide, and thus cell divisions can be tracked in vivo using flow cytometry (33, 34). Nylon wool-passed, CFSE-labeled C57BL/6 T cells were adoptively transferred into lethally irradiated BALB/c recipients that were treated with anti-CD25 Ab, rat Ig control Ab, CTLA4-Ig and anti-CD40L, or CTLA4-Ig, anti-CD40L, and anti-CD25 Ab. Recipient splenocytes were harvested 3 days after adoptive transfer and analyzed by flow cytometry for CD4 or CD8 expression and CFSE fluorescence.
For both CD4+ and CD8+ populations, only populations with fluorescence corresponding to greater than four divisions were included in the analysis, as syngeneic T cells transfers undergo up to three divisions, but not detectably more, in the same time period. The precursor frequency of cells undergoing more than four divisions was calculated as described in Materials and Methods.
As shown in the histograms in Fig. 5, costimulation blockade and anti-CD25-treated animals show reduced
numbers of cells in the highly divided population of
CD4+ and CD8+ cells
compared with isotype controls. Combination of these therapies further
reduced the frequency of highly divided cells. This is reflected in the
precursor frequency of cells that undergo greater than four divisions
(Fig. 6). Control treated animals had a
precursor frequency of 6.7 ± 0.52% of transferred CD4 cells and
4.1 ± 0.10% of transferred CD8 cells. Both costimulation
blockade and CD25 blockade alone significantly reduce the precursor
frequency of divided cells (CD4, 1.95 ± 0.092% with CB and
3.85 ± 0.69% with anti-CD25; CD8, 2.34 ± 0.17% with
CB and 2.67 ± 0.64% with anti-CD25; p <
0.05 for both CD4 and CD8). A combination of the two treatments
together further decreases proliferation (CD4, 0.96 ± 0.11%
(p < 0.0001); CD8, 1.42 ± 0.087%
(p < 0.005 vs CB alone)).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain, a
clinically approved therapeutic modality, to be an effective supplement
to costimulation blockade regimens that simultaneously target CD28-B7
and CD40L-CD40 interactions. In contrast to our initial hypothesis, the beneficial effect of anti-CD25 therapy is not mediated solely by an inhibitory or depleting effect on activated CD8+ T cells, but also perhaps through a synergistic action with costimulation blockade to inhibit both CD4+ and CD8+ T cell responses. Two observations argue against an underlying depleting mechanism for the effect of anti-CD25 therapy. First, neutralization of IL-2 cytokine has a similar effect in prolonging allograft survival. Second, CD8+ T cells reject allografts with near-normal kinetics in CD4+-depleted animals receiving treatment with anti-CD25 Ab, establishing that the allospecific cells remain present and capable of effector function.
Multiple previous experiments have shown the IL-2 pathway to be
dispensable in allograft rejection, an observation largely attributed
to the redundancy inherent in the IL-2R system (21, 23).
The c component of the IL-2R, which mediates
intracellular signaling following cytokine binding through the Janus
kinase-STAT signaling cascade, is shared by the cytokines IL-4, -7, -9,
-13, and -15 (25). IL-15 also shares the IL-2R 
-chain,
a target that is now of considerable interest. Others have shown
prolonged allograft survival through blockade of the
c and through selective blockade of IL-15
(35, 36, 37, 38). Our findings confirm that anti-CD25 therapy
alone has little effect on skin allograft survival, but when used in
concert with costimulation blockade at the time of engraftment, it
achieves significant prolongation in survival. The absence of
indefinite survival in our model may be due to activation signals
delivered by any of the cytokines sharing the
c or other as yet to be defined costimulatory
pathways. The recent findings of Ferrari-Lacraz et al.
(38) demonstrate that IL-15 blockade inhibits
costimulation blockade-resistant rejection in allografts. Thus, it
appears that both IL-2 and IL-15 are important in the alloimmune
response. Indeed, the recent work of Li et al. (39)
demonstrates a staggered role for the two cytokines, in which IL-15 is
critical to early rounds of T cell proliferation, and IL-2 is critical
to late proliferation and accompanying apoptosis. Therefore, combined
or staggered treatments focused on both the IL-2 and IL-15 pathways may
prove beneficial.
Our findings are somewhat surprising in light of the studies by Lakkis
et al. (31) where blockade or absence of IL-2 eliminated
an extension in heart allograft survival mediated by CTLA4-Ig. In this
model the antagonistic effects were attributed to the role of IL-2 in
preparing CD8+ cells for activation-induced cell
death (AICD), which was later shown to be mediated by up-regulation of
Fas ligand and down-regulation of c
(40). On the surface, it would appear that if this model
held in our experiments, we would expect anti-CD25 therapy to
exacerbate costimulation blockade-resistant rejection, but this was
clearly not the case. There are several differences in the experimental
design of the earlier experiments and our current results, including
the strain combinations used, tissue types transplanted, and timing of
costimulation blockade, any of which might account for the differential
effects of anti-IL-2R Abs in our studies. Further, Lakkis et al.
(31) made use of knockout animals, in which IL-2 signaling
is never possible, whereas blockade of these signals with Ab in our
experiments may be incomplete. Such differences in design preclude
direct comparisons between these studies; however, the results of these
experiments would be consistent if IL-2 blockade in our model results
in passive T cell death through IL-2 starvation, vs the active
IL-2R-dependent AICD required for tolerance in IL-2-deficient animals,
where alternative pathways may substitute for IL-2 to prevent
starvation.
Like many cytokines, it is apparent that IL-2 plays multiple roles, including involvement in both expansion and death of activated T cells. Our data indicate that concurrent blockade of costimulatory signals and the IL-2 pathway effectively prevents the generation of alloreactive effector cells. This highlights IL-2’s role in the activation and expansion of costimulation blockade-resistant CD8+ T cells. In contrast, the absence of the IL-2 pathway has a very different effect. In this setting, where IL-2’s role in proliferation may be diminished, IL-2 signals appear to play an important role in the elimination of alloreactive T cells by enhancing AICD (31). Taken together these findings indicate that combinations of costimulation blockade and anti-CD25 mAbs may be used to inhibit undesirable immune responses. However, it appears that concomitant therapy must be given at the outset of the response. Extrapolation of these approaches to treatment of ongoing responses, such as autoimmunity, may exacerbate, rather than ameliorate, disease. Further study of this issue is clearly warranted.
Our data showing diminished survival when CD4+ T cells are depleted from recipients treated with CB and anti-CD25 are consistent with a growing body of data implicating an important role for regulatory CD4+ T cells in alloresponses. CD25+ CD4+ regulatory cells have received considerable attention and are particularly relevant in light of our use of anti-CD25 mAbs. However, the role of IL-2 signaling in these regulatory cells has not been elucidated (41, 42). Our laboratory is currently pursuing the role of regulatory CD4+ T cells in this and other models of costimulation blockade-based therapies. Further study of this incompletely understood phenomenon may allow targeted utilization of regulatory effects to ameliorate allograft rejection.
Our data clearly demonstrate that blockade of the IL-2 pathway inhibits costimulation blockade-resistant rejection. Clinically available anti-CD25 mAbs may therefore serve as a useful adjunct to emerging costimulation blockade strategies. Future study of the effect of the IL-2 pathway may also elucidate the role of regulation in certain rejection responses.
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Acknowledgments |
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Footnotes |
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2 T.R.J. and J.H. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Christian P. Larsen or Dr. Thomas C. Pearson, Transplantation Immunology Laboratory, Emory University, Suite 5105, WMB, 1639 Pierce Drive, Atlanta, GA 30322. E-mail addresses: clarsen@emory.org or tpearson{at}emory.org
4 Abbreviations used in this paper: CD40L, CD40 ligand; AICD, activation-induced cell death; CB, costimulation blockade; c, common -chain; MST, mean survival time.
Received for publication September 19, 2001. Accepted for publication November 20, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain enhances cardiac allograft survival. J. Immunol. 165:3444.-chain of cytokine receptors induces T cell apoptosis and long-term islet allograft survival. J. Immunol. 164:1193.-chain in regulating IL-2- dependent, activation-induced CD8+ T cell death. J. Immunol. 163:3131.This article has been cited by other articles:
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  B. E. Burrell, K. Csencsits, G. Lu, S. Grabauskiene, and D. K. Bishop CD8+ Th17 Mediate Costimulation Blockade-Resistant Allograft Rejection in T-bet-Deficient Mice J. Immunol., September 15, 2008; 181(6): 3906 - 3914. [Abstract] [Full Text] [PDF]
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 A. Izawa, K. Sano, M. Takehara, M. Inobe, J.-i. Suzuki, H. Imamura, M. Takahashi, U. Ikeda, M. Isobe, and T. Uede Cre/loxP-mediated CTLA4IgG gene transfer induces clinically relevant immunosuppression via on-off gene recombination in vivo Cardiovasc Res, January 1, 2006; 69(1): 289 - 297. [Abstract] [Full Text] [PDF]
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G. Demirci, F. Amanullah, R. Kewalaramani, H. Yagita, T. B. Strom, M. H. Sayegh, and X. C. Li Critical Role of OX40 in CD28 and CD154-Independent Rejection J. Immunol., February 1, 2004; 172(3): 1691 - 1698. [Abstract] [Full Text] [PDF]
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G. Demirci, W. Gao, X. X. Zheng, T. R. Malek, T. B. Strom, and X. C. Li On CD28/CD40 Ligand Costimulation, Common {gamma}-Chain Signals, and the Alloimmune Response J. Immunol., May 1, 2002; 168(9): 4382 - 4390. [Abstract] [Full Text] [PDF]
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), 250 µg rat IgG (
), 500 µg each of CTLA4-Ig and
anti-CD40L (costimulation blockade; 
), costimulation blockade
and 250 µg rat IgG (
), or costimulation blockade and 250 µg
anti-CD25 (
) on days 0, 2, 4, and 6 following engraftment
(n = 5 for each group). Grafts were monitored by
visual inspection until rejection occurred. Animals receiving only
costimulation blockade or costimulation blockade with the rat IgG
isotype control reject skin grafts relatively quickly (MST, 34 and 32
days, respectively). Animals receiving additional anti-CD25 have
significantly prolonged allograft survival (MST, 76 days).
B, C57BL/6 recipients of BALB/C skin grafts were treated
with costimulation blockade (
) on days 0, 2, 4, and 6 following engraftment
(n = 6 for each group). Animals treated with
costimulation blockade reject with a MST of 29 days. Additional
anti-IL-2 significantly prolongs skin graft survival (MST, 61
days), as does additional anti-CD25 (MST, 76 days).
), costimulation blockade (
) on days 0 and 2
after infusion. Cells were harvested at 70 h and were analyzed by
flow cytometry for diminution of CFSE signal and CD4+ or
CD8+ cells. Data represent the precursor frequency of cells
undergoing more than four divisions.