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The Journal of Immunology, 2002, 169: 3744-3751.
Copyright © 2002 by The American Association of Immunologists

Physiological Mechanisms of Regulating Alloimmunity: Cytokines, CTLA-4, CD25+ Cells, and the Alloreactive T Cell Clone Size1

Masayuki Sho*,{dagger}, Akira Yamada{dagger}, Nader Najafian{dagger}, Alan D. Salama{dagger}, Hiroshi Harada*, Sigrid E. Sandner*, Alberto Sanchez-Fueyo{ddagger}, Xin Xiao Zheng{ddagger}, Terry B. Strom{ddagger} and Mohamed H. Sayegh2,*,{dagger}

* Department of Medicine, Children’s Hospital, {dagger} Laboratory of Immunogenetics and Transplantation, Brigham and Women’s Hospital, and {ddagger} Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanisms underlying physiological regulation of alloimmune responses remain poorly defined. We investigated the roles of cytokines, CTLA-4, CD25+ T cells, and apoptosis in regulating alloimmune responses in vivo. Two murine cardiac transplant models were used, B10.D2 (minor mismatch) and C57BL/6 (major mismatch), into BALB/c recipients. Recipients were wild type, STAT4-/- (Th1 deficient), or STAT6-/- (Th2 deficient) mice. Minor mismatched allografts were accepted spontaneously in ~70% of wild type and STAT4-/- mice. By contrast, there was significantly shorter graft survival in minor mismatched STAT6-/- mice. Either the adoptive transfer of STAT4-/- splenocytes or the administration of IL-4Fc fusion protein into STAT6-/- mice resulted in long term graft survival. Blocking CTLA-4 signaling accelerated the rejection in all recipients, but was more pronounced in the minor combination. This was accompanied by an increased frequency of alloreactive T cells. Furthermore, CTLA-4 blockade regulated CD4+ or CD8+ as well as Th1 or Th2 alloreactive T cells. Finally, while anti-CD25 treatment prolonged graft survival in the major mismatched combination, the same treatment accelerated graft rejection in the minor mismatched group. The latter was associated with an increased frequency of alloreactive T cells and inhibition of T cell apoptosis. These data demonstrate that cytokine regulation, CTLA-4 negative signaling, and T cell apoptosis play critical roles in regulating alloimmunity, especially under conditions where the alloreactive T cell clone size is relatively small.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Three principles exist for physiologic termination of T cell responses following Ag activation: clonal anergy, cytokine-mediated regulation, and clonal deletion by apoptosis (1). Although these mechanisms have been demonstrated in autoimmunity, they are poorly defined in alloimmune responses in vivo.

CTLA-4 (CD152) is a physiologic terminator of immune responses to pathogens and self-Ags. CTLA-4 is induced on activated T cells and provides a negative regulatory signal by inhibiting IL-2 transcription and cell cycle progression (2, 3, 4). CTLA4 also plays an important role in acquired tolerance to nominal (5, 6, 7) and self Ags (8). More recently, it has been suggested that CTLA4 may play a role in the function of CD4+CD25+ regulatory T cells (9, 10). Thus, its underlying mechanism of immunological down-regulation may be not only induction of anergy, but also active regulation. Although the importance of CTLA-4 negative signaling has been suggested to play an important role in acquired tolerance in some transplant models (11, 12, 13), its precise physiologic role in regulating alloimmune responses in vivo remains unclear.

Cytokine-mediated regulation is a second important mechanism for terminating and regulating immune responses (1). The role of cytokines in alloimmune responses has been extensively investigated. Th1 cells play an important role in allograft rejection, whereas Th2 cells are associated with prolongation of graft survival and tolerance induction in some models (14). However, this simple Th1/Th2 paradigm belies a more complex interaction between the cytokine environment and allograft survival. Recent data demonstrate that in certain transplant models Th1 cytokine knockout mice may reject their allografts (15) and that while some Th1 cytokines are required for tolerance induction (16, 17), Th2 cytokines such as IL-4 are not necessary (18, 19). Thus, the physiological role of cytokines in regulation of alloimmune responses has not been clearly elucidated.

Finally, T cell apoptosis is yet another major mechanism for the termination of immune responses (1). Fas-mediated activation-induced cell death (AICD)3 is critical for the elimination of Ag-activated cells. Since this process is potentiated by IL-2, both IL-2- and IL-2R- ({alpha}- or {beta}-chain) deficient mice have impaired AICD and develop massive lymphoproliferative disorders with associated autoimmune disease (20, 21, 22). Furthermore, it appears that deletion of alloreactive T cells through passive and AICD mechanisms is required for the development of peripheral transplantation tolerance across MHC barriers, possibly by allowing a reduction in alloreactive T cell clone size (23, 24).

In this study we investigated the physiological regulatory role of CTLA-4, Th1/Th2 cytokines, CD25+ T cells, and apoptosis in allograft acceptance and rejection. To this end we used two different mouse heart transplantation models: a fully MHC-mismatched combination (major mismatched combination; C57BL/6 donors into BALB/c recipients) as a model with a large alloreactive T cell clone size and a multiple minor histocompatibilityAg-mismatched combination (minor mismatched combination; B10.D2 donors into BALB/c recipients) as a model with relatively small alloreactive T cell clone size. Wild-type (WT) BALB/c, STAT4-deficient (STAT4-/-, impaired Th1 response) or STAT6-deficient (STAT6-/-, impaired Th2 response) mice were used to investigate the physiologic role of the cytokine environment in alloimmune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

C57BL/6 (H-2b) and BALB/c (H-2d) mice, aged 6–8 wk, were purchased from Taconic Farms (Germantown, PA). STAT4-/-, STAT6-/-, and B10.D2 (H-2d H2-T18c Hc1/nSnJ) mice, aged 6–8 wk, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice homozygous for a targeted disruption of the STAT4 gene (STAT4-/- mice) and the STAT6 gene (STAT6-/- mice) have been previously described (25, 26). We have also previously reported the rejection tempo and cytokine pattern of these mice in response to alloantigen stimulation (27).

Fusion proteins and Abs

Anti-CTLA-4 mAb (4F10, a gift of Dr. J. Bluestone, San Francisco, CA) and anti-IL-2R mAb (PC-61, a gift of Dr. L. Turka, Philadelphia, PA) were manufactured from their respective hybridomas by Bioexpress Cell Culture Services (West Lebanon, NH). Recipients received anti-CTLA-4 mAb or anti-IL-2R mAb (500 µg i.p. on day 0 plus 250 µg i.p. on days 2, 4, 6, 8, and 10). Some recipients received anti-CTLA-4 mAb (500 µg i.p. on day 30 plus 250 µg i.p. on days 32, 34, 36, 38, and 40). Anti-CD4- and anti-CD8-depleting mAbs were prepared from hybridomas GK1.5 (rat anti-mouse CD4) and 2.43 (rat anti-mouse CD8), respectively, obtained from American Type Culture Collection (Manassas, VA). All treated mice received 0.1 ml i.p. unpurified ascites of the appropriate Ab (roughly equivalent to 100 µg purified Ab) 6, 3, and 1 days before transplantation (28, 29). This regimen insures >95% depletion of the respective cell type in the peripheral blood on the day of transplantation (28, 29). Murine IL-4/Fc fusion protein was prepared as previously described (18). Murine IL-4/Fc was given on days 0–14 (0.2 µg i.p.).

Transplantation

BALB/c mice were used as recipients, and B10.D2 or C57BL/6 mice were used as donors. The cardiac allografts were placed in an intra-abdominal location, as previously described (30). Cardiac graft function was assessed by daily palpation. Rejection was defined as complete cessation of a palpable beat and was confirmed by direct visualization after laparotomy (27, 29). Furthermore, cardiac grafts from rejecting or long term survivors (>100 days) were evaluated histologically for evidence of acute and/or chronic rejection. Grafts were fixed in 10% buffered formalin, embedded in paraffin, coronally sectioned, and stained with H&E for evaluation of cellular infiltrates, Verhoeff’s elastin for vessel arteriosclerosis scoring, or Masson’s Trichrome stain for evaluation of fibrosis by light microscopy. Arteriosclerosis was assessed using light microscopy, and the percentage of luminal occlusion by intimal thickening was determined using the scoring system previously described (27).

ELISPOT

The technique for ELISPOT analysis has been described by our group and others (27, 29, 31, 32). Briefly, Immunospot plates (Cellular Technology, Cleveland, OH) were coated with 4 µg/ml rat anti-mouse IFN-{gamma} capture mAb (R4-6A2) in sterile PBS overnight. The plates were then blocked for 1 h with sterile PBS containing 1% BSA (fraction V) and washed three times with sterile PBS. Splenocytes (1 x 106 in 200 µl HL-1 medium containing 1% L-glutamine) were then placed in each well in the presence of 1 x 106 irradiated (3000 rad) syngeneic or allogeneic splenocytes and cultured for 24 h at 37°C in 5% CO2. After washing with PBS, followed by PBS containing 0.05% Tween (PBST), 2 µg/ml biotinylated rat anti-mouse IFN-{gamma} detection mAb (XMG1.2) was added overnight. The plates were then washed four times in PBST, followed by 2-h incubation with streptavidin-HRP (Dako, Carpenteria, CA) diluted at 1/2000 in PBS/1% BSA. All mAbs were purchased from BD PharMingen (San Diego, CA). After washing three times with PBST/followed by PBS, the plates were developed using 800 µl 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO; 10 mg dissolved in 1 ml N,N-dimethylformamide) mixed in 24 ml 0.1 M sodium acetate (pH 5.0) plus 12 µl H2O2. The resulting spots were counted on a computer-assisted ELISPOT image analyzer (T Spot Image Analyzer; Cellular Technology).

Apoptosis assay

Spleens and lymph nodes were harvested from BALB/c recipient mice 5 days after transplantation. A single-cell suspension of spleens and lymph nodes was prepared in HBSS. RBC were lysed by hypotonic shock. Lymphocytes were washed and resuspended in HBSS. Cells were first incubated with the FITC-conjugated anti-mouse CD4 or CD8 mAb for 30 min at 4°C. Then cells were stained with PE-conjugated annexin V and the vital dye 7-amino-actinomycin D (7-AAD) (annexin V apoptosis detection kit; BD PharMingen). Cells were analyzed immediately by flow cytometry. Apoptosis was determined as the percentage of live (7-AAD negative), CD4+ or CD8+ cells staining positively with annexin V.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Allograft survival and frequency of alloreactive T cell in major and minor Ag-mismatched transplant models

In the fully MHC-mismatched combination (C57BL/6 into BALB/c) heart grafts were rejected promptly within 10 days in WT recipients, whereas in the multiple minor histocompatibility Ag-mismatched combination (B10.D2 into BALB/c) there was spontaneous long term allograft survival without any manipulation in ~70% of animals (Fig. 1GoA). Interestingly, long term surviving grafts were free of pathological features of chronic rejection, similar to what was observed in syngeneic BALB/c grafts transplanted into BALB/c recipients. The frequency of alloreactive IFN-{gamma}-producing recipient splenocytes was measured on day 10 post-transplantation in unmodified recipients of each model. The frequency of donor-specific alloreactive IFN-{gamma}-producing cells in the minor mismatched combination was significantly smaller (5.8 ± 2.8 times) than that in the major mismatched combination (Fig. 1GoB). These data suggest that there may be correlation between spontaneous allograft survival and alloreactive T cell clone size.



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  		FIGURE 1. Cardiac graft survival and alloreactive clone size in major vs minor mismatched combinations. A, In the fully MHC-mismatched combination (C57BL/6 into BALB/c, major mismatch), allogeneic heart graft were rejected within 10 days ({blacksquare}; n = 5, MST, 7 days), whereas in the multiple minor histocompatibility Ag-mismatched combination (B10. D2 into BALB/c, minor mismatch), ~70% of cardiac grafts were accepted spontaneously ({square}; n = 7; MST, >100 days). B, Frequency of IFN-{gamma}-producing, donor-specific T cells in BALB/c mice following allogeneic (either C57BL/6 or B10. D2) cardiac transplantation. Recipient mice were sacrificed, and splenocytes were harvested on day 10. Splenocytes (1 x 106 cells/well) were incubated with donor irradiated splenocytes. The frequencies were then determined by ELISPOT assay. Data are expressed as the mean ± SEM of triplicate wells. The results represent five independent experiments (p < 0.0001, major vs minor mismatched combination).

 
Role of cytokines in physiological regulation of alloimmune responses

As we previously reported, both STAT4-/- and STAT6-/- mice reject fully mismatched cardiac allografts with a similar tempo as WT recipients (27), with all grafts being rejected within 10 days (Fig. 2GoA). By contrast, in the minor mismatched combination, graft survival was significantly shorter in STAT6-/- recipients compared with STAT4-/- and WT recipients (p < 0.05; Fig. 2GoB). These data suggest that a predominant Th1 environment is less permissive of long term allograft survival than a Th2 environment in the presence of a small alloreactive T cell clone size. To confirm the regulatory functions of Th2 cytokines in the minor mismatched model we used two approaches. First, we adoptively transferred whole naive splenocytes from STAT4-/- mice into STAT6-/- recipients on the day of transplantation. Interestingly, grafts were accepted indefinitely without any further manipulation (Fig. 2GoC). We also tested the efficacy of systemic administration of IL-4Fc fusion protein (to supply the deficient Th2 cytokine) in STAT6-/- recipients. Graft survival was also restored to the same as in WT or STAT4-/- recipients (Fig. 2GoC). Both these experiments suggest that Th2 cytokines play an important physiological role in spontaneous graft acceptance across minor histocompatibility barriers.



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  		FIGURE 2. Roles of cytokines in physiological regulation of alloimmune responses. A, In the fully MHC-mismatched combination, all WT, STAT4-/-, and STAT6-/- recipients rejected the cardiac allograft in 10 days at the same tempo. B, Th1 environment is less permissive to long term allograft survival in the presence of a small alloreactive T cell clone size. Cardiac allografts from B10.D2 donor mice were transplanted into WT of BALB/c (n = 7), STAT4-/- (n = 9), or STAT6-/- (n = 7) recipients. Minor mismatched cardiac allografts were accepted spontaneously in ~70% of WT and STAT4-/- mice (MST, >100 days). By contrast, there was significantly shorter graft survival in minor mismatched STAT6-/- mice (MST, 14 days; p < 0.05 vs WT or STAT4-/-). C, Th2 cytokines play an important role in regulating allograft acceptance across minor histocompatibility barriers. B10.D2 donor hearts were transplanted into BALB/c background STAT6-/- recipients. Naive splenocytes (5 x 107) from STAT4-/- mice were adoptively transferred into STAT6-/- recipients on the day of cardiac transplantation (n = 6). All grafts were accepted without any further manipulation (MST, >100 days; p = 0.004 compared with untreated STAT6-/-). IL-4/Fc fusion protein was given (0.2 µg i.p.) on days 0–14 after cardiac transplantation (n = 6). Five of six grafts were accepted (MST, >100 days; p = 0.018 compared with untreated STAT6-/-).

 
Role of CTLA-4 in physiological regulation of alloimmune responses

To investigate the importance of CTLA-4 in regulating alloimmune responses, we used a blocking anti-CTLA-4 mAb in both MHC and minor mismatched combinations. In the former combination anti-CTLA-4 mAb treatment caused a small, but significant, acceleration of allograft rejection (median survival time (MST), 6; n = 7; p = 0.0018 compared with untreated WT recipients; Fig. 3GoA). To further address the mechanism by which CTLA-4 accelerated rejection, we employed depleting anti-CD4 or anti-CD8 mAbs using a previously established protocol (28, 29). Transient depletion of CD4+ (MST, 34.5 days; n = 6) and CD8+ (MST, 14 days; n = 6) T cells resulted in significant prolongation of graft survival compared with that of untreated WT mice (MST, 7 days; n = 5; p < 0.005). CTLA-4 blockade accelerated rejection in both CD4+ and CD8+ depleted mice (Fig. 3Go, B and C). These data establish an essential physiological role for CTLA-4 in regulating allograft rejection mediated by either CD4 or CD8+ T cells.



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  		FIGURE 3. CTLA-4 regulates both CD4+ and CD8+ alloreactive T cells in the alloimmune response. A, BALB/c recipients of major mismatched C57BL/6 cardiac grafts were treated with six doses of anti-CTLA-4 mAb on days 0, 2, 4, 6, 8, and 10. CTLA-4 blockade accelerated cardiac allograft rejection in the major mismatched combination (MST, 6 days; p = 0.0018 compared with untreated WT control). B, Depleting anti-CD8 mAb was given on days 6, 3, and 1 before transplantation. Anti-CTLA-4 mAb treatment accelerated cardiac allograft rejection in CD8-depleted recipients (MST, 6 days; n = 4; p = 0.0013) compared with CD8-depleted control recipients (MST, 14 days; n = 6). C, Depleting anti-CD4 mAb was given on days 6, 3, and 1 before transplantation. Anti-CTLA-4 mAb treatment accelerated rejection in CD4-depleted recipients (MST, 18.5 days; n = 4; p = 0.0018) compared with CD4-depleted control recipients (MST, 34.5 days; n = 6).

 
In minor mismatched WT recipients there was a more pronounced acceleration of allograft rejection (Fig. 4GoA). All anti-CTLA-4-treated recipients rejected their allografts by 12 days after transplantation, whereas 70% of untreated recipients accepted allograft spontaneously for >100 day (p < 0.001). In addition, consistent with the in vivo data, the frequency of IFN-{gamma}-producing donor-specific T cells determined by ELISPOT was significantly increased after anti-CTLA-4 mAb treatment (Fig. 4GoB). However, late introduction of anti-CTLA-4 starting day on 30 after transplantation did not cause rejection (MST, >100 days; n = 4).



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  		FIGURE 4. CTLA-4 regulates both Th1 and Th2 alloreactive cells in the alloimmune response. A, BALB/c background WT, STAT4-/-, or STAT6-/- recipients of minor mismatched B10.D2 cardiac grafts were treated with six doses of anti-CTLA-4 mAb on days 0, 2, 4, 6, 8, and 10. There was profound acceleration of allograft rejection in all recipients (p < 0.001 in WT or STAT4-/-, p < 0.005 in STAT6-/- compared with each untreated control recipient). B, Frequency of IFN-{gamma}-producing, donor-specific splenocytes in WT BALB/c recipients treated with none or anti-CTLA4 mAb following minor mismatched B10.D2 allogeneic heart transplantation. Recipient mice were sacrificed, and splenocytes were harvested on day 10. Splenocytes (1 x 106 cells/well) were incubated with irradiated donor splenocytes. The frequencies were then determined by ELISPOT assay. Data are expressed as the mean ± SEM of triplicate wells. The results represent five independent experiments (p < 0.0001, untreated vs anti-CTLA4 mAb-treated recipients).

 
Furthermore, to address the regulatory role of CTLA4 in Th1-predominant or Th2-predominant environments, we treated STAT4-/- and STAT6-/- recipients of minor mismatched grafts with anti-CTLA-4 mAb. Both STAT4-/- and STAT6-/- CTLA-4-treated recipients rejected their allografts within 10 days of transplantation, indicating that CTLA-4 regulates both Th1 and Th2 alloreactive T cells (Fig. 4GoA). In all experiments rejected grafts from CTLA4 mAb-treated animals showed extensive and massive interstitial infiltration. Taken together these data confirm the important regulatory functions of CTLA-4 in alloimmune responses, especially across minor histocompatibility barriers.

Role of CD25+ T cells in physiological termination of alloimmune responses

Next, we studied the physiological role of CD25+ T cells in alloimmune responses. We used the lytic anti-CD25 mAb (PC61) in vivo. While ~6% of CD4+ T cells expressed CD25 in the peripheral blood of naive BALB/c mice, an increased number of CD25+ T cells was seen at 10 days after cardiac transplantation in both major mismatched and minor mismatched recipients (11.3 and 8%, respectively). After anti-CD25 mAb treatment, the number of cells expressing CD25 was markedly reduced in major and minor mismatched recipients (1.2 and 1.3%, respectively). Thus, as previously reported, the mAb (PC-61) used in this study markedly reduced the number of CD25+ T cells in peripheral blood (33). Consistent with previous studies (34, 35, 36), anti-CD25 mAb treatment significantly prolonged allograft survival in the MHC-mismatched combination (Fig. 5GoA). By sharp contrast, this treatment accelerated graft rejection in the minor mismatched combination (Fig. 5GoC). Interestingly, these data suggest a dual role of CD25+ cells according to alloreactive T cell clone size. These in vivo data were confirmed by ELISPOT. While lower frequencies of donor-specific, IFN-{gamma}-producing cells were seen after anti-CD25 mAb treatment in recipients of major mismatched grafts, significantly elevated frequencies were observed after anti-CD25 mAb treatment in recipients of minor mismatched grafts compared with controls (Fig. 5Go, B and D).



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  		FIGURE 5. The differential effect of anti-CD25 mAb on alloimmune response. A, MHC-mismatched C57BL/6 donor hearts were transplanted into BALB/c recipients. Recipients were treated with none or anti-CD25 mAb (500 µg i.p. on day 0 plus 250 µg i.p. on days 2, 4, 6, 8, and 10). Anti-CD25 mAb treatment prolonged allogeneic heart graft survival ({blacksquare}; n = 4; MST, 17 days; p < 0.001) compared with untreated control recipient ({square}; n = 5; MST, 7 days). B, Frequency of IFN-{gamma}-producing, donor-specific T cells in BALB/c mice following allogeneic C57BL/6 cardiac transplantation. Recipient mice were sacrificed, and splenocytes were harvested on day 10. Splenocytes (1 x 106 cells/well) were incubated with donor irradiated splenocytes. The frequencies were then determined by ELISPOT assay. Data are expressed as the mean ± SEM of triplicate wells. The results represent three independent experiments (p < 0.005, untreated vs treated with anti-CD25 mAb). C, Minor mismatched B10.D2 donor hearts were transplanted into BALB/c recipients. Anti-CD25 mAb treatment induced allogeneic heart graft rejection ({blacksquare}; n = 5; MST, 12 days; p < 0.001) compared with untreated control recipient ({square}; n = 7; MST, > 100 days). D, Frequency of IFN-{gamma}-producing, donor-specific T cells in BALB/c mice following allogeneic B10.D2 cardiac transplantation. The frequencies were then determined by ELISPOT assay. Data are expressed as the mean ± SEM of triplicate wells. The results represent three independent experiments (p < 0.001, untreated vs treated with anti-CD25 mAb).

 
Role of apoptosis in physiological termination of alloimmune responses

Finally, to investigate the role of apoptosis in terminating the alloimmune response in vivo, leukocytes were isolated from spleens and lymph nodes of transplanted mice. Cells were stained with annexin V, 7-AAD, and CD4 or CD8 mAb as previously described and were analyzed by flow cytometry. Interestingly, in the minor mismatched combination, a significant number of both CD4 and CD8 cells from untreated transplanted mice expressed annexin V. Following anti-CD25 mAb treatment the number of cells undergoing apoptosis was markedly reduced (Fig. 6Go). However, unlike in the minor combinations, in at least five different experiments we could not detect any significant and consistent changes when comparing apoptosis results before and after anti-CD25 mAb treatment in recipients of major mismatched grafts. These data suggest that anti-CD25 mAb treatment inhibited AICD and caused accelerated graft rejection in the minor combination. Thus, apoptosis may be a key mechanism contributing to spontaneous graft acceptance in the minor mismatched combination.



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  		FIGURE 6. Anti-CD25 mAb treatment inhibits apoptosis in minor mismatched combinations. B10.D2 donor hearts were transplanted into BALB/c recipients. Spleens and lymph nodes were harvested from BALB/c recipient mice 4 days after transplantation. A single-cell suspension of spleens and lymph nodes was prepared in HBSS. Cells were first incubated with the FITC-conjugated anti-mouse CD4 or CD8 mAb. Then cells were stained with PE-conjugated annexin V. Cells were immediately analyzed by flow cytometry. Apoptosis was determined as the percentage of annexin V+ cells that excluded 7-AAD+ cells. Proportion of double staining positive either CD4+ or CD8+ T cells (%) = % of costained T cells/% of total either CD4+ or CD8+ T cells. After anti-CD25 treatment in minor mismatched combinations, the significant reduction of annexin V+ cells was seen in both CD4+ and CD8+ T cells. The results represent four independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The B7-CD28/CTLA4 pathway is the best-characterized T cell costimulatory pathway and is central to both T cell activation and anergy. CTLA-4/B7 interactions provide a critical negative signal for T cell activation, and we have recently shown that CTLA-4 signaling can also inhibit T cell responses independently of CD28 (29, 37). However, the precise role of CTLA-4 in regulating alloreactive T cell responses under physiological conditions in vivo remains unclear (2, 3). In addition, CTLA4 regulation of CD4+ T cell responses has been clearly demonstrated, and its function to regulate CD8+ T cells has been debated (38, 39, 40). Furthermore, the effect of CTLA4 engagement on Th1 and Th2 cell differentiation appears complex. It has been suggested that CTLA-4 serves to counter CD28-mediated activation by preventing Th2 and promoting Th1 differentiation. Moreover, anti-CTLA-4 mAb treatment was shown to enhance Th2 cell development (41). However, it has also been reported that both Th1 and Th2 cells express CTLA-4 and that CTLA-4 engagement suppresses cytokine production by both cell types (42).

In the present studies we have shown that a blocking anti-CTLA-4 mAb accelerated allograft rejection mediated by either CD4+ or CD8+ T cells and in a predominantly Th1 or Th2 environment. These data, therefore, clearly demonstrate that CTLA-4 is a critical physiologic regulator of in vivo alloimmune responses and is capable of regulating both CD4+ and CD8+ T cells regardless of their cytokine milieu, especially when the alloreactive T cell clone size is relatively small. Indeed, based on our results in the minor mismatched combination, intact CTLA-4 signaling appears to be critical for spontaneous allograft acceptance. Recent studies have demonstrated the constitutive expression of CTLA-4 on CD4+CD25+ regulatory T cells and have thus suggested a central role for the molecule in the action of these cells (9, 10, 43). In addition, CD4+CD25+ T cells have been reported to prevent the development of autoimmunity and alloimmunity (44, 45, 46, 47, 48). However, since CD4+CD25+ T cells from CTLA-4-deficient mice also demonstrate some regulatory activity, the precise role for CTLA-4 in the function of regulatory T cells remains to be defined (10). Interestingly, we found that the late introduction (starting on day 30 after transplantation) of anti-CTLA-4 mAb did not result in rejection in the minor Ag-mismatched combination, in which spontaneous allograft acceptance occurred in the majority of cases. This suggests that while CTLA4 plays a vital role in the induction phase of the alloimmune response, it plays little regulatory role in the maintenance phase of the alloimmune response. Therefore, early CTLA4 blockade in our model may, on the one hand, be inhibiting the functions of a regulatory T cell population or preventing alloreactive T cells from being rendered anergic, thus leading to a higher frequency of activated/effector alloreactive T cells (as demonstrated by ELISPOT) that mediate accelerated allograft rejection.

We were intrigued with the novel data showing that that there is a discrepancy in the effect of anti-CD25 mAb treatment on graft survival in recipients of fully allogeneic vs minor mismatched allografts. In sharp contrast to the MHC-mismatched combination, in which graft survival was prolonged, there was remarkable acceleration of graft rejection in the minor Ag-mismatched combination. The mechanisms of this observation are probably complex and not necessarily mutually exclusive. On the one hand, the anti-CD25 mAb may be reducing the number of CD25+ alloreactive effector T cells, while, on the other, the same treatment may be depleting CD25+ regulatory T cells. This is supported by our data showing markedly decreased number of peripheral CD25+ T cells after mAb therapy. In addition, our apoptosis data suggest that the mechanism underlying the accelerated rejection following anti-CD25 mAb treatment in the minor mismatched combination may have been at least in part related to the inhibition of programmed cell death. This is probably related to decreased IL-2 secretion. Therefore, the net result may depend on the balance between effector and regulatory cells as a function of the size of the alloreactive T cell. In the major mismatched combination, in which T cell death and regulatory cells play minor roles in physiologically regulating the alloimmune response, a dominant effect of anti-CD25 mAb was reduction of the alloreactive T cell clone size and prolongation of graft survival. In the minor mismatched combination, in which apoptosis and regulation play critical roles in regulating alloimmunity, anti-CD25 mAb therapy accelerated allograft rejection. These findings may have relevant clinical implications. First, recent data from our group showed that calcineurin inhibitors or anti-CD25 mAb therapy abrogated the beneficial effects of CD154 blockade in promoting long term allograft survival (49). Second, the use of anti-CD25 mAb for induction therapy may have a detrimental effect in cases of transplantation using well-matched donor-recipient combinations. Since the use of anti-CD25 mAb has recently become more common in clinical transplantation, further careful evaluation of its impact on graft survival under these circumstances is warranted.

Accumulating data have confused the role of Th1 cells as solely being mediators of allograft rejection (15). Previous studies using cytokine gene knockout mice clearly showed that certain Th1 cytokines were required for peripheral tolerance induction (16, 17). Moreover, data on the role of Th2 cells in tolerance induction have been conflicting (32). Some studies have shown that tolerance correlated with increases in Th2 cytokines, and that Th2 cytokines were necessary for neonatal tolerance (14, 50). However, there is no direct evidence that Th2 cells are involved in alloimmune tolerance in adult animals (46). Li et al. (51) reported that Th1 to Th2 immune deviation using anti-IL-12 mAb in an islet transplantation model induced prolonged engraftment only when there was multiple minor Ag mismatches, but not in fully MHC-mismatched grafts. Our study corroborates these findings in a physiological model. We found that a Th1 environment was less permissive for allograft acceptance and that Th2 cytokines are critical in minor Ag-mismatched combinations. These conclusions were highlighted by the results of our adoptive transfer experiments with either splenocytes of STAT4-/- mice or administration of IL-4Fc fusion protein into STAT6-/- mice. The restoration of immune deviation from a Th1 to a Th2 environment resulted in spontaneous graft acceptance. Hence, in an analogous manner to CD25+ T cells, cytokine-mediated regulation may play a more significant role when the alloreactive T cell clone size is limited. This is further supported by recent work from our laboratory using a model of experimental autoimmune encephalomyelitis in which the clone size of pathogenic CD4 cells is of a similar magnitude to the minor mismatched transplant model and in which disease is only inducible in a Th1 (WT and STAT6-/-), but not Th2 (STAT4-/-), environment (52).

In conclusion, we have provided important principles for the physiologic regulation of alloimmune responses in vivo. In the presence of low frequency of alloreactive T cell CTLA-4 negative signaling, Th2 cytokines, CD25+ cells, and T cell apoptosis play critical roles in the physiological termination of the alloimmune response. Of course, these mechanisms may not be mutually exclusive. These principles have important clinical implications for future design of novel strategies for the induction of transplantation tolerance (53).


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI34965 and AI40152. Back

2 Address correspondence and reprint requests to Dr. Mohamed H. Sayegh, Laboratory of Immunogenetics and Transplantation, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: msayegh{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: AICD, activation-induced cell death; MST, median survival time; WT, wild type; 7-AAD, 7-amino-actinomycin D. Back

Received for publication May 21, 2002. Accepted for publication August 1, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Van Parijs, L., A. K. Abbas. 1998. Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science 280:243.[Abstract/Free Full Text]
  2. Chambers, C. A., M. S. Kuhns, J. G. Egen, J. P. Allison. 2001. CTLA-4-mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Annu. Rev. Immunol. 19:565.[Medline]
  3. Oosterwegel, M. A., R. J. Greenwald, D. A. Mandelbrot, R. B. Lorsbach, A. H. Sharpe. 1999. CTLA-4 and T cell activation. Curr. Opin. Immunol. 11:294.[Medline]
  4. Salomon, B., J. A. Bluestone. 2001. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu. Rev. Immunol. 19:225.[Medline]
  5. Perez, V. L., L. Van Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6:411.[Medline]
  6. Van Parijs, L., V. L. Perez, A. Biuckians, R. G. Maki, C. A. London, A. K. Abbas. 1997. Role of interleukin 12 and costimulators in T cell anergy in vivo. J. Exp. Med. 186:1119.[Abstract/Free Full Text]
  7. Greenwald, R. J., V. A. Boussiotis, R. B. Lorsbach, A. K. Abbas, A. H. Sharpe. 2001. CTLA-4 regulates induction of anergy in vivo. Immunity 14:145.[Medline]
  8. Issazadeh, S., M. Zhang, M. H. Sayegh, S. J. Khoury. 1999. Acquired thymic tolerance: role of CTLA4 in the initiation and maintenance of tolerance in a clinically relevant autoimmune disease model. J. Immunol. 162:761.[Abstract/Free Full Text]
  9. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
  10. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.[Abstract/Free Full Text]
  11. Markees, T. G., N. E. Phillips, E. J. Gordon, R. J. Noelle, L. D. Shultz, J. P. Mordes, D. L. Greiner, A. A. Rossini. 1998. Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4+ T cells, interferon-{gamma}, and CTLA4. J. Clin. Invest. 101:2446.[Medline]
  12. Zheng, X. X., T. G. Markees, W. W. Hancock, Y. Li, D. L. Greiner, X. C. Li, J. P. Mordes, M. H. Sayegh, A. A. Rossini, T. B. Strom. 1999. CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment. J. Immunol. 162:4983.[Abstract/Free Full Text]
  13. Judge, T. A., Z. Wu, X. G. Zheng, A. H. Sharpe, M. H. Sayegh, L. A. Turka. 1999. The role of CD80, CD86, and CTLA4 in alloimmune responses and the induction of long-term allograft survival. J. Immunol. 162:1947.[Abstract/Free Full Text]
  14. Sayegh, M. H., E. Akalin, W. W. Hancock, M. E. Russell, C. B. Carpenter, P. S. Linsley, L. A. Turka. 1995. CD28–B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2. J. Exp. Med. 181:1869.[Abstract/Free Full Text]
  15. Steiger, J., P. W. Nickerson, W. Steurer, M. Moscovitch-Lopatin, T. B. Strom. 1995. IL-2 knockout recipient mice reject islet cell allografts. J. Immunol. 155:489.[Abstract]
  16. Konieczny, B. T., Z. Dai, E. T. Elwood, S. Saleem, P. S. Linsley, F. K. Baddoura, C. P. Larsen, T. C. Pearson, F. G. Lakkis. 1998. IFN-{gamma} is critical for long-term allograft survival induced by blocking the CD28 and CD40 ligand T cell costimulation pathways. J. Immunol. 160:2059.[Abstract/Free Full Text]
  17. Dai, Z., B. T. Konieczny, F. K. Baddoura, F. G. Lakkis. 1998. Impaired alloantigen-mediated T cell apoptosis and failure to induce long-term allograft survival in IL-2-deficient mice. J. Immunol. 161:1659.[Abstract/Free Full Text]
  18. Nickerson, P., X. X. Zheng, J. Steiger, A. W. Steele, W. Steurer, P. Roy-Chaudhury, W. Muller, T. B. Strom. 1996. Prolonged islet allograft acceptance in the absence of interleukin 4 expression. Transplant. Immunol. 4:81.[Medline]
  19. Lakkis, F. G., B. T. Konieczny, S. Saleem, F. K. Baddoura, P. S. Linsley, D. Z. Alexander, R. P. Lowry, T. C. Pearson, C. P. Larsen. 1997. Blocking the CD28–B7 T cell costimulation pathway induces long term cardiac allograft acceptance in the absence of IL-4. J. Immunol. 158:2443.[Abstract]
  20. Sadlack, B., H. Merz, H. Schorle, A. Schimpl, A. C. Feller, I. Horak. 1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75:253.[Medline]
  21. Willerford, D. M., J. Chen, J. A. Ferry, L. Davidson, A. Ma, F. W. Alt. 1995. Interleukin-2 receptor {alpha} chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521.[Medline]
  22. Suzuki, H., T. M. Kundig, C. Furlonger, A. Wakeham, E. Timms, T. Matsuyama, R. Schmits, J. J. Simard, P. S. Ohashi, H. Griesser, et al 1995. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor {beta}. Science 268:1472.[Abstract/Free Full Text]
  23. Wells, A. D., X. C. Li, Y. Li, M. C. Walsh, X. X. Zheng, Z. Wu, G. Nunez, A. Tang, M. Sayegh, W. W. Hancock, et al 1999. Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat. Med. 5:1303.[Medline]
  24. Li, Y., X. C. Li, X. X. Zheng, A. D. Wells, L. A. Turka, T. B. Strom. 1999. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat. Med. 5:1298.[Medline]
  25. Kaplan, M. H., Y. L. Sun, T. Hoey, M. J. Grusby. 1996. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature 382:174.[Medline]
  26. Kaplan, M. H., U. Schindler, S. T. Smiley, M. J. Grusby. 1996. Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4:313.[Medline]
  27. Kishimoto, K., V. M. Dong, S. Issazadeh, E. V. Fedoseyeva, A. M. Waaga, A. Yamada, M. Sho, G. Benichou, Jr H. Auchincloss, M. J. Grusby, et al 2000. The role of CD154-CD40 versus CD28–B7 costimulatory pathways in regulating allogeneic Th1 and Th2 responses in vivo. J. Clin. Invest. 106:63.[Medline]
  28. Ghobrial, R. R., M. Boublik, H. J. Winn, Jr H. Auchincloss. 1989. In vivo use of monoclonal antibodies against murine T cell antigens. Clin. Immunol. Immunopathol. 52:486.[Medline]
  29. Yamada, A., K. Kishimoto, V. M. Dong, M. Sho, A. D. Salama, N. G. Anosova, G. Benichou, D. A. Mandelbrot, A. H. Sharpe, L. A. Turka, et al 2001. CD28-independent costimulation of T cells in alloimmune responses. J. Immunol. 167:140.[Abstract/Free Full Text]
  30. Corry, R. J., H. J. Winn, P. S. Russell. 1973. Primarily vascularized allografts of hearts in mice: the role of H-2D, H-2K, and non-H-2 antigens in rejection. Transplantation 16:343.[Medline]
  31. Valujskikh, A., D. Matesic, A. Gilliam, D. Anthony, T. M. Haqqi, P. S. Heeger. 1998. T cells reactive to a single immunodominant self-restricted allopeptide induce skin graft rejection in mice. J. Clin. Invest. 101:1398.[Medline]
  32. Matesic, D., A. Valujskikh, E. Pearlman, A. W. Higgins, A. C. Gilliam, P. S. Heeger. 1998. Type 2 immune deviation has differential effects on alloreactive CD4+ and CD8+ T cells. J. Immunol. 161:5236.[Abstract/Free Full Text]
  33. Onizuka, S., I. Tawara, J. Shimizu, S. Sakaguchi, T. Fujita, E. Nakayama. 1999. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor {alpha}) monoclonal antibody. Cancer Res. 59:3128.[Abstract/Free Full Text]
  34. Isobe, M., J. Suzuki, Y. Okubo, S. Horie, T. Tanaka, M. Miyasaka, M. Sekiguchi. 1997. Treatment of murine cardiac allograft by monoclonal antibodies to IL-2 receptor {alpha} chain and {beta} chain. Transplant. Proc. 29:2301.[Medline]
  35. Jr Brown, P. S., G. L. Parenteau, F. M. Dirbas, R. J. Garsia, C. K. Goldman, M. A. Bukowski, R. P. Junghans, C. Queen, J. Hakimi, W. R. Benjamin, et al 1991. Anti-Tac-H, a humanized antibody to the interleukin 2 receptor, prolongs primate cardiac allograft survival. Proc. Natl. Acad. Sci. USA 88:2663.[Abstract/Free Full Text]
  36. Tanaka, K., W. W. Hancock, H. Osawa, K. G. Stunkel, T. V. Alberghini, T. Diamantstein, N. L. Tilney, J. W. Kupiec-Weglinski. 1989. Mechanism of action of anti-IL-2R monoclonal antibodies. ART-18 prolongs cardiac allograft survival in rats by elimination of IL-2R+ mononuclear cells. J. Immunol. 143:2873.[Abstract]
  37. Lin, H., J. C. Rathmell, G. S. Gray, C. B. Thompson, J. M. Leiden, M. L. Alegre. 1998. Cytotoxic T lymphocyte antigen 4 (CTLA4) blockade accelerates the acute rejection of cardiac allografts in CD28-deficient mice: CTLA4 can function independently of CD28. J. Exp. Med. 188:199.[Abstract/Free Full Text]
  38. Bachmann, M. F., P. Waterhouse, D. E. Speiser, K. McKall-Faienza, T. W. Mak, P. S. Ohashi. 1998. Normal responsiveness of CTLA-4-deficient anti-viral cytotoxic T cells. J. Immunol. 160:95.[Abstract/Free Full Text]
  39. Frauwirth, K. A., M. L. Alegre, C. B. Thompson. 2001. CTLA-4 is not required for induction of CD8+ T cell anergy in vivo. J. Immunol. 167:4936.[Abstract/Free Full Text]
  40. Chambers, C. A., T. J. Sullivan, J. P. Allison. 1997. Lymphoproliferation in CTLA-4-deficient mice is mediated by costimulation-dependent activation of CD4+ T cells. Immunity 7:885.[Medline]
  41. Walunas, T. L., J. A. Bluestone. 1998. CTLA-4 regulates tolerance induction and T cell differentiation in vivo. J. Immunol. 160:3855.[Abstract/Free Full Text]
  42. Alegre, M. L., H. Shiels, C. B. Thompson, T. F. Gajewski. 1998. Expression and function of CTLA-4 in Th1 and Th2 cells. J. Immunol. 161:3347.[Abstract/Free Full Text]
  43. Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, J. A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12:431.[Medline]
  44. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
  45. Suri-Payer, E., A. Z. Amar, A. M. Thornton, E. M. Shevach. 1998. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J. Immunol. 160:1212.[Abstract/Free Full Text]
  46. Hara, M., C. I. Kingsley, M. Niimi, S. Read, S. E. Turvey, A. R. Bushell, P. J. Morris, F. Powrie, K. J. Wood. 2001. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J. Immunol. 166:3789.[Abstract/Free Full Text]
  47. Hall, B. M., N. W. Pearce, K. E. Gurley, S. E. Dorsch. 1990. Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. III. Further characterization of the CD4+ suppressor cell and its mechanisms of action. J. Exp. Med. 171:141.[Abstract/Free Full Text]
  48. Zhai, Y., J. W. Kupiec-Weglinski. 1999. What is the role of regulatory T cells in transplantation tolerance?. Curr. Opin. Immunol. 11:497.[Medline]
  49. Sho, M., S. E. Sandner, N. Najafian, A. D. Salama, V. Dong, A. Yamada, K. Kishimoto, H. Harada, I. Schmitt, and M. H. Sayegh. 2002. New insights into the interactions between T cell costimulatory blockade and conventional immunosuppressive drugs. Ann. Surg. In press.
  50. Donckier, V., M. Wissing, C. Bruyns, D. Abramowicz, M. Lybin, M. L. Vanderhaeghen, M. Goldman. 1995. Critical role of interleukin 4 in the induction of neonatal transplantation tolerance. Transplantation 59:1571.[Medline]
  51. Li, X. C., M. S. Zand, Y. Li, X. X. Zheng, T. B. Strom. 1998. On histocompatibility barriers, Th1 to Th2 immune deviation, and the nature of the allograft responses. J. Immunol. 161:2241.[Abstract/Free Full Text]
  52. Chitnis, T., N. Najafian, C. Benou, A. D. Salama, M. J. Grusby, M. H. Sayegh, S. J. Khoury. 2001. Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis. J. Clin. Invest. 108:739.[Medline]
  53. Salama, A. D., G. Remuzzi, W. E. Harmon, M. H. Sayegh. 2001. Challenges to achieving clinical transplantation tolerance. J. Clin. Invest. 108:943.[Medline]



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