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* Department of Medicine and Department of Surgery, Transplant Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115; and
University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Bulk TReg populations are capable of establishing a variety of interactions with both self and foreign MHC:peptide complexes (11, 12, 13). Given the heterogeneity of TReg Ag recognition, monospecific TCR transgenic (Tg) systems have been critical to understanding the specificity of TReg function in response to nominal Ags. For instance, the demonstration that after specific TCR stimulation, TReg suppression in vitro can be extended to bystander effector T cells (TEff) bearing different specificities was facilitated by the use of influenza hemagglutinin-specific TCR Tg TRegs (14). Similarly, the Ag-specific nature of TReg proliferation (15, 16) and suppressive function (17) in vivo was also demonstrated through the use of TCR Tg systems. In contrast to immune responses against nominal Ags, in the absence of a suitable TCR Tg alloreactive system, the elucidation of TReg specificity in transplantation has been more difficult to achieve and is still controversial. A source for confusion has been the widespread use of lymphopenic adoptive transfer systems in which nonspecific suppression of homeostatic proliferation can mask the regulatory effects of polyclonal TRegs (18). In addition, in these models TRegs harvested from naive, alloantigen-inexperienced mice are capable of preventing TEff from rejecting MHC-mismatched allografts when cell transfer is performed at high ratios of TReg to TEff (10, 19, 20). This finding, which most likely reflects the inherent alloantigen cross-reactivity of TReg TCRs, can also be interpreted as indicating that alloantigen-specific TRegs are not required in transplantation tolerance. In contrast, we and others have shown that TRegs exhibit donor specificity, but only after alloantigen exposure in the presence of a tolerizing regimen (6, 8, 10), a phenomenon that is crucial for the induction of transplantation tolerance. It must be acknowledged, however, that these later experiments were not performed using a criss-cross design (21) and therefore cannot be considered unambiguous proof of specificity. Thus, elucidation of whether TRegs can mediate alloantigen-specific suppressive effects, which would be critical as a first step to understanding the mechanisms of donor specificity in transplantation tolerance, remains an unsolved question.
The ABM TCR Tg mouse is a C57BL/6 (I-Ab)-derived strain that expresses a V
2.1 and a V
8.1 TCR specific for the intact class II molecule I-Abm12 (expressed on a variant strain of C57BL/6 called B6.C-H2bm12/KhEg, hereafter referred to as bm12) and does not recognize other alloantigens (22, 23, 24). This is, therefore, a CD4+ TCR Tg model of direct alloantigen presentation. I-Abm12 and I-Ab differ only at three amino acids in a span of five amino acids (25). Hence, bm12 and C57BL/6 mice have only a limited MHC class II mismatch, which is, nonetheless, sufficient to prompt rejection of bm12 skin allografts by C57BL/6 mice (26). In contrast, bm12 hearts are not acutely rejected by C57BL/6 recipients, although the grafts eventually develop severe arterial disease (chronic rejection) (24).
We have previously determined that ABM mice, in which 90–95% of peripheral CD4+ T cells express the V2.1/V8.1 TCR Tg (24), spontaneously accept bm12 heart allografts, provided recipients have not been previously sensitized by the placement of bm12 skin allografts (24). In addition, long-term surviving bm12 heart allografts from ABM recipients exhibit only minimal signs of chronic rejection. Thus, despite the very high frequency of alloreactive T cells, ABM recipients fail to acutely or chronically reject bm12 heart allografts. We report in this article that in ABM mice a small fraction of TCR Tg CD4+ T cells constitutively express CD25 and are bona fide TRegs. These allospecific regulatory T cells are powerfully suppressive both in vitro and in vivo and are responsible for the capacity of ABM mice to spontaneously accept bm12 hearts. Using this system we show that during alloimmune responses, TReg suppressive function is dependent on specific TCR stimulation. This suggests that one of the mechanisms contributing to the exquisite specificity of allograft tolerance could be the preferential activation of alloantigen-specific TRegs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The ABM (anti-bm12) TCR Tg mice were generated by Dr. E. Palmer (University Hospital, Basel, Switzerland) (22). TEa CD4+ TCR Tg mice were provided by Dr. R. J. Noelle (Dartmouth Medical School, Lebanon, NH). The TEa TCR recognizes the I-E-derived peptide ASFEAQGLANIAVDKA in the context of I-Ab, which is expressed in all APCs from H-2b/I-E+ strains (e.g., CB6F1, an F1 hybrid of C57BL/6 and BALB/c) (27). Bm12, CB6F1, BALB/c, C57BL/6, and C57BL/6 nude mice were purchased from The Jackson Laboratory. F1 (BALB/c x bm12) hybrids and ABM backcrossed into the Rag-2-deficient (Rag–/–) background were generated in our laboratory. Only Rag+/+ ABM mice were used to isolate TCR Tg CD4+CD25+ T cells. Mice were maintained under pathogen-free conditions at Beth Israel Deaconess Medical Center and were used at 6–8 wk of age. Animal experiments were approved by the Beth Israel Deaconess Medical Center institutional animal care committee.
Cell sorting
Single-cell suspensions, prepared from lymph nodes and spleens, were enriched for T cells using T cell enrichment columns (R&D Systems), and T cell subset sorting was achieved using a MoFlo cell sorter (DakoCytomation) after staining with fluorochrome-conjugated anti-CD25, anti-CD4, anti-V2.1, and anti-V8.1 mAbs (all mAbs from BD Pharmingen). Purity was consistently >95% for CD4+CD25– and CD4+CD25+ T cell preparations and >90% for V2.1 and V8.1 double-positive cells. T cell subsets from C57BL/6 or TEa mice were sorted based on CD4 and CD25 markers only.
Cell culture experiments
CD4+CD25– T cells (5 x 104) were cultured with 3 x 105 irradiated allogeneic splenocytes or 104 allogeneic bone marrow-derived mature DCs, with or without 5 x 104 of CD4+CD25+ T cells, and proliferation was measured by [3H]TdR incorporation. DCs were derived from bone marrow by culture for 6 days in RPMI 1640 plus 10% FCS, antibiotics, 50 µM 2-ME, and 10 ng/ml GM-CSF, with addition of LPS during the last 12 h, and were sorted based on high CD86 expression.
Real-time PCR
Real-time PCR was performed with the ABI 7700 sequence detector system using commercially designed primer/probe sets (Applied Biosystems). The expression of the target genes was normalized to that of the housekeeping gene GAPDH, and data were expressed as the relative fold difference between cDNA from the study samples and that from a calibrated sample.
Heterotropic cardiac transplantation
Cardiac transplants were performed in ABM recipients as previously described (28). In some cases thymectomized recipients were given 200 µg of rat anti-mouse CD25 mAb (PC61, 5.3, IgG1; ATCC TB222) i.p. 4 wk before transplantation. We have previously determined that at such doses, anti-CD25 mAb eliminates >80% of CD4+CD25+ T cells in secondary lymphoid tissues.
Adoptive cell transfer and skin transplantation
Lymphopenic C57BL/6 nude mice were injected with sorted CD4+CD25+ and/or CD4+CD25– T cells transferred at different cell ratios 1 day before skin allograft transplantation. Full-thickness trunk skin grafts from donor mice were then grafted onto the dorsum of adoptively transferred recipient mice.
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Results |
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In ABM mice, a small fraction (mean, 5%; n > 20) of CD4+ T cells from secondary lymphoid tissues constitutively expressed CD25 as well as other markers typical of the TReg phenotype (Fig. 1). The proportion of CD4+CD25+ T cells was smaller than that observed in C57BL/6 controls (mean, 8.6%; n > 20; p < 0.005). Most of the CD4+CD25+ T cells present in ABM mice bore the V2.1+/V8.1+ TCR (mean, 71%; n > 20), indicating that they expressed the anti-bm12 TCR Tg, although this proportion was lower than that in ABM CD4+CD25– T cells (mean, 84%; n > 20; p < 0.004; Fig. 1A). As previously reported for other mice carrying Tg TCRs (14, 29, 30), in ABM mice, CD4+CD25+ T cells were only found in conventional, not in Rag–/–, backgrounds (data not shown). This presumably reflects the need for endogenous TCR -chain rearrangement for the thymic development of CD4+CD25+ Tg cells (30). To characterize ABM Tg CD4+CD25+ T cells, we quantified the expression of genes associated with TReg function. Resting CD4+CD25+, but not CD4+CD25–, ABM Tg T cells expressed high levels of CTLA4, Forkhead/winged helix transcription factor gene (FoxP3), and CD103 (Fig. 1B). No significant differences were found in the expression of these genes between Tg and control C57BL/6 CD4+CD25+ T cells (data not shown). Together, our results indicate that allospecific anti-bm12 Tg CD4+CD25+ T cells are present in ABM mice, and that these cells exhibit a similar phenotype to conventional TRegs.
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ABM Tg TRegs, but not ABM Tg CD4+CD25– TRegs, were anergic in vitro after direct stimulation with bm12 splenocytes (Fig. 2A). In addition, ABM Tg TRegs, but not wild-type C57BL/6 TRegs, powerfully suppressed the allospecific proliferation of ABM Tg CD4+CD25– TEff (Fig. 2B). To study the allospecificity of ABM Tg TRegs in vitro, we took advantage of the capacity of TRegs to proliferate if cultured with mature DCs (16). ABM Tg TRegs proliferated in response to mature bm12, but not third-party, bone marrow-derived mature DCs (Fig. 2C). Highly specific effects were also elicited when ABM Tg CD4+CD25– T cells were challenged with the two populations of mature DCs, albeit the effector T cell proliferation was significantly higher than that of TRegs (Fig. 2C). To determine whether ABM Tg TRegs could suppress the proliferation of T cells bearing different TCR specificities, we used TEa Tg CD4+CD25– T cells, which mount strong proliferative responses when cultured with CB6F1, but not with bm12, irradiated splenocytes. ABM Tg TRegs did not suppress the proliferation of TEa Tg CD4+CD25– T cells in response to CB6F1 stimulators (Fig. 2D, center column). In contrast, in the presence of mixed CB6F1 and bm12 stimulators, ABM Tg TRegs markedly inhibited TEa Tg CD4+CD25– T cell proliferation (Fig. 2D, right column). Taken together, our results indicate that ABM Tg TRegs are absolutely dependent on their cognate alloantigen for activation and proliferation. However, once activated, they can suppress the proliferation of TEff specific for alloantigens expressed on different APCs (bystander suppression).
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To determine the role of ABM TRegs in the prevention of both acute and chronic bm12 heart allograft rejection, we performed a set of heart transplants in ABM Tg recipients that had been thymectomized and depleted of CD4+CD25+ TRegs by anti-CD25 mAb treatment. ABM mice universally rejected bm12 heart allografts in the absence of TRegs (Fig. 3A). Thus, allospecific TRegs powerfully suppress cytopathic alloreactive T cells in vivo and prevent both acute and chronic allograft rejection.
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To test the capacity of ABM Tg TRegs to mediate alloantigen-specific effects in vivo, we conducted adoptive transfer experiments using lymphopenic skin allograft recipients. In this model, the transfer of as few as 1 x 105 CD4+CD25– or CD8+ wild-type naive T cells into skin allograft recipients results in rapid graft rejection, whereas transferred TRegs cells do not induce rejection and prevent CD4+CD25– TEff populations from destroying the grafts (10). The median survival time of bm12 skin grafts challenged with 105 ABM Tg CD4+CD25– T cells was 10 days (Fig. 3B). C57BL/6 TRegs (105) did not prevent 105 ABM Tg CD4+CD25– T cells from rapidly rejecting bm12 allografts. In contrast, ABM Tg TRegs significantly delayed the occurrence of graft rejection (median survival time, 12 vs 40 days; p < 0.01; Fig. 3B). C57BL/6 TRegs only prevented skin allograft rejection when transferred at a high ratio (3:1) of TReg to ABM Tg CD4+CD25– T cell, albeit this protective effect was less marked than after administering an equivalent number of ABM Tg TRegs (data not shown). The need to transfer very high TReg to TEff ratios to ensure effective suppression when using naive polyclonal TRegs has been previously reported (10). ABM mice have a 30-fold higher frequency of I-Abm12-reactive CD4+ T cells than polyclonal C57BL/6 mice (24). Hence, our data indicate that the net suppressive effects exerted by bulk TReg populations in transplantation critically depend on the frequency of alloreactive TRegs among them.
ABM Tg TRegs do not prevent the rejection of third-party skin allografts
To elucidate the fine specificity of TReg function in vivo, we performed additional experiments transferring C57BL/6 CD4+CD25– T cells together with ABM Tg TRegs into recipients of bm12 or third-party (BALB/c) skin allografts. Polyclonal CD4+CD25– T cells are capable of rejecting any MHC-mismatched allogeneic skin allograft (10). ABM Tg TRegs, in contrast, do not mediate immunosuppressive effects in vitro unless direct recognition of intact bm12 alloantigens takes place (Fig. 2). Furthermore, ABM Tg CD4+CD25– T cells fail to reject third-party BALB/c skin allografts (our unpublished observations). Hence, we hypothesized that ABM Tg TRegs would prevent wild-type CD4+CD25– T cells from rejecting bm12, but not third-party strain, allografts. As predicted, the cotransfer of ABM Tg TRegs had no effect on the capacity of C57BL/6 CD4+CD25– T cells to reject BALB/c skin allografts (Fig. 4A), whereas a protective effect was exerted upon bm12 allografts (Fig. 4B). The failure of transferred ABM Tg TRegs to delay BALB/c skin allograft rejection persisted even after markedly increasing the ratio of TReg to TEff, at variance with the effect of transferring polyclonal C57BL/6 TRegs (Fig. 5C). These findings indicate that adoptively transferred TRegs suppress cytopathic alloimmune responses only when TRegs are stimulated by allografts expressing their cognate Ags.
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Linked suppression, a phenomenon in which TRegs can suppress the rejection of third-party alloantigens provided they are expressed on the same APC as the tolerated Ags (4), is considered one of the hallmarks of peripheral allograft tolerance. To test the capacity of TRegs to mediate this process, we used F1 (BALB/c x bm12) allografts as a source of APCs expressing both bm12 and third-party (BALB/c) alloantigens. The cotransfer of ABM Tg TRegs and C57BL/6 CD4+CD25– T cells at a 1:1 ratio into hosts grafted with F1 skin did not delay the occurrence of allograft rejection (Fig. 5A). Nonetheless, the administration of a higher TReg to CD4+CD25– T cell ratio resulted in significant prolongation of F1 allograft survival (Fig. 5B). These results are at variance with those of experiments performed using BALB/c allografts (Fig. 5C). Taken together, our findings indicate that TReg suppressive effects can extend to TEff responding to third-party alloantigens present on the same graft that stimulates the TRegs (linked suppression).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Our data also show that the spontaneous acceptance of bm12 heart allografts by ABM TCR Tg recipients is absolutely dependent on the presence of anti-I-Abm12 TCR Tg TRegs. This is remarkable given that in ABM mice, the overall anti-I-Abm12 TEff responder frequency is 
70% (24), whereas only 5% of TCR Tg CD4+ T cells express TReg markers. These findings indicate that in nonlymphopenic situations, natural TRegs with a defined specificity exhibit a very strong capacity to prevent allograft rejection when their cognate Ag is expressed on the graft. This is in keeping with a recent report using mice expressing a Tg TCR directed against the minor histocompatibility Ag, HY (31). Taken together, these studies, using nonlymphopenic hosts, suggest that both in vitro assays and in vivo adoptive transfer systems, in which unphysiologic ratios of TReg to TEff are commonly required to ensure effective suppression, most likely underestimate the regulatory properties of natural TRegs. These are clinically relevant observations, suggesting that administration of a limited number of allospecific TRegs to nonlymphopenic transplant recipients might be an effective strategy to induce graft acceptance.
The use in our experiments of natural, alloantigen-inexperienced, ABM TCR Tg TRegs (i.e., TRegs obtained from naive unmanipulated ABM mice) precludes us from directly addressing the current controversy of whether tolerizing regimens result in the generation of allospecific TRegs (5, 6, 10, 21). Our observation that regulation of transplant rejection by TRegs critically depends on specific TCR stimulation raises the possibility that tolerance-inducing strategies might be acting, at least in part, by preferentially expanding alloantigen-specific TRegs. However, the use of polyclonal TRegs harvested from tolerized recipients to assess TReg specificity has resulted in much less clear-cut results (5, 6, 10, 21, 32). In a polyclonal population of T cells, the expression of two TCR heterodimers by a single cell or Ag cross-reactivity by a given TCR may create a situation in which a TReg with alloantigen-specificity may be activated by another Ag. Moreover, the use of adoptive transfer systems involving lymphopenic hosts may exacerbate these effects. Alternatively, other regulatory T cell subsets might also be participating in ensuring transplantation tolerance allospecificity. Additional studies are required to completely elucidate these hypotheses.
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by grants from the National Institutes of Health: RO1AI51559 (to M.H.S.), R21HL079450 (to L.A.T. and M.H.S.), R01AI37691 (to M.H.S. and L.A.T.), and PO1AI41521 (to L.A.T., M.H.S., and T.B.S.). 
2 A.S.-F. and S.S. contributed equally to this work and should be considered as co-first authors.
3 L.A.T. and M.H.S. share senior authorship.
4 Address correspondence and reprint requests to Dr. Mohamed H. Sayegh, Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115. E-mail address: msayegh{at}rics.bwh.harvard.edu
5 Abbreviations used in this paper: TReg, regulatory T cell; TEff, effector T cell; Tg, transgenic.
Received for publication September 12, 2005. Accepted for publication October 21, 2005.
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T cell receptor. Science 281: 835-838. bm12 gene is evidence for genetic exchange between mouse immune response genes. Nature 308: 551-553. [Medline]This article has been cited by other articles:
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  C. Becker, C. Taube, T. Bopp, C. Becker, K. Michel, J. Kubach, S. Reuter, N. Dehzad, M. F. Neurath, K. Reifenberg, et al. Protection from graft-versus-host disease by HIV-1 envelope protein gp120-mediated activation of human CD4+CD25+ regulatory T cells Blood, August 6, 2009; 114(6): 1263 - 1269. [Abstract] [Full Text] [PDF]
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F. Casiraghi, N. Azzollini, P. Cassis, B. Imberti, M. Morigi, D. Cugini, R. A. Cavinato, M. Todeschini, S. Solini, A. Sonzogni, et al. Pretransplant Infusion of Mesenchymal Stem Cells Prolongs the Survival of a Semiallogeneic Heart Transplant through the Generation of Regulatory T Cells J. Immunol., September 15, 2008; 181(6): 3933 - 3946. [Abstract] [Full Text] [PDF]
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A. D. Griesemer, J. C. LaMattina, M. Okumi, J. D. Etter, A. Shimizu, D. H. Sachs, and K. Yamada Linked Suppression across an MHC-Mismatched Barrier in a Miniature Swine Kidney Transplantation Model J. Immunol., September 15, 2008; 181(6): 4027 - 4036. [Abstract] [Full Text] [PDF]
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S. Hao, J. Yuan, S. Xu, M. A. Munegowda, Y. Deng, J. Gordon, Z. Xing, and J. Xiang Antigen Specificity Acquisition of Adoptive CD4+ Regulatory T Cells via Acquired Peptide-MHC Class I Complexes J. Immunol., August 15, 2008; 181(4): 2428 - 2437. [Abstract] [Full Text] [PDF]
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 J. Yang, M. O. Brook, M. Carvalho-Gaspar, J. Zhang, H. E. Ramon, M. H. Sayegh, K. J. Wood, L. A. Turka, and N. D. Jones Allograft rejection mediated by memory T cells is resistant to regulation PNAS, December 11, 2007; 104(50): 19954 - 19959. [Abstract] [Full Text] [PDF]
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A. Habicht, S. Dada, M. Jurewicz, B. T. Fife, H. Yagita, M. Azuma, M. H. Sayegh, and I. Guleria A Link between PDL1 and T Regulatory Cells in Fetomaternal Tolerance J. Immunol., October 15, 2007; 179(8): 5211 - 5219. [Abstract] [Full Text] [PDF]
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J. A. Kapp, K. Honjo, L. M. Kapp, K. Goldsmith, and R. P. Bucy Antigen, in the Presence of TGF-beta, Induces Up-Regulation of FoxP3gfp+ in CD4+ TCR Transgenic T Cells That Mediate Linked Suppression of CD8+ T Cell Responses J. Immunol., August 15, 2007; 179(4): 2105 - 2114. [Abstract] [Full Text] [PDF]
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 D. D. Kish, A. V. Gorbachev, and R. L. Fairchild Regulatory function of CD4+CD25+ T cells from Class II MHC-deficient mice in contact hypersensitivity responses J. Leukoc. Biol., July 1, 2007; 82(1): 85 - 92. [Abstract] [Full Text] [PDF]
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C. J. Callaghan, F. J. Rouhani, M. C. Negus, A. J. Curry, E. M. Bolton, J. A. Bradley, and G. J. Pettigrew Abrogation of Antibody-Mediated Allograft Rejection by Regulatory CD4 T Cells with Indirect Allospecificity J. Immunol., February 15, 2007; 178(4): 2221 - 2228. [Abstract] [Full Text] [PDF]
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 C. Fozza, E. Nadal, M. Longinotti, and F. Dazzi T-cell receptor repertoire usage after allografting differs between CD4+CD25+ regulatory T cells and their CD4+CD25 counterpart Haematologica, February 1, 2007; 92(2): 206 - 214. [Abstract] [Full Text] [PDF]
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