A Key Role for TGF-Signaling to T Cells in the Long-Term Acceptance of Allografts 1 Stephen R. Daley, 2 Jianbo Ma, Elizabeth Adams, Stephen P. Cobbold, 3 and Herman Waldmann 3,4 TGF-is a key immunoregulatory cytokine which supports self-tolerance by signaling to T cells. In this report, we show a crucial role for TGF-signaling to T cells in enabling the long-term acceptance of allografts, whether natural or induced therapeutically by coreceptor and costimulation blockade. The requirement for TGF-appears most pronounced during the initial exposure to alloantigens. We demonstrate the ability of TGF-to direct the development in vitro of regulatory cells that suppress graft rejection in vivo. Such suppression was not affected by anti-TGF-treatment of the recipient mice. Despite this, TGF-may still have a role in CD4 cell-mediated suppression of antiallograft responses in vivo, since its neutralization can, in some cases, abrogate suppression. These results show that TGF-signaling to T cells is dispensable for mounting destructive responses against skin allografts while appearing to be an essential intermediary in establishing long-term tolerance. The Journal of Immunology, 2007, 179: 3648 –3654. T ransforming growth factor has been shown to have a crucial role in immunological self-tolerance (1, 2). Re- cently, a major checkpoint for its protective activity has been narrowed to that of T cell signaling, since ablation of the TGF-receptor type II (Tgfbr2) 5 gene in T cells produces cata- strophic autoimmunity (3, 4) associated with increased turnover and maturation of CD4 + and CD8 + T cells and decreased “com- petitive fitness” of Foxp3 + regulatory T cells (Treg) (3). Ablation of the TGFRII gene in B cells heightens the responsiveness of IgM + and IgG + B cells and blocks isotype switching to IgA (5), suggesting that TGF-serves parallel purposes in controlling the differentiation of both T and B cells. Interference with TGF- signaling in CD11c + cells using a dominant-negative Tgfbr2 trans- gene resulted in the accumulation of an abnormally high frequency of NK cells, most of which synthesized IFN-in response to stim- ulation, relative to wild type (6). Thus, a common thread is that TGF-regulates the homeostasis of several leukocyte subsets in vivo and it clearly has the capacity to regulate immunity. TGF-is not, however, an obligate anti-inflammatory cytokine, because TGF-was identified as a component of in vitro milieu that favor Th17 commitment in CD4 + T cells (7–9). Its activities for good or bad must then be contextual and cannot always be predicted. The first indication that TGF-may have a role in transplanta- tion tolerance came from studies examining tolerance induced by donor-specific transfusions (10). More recently, a role for TGF- has been highlighted in the induction of tolerance by coreceptor blockade with a CD4-specific mAb. This study used a TCR- trans- genic mouse (A1.RAG1 -/- ) where all T cells bear a TCR specific for a male transplantation Ag (Dby presented by H2-E k ) (11). Fe- male A1.RAG1 -/- mice normally reject male skin grafts, but non- depleting anti-CD4 treatment permits life-long graft survival (11). We showed that TGF-, in conjunction with coreceptor blockade, had a clear role in conversion of naive CD4 + T cells to Foxp3 + Treg (12). We showed that neutralization of TGF-prevented tol- erance induction to male grafts by CD4 coreceptor blockade (12). These, along with the finding that transplanted tumors survived in wild-type hosts but were rejected in hosts where CD8 + T cells were deficient in TGF-signaling (13), all argue for TGF-as a protective factor against allograft rejection. Building on our previous studies using systemic TGF-neu- tralization, we use here recipient mice that express a T cell-specific dominant-negative TGF-receptor type II (dnTgfbr2). By intro- ducing this transgene into the A1.RAG1 -/- model, we examine whether one activity of TGF-in supporting allograft tolerance is by signaling to T cells. We use dnTgfbr2 + mice as a tool to seek tolerance checkpoints bypassed by dnTgfbr2 + T cells. We also study the interval during which TGF-is required to act: before exposure, during the initial exposure, or after prolonged exposure to alloantigens shed by skin grafts. Materials and Methods Mice CBA/Ca (CBA), B10.BR, BALB/k, hCD52-Tg (14), A1.RAG1 -/- (11), and CD4dnTgfbr2 (dnTgfbr2 + ) (15) mice have been described. dnTgfbr2 + mice had been backcrossed to C57BL/6 (B6) for 10 generations. Wild- type and dnTgfbr2 + mice were offspring of dnTgfbr2 + males hemizygous for this transgene. We crossed dnTgfbr2 + mice with B6.RAG1 -/- mice for two generations to produce dnTgfbr2 + RAG1 -/- animals that were then crossed with A1.RAG1 -/- to introduce the Dby-specific TCR transgene. Genotyping of dnTgfbr2 + vs wild-type mice used PCR on genomic DNA Therapeutic Immunology Group, Sir William Dunn School of Pathology, Oxford, United Kingdom Received for publication April 30, 2007. Accepted for publication July 13, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work supported by a Medical Research Council U.K. Program Grant “Thera- peutic Immunoregulation” and the European Union FP6 “RISET” Consortium. S.D. was supported by the Rhodes Trust. 2 Current address: John Curtin School of Medical Research, Building 54, ANU, Mills Road, Acton, Australia. 3 S.C. and H.W. contributed equally as joint senior authors. 4 Address correspondence and reprint requests to Prof. Herman Waldmann, Sir Wil- liam Dunn School of Pathology, South Parks Road, Oxford, U.K. E-mail address: herman.waldmann@path.ox.ac.uk 5 Abbreviations used in this paper: Tgfbr2, TGF-receptor type II; dnTgfbr2, dom- inant-negative TGF-receptor type II; Treg, regulatory T cell. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 The Journal of Immunology www.jimmunol.org on March 17, 2011 www.jimmunol.org Downloaded from