Regulatory T cells maintain peripheral tolerance to islet allografts induced by intrathymic injection of MHC class I allopeptides

Although transplantation remains the treatment of choice for diabetes melli tus, immunological rejection of allografts continues to be a major problem. The search for strategies to prevent graft rejection led us to examine if the fate of developing T cells may be influenced by the presence of allo MH C class I peptides in the thymus because T cell receptor-MHC class I/self-p eptide interaction regulates thymocyte development. We studied the effects of intrathymic (IT) injection of a short segment of a synthetic immunogenic MHC class I peptide (peptide 2, residues 67-85) of the hypervariable domai n of RT1.A derived from WAG rat (RT1(U)) on islet graft survival in the WF( RT1(U))-to-ACI combination. Adult diabetic male recipients were treated wit h IT injection of a single WAG-derived MHC class I peptide 7 days before in traportal islet transplantation. Long-term unresponsive islet recipients we re examined for the development of alloantigen (Ag)-specific regulatory cel ls. The results showed that while IT injection of 150 mu g peptide 2 on day -7 did not prolong graft survival in naive recipients [median survival tim e (MST) of 14.0 days vs. 9.6 in controls], IT injection of 300 or 600 mu g peptide 2 led to normoglycemia and permanent islet survival (> 200 days) in 4/6 and 3/5 STZ-induced diabetic ACI recipients, respectively. IT injectio n of 150, 300, or 600 mu g peptide 2 combined with 0.5 antilymphocyte serum (ALS) immunosuppression on day -7 led to 100% permanent islet allograft su rvival (> 200 days) compared to MST of 15.0 +/- 2.3 days in ALS alone-treat ed controls. Similarly prepared animals rejected third-party Brown Norway ( BN) islets in an acute fashion, thus demonstrating donor specificity. Intra venous injection of 300 mu g peptide 2 combined with 0.5 mi ALS did not pro long islet allograft survival. The long-term unresponsive islet allograft r ecipients challenged with second set grafts accepted permanently 100% donor -type cardiac allografts while rejecting third-party (BN) hearts without re jecting the primary Wistar Furth (WF) islets. In analyzing the underlying m echanisms of acquired systemic tolerance, we found no suppressor/regulatory cells in adoptive transfer studies in tolerant animals at 30 days after IT injection of allopeptides. In contrast, adoptive transfer of 5 x 10(7) uns eparated spleen cells from tolerant animals at 60 and 100 days after islet transplantation into lightly irradiated [200 rad total body irridation (TBI )] ACI recipients led to donor-specific permanent islet graft survival in 2 /3 and 4/5 secondary recipients, respectively, compared to an MST of 13.8 d ays in Lightly irradiated ACI given unmodified syngeneic spleen cells. In a ddition, adoptive transfer of 2 x 10(7) purified T cells obtained from long -term functioning islet recipients led to permanent donor-specific islet su rvival in secondary recipients. The finding that IT injection of a short se gment of a synthetic immunodominant MHC class I peptide derived from WAG th at shares the RT1.A(U) domain with the graft donor is capable of inducing a cquired systemic tolerance to WF islets suggests that linked recognition or epitope suppression may be involved in the induction of unresponsiveness. Generation of peripheral Ag-specific regulatory cells that suppress Ag-spec ific alloreactive T cells is, in part, responsible for the maintenance of t olerance in this model.