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 mellitus, 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 MHC class I peptides in the thymus because T cell receptor-MHC class I/self-peptide 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 domain of RT1.A derived from WAG rat (RT1U) on islet graft survival in the WF(RT1U)-to-ACI combination. Adult diabetic male recipients were treated with IT injection of a single WAG-derived MHC class I peptide 7 days before intraportal islet transplantation. Long-term unresponsive islet recipients were examined for the development of alloantigen (Ag)-specific regulatory cells. The results showed that while IT injection of 150 microg peptide 2 on day -7 did not prolong graft survival in naive recipients [median survival time (MST) of 14.0 days vs. 9.6 in controls], IT injection of 300 or 600 microg peptide 2 led to normoglycemia and permanent islet survival (> 200 days) in 4/6 and 3/5 STZ-induced diabetic ACI recipients, respectively. IT injection of 150, 300, or 600 microg peptide 2 combined with 0.5 antilymphocyte serum (ALS) immunosuppression on day -7 led to 100% permanent islet allograft survival (> 200 days) compared to MST of 15.0 +/- 2.3 days in ALS alone-treated controls. Similarly prepared animals rejected third-party Brown Norway (BN) islets in an acute fashion, thus demonstrating donor specificity. Intravenous injection of 300 microg peptide 2 combined with 0.5 ml ALS did not prolong islet allograft survival. The long-term unresponsive islet allograft recipients challenged with second set grafts accepted permanently 100% donor-type cardiac allografts while rejecting third-party (BN) hearts without rejecting the primary Wistar Furth (WF) islets. In analyzing the underlying mechanisms 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) unseparated spleen cells from tolerant animals at 60 and 100 days after islet transplantation into lightly irradiated [200 rad total body irradiation (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 days in lightly irradiated ACI given unmodified syngeneic spleen cells. In addition, adoptive transfer of 2 x 10(7) purified T cells obtained from long-term functioning islet recipients led to permanent donor-specific islet survival in secondary recipients. The finding that IT injection of a short segment of a synthetic immunodominant MHC class I peptide derived from WAG that shares the RT1.A(U) domain with the graft donor is capable of inducing acquired 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-specific alloreactive T cells is, in part, responsible for the maintenance of tolerance in this model.