COMPARATIVE STUDIES OF SPECIFIC ACQUIRED SYSTEMIC TOLERANCE INDUCED BY INTRATHYMIC INOCULATION OF A SINGLE SYNTHETIC WISTAR-FURTH (RT1U) ALLO-MHC CLASS I (RT1.AU) PEPTIDE OR WAG (RT1U)-DERIVED CLASS I PEPTIDE1

Background. Because T cell receptor-MHC class I/self-peptide interactions regulate T-cell development, the presence of MHC allopeptides in the thymus may influence T-cell tolerance to alloantigens. This hypothesis is supported by our most recent finding that intrathymic (IT) inoculation of nonimmunogenic synthetic peptides derived from WAG RT1.A induces tolerance to cardiac allografts in the Wistar-Furth (WF)-to-ACI model. To evaluate whether in vivo immunogenicity of MHC peptides is relevant to tolerance induction and to examine the effect of peptide specificity, we compared the effects on graft survival of well-defined, strain-specific immunogenic WF MHC class I peptides (RT1.A U ) with closely related but non-strain-specific class I peptides derived from WAG (RT1 U ). Methods. In vivo immunization of seven MHC class I peptides synthesized from RT1.A U sequences showed that two (u-5 and u-7) were immunogenic, whereas five others were not immunogenic in ACI recipients. We then examined the effects on cardiac allograft survival in the WF-to-ACI model of the two immunogenic RT1.A U peptides (u-5 and u-7) and three immunogenic WAG-derived peptides (peptides 1, 2, and 5). Results. A combination of equal amounts (150 μg or 300 μg) of u-5 or u-7 each with 0.5 ml of antilymphocyte serum (ALS) on day -7 led to 60% and 100% permanent graft survival (>150 days), respectively. IT injection of the individual peptides on day -7 showed that only 300 μg of u-5 significantly prolonged graft survival to a median survival time of 17.3 days from 10.5 days in naive recipients. IT injection of 150, 300, and 600 μg of u-5 combined with 0.5 ml of ALS on day -7 led to permanent graft survival (> 150 days) in four of six, nine of nine, and six of six ACI recipients, respectively, compared with a median survival time of 15.4 days in ALS alone-treated controls. In contrast, similar treatments with peptide u-7 with or without 0.5 ml of ALS did not prolong graft survival, thus demonstrating s that peptide u-5 alone mediates the observed effects on graft prolongation. A total of 300 μg of u-5 injected IT combined with ALS led to acute rejection of third; party (Lewis) grafts. Intravenous injection of 300 μg of : u-5 with ALS also did not prolong WF graft survival in ACI recipients. The long-term unresponsive ACI recipients accepted permanently donor-type (WF) but not third-party (Lewis) second-set cardiac and islet allografts. Similarly, we showed that although IT injection of 600 and 1200 μg of a mixture of immunogenic WAG-derived peptides 1, 2, and 5 combined with 0.5 ml of ALS on day -7 led to permanent WF graft survival in ACI, only IT injection of 300 μg of peptide 2 combined with ALS led to permanent graft survival (>150 days) in four of five animals. To define the underlying mechanisms of tolerance, we examined in vitro the mixed lymphocyte reaction (MLR), cell-mediated lymphocytotoxicity, and cytokine profile of unresponsive recipients. Although the results showed nonspecific T-cell suppression in the MLR at 25 days after transplantation, which correlated with the persistence of ALS immunosuppression, long-term unresponsive animals showed normal MLR to donor and third-party antigens. In contrast, the donor-specific reactive cytotoxic T lymphocytes remained suppressed in short-term and long-term unresponsive rats.