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

Background. Because T cell receptor-MHC class I/self-peptide interacti ons regulate T-cell development, the presence of MHC allopeptides in t he thymus may influence T-cell tolerance to alloantigens. This hypothe sis is supported by our most recent finding that intrathymic (IT) inoc ulation 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 peptid e specificity, we compared the effects on graft survival of well-defin ed, strain-specific immunogenic WF MHC class I peptides (RT1.A(U)) wit h closely related but non-strain-specific class I peptides derived fro m WAG (RT1(U)). Methods. In vivo immunization of seven MHC class I pep tides synthesized from RT1.A(U) sequences showed that two (u-5 and u-7 ) were immunogenic, whereas five others were not immunogenic in ACI re cipients. We then examined the effects on cardiac allograft survival i n the WF-to-ACI model of the two immunogenic RT1.A(U) peptides (u-5 an d u-7) and three immunogenic WAG-derived peptides (peptides 1, 2, and 5). Results. A combination of equal amounts (150 mu g or 300 mu g) of u-5 or u-7 each with 0.5 ml of antilmyphocyte serum (ALS) on day -7 le d to 60% and 100% permanent graft survival (>150 days), respectively. IT injection of the individual peptides on day -7 showed that only 300 Erg of u-5 significantly prolonged graft survival to a median surviva l time of 17.3 days from 10.5 days in naive recipients. IT injection o f 150, 300, and 600 mu g of u-5 combined with 0.5 mi of ALS on day -7 led to permanent graft survival (> 150 days) in four of six, nine of n ine, and six of six ACI recipients, respectively compared with a media n survival time of 15.4: days in ALS alone-treated controls. In contra st, similar treatments with peptide u-7 with or without 0.5 mi of ALS did not prolong graft survival, thus demonstrating that peptide u-5 al one mediates the observed effects on graft prolongation. A total of 30 0 mu g of u-5 injected IT combined with ALS led to acute rejection of third-party (Lewis) grafts. Intravenous injection of 300 mu g of u-5 w ith ALS also did not prolong WP 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 allograf ts. Similarly, we showed that although IT injection of 600 and 1200 mu g of a mixture of immunogenic WAG-derived peptides 1, 2, and 5 combin ed with 0.5 mi of ALS on day -7 led to permanent WF graft survival in ACI, only IT injection of 300 mu g of peptide 2 combined with BLS led to permanent graft survival (>150 days) in four of five animals. To de fine 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 sho wed nonspecific T-cell suppression in the MLR at 25 days after transpl antation, which correlated with the persistence of ALS immunosuppressi on, long-term unresponsive animals showed normal MLR to donor and thir d-party antigens. In contrast, the donor-specific reactive cytotoxic T lymphocytes remained suppressed in short-term and long-term unrespons ive rats. Conclusion. Of interest is our finding that IT injection of a short segment of WAG-derived MHC class I peptide induces active acqu ired tolerance similar to results obtained with the use of pure WE-der ived peptide u-5 in the WF-to-ACI rat combination. It is noteworthy th at we could not confirm the T helper (Th)1/Th2 paradigm in this model by initial cytokine analysis. Whether induction of tolerance by IT inj ection of allo-MHC peptides will have clinical usefulness must await r esults of similar studies in large animals. However, of major interest is the finding that a short segment of RT1.A(U) represents the tolero genic epitope that induces tolerance.