We have recently shown that a single intrathymic injection of synthetic 25mer peptides, representing full sequences of the hypervariable domain of RT1.BuB (4 peptides) and RT1.Du beta (4 peptides) WF class II MHC molecules, 48 hr before transplantation induces donor-specific unresponsiveness to WF rat renal allografts in adult LEW recipients. The induction of unresponsiveness was abrogated by the recipient's thymectomy within the first week after intrathymic injection. Peripheral T cells of long-term survivors exhibited antigen-specific hyporesponsiveness in the LEW x WF MLR. Studies on the mechanisms of induction of acquired thymic unresponsiveness to alloantigen in vivo and in vitro are now reported. First, since we have previously demonstrated in LEW responders that only 4 of the 8 synthetic 25mer peptides, 2 RT1.Du beta and 2 RT1.Bu beta sequences, were immunogenic in vitro and in vivo, we compared the tolerogenicity of the immunogenic versus the nonimmunogenic peptides. While LEW rats intrathymically injected with the nonimmunogenic peptides acutely rejected their renal allografts within 6-10 days, animals injected with the immunogenic peptides did not reject their grafts and are surviving > 100 days with normal allograft function. In vitro studies established that peripheral T cells from intrathymically tolerized animals exhibited antigen-specific hyporesponsiveness in the LEW x WF MLR starting as early as 1 week posttransplant. Immunohistological evaluation of renal allografts from intrathymically tolerized animals 1 week postengraftment showed marked reduction in mononuclear cell infiltrates with no evidence of tubulitis, and marked reduction in intragraft staining for activation and inflammatory cytokines and alloantibodies, as compared with acutely rejecting controls. Systemic administration of 1000 U of rIL-2 daily for 5 days starting on the day of transplantation abrogated the tolerogenic effect of intrathymic MHC allopeptides. Injection of 100 micrograms of a single immunogenic peptide, RT1.Du beta 2 (residues 20-44), into the thymus of responder LEW rats 48 hrs before immunization with RT1.Du beta 2 effected significant reduction of in vitro proliferation of primed lymphocytes to RT1.Du beta 2, an effect that was abrogated by addition of rIL-2 in vitro. In contrast, thymectomy beyond 2 weeks and administration of rIL-2 at 4-6 weeks after transplantation failed to cause rejection. These observations indicate that thymic recognition of immunodominant class II MHC allopeptides leads to peripheral T cell anergy that mediates the induction phase of systemic unresponsiveness to renal allografts. The maintenance phase appears to be mediated by dense anergy or clonal deletion.(ABSTRACT TRUNCATED AT 400 WORDS)