MECHANISMS OF ACQUIRED THYMIC UNRESPONSIVENESS TO RENAL-ALLOGRAFTS - THYMIC RECOGNITION OF IMMUNODOMINANT ALLO-MHC PEPTIDES INDUCES PERIPHERAL T-CELL ANERGY

We have recently shown that a single intrathymic injection of syntheti c 25mer peptides, representing full sequences of the hypervariable dom ain of RT1.B-u beta (4 peptides) and RT1.D-u beta (4 peptides) WF clas s II MHC molecules, 48 hr before transplantation induces donor-specifi c unresponsiveness to WF rat renal allografts in adult LEW recipients. The induction of unresponsiveness was abrogated by the recipient's th ymectomy within the first week after intrathymic injection. Peripheral T cells of long-term survivors exhibited antigen-specific hyporespons iveness 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.D-u be ta and 2 RT1.B-u beta sequences, were immunogenic in vitro and in vivo , we compared the tolerogenicity of the immunogenic versus the nonimmu nogenic peptides. While LEW rats intrathymically injected with the non immunogenic 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 functi on. In vitro studies established that peripheral T cells from intrathy mically tolerized animals exhibited antigen-specific hyporesponsivenes s in the LEW x WF MLR starting as early as 1 week posttransplant. Immu nohistological evaluation of renal allografts from intrathymically tol erized animals 1 week postengraftment showed marked reduction in monon uclear cell infiltrates with no evidence of tubulitis, and marked redu ction in intragraft staining for activation and inflammatory cytokines and alloantibodies, as compared with acutely rejecting controls. Syst emic administration of 1000 U of rIL-2 daily for 5 days starting on th e day of transplantation abrogated the tolerogenic effect of intrathym ic MHC allopeptides. Injection of 100 mu g of a single immunogenic pep tide, RT1.D-u beta 2 (residues 20-44), into the thymus of responder LE W rats 48 hrs before immunization with RT1.D-u beta 2 effected signifi cant reduction of in vitro proliferation of primed lymphocytes to RT1. D-u 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 a t 4-6 weeks after transplantation failed to cause rejection. These obs ervations indicate that thymic recognition of immunodominant class II MHC allopeptides leads to peripheral T cell anergy that mediates the i nduction phase of systemic unresponsiveness to renal allografts. The m aintenance phase appears to be mediated by dense anergy or clonal dele tion. These findings also confirm the role of indirect T cell recognit ion of alloantigen in vascularized allograft rejection, since function al inactivation/deletion of T cells in this pathway is sufficient to p revent rejection.