Chronic immunosuppressive therapy currently required for human transplantation is associated with high risks of infection and malignancies. In contrast, the state of donor-specific tolerance (DST) would not be associated with such risks. DST has been achieved in mixed chimeras, prepared with allogeneic bone marrow transplantation (BMT) following conditioning with a nonmyeloablative, or nonlethal regimen (NLR) consisting of anti-T cell mAbs, low dose (3 Gy) whole body irradiation, and high-dose (7 GY) thymic irradiation. Chimeras, created by transplanting b10.A(K(k), IE(k), IA(k), D(d)) bond marrow cells into B10 (K(b), IE-, IA(b), D(b)) mice conditioned with the NLR, show clonal deletion of donor-reactive T cells, specifically Vbeta11+ T cells, which are present in normal B10 mice, but deleted in normal B10.A mice due to expression of an endogenous superantigen in association with I-E. In these chimeras, loss of tolerance to donor-type (B10.A) skin grafts is observed when donor antigen is depleted using a class I-specific anti- donor mAb. Such loss of tolerance appears dependent on the host thymus, suggesting that, in the absence of donor antigens, donor-reactive T cells emerge from the thymus, thus implicating a central mechanism. However, contribution of peripheral tolerance mechanisms to DST induced by the NLR is not known. Phase I of the application will consist of didactic instruction of the PI and specific aims #1-3. The first specific aim will determine the relative contributions of clonal deletion, T cell anergy, and suppression, to the maintenance of DST induced in this model. Reappearance of Vbeta11+ T cells will be evaluated in euthymic and thymectomized chimeras following depletion of donor antigens, and correlated with rejection of donor-type skin grafts. The presence of T cell anergy or suppression, not dependent upon the presence of donor-type antigen, will be evaluated if skin grafts are accepted by donor antigen- depleted, thymectomized chimeras. The second specific aim will determine which donor cell lineages (thymocytes, thymic B cells, or thymic dendritic cells) maintain central tolerance. Splenocytes and thymocytes from chimeric animals will be adoptively transferred into B10 (host-type) nude mice at various times after beginning anti-donor mAb treatment. The tolerance status of the nude recipients will then be correlated with chimerism of different lineages and tissues, and with Vbeta11+ T cell deletion in the original chimeras at the time of adoptive transfer. The third specific aim will delineate the role of T cell anergy or suppression in the induction of DST. Splenocytes from chimeras, not completely deleted of Vbeta11+ host-type T cells, will be injected into B10 nudes at early time points following BMT. Acceptance or rejection of donor-type (B10.A) skin grafts in each experiment will demonstrate whether or not host T cells are tolerant. Evidence for tolerance will implicate anergy or suppression, and continued tolerance after administration of non-tolerant, normal B10 (host-type) spleen cells will isolate suppression as the mechanism. Phase 2 will consist of specific aims #4-5.
Under specific aim #4, T-cell receptor (TCR) transgenic mice will receive marrow expressing MHC antigens recognized by the transgenic TCR-bearing T cells. Deletion of these T cells in bone marrow recipients will establish clonal deletion as a mechanism of DST for antigens known to serve as transplantation antigens.
Under specific aim #5, this transplantation antigen-induced tolerant state will be subjected to infection by N. Brasiliensis and murine CMV infection in order to evaluate its stability. If donor-specific tolerance is not disrupted, then the NLR and allogeneic BMT could be used to induce similar tolerance in humans.
