PROJECT SUMMARY / ABSTRACT Achieving a state of tolerance in organ transplant recipients could lead to indefinite graft survival without chronic immunosuppression and its associated morbidity/mortality. Tolerance of kidney allografts has been achieved in non-human primates (NHPs) and in humans by using a combination of nonmyeloablative conditioning and donor bone marrow transplantation that results in transient mixed hematopoietic chimerism. However, identical mixed chimerism protocols have failed to induce tolerance in NHP heart allograft recipients. It is well known that there are organ-specific differences in tolerance induction with kidney and liver allografts being “tolerance-prone” and heart and lung grafts being “tolerance-resistant.” Despite the immune barriers posed by heart allografts, our laboratory has recently developed a novel protocol that has, for the first time, achieved long-term, stable tolerance of fully MHC mismatched heart allografts in cynomolgus monkeys. This remarkable result was attained in heart recipients by combining a transient mixed chimerism protocol with donor kidney cotransplantation, which promoted the contributions of host regulatory T cells (Tregs). While a landmark result, this strategy does not have wide clinical feasibility due to the use of experimental agents and the ethical barrier associated with sacrificing a kidney simply to induce heart tolerance. Although lung allografts will not be studied in this project, our recent findings in lung recipients provide proof of principle that tolerance of resistant thoracic organs can also be achieved by inducing a durable (for the life of the allograft) state of mixed chimerism; these were the first NHPs to become tolerant of lung allografts. Thus, we hypothesize that innovative mixed chimerism strategies that either 1) amplify host regulatory mechanisms in transient mixed chimerism protocols or 2) achieve a state of durable donor chimerism will induce long-lasting tolerance in recipients of hearts allografts transplanted alone. We will test this hypothesis by 1) using chimeric antigen receptors (CAR) Tregs to enhance the early contributions of Tregs in conditioning regimens which induce transient chimerism, 2) developing a novel conditioning regimen able to induce durable mixed chimerism, and 3) testing the ability of two FDA-approved drugs, venetoclax (a Bcl-2 inhibitor) and tocilizumab (anti-IL-6R mAb) to eliminate the need of donor kidney transplantation to achieve heart allograft tolerance. We anticipate that by the end of the funding period, we will have generated one or more effective and safe tolerance protocol that can be immediately trialed in human recipients receiving hearts alone.