Despite improvements in short-term survival, long-term results of organ transplantation have not been satisfactory due to the various side effects of life-long administration of potent immunosuppressive drugs as well as to chronic rejection. Therefore, induction of allograft tolerance remains an important goal of organ transplantation. Induction of chimerism through donor bone marrow transplantation has so far been the only approach that reproducibly induced tolerance in clinical trials. However, widespread application of this approach is hampered by 1) severe myelosuppression associated with the conditioning regimen needed to achieve engraftment of hematopoietic stem cells, and 2) inconsistent induction of tolerance due to limited chimerism resulting from the current non-myeloablative conditioning regimens. To address these obstacles, a novel protocol using a B cell lymphoma 2 (Bcl-2) inhibitor and costimulatory blockade to induce persistent mixed chimerism and allograft tolerance without myelosuppressive treatments has been developed in mice. Our preliminary studies to evaluate this approach in nonhuman primates (NHPs) using a clinically available selective Bcl-2 inhibitor, ABT-199 (Venetoclax), have shown encouraging results. Extended duration of mixed chimerism and long-term immunosuppression-free renal allograft survival were achieved without myelosuppressive complications. Our preliminary study of a second Bcl-2 inhibitor, ABT-263, which has affinities to Bcl-2, Bcl-xL and Bcl-w showed greater capacity to delete conventional T cells while sparing regulatory T cells, which suggests that it may be even more tolerogenic than ABT-199. The objectives of this research proposal are 1) to establish a clinically applicable nontoxic conditioning regimen for induction of tolerance based on Bcl-2 family protein inhibition and 2) to investigate the mechanisms by which Bcl-2 inhibition promotes mixed chimerism and renal allograft tolerance in NHPs. We will first test the ABT-199 regimen in a larger cohort of animals to prove the reliability and efficacy of this approach as well as to identify the optimal costimulatory blockade. We will next determine whether ABT-263 is more effective than its counterpart ABT-199 and can induce chimerism and tolerance without any myelosuppressive treatment (e.g. total body irradiation). Finally, we will investigate the mechanisms by which Bcl-2 with or without Bcl-xL/Bcl-w inhibition promote mixed chimerism and renal allograft tolerance in the absence of myelosuppression. Extensive in vitro and in vivo experiments are planned to elucidate the mechanistic pathways of deletional or regulatory tolerance.