Project 1 Summary: The overall goal of Project 1 is to induce tolerance of porcine kidneys in baboons by co-transplantation (Tx) of vascularized donor thymus. We have made significant progress during the current project period (2016-2020). First, we have identified the mechanism responsible for proteinuria, a significant obstacle in our GalT-KO pig-to-baboon kidney xenotransplant (XTx) model for the long-term survival of life-supporting kidney xenografts, and developed effective treatment strategies for overcoming it. Down-regulation of sphingomyelin phosphodiesterase acid-like 3b and up-regulation of CD80 on pig podocytes were found to play critical roles in proteinuria and species incompatibility between pig CD47 and baboon SIRPα, which caused phagocytosis of pig endothelial cells and podocytes by baboon macrophages, was observed. We learned that either the administration of CTLA4-Ig and rituximab mAb for GalT-KO grafts or the use hCD47 transgenic (Tg) GalT-KO kidney grafts inhibited the development of post-transplant proteinuria, and markedly prolonged baboon survival up to 193 days. While the exponential growth of pig grafts or drug-related side effects triggered the euthanasia of recipient baboons, no evidence of graft rejection was seen and both pig-specific unresponsiveness in vitro, and the development of new baboon T cells were observed in multiple recipients VT+K XTx. Second, in collaboration with Project 2, we have achieved prolonged peripheral blood macrochimerism lasting >60 days accompanied by gradual loss of anti-non-Gal antibodies (abs) in baboons following hCD47+ pig intra-bone bone marrow Tx. Such durable porcine chimerism has not been previously achieved in any primate. Our renewal is focused on achieving two aims. Aim 1: Optimize the protocol for pig kidney XTx with vascularized thymic grafts by determining: (1) acceptable non-Gal ab levels; (2) requirement for human complement regulatory protein Tg for recipients with high non-Gal ab levels; (3) a strategy to prevent rejection in recipients with high non-Gal abs; (4) acceptable preservation times of pig kidney and thymus grafts using an FDA-approved continuous organ preservation machine; and (5) the ability to completely terminate immunosuppression. Aim 2: Determine the ability of combined VT+KTx and IBBMTx using hCD47+ human IL3 receptor (hIL3r) transgenic (Tg) pigs as BM donors on (achieve robust, durable T cell tolerance by combined deletional and regulatory mechanisms. Additionally, we will also explore the ability of this combined approach to permit long-term kidney xenograft tolerance in baboons with high non-Gal natural antibody levels. We anticipate that the above studies will lead to achievement of the short- term goal of extending pig VTL+KTx grafts to clinical applications in the next five years, and the long- term goal of inducing robust tolerance of pig xenografts, respectively.