Project Summary: Project 2 is specifically designed toward the development of a tolerance induction strategy for curative treatment of end-stage diabetic nephropathy, using living donor composite Islet-Kidney (IK) transplantation (Tx). Many diabetic patients in renal failure, especially children, have potential donors willing to provide both a kidney and islets for transplantation. Unfortunately, the quantity of islets obtained through partial pancreatectomy from living donors is often insufficient to achieve insulin independence following isolated islet Tx. We have previously demonstrated that the strategy of transplanting pre-vascularized islets as part of composite IKs in large animal models requires far fewer islets to achieve insulin independence than Tx of free, non-vascularized islets. Both renal and islet function were restored by IK tx across fully allogeneic barriers in nephrectomized diabetic baboons using a clinically relevant immunosuppression protocol. More recently, we reported the successful induction of tolerance of IKs in rhesus monkeys using a novel, reduced intensity, hematopoietic cell Tx protocol in a “parent-to-offspring” combination. These data demonstrated “proof of principle” for the approach of induction of tolerance of allogeneic islet-kidneys. However, although allograft tolerance was achieved, chimerism was transient and insulin supplementation was required early post transplantation. We hypothesize that components of the conditioning regimen and/or donor cell source may have had an early negative impact on islet function. We also hypothesize that induction of tolerance through durable mixed chimerism may be more effective than transient chimerism in reversing autoimmunity associated with T1D, as has been demonstrated recently in an NOD mouse model. We therefore propose here to: 1) optimize the conditioning protocol and mobilized cell product in ways that are expected to improve preservation of islet function during the tolerance induction phase (Aim 1), including replacement of CyA with Rapamycin and anti-CD40 mAb and. If needed, use of purifed HSC; 2) add ex-vivo expanded donor-specific Tregs to the induction regimen, in order to facilitate the establishment of durable mixed chimerism (Aim 2). This approach is possible with the use of living-related donors, from whom one can generate donor-specific Tregs in advance of the transplant; and 3) study the fate of both effector and regulatory T cells in recipients of IKs (Aim 3), in order to determine the mechanism of tolerance and identify potential biomarkers predicting tolerance vs rejection. These studies will involve assessment of the deletion or expansion of effector and regulatory T cells using a high-throughput TCR sequencing approach developed in Core B. Islets will be provided by Core A and collaboration with Project 1 and both cores will be coordinated through Core C.