PROJECT SUMMARY / ABSTRACT The objective of this project is to induce localized immune tolerance to transplanted human beta cells derived from renewable stem cell sources as a treatment for type 1 diabetes. We will test our approach to prevent immune rejection of grafted human stem cell derived beta cells in diabetic mice with elements of human immune systems. In addition, we seek to explain the detailed biological mechanisms by which the therapy works by conducting in vitro studies using islet- reactive human T cells. Recent advances in the generation of stem cell derived beta-like cells (sBCs) have raised the possibility of providing a renewable source of functional beta cells for transplantation, effectively overcoming the severe shortage of human donor islets. We have generated simplified culture conditions that accurately and efficiently generate unlimited quantities of glucose-responsive insulin-expressing beta cells in vitro. The stem cell derived beta cells generated have already been used successfully for beta cell replacement therapy in animal models. By combining cell engineering with biomaterials engineering to display negative regulators of immunity (e.g. programmed death-ligand 1, PD-L1; and tumor necrosis factor (TNF)- related apoptosis-inducing ligand, TRAIL), we will functionalize stem cell derived beta cells (sBCs) to counter autoimmunity upon transplantation. A major strength of our approach is that the immunotherapy is strongly localized to grafted beta cells, which increases specificity and avoids the negative side effects of systemic immunotherapy. We hypothesize that stem cell derived human pseudo islets transplanted with PD-L1 and/or TRAIL will be resistant to autoimmune destruction and exhibit enhanced engraftment / survival in an in vivo model of beta cell graft rejection using humanized mice. Our strategy will induce localized tolerance to beta cell antigens while simultaneously promoting sBC graft vascularization.