PROJECT SUMMARY Dysregulation of blood glucose due to T1D leads to both short term consequences such as hypoglycemia induced injury and death, and long-term complications including amputation, blindness, kidney failure, and neuropathy. Islet transplantation has the potential to provide biologic glycemic regulation for all T1D patients, effectively curing the disease. However, this promising therapy is plagued by the requirement for ineffective immunosuppressive therapies. Currently, only 50 to 70% of grafts remain viable at 5 years post-transplantation, largely due to the immunosuppressive therapy causing toxicity and failing to fully protect the graft. Currently, a costly cocktail of immunosuppressive drugs is given in a rarely successful attempt to provide broad immune coverage with side effect mitigation. Thus, a need exists for simpler, lower cost, and more effective alternatives to these immunosuppressive regimens that can maintain islet transplant survival without off-target side-effects. The objective of this proposal is to mechanistically understand how repurposing a single component of these immunosuppressive cocktails, rapamycin, using engineered nanoscale drug carriers can provide sustained islet protection. Vesicular polymeric nanocarriers (i.e. polymersomes, PS) encapsulating rapamycin (rPS) were found to uniquely change the cellular biodistribution of rapamycin to avoid side-effects and significantly improve efficacy. Importantly, rapamycin normally has a wide cellular biodistribution and functions by directly inhibiting T cell proliferation, but rPS completely avoids T cells and instead switches the immunosuppressive mechanism to a selective and potent costimulation blockade of antigen presenting cells (APCs). This novel cell-selective nanocarrier-enhanced costimulation blockade was characterized by an upregulation of CD8+ regulatory T cells and double positive CD4+CD8+ T cells, achieving sustained normoglycemia in a rigorous fully major histocompatibility complex (MHC) mismatched allogenic intraportal (liver) islet transplantation mouse model. Here, this proposal will investigate and benchmark the immunological mechanism of rPS against the clinically relevant therapeutic belatacept, a CTLA4-IgG fusion protein that induces costimulation blockade via an alternative method: the blocking of CD80/86 coreceptors on APCs. The following aims will be achieved: Aim 1: Determine whether rPS induces general immunosuppression or antigen-specific tolerance. It is currently not known whether rPS induce antigen-specific or general systemic immunosuppression. A novel ex vivo mixed lymphocyte reaction assay and in vivo dual antigen-specific / non-specific islet transplantation will address this question. Aim 2: Compare the immunomodulatory mechanisms of costimulation blockade induced by rPS vs. CTLA4-IgG for enhanced allogenic islet graft survival. High parameter flow cytometry using T-distributed Stochastic Neighbor Embedding (tSNE) analysis and a...