PROJECT SUMMARY Extracellular vesicles (EVs) are membrane bound nanoparticles that can interact with other cells as a means of cell:cell communication. Emerging data suggest that β cell-derived exosomes, an EV subtype released by exocytosis of multivesicular bodies, may act as paracrine effectors in islet health. Given the potential for exosomes to play a role in β cell communication with surrounding cells in the islet microenvironment, a critical need exists for deliberate research defining mechanisms of β cell exosome biology under physiologic and pathophysiologic circumstances. β cell expression of the transmembrane immune checkpoint protein death-ligand 1 (PD-L1) plays a key role to support β cell survival in type 1 diabetes (T1D). Preliminary data suggest that β cell exosome membranes carry PD-L1, and that exosomal PD-L1 is upregulated by cytokine treatment of parent β cells. Our central hypothesis is that stressed β cells in the T1D microenvironment alter their exosomal contents to include protective mechanisms aimed at evading β cell destruction associated with autoimmunity. Aim 1 will elucidate the mechanistic etiology of altered β cell exosome PD-L1 cargo. We hypothesize that proinflammatory interferon signaling activates molecular regulators of intracellular β cell PD-L1 that, in concert with mediators of exosome biogenesis, increase total exosomal PD-L1. Chemical and genetic manipulation will be used to test impacts of these pathways on β cell exosomal PD-L1. Aim 2 will test the hypothesis that via transfer to or interaction with surrounding β cells and T cells, β cell exosomal PD-L1 loading is a protective mechanism shielding β cells from autoimmune destruction. Use of β cells differentiated from a human induced pluripotent stem cell line overexpressing GFP-tagged PD-L1 will allow for direct tracking of PD-L1 transfer and binding to other β cells and CD8+ T cells. Aim 3 will employ a novel microfluidic device to test the hypothesis that PD-L1+ exosome release will be increased in humans with or at risk for T1D. We will quantify differences in total and islet-derived EV PD-L1 in human plasma or pancreas slice media and compare to nondiabetic controls. Testing will also be performed in plasma from individuals with longstanding T1D with or without residual detectable C-peptide to determine if plasma islet-derived EV PD-L1 is linked to functional β cell survival. This work will lead to a paradigm shift in the field’s understanding of β cell communication with surrounding cells, and determine the clinical potential of islet-derived PD-L1 exosome cargo as a therapeutic targeting β cell survival or a biomarker to dissect T1D disease-related heterogeneity.