1171-P: Cytokines Do Not Consistently Change Proinsulin (PI) Cargo in Global Human Islet-Derived Extracellular Vesicle (EV) Populations

Introduction and Objective: Multiple factors may trigger or exacerbate progression of Type 1 diabetes (T1D). EVs, nanoparticles housing molecular cargo, have been implicated in many diseases, including diabetes. β-cell EVs can present antigens and carry PI as cargo. Cytokine exposure leads to β-cell ER stress, reduces PI processing enzyme expression, and increases PI secretion relative to insulin or C-peptide. Cytokine-induced stress is also associated with changes in EV cargo, as well as intracellular formation of immunogenic peptides, including modified PI. We sought to understand if β-cell EV PI cargo might be changed under cytokine exposure. Methods: INS1 or EndoC-βH1 cells and human islets were treated with 5 ng/mL IL-1β, or IL-1β + 100 ng/mL IFNγ + 10 ng/mL TNFα, for 48 hrs. Plasma (450 µL) from five children with recent onset type 1 diabetes and age, sex, and BMI-matched nondiabetic controls was also assayed. EVs were isolated by ultracentrifugation (30000 x g, 1.5 hrs) or size exclusion chromatography. EV proteins were precipitated by the TCA-acetone method and PI was quantified by immunoblotting or ELISA for human samples. Results: Cytokine treatment did not consistently change EV concentration or EV PI cargo in INS1, EndoC-βH1, or islet-derived EVs. Tunicamycin, but not thapsigargin treatment of cells yielded an increase in EV PI content, suggesting a potential link to protein trafficking rather than ER stress. EV PI content in human plasma was frequently undetectable, and when detectable, was not significantly different in plasma from children with T1D vs. controls. Conclusion: Global changes in human islet EV PI cargo were not detected under conditions of cytokine stress or recent onset T1D. These findings point away from EV-derived PI as a large-scale or frequent contributor to the development of neoantigens in T1D.