80-OR: STAT-Dependent β-Cell Extracellular Vesicle (EV) Programmed Death-Ligand 1 (PD-L1) Shuttling Abrogates CD4+ T Cell Activation in Type 1 Diabetes (T1D)

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Introduction and Objective: EVs are membrane-bound nanoparticles that contribute to cell:cell communication. β cell EV PD-L1 protein is increased by interferon (IFN) exposure and is able to bind and inactivate CD8+ T cells, suggesting a role in the interplay between β cell destruction and survival in T1D. However, mechanisms underlying β cell PD-L1 incorporation into EVs and its physiological effects are not completely understood.Methods: NIT-1 β cells were transfected with siRNA for STAT1+2 (vs. scramble control), then treated with 24h 2000 U/mL IFN-α (vs. vehicle). Murine NIT-1 β cells were transfected with PD-L1 to make PD-L1 overexpressing (PD-L1 OE) EVs. Anti-CD3 and CD28-activated nonobese diabetic mouse (NOD) splenocytes were treated with PD-L1 OE EVs. Flow cytometry-based assays tested functional impacts on CD4+ T cells. To test translational relevance, EV PD-L1 was quantified in pancreas slice perifusate from human organ donors with (n=11) or without (n=12) T1D using the ExoView-R200 imaging platform.Results: IFN-α treatment doubled β cell EV PD-L1 cargo without increasing EV numbers; this increase was abrogated by genetic STAT1/STAT2 inhibition. Compared to wild-type EVs, PD-L1 OE EVs suppressed CD4+ T cell proliferation (0.4-fold reduction in CTV dye dilution) and decreased CD69 and CD25 activation marker expression. Anti-PD-L1 antibody pre-treatment reversed these effects, confirming a specific role for EV PD-L1 in CD4+ T cell suppression. Pancreas slice EVs exhibited significant heterogeneity in EV PD-L1 content, but overall EV PD-L1 was increased in donors with T1D vs. nondiabetic controls.Conclusion: β cell EV PD-L1 is increased in pancreas slice perifusate from donors with T1D. STAT pathway activation is required for IFN-induced β cell EV PD-L1 shuttling. Increased β cell EV PD-L1 modulates CD8+ and CD4+ T cells, suggesting a key role in the β cell:immune system dialogue surrounding β cell destruction in T1D.

Disclosure

I. Amalaraj: None. C. Rao: None. S. Roy: None. J. Piganelli: None. E.K. Sims: Consultant; Sanofi. Speaker’s Bureau; Med Learning Group. Other Relationship; American Diabetes Association.



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