ABSTRACT: Introduction/Objective: Type 1 diabetes (T1D) is an autoimmune disease characterized by the loss of insulin-producing beta cells and has no cure. The role of cell-cell interactions in immune recognition of beta cells remains poorly understood. Beta cells develop adjacent to ductal cells, which undergo changes during T1D progression, suggesting crosstalk between the two cell types. Extracellular vesicles (EVs) mediate intercellular communication through their cargo, but whether human ductal cells secrete EVs that modulate beta cells is unknown. We hypothesize that exposure of human ductal cells to T1D-associated cytokines alters their EV cargo, with consequence in beta cells. Methods: Human exocrine tissues from non-diabetic cadaveric donors were cultured in a 3D suspension to support ductal cell survival. Cells were treated with TNF-α, IL-1β, and IFN-γ or vehicle control for 48 hours and analyzed for proinflammatory-related gene expression and viability by qRT-PCR and Trypan blue exclusion, respectively. EVs from cytokine- and vehicle-treated ductal cells were isolated using size exclusion chromatography and characterized by dot blot, transmission electron microscopy, and nanoparticle tracking. The effects of EVs on EndoC-βH1 human beta cells were assessed with qRT-PCR, RNA-sequencing and flow cytometry, and EV cargo was analyzed by proteomics and transcriptomics. Results: EVs from cytokine-treated ductal cells were internalized by EndoC-βH1 beta cells and, compared to control ductal EVs, increased HLA Class I gene and protein expression in EndoC-βH1 cells, as shown by qRT-PCR, bulk RNA sequencing, and flow cytometry. EV cargo from cytokine-treated versus control ductal cells, was enriched for transcripts associated with inflammatory and antigen-presentation-related pathways, including CXCL9, CXCL10, CXCL11, IDO1, and GBP4. Conclusion: Primary human ductal cells secrete EVs that, under T1D-associated cytokine exposure, alter their cargo and upregulate HLA Class I gene and protein expression in human EndoC-βH1 beta cells. Because HLA Class I is critical for beta cell recognition by autoimmune cells, our results reveal a previously unknown ductal cell communication pathway with implication for modulating beta cells during T1D progression.
