Aims/hypothesis <p>Type 1 diabetes is an autoimmune disease marked by the destruction of beta cells in pancreatic islets, with an incomplete picture of disease progression and a lack of a definitive cure. A recent finding linked pancreatic ductal cells of type 1 diabetic donors with elevated levels of human leukocyte antigen (HLA) class II molecules; however, the causal relationship and functional significance of this finding remain unknown. Because HLA class II molecules are typically expressed by professional antigen-presenting cells (APCs), this raises the possibility of ductal cells functioning as non-professional APCs. In this study, we test the hypothesis that ductal cells are responsive to type 1 diabetes-associated proinflammatory cytokines, TNF-α, IL-1β and IFN-γ, and can act as non-professional APCs.</p> Methods <p>Pancreatic exocrine cells were obtained from cadaveric donors without diabetes following islet removal. Cells were cryopreserved and thawed into a defined culture medium tailored to support ductal cell survival in a 3D suspension culture system. Ductal cells were exposed to various doses of cytokines for 48 h and analysed for gene and protein expression, using quantitative PCR with reverse transcription, bulk RNA-seq, flow cytometry and western blot analyses. Correlation between cytokine response and APC-related gene expression was evaluated using publicly available single-cell RNA-seq datasets from 86 donors. The functional ability of cytokine-treated ductal cells to present an exogenous autoantigen (glutamic acid decarboxylase 65 kDa isoform [GAD65]) to T cells was tested using a GAD65-specific autoreactive CD4⁺ T cell clone (BRI-4.13) isolated from a type 1 diabetic donor.</p> Results <p>Within 48 h, a combination of TNF-α, IL-1β and IFN-γ stimulated mRNA and protein expression of HLA class II, co-stimulatory and antigen-processing molecules in non-diabetic ductal cells. Bulk RNA-seq analysis showed that cytokines significantly upregulated biological pathways in ‘antigen processing and presentation’ and ‘type 1 diabetes’. Single-cell RNA-seq analysis revealed a positive correlation between cytokine response and APC gene expression in human pancreatic ductal cells. Cytokine-treated ductal cells pulsed with exogenous GAD65 peptide activated and induced proliferation of BRI-4.13 T cells. Unexpectedly, ~0.9% of KRT19⁺ ductal cells expressed GAD protein endogenously, and 26.6% of KRT19⁺GAD⁺ ductal cells expressed the endocrine marker CHGA.</p> Conclusions/interpretation <p>These results demonstrate that non-diseased primary ductal cells respond to TNF-α, IL-1β and IFN-γ by upregulating APC molecules and presenting antigen to autoreactive CD4⁺ T cells. To the best of our knowledge, our results provide the first evidence that non-diabetic human ductal cells can present antigen to T cells, which implicates ductal cells in contributing to type 1 diabetes progression.</p> Graphical Abstract <p></p>