2393-P: Stage Specific–Macrophage States and Lineage Trajectories in Type 1 Diabetes Revealed by Single-Cell RNA Sequencing

Introduction and Objective: Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-producing pancreatic β-cells by immune cells, yet the cellular programs driving disease progression remain incompletely defined. To understand macrophage heterogeneity and their role during T1D, pancreatic macrophages were analyzed. Methods: Single-cell RNA sequencing of sorted pancreatic macrophages from T1D-prone NOD.hCD68-GFP mice during spontaneous T1D were performed. Macrophages were analyzed across various T1D stages including 6-, 9-, 21-weeks of age and diabetic. Results: Based on established macrophage markers, Il1b+, Csf1r+, Ccr9+, Ptpn22+ and H2-DMb2+ clusters were designated as pro-inflammatory, whereas clusters expressing Tgfb2+ and Wnt4+ were designated as anti-inflammatory macrophages. A distinct proinflammatory Ccr9+ macrophage emerged at 21 weeks and persisted into the diabetic stage. In overtly diabetic mice, we identified Ptpn22+ macrophage subset, suggesting a disease-associated inflammatory state. Chemokine signaling was dynamic with increased expression of immune cell recruiting chemokines Ccl4 and Ccl5 at diabetes onset and elevated Ccl8 expression during overt disease. Reg1, a gene associated with β-cell stress and regeneration, exhibited significantly higher expression during the diabetic stage compared to earlier time points. Increased Reg1 expression during diabetes was confirmed by flow cytometry, confirming its disease-stage enrichment. Stemness analyses revealed that pro-inflammatory macrophages arise from a Csf1r+macrophage progenitor-like population, defining a developmental continuum toward inflammatory states during T1D progression. Conclusion: Together, these findings uncover disease stage-specific macrophage states that likely contribute to immune-mediated β-cell destruction in T1D. Disclosure H. Shinde: None. K.H. Burnette: None. H. Tse: None. Funding American Diabetes Association (1-25-PDF-107), NIH (DK126456, DK127497, DK131716, DK138469NSF: DMR2208831), Breakthrough T1D (2-SRA-2024-1509-S-B)