W130 - Islet Autoreactive Regulatory and Conventional T Cells Have Distinct TCR Repertoires and Differ Transcriptionally from Foreign Antigen-reactive T Cells

Abstract Text: Regulatory T cell (Treg) dysfunction has been previously implicated in the pathogenesis of type 1 diabetes (T1D). Despite growing knowledge of the conventional T cells (Tconv) that target the islet, little is known about islet autoreactive (IAR) Tregs. We established an AIM assay to isolate antigen-reactive Tregs and Tconv cells from three healthy individuals and three individuals with established T1D and analyzed them using multimodal 10X sequencing to determine whether IAR Tregs had unique features compared to those specific for foreign antigens. Antigen-reactive cells were isolated by stimulating PBMC with islet or CEFX microbial peptide pools, CD3/CD28 antibodies, or vehicle overnight. Each sample was then barcoded, combined, and enriched for CD154+ and CD137+ activated cells. Enriched cells were stained with CITE-seq and flow antibodies, sorted purified, and processed for 10X sequencing. After barcode demultiplexing, antigen-reactive cells were defined as CD154-CD137+CD25+CD127low for Tregs and CD154+CD69+ for Tconv cells using CITE-seq antibody expression. IAR Tregs were transcriptionally distinct from foreign antigen-reactive Tregs, with enrichment of cells expressing metabolism genes and fewer classic HLA-DR+CD39+ Tregs than foreign antigen-reactive Tregs. IAR Tconv cells differed from foreign antigen-reactive Tconvs with enrichment of cells with effector memory gene expression compared with cells enriched in interferon response gene expression in foreign antigen-reactive Tconv cells. Furthermore, IAR Treg and IAR Tconv had greater clonal expansion compared to foreign antigen-reactive cells. Notably, the TCR repertoires of Tregs and Tconv cells were distinct. This study identifies unique differences in IAR Tregs that may be important in T1D development.