Single cell mapping identifies expansion of Cytotoxic CD4 T cells in the atherosclerotic plaque of subjects with Type 2 Diabetes

Abstract Patients with Type 2 diabetes (T2D) are at greater risk of atherosclerotic cardiovascular disease (ASCVD) and fail to fully respond to standard lipid lowering drugs. While the role of macrophages in T2D plaque pathology has been investigated, no studies have systematically investigated the heterogeneity of CD4 T cells in human plaques and how they respond to cholesterol reduction. To identify the specific T cell alterations in plaques from patients with T2D, we performed scRNAseq of human carotid plaques from 22 subjects (54.5% T2D) were being treated with cholesterol lowering drugs (average LDL cholesterol of 1.2 mmol/L) undergoing carotid endarterectomy. Data were processed and integrated using Seurat and Slingshot algorithms. We identified a total of 24 immune cell clusters by unbiased clustering of CD45+ cells, including CD4 T cells that presented similar frequencies between T2D (~11%) and non-T2D patients (~10%). CD4 T cells comprised of 13 sub clusters: naïve, central memory (CM), effector memory (EM), T regulatory (Tregs), cytotoxic (CTL), and gamma delta (γδ) T cells. One cluster of CD4 CTLs (cluster 5) expressing TRDC and TRGC2 surface markers along with cytotoxicity markers GZMB, GNLY, and PRF1 was significantly expanded in T2D plaques (p=0.032). This CD4CTL expressed high levels of transcription factors (TFs) involved in T cell differentiation such as RUNX3 and ZEB2 and contained sets of genes involved in cytotoxicity, chemotaxis and IL-1 signaling. Trajectory analysis using Slingshot inferred that CD4 CTLs clusters in the plaques differentiate from CD4 EM T cells. Plaques from patients with T2D are highly enriched in CTL/γδ T cells and suggest that their cytotoxic potential contributes to the increased cardiovascular risk. This Work is supported by AHA SFRN 20SFRN35210252 and the AHA-SFRN Training Program.