Abstract 101: Single-cell Profiling of Atherosclerotic Tissue Identifies T Cell Subsets Associated with Cerebrovascular Events

Atherogenesis is driven by the infiltration of immune cells into the arterial wall where inflammation occurs and promotes plaque growth. While some plaques are stable, others are vulnerable and may rupture and lead to stroke or myocardial infarction. The contribution of specific immune cell types to plaque stability is obscure. In our present study, we aim to characterize the immune cell repertoire of human atherosclerosis using innovative single cell methods. We used mass cytometry (CyTOF) to study plaque tissue and paired blood from symptomatic (TIA or stoke < 6 months) or asymptomatic (no TIA or stroke) patients undergoing carotid endarterectomy. Using unbiased MetaLouvain clustering analysis, we characterized cell populations from two independent cohorts to (1) define major immune populations (cohort1; n=15), and (2) detail the adaptive immune compartment (cohort2; n=23), which was predominant in cohort 1. Moreover, we used single cell RNAseq to obtain the transcriptional signatures of immune infiltrates of atherosclerotic tissue. We found that T cells dominate advanced plaques. Atherosclerotic tissue was enriched in CD8 ⁺ Effector Memory (EM) T cells that were CD69 ^hi , CCR5 ^hi and PD-1 ^hi vs. their blood counterparts. Our transcriptional profiling confirmed that T cells were the majority of all immune populations, and that T cells expressed genes that represented an activated and largely exhausted phenotype. Our CyTOF results identified differences in T cell subsets between patient types. Symptomatic patients had a higher frequency of a subset of CD4 ⁺ EM T cells (CD69 ^hi CCR5 ^hi PD1 ^hi CD27 ^lo CD28 ^lo ), and displayed a more differentiated phenotype of CD27 ^lo GZMB ^lo CD38 ^lo CD8 ⁺ EM T cells. Our study is the first to provide a single cell immune atlas of advanced atherosclerotic lesions and to identify specific subsets associated with clinical outcomes of atherosclerotic disease. This study will be valuable for the future design of precise tissue-targeted immunotherapies.