Single-cell RNA-sequencing to interrogate TCR-Repertoire and antigen-specificity in human atherosclerosis

Abstract Background Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of distinct immune cells and lipids in the arterial wall. Despite recent advances in immunophenotyping, the molecular characteristics of T cells and their T cell receptor (TCR) repertoire in patients with atherosclerosis remain elusive. Studies using bulkRNA-sequencing of the TCR in both mice and humans have suggested a significant proportion of T cells in atherosclerotic plaques arises from a small number of antigen-specific T cells that express a limited TCR repertoire. However, it is unknown whether this restriction of TCR usage is caused by CD8+ or CD4+ T cells. Methods Here, we performed paired Single-cell RNA-sequencing (scRNAseq) and TCR-sequencing on atherosclerotic plaques (n=8) and peripheral blood mononuclear cells (PBMCs) from patients with and without coronary artery disease (CAD: n=31, noCAD: n=15). To analyze the paired scRNAseq and TCR sequencing data, we developed a bioinformatic tool that enables the identification of clonally expanded T cells in scRNAseq datasets. This tool allowed to investigate the T cell response in human atherosclerosis at a single-cell level and link clonal expansion with specific gene expression. Results Our analysis revealed that CD8+, rather than CD4+ T cells, exhibit an oligoclonal phenotype in the atherosclerotic plaques. By applying bioinformatic pseudotime analysis, we were able to recapitulate the developmental relationships within the T cell compartment. Our results demonstrate that clonally expanded CD8+ T cells show a TEMRA phenotype (Effector Memory–Expressing CD45RA) and originate from naive T cells within the plaque microenvironment. Pathway analyses of enriched CD8+ Plaque TEMRAs revealed increased signalling for cellular activation and migration, response to stress, and inflammation. In addition, clonally expanded CD8+ cells were also detected in PBMCs from patients with CAD and appear to be overrepresented in comparison to patients without CAD. Conclusion Our study provides new insights into the T cell response in human atherosclerosis. We demonstrate that CD8+ T cells show an oligoclonal phenotype in the atherosclerotic plaque and are also present in PBMCs from patients suffering from CAD. Furthermore, these cells appear to be dysregulated, suggesting a possible mechanism by which they contribute to disease progression. These findings highlight the power of paired scRNAseq and TCR sequencing whic