Abstract 004: Comprehensive Assessment of Immune Cells in Mouse and Human Atherosclerosis by Single-cell RNA-sequencing and Mass Cytometry

Atherosclerosis, an inflammatory disease of large arteries, is - through its clinical manifestations stroke and myocardial infarction - globally the leading cause of morbidity and mortality. The interplay of pro- and anti-inflammatory leukocytes in the aorta modulates and drives atherosclerosis. Although cells of the innate and adaptive immune system are found in atherosclerotic plaques, their phenotypic and functional diversity is poorly understood. Here, we applied single cell RNA-sequencing (scRNAseq) and mass cytometry (CyTOF) to assess leukocyte diversity in depth, thus defining an immune cell atlas in atherosclerosis. Single cell transcriptional profiling of aortic leukocytes from 20-week old chow (CD) and western diet (WD) fed Apoe -/- and Ldlr -/- mice revealed 11 phenotypically different leukocyte clusters. Atherosclerotic aortas exhibited enhanced leukocyte diversity, whilst WD further changed the abundance of leukocyte subpopulations. Gene set enrichment analysis of single cells established that multiple pathways, e.g. for lipid metabolism, proliferation, and cytokine secretion, pertained to particular leukocyte clusters. Applying a novel 35-marker CyTOF panel with metal-labelled antibodies confirmed the phenotypic diversity of aortic leukocytes. Among lymphocytes, we detected three principal B-cell subsets defined by scRNAseq, CyTOF, and flow cytometry. These B cell subsets harbor distinct surface marker expression, functional gene pathways, and ex vivo cytokine production. Finally, we used leukocyte cluster gene signatures to assess leukocyte frequencies in 121 human plaques by a transcriptomic deconvolution strategy. This approach revealed a similar immune cell complexity in human carotid plaques with a higher percentage of monocytes and macrophages. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunological mechanisms and cell-type specific pathways, and may result in novel diagnostic risk stratification tools.