Background and Aims: Atherosclerosis and its clinical sequelae, myocardial infarction and stroke, represent the main causes of death worldwide. Although preclinical evidence has suggested the existence of a sustained inflammatory and immune response driving disease and complications, the extend of cellular alterations in human atherosclerosis remains enigmatic. Here, we employ single cell RNA-sequencing (scRNA-seq) on peripheral blood mononucleated  cells (PBMCs) in a well-defined cardiovascular risk cohort from the The Ludwigshafen Risk and Cardiovascular Health (LURIC) trial to define changes in the immune cell landscape in atherosclerotic patients.

Methods and Results: Of 3317 patients enrolled in the LURIC trial, we selected individuals with stable coronary-artery disease (CAD) (n=31) and healthy patients (no CAD) (n=16) by propensity score matching, adjusted for CRP, NT-proBNP, Troponin T, LDL-C, and Cystatin-C, in a case-control design. ScRNA-seq was performed by droplet sequencing on PBMCs stored in liquid nitrogen. Individual data sets were integrated and changes between healthy individuals and patients with CAD were evaluated on numeric and transcriptional levels. Single cell transcriptomes were annotated using an established CITE-seq reference atlas. We identified several regulated leukocyte populations that were differentially regulated between both conditions. Interestingly, NK cells (>1.4-fold more), and small cell populations like Tregs (>1.4-fold more), platelets (>10-fold more), stem cells (>3-fold more), and proliferating cells (>2-fold-more) were significantly increased in PBMCs from patients suffering from CAD. On the contrary, distinct B cell and naïve T cell populations were considerably overrepresented in healthy control patients (>1.4- and >1.7-fold more respectively). Pathway analyses of distinct clusters in patients with CAD revealed increased signaling for cellular exhaustion, response to stress, and leukocyte activation. Especially T cell subsets presented an enrichment of cytokine-, hypoxia- and TCR-signaling markers in patients with CAD. In contrast, bulk RNA-sequencing revealed only minor changes between both conditions, suggesting full resolution of transcriptional regulation only at the single cell level.

Conclusion: Employing scRNAseq, we demonstrate that atherosclerosis is associated with a profound change in the circulating immune cell landscape even in the absence of measurable differences in inflammatory biomarkers. These findings propose the usage of scRNAseq for the discovery of outcome-relevant cellular biomarkers in the future.