ABSTRACT Background Despite the decades-old knowledge that diabetes mellitus (DM) is a major risk factor for cardiovascular disease (CVD), the reasons for this association are only partially understood. Among the immune cells involved in CVD development, accumulating evidence supports the critical role of T cells as drivers and modifiers of this condition. CD4+ T cells are commonly found in atherosclerotic plaques. The activity and distribution of CD4+ T cell subsets differs between the sexes. Methods Peripheral blood mononuclear cells (PBMCs) of 61 men and women who underwent cardiac catheterization were interrogated by single cell RNA sequencing (scRNA-Seq, ∼200,000 cells) combined with 49 protein markers (CITE-Seq). Coronary artery disease (CAD) was quantified using Gensini scores, with scores above 30 considered CAD+ and below 6 considered CAD-. Four pairs of groups were matched for clinical and demographic parameters. To test how DM changed cell proportions and gene expression, we compared matched groups of diabetic and non-diabetic subjects. We analyzed 41,782 single CD4+ T cell transcriptomes for sex differences in 61 mostly statin-treated coronary artery disease patients with and without DM. Results We identified 16 clusters in CD4 T cells. The proportion of cells in CD4 cluster 8 (CD4T8, CCR2+ Em) was significantly decreased in CAD+, especially among DM+ participants. The proportions of cells in CD4T2, CD4T11, CD4T16 were increased and CD4T13 was decreased in CAD+ among DM+Statin+ participants. CD4T12 was increased in DM+ participants. In female participants, CD4T8, 12, and 13 were decreased compared to in male participants. In CD4 T cells, 31 genes showed significant and coordinated upregulation in both CAD and DM. The DM gene signature was partially additive to the CAD gene signature. Conclusions We conclude that CAD and DM are clearly reflected in PBMC transcriptomes and that significant differences exist between women and men and between subjects treated with statins or not.

Sex differences in coronary artery disease and diabetes revealed by scRNA-Seq and CITE-Seq of human CD4+ T cells