Monocytes are associated with human cardiovascular disease progression. Monocytes are segregated into three major subsets: classical (cMo), intermediate (iMo), and nonclassical (nMo). Recent studies have identified heterogeneity within each of these main monocyte classes, yet the extent to which these subsets contribute to heart disease progression is not known. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects within the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using the Gensini Score (GS). We employed high-dimensional single-cell transcriptome and protein methods to define how human monocytes differ in subjects with low to severe coronary artery disease. We analyzed 487 immune-related genes and 49 surface proteins at the single-cell level using Antibody-Seq (Ab-Seq). We identified six subsets of myeloid cells (cMo, iMo, nMo, plasmacytoid DC, classical DC, and DC3) at the single-cell level based on surface proteins, and we associated these subsets with coronary artery disease (CAD) incidence based on Gensini score (GS) in each subject. Only frequencies of iMo were associated with high CAD (GS > 32), adj.p = 0.024. Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r = 0.314, p = 0.014) and further showed a robust sex-dependent positive correlation in female subjects (r = 0.663, p = 0.004). cMo frequencies did not correlate with CAD severity. Key gene pathways differed in iMo among low and high CAD subjects and between males and females. Further single-cell analysis of iMo revealed three iMo subsets in human PBMC, distinguished by the expression of HLA-DR, CXCR3, and CD206. We found that the frequency of immunoregulatory iMo_HLA-DR⁺CXCR3⁺CD206⁺ was associated with CAD severity (adj.p = 0.006). The immunoregulatory iMo subset positively correlated with GS in both females (r = 0.660, p = 0.004) and males (r = 0.315, p = 0.037). Cell interaction analyses identified strong interactions of iMo with CD4⁺ effector/memory T cells and Tregs from the same subjects. This study shows the importance of iMo in CAD progression and suggests that iMo may have important functional roles in modulating CAD risk, particularly among females.

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