SUMMARY - Subclinical atherosclerotic cardiovascular disease (ASCVD) occurs in more than half of adults over 50 and progresses in many to clinical ASCVD events such as myocardial infarction (MI), stroke, and limb ischemia. A key contributor to ASCVD progression is excess inflammation, which persists in 1/3rd of people on otherwise optimal therapy for ASCVD and confers twice the risk for recurrent ASCVD and death. Yet, although therapies bluntly targeting inflammation can modestly reduce ASCVD events, off-target effects resulting from broad and sustained systemic inflammation reduction, such as infection, limit use. These results highlight the therapeutic potential of inflammation modulation in ASCVD, but also the need for more precise, nuanced immunologic targets. Experimental findings from our group and others revealed dynamic ways in which immune cells polarize to favor inflammatory versus inflammation-resolving functions, including via metabolic and epigenetic rewiring to alter gene expression. Yet, these have largely been limited to experimental models and small cross-sectional human studies utilizing convenience samples. Meanwhile, studies by our group and others in large population-based cohorts indicated associations of broad immune cell phenotypes in the blood with ASCVD, but lacked granular data on cell-level functional heterogeneity within these broad cell subsets. Thus, there is a critical gap in human data, particularly longitudinal data, on relationships between immune cell functional polarization and ASCVD; this limits precise insights into immune cell-level features that could be targeted to modulate inflammation and reduce ASCVD. We propose a translational epidemiologic study investigating single-cell transcriptomic and gene-regulatory profiles of immune cells in human peripheral blood mononuclear cells (PBMCs) and plaque associated with coronary, carotid, and lower extremity manifestations of ASCVD. Based on our pilot and prior data, we hypothesize that inflammatory monocyte polarization, as well as linked epigenetic regulatory elements and glycolytic metabolic preference: (1) precede clinical ASCVD onset and (2) associate with high-risk plaque morphology and clinical presentation. We investigate this in Aim 1 with single-cell sequencing and functional metabolic analyses of PBMCs viably preserved from Multi-Ethnic Study of Atherosclerosis (MESA) participants with subclinical ASCVD at baseline who progressed to (cases) versus remained free from (controls) clinical ASCVD over follow-up. In Aim 2, we analyze immune cells isolated from the blood and plaque of patients with prevalent clinical ASCVD undergoing carotid or femoral endarterectomy; we will determine single-cell gene expression, epigenetic regulation, and metabolic polarization associated with high-risk plaque morphology and clinical presentation, as well as whether these features in plaque are imprinted in blood. Our pilot data in human PBMCs and plaque inform our approach...