Objective: Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in composition. Approach and Results: We utilized the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). In order to study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16+ nonclassical monocyte population and 4 subsets belong to the CD14+ classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan+CXCR6+ nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an athero-protective role. Conclusions: Our data demonstrates that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.

Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here we show that during atherogenesis Treg cells lose Foxp3 expression and their immunosuppressive function, leading to the conversion of a fraction of these cells into T follicular helper (Tfh) cells. We show that Tfh cells are pro-atherogenic and that their depletion reduces atherosclerosis. Mechanistically, the conversion of Treg cells to Tfh cells correlates with reduced expression of IL-2Rα and pSTAT5 levels and increased expression of IL-6Rα. In vitro, incubation of naive T cells with oxLDL prevents their differentiation into Treg cells. Furthermore, injection of lipid-free Apolipoprotein AI (ApoAI) into ApoE−/− mice reduces intracellular cholesterol levels in Treg cells and prevents their conversion into Tfh cells. Together our results suggest that ApoAI, the main protein in high-density lipoprotein particles, modulates the cellular fate of Treg cells and thus influences the immune response during atherosclerosis.

Rationale B cells contribute to atherosclerosis through subset specific mechanisms. Whereas some controversy exists about the role of B-2 cells, B-1a cells are atheroprotective due to secretion of atheroprotective IgM antibodies independent of antigen. B-1b cells, a unique subset of B-1 cells that respond specifically to T cell-independent antigens, have not been studied within the context of atherosclerosis. Objective To determine whether B-1b cells produce atheroprotective IgM antibodies and function to protect against diet induced atherosclerosis. Methods and Results We demonstrate that B-1b cells are sufficient to produce IgM antibodies against oxidation specific epitopes (OSE) on LDL both in vitro and in vivo. Additionally, we demonstrate that B-1b cells provide atheroprotection after adoptive transfer into B and T cell deficient (Rag1−/−Apoe−/−) hosts. We implicate Id3 in the regulation of B-1b cells as B cell-specific Id3 knockout mice (Id3BKOApoe−/−) have increased numbers of B-1b cells systemically, increased titers of OSE-reactive IgM antibodies, and significantly reduced diet-induced atherosclerosis compared to Id3WTApoe−/− controls. Finally, we report that the presence of a homozygous SNP in ID3 in humans that attenuates Id3 function is associated with an increased percentage of circulating B-1 cells and anti-MDA-LDL IgM suggesting clinical relevance. Conclusions These results provide novel evidence that B-1b cells produce atheroprotective OSE-reactive IgM antibodies and protect against atherosclerosis in mice, and suggest that similar mechanisms may occur in humans.

Objective Human monocyte subsets are defined as classical (CD14++CD16−), intermediate (CD14++CD16+), and nonclassical (CD14+CD16+). Alterations in monocyte subset frequencies are associated with clinical outcomes, including cardiovascular disease, in which circulating intermediate monocytes independently predict cardiovascular events. However, delineating mechanisms of monocyte function is hampered by inconsistent results among studies. Approach and Results We utilize CyTOF mass cytometry to profile human monocytes using a panel of 36 cell surface markers. Using the dimensionality reduction approach viSNE, we define monocytes by incorporating all cell surface markers simultaneously. Using viSNE, we find that although classical monocytes are defined with high purity using CD14 and CD16, intermediate and nonclassical monocytes defined using CD14 and CD16 alone are frequently contaminated, with average intermediate and nonclassical monocyte purity of approximately 86.0% and 87.2% respectively. To improve the monocyte purity, we devised a new gating scheme that takes advantage of the shared coexpression of cell surface markers on each subset. In addition to CD14 and CD16, CCR2, CD36, HLA-DR and CD11c are the most informative markers that discriminate among the three monocyte populations. Using these additional markers as filters, our revised gating scheme increases the purity of both intermediate and nonclassical monocyte subsets to 98.8% and 99.1% respectively. We demonstrate the utility of this new gating scheme using conventional flow cytometry of PBMCs from subjects with cardiovascular disease. Conclusions Using CyTOF mass cytometry we have identified a small panel of surface markers that can significantly improve monocyte subset identification and purity in flow cytometry. Such a revised gating scheme will be useful for clinical studies of monocyte function in human cardiovascular disease.