Nonclassical Monocytes in Health and Disease.

Monocytes are innate blood cells that maintain vascular homeostasis and are early responders to pathogens in acute infections. There are three well-characterized classes of monocytes: classical (CD14+CD16- in humans and Ly6Chi in mice), intermediate (CD14+CD16+ in humans and Ly6C+Treml4+ in mice), and nonclassical (CD14-CD16+ in humans and Ly6Clo in mice). Classical monocytes are critical for the initial inflammatory response. Classical monocytes can differentiate into macrophages in tissue and can contribute to chronic disease. Nonclassical monocytes have been widely viewed as anti-inflammatory, as they maintain vascular homeostasis. They are a first line of defense in recognition and clearance of pathogens. However, their roles in chronic disease are less clear. They have been shown to be protective as well as positively associated with disease burden. This review focuses on the state of the monocyte biology field and the functions of monocytes, particularly nonclassical monocytes, in health and disease.

Coexpression of CD71 and CD117 Identifies an Early Unipotent Neutrophil Progenitor Population in Human Bone Marrow.

Neutrophils are the most abundant peripheral immune cells and thus, are continually replenished by bone marrow-derived progenitors. Still, how newly identified neutrophil subsets fit into the bone marrow neutrophil lineage remains unclear. Here, we use mass cytometry to show that two recently defined human neutrophil progenitor populations contain a homogeneous progenitor subset we term "early neutrophil progenitors" (eNePs) (Lin-CD66b+CD117+CD71+). Surface marker- and RNA-expression analyses, together with in vitro colony formation and in vivo adoptive humanized mouse transfers, indicate that eNePs are the earliest human neutrophil progenitors. Furthermore, we identified CD71 as a marker associated with the earliest neutrophil developmental stages. Expression of CD71 marks proliferating neutrophils, which were expanded in the blood of melanoma patients and detectable in blood and tumors from lung cancer patients. In summary, we establish CD117+CD71+ eNeP as the inceptive human neutrophil progenitor and propose a refined model of the neutrophil developmental lineage in bone marrow.

Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal.

The functions of Nr4a1-dependent Ly6C(low) monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid "danger" signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6C(low) monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6C(low) monocyte development, crawling, or retention in Nr4a1(-/-), Itgal(-/-), and Tlr7(host-/-BM+/+) and Cx3cr1(-/-) mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7(host+/+BM-/-) mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6C(low) monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.

Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.

The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.

Deleting an Nr4a1 Super-Enhancer Subdomain Ablates Ly6Clow Monocytes while Preserving Macrophage Gene Function.

Mononuclear phagocytes are a heterogeneous family that occupy all tissues and assume numerous roles to support tissue function and systemic homeostasis. Our ability to dissect the roles of individual subsets is limited by a lack of technologies that ablate gene function within specific mononuclear phagocyte sub-populations. Using Nr4a1-dependent Ly6Clow monocytes, we present a proof-of-principle approach that addresses these limitations. Combining ChIP-seq and molecular approaches we identified a single, conserved, sub-domain within the Nr4a1 enhancer that was essential for Ly6Clow monocyte development. Mice lacking this enhancer lacked Ly6Clow monocytes but retained Nr4a1 gene expression in macrophages during steady state and in response to LPS. Because Nr4a1 regulates inflammatory gene expression and differentiation of Ly6Clow monocytes, decoupling these processes allows Ly6Clow monocytes to be studied independently.

Bone Marrow Transplantation Rescues Monocyte Recruitment Defect and Improves Cystic Fibrosis in Mice.

Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multiorgan inflammation that affects tens of thousands of people worldwide. The causative gene, cystic fibrosis transmembrane conductance regulator (CFTR), is mutated in CF patients. CFTR functions in epithelial cells have traditionally been thought to cause the disease symptoms. Recent work has shown an additional defect: monocytes from CF patients show a deficiency in integrin activation and adhesion. Because monocytes play critical roles in controlling infections, defective monocyte function may contribute to CF progression. In this study, we demonstrate that monocytes from CFTRΔF508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type (WT) bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with WT monocytes, significantly improved survival, and reduced inflammation. WT/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. WT mice reconstituted with CF bone marrow also show lethality, suggesting that the CF defect in monocytes is not only necessary but also sufficient to cause disease. We also show that monocyte-specific knockout of CFTR retards weight gains and exacerbates dextran sulfate sodium-induced colitis. Our findings show that providing WT monocytes by bone marrow transfer rescues mortality in CF mice, suggesting that similar approaches may mitigate disease in CF patients.

Publisher Correction: Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.

In this Letter, affiliation number 1 was originally missing from the HTML; the affiliations were missing for author Ming-Yow Hung in the HTML; and the Fig. 4 legend erroneously referred to panels a-h, instead of a-g. These errors have been corrected online.

Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.

Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Artery Disease.

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.

The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes.

The transcription factors that regulate differentiation into the monocyte subset in bone marrow have not yet been identified. Here we found that the orphan nuclear receptor NR4A1 controlled the differentiation of Ly6C- monocytes. Ly6C- monocytes, which function in a surveillance role in circulation, were absent from Nr4a1-/- mice. Normal numbers of myeloid progenitor cells were present in Nr4a1-/- mice, which indicated that the defect occurred during later stages of monocyte development. The defect was cell intrinsic, as wild-type mice that received bone marrow from Nr4a1-/- mice developed fewer patrolling monocytes than did recipients of wild-type bone marrow. The Ly6C- monocytes remaining in the bone marrow of Nr4a1-/- mice were arrested in S phase of the cell cycle and underwent apoptosis. Thus, NR4A1 functions as a master regulator of the differentiation and survival of 'patrolling' Ly6C- monocytes.

Combined protein and transcript single-cell RNA sequencing in human peripheral blood mononuclear cells.

BACKGROUND: Cryopreserved peripheral blood mononuclear cells (PBMCs) are frequently collected and provide disease- and treatment-relevant data in clinical studies. Here, we developed combined protein (40 antibodies) and transcript single-cell (sc)RNA sequencing (scRNA-seq) in PBMCs. RESULTS: Among 31 participants in the Women's Interagency HIV Study (WIHS), we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs (tool for visualizing high-dimensional data) and Louvain clustering, we identified 50 subsets among CD4+ T, CD8+ T, B, NK cells, and monocytes. This resolution was superior to flow cytometry, mass cytometry, or scRNA-seq without antibodies. Combined protein and transcript scRNA-seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportions. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease. CONCLUSIONS: In conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs.

Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2+ foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, Pf4, which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.

Atherosclerosis Impairs Naive CD4 T-Cell Responses via Disruption of Glycolysis.

Objective: CD4 T cells are important regulators of atherosclerotic progression. The metabolic profile of CD4 T cells controls their signaling and function, but how atherosclerosis affects T-cell metabolism is unknown. Here, we sought to determine the impact of atherosclerosis on CD4 T-cell metabolism and the contribution of such metabolic alterations to atheroprogression. Approach and Results: Using PCR arrays, we profiled the expression of metabolism genes in CD4 T cells from atherosclerotic apolipoprotein-E knockout mice fed a Western diet. These cells exhibited dysregulated expression of genes critically involved in glycolysis and fatty acid degradation, compared with those from animals fed a standard laboratory diet. We examined how T-cell metabolism was changed in either Western diet­fed apolipoprotein-E knockout mice or samples from patients with cardiovascular disease by measuring glucose uptake, activation, and proliferation in CD4 T cells. We found that naive CD4 T cells from Western diet­fed apolipoprotein-E knockout mice failed to uptake glucose and displayed impaired proliferation and activation, compared with CD4 T cells from standard laboratory diet­fed animals. Similarly, we observed that naive CD4 T-cell frequencies were reduced in the circulation of human subjects with high cardiovascular disease compared with low cardiovascular disease. Naive T cells from high cardiovascular disease subjects also showed reduced proliferative capacity. Conclusions: These results highlight the dysfunction that occurs in CD4 T-cell metabolism and immune responses during atherosclerosis. Targeting metabolic pathways within naive CD4 T cells could thus yield novel therapeutic approaches for improving CD4 T-cell responses against atheroprogression.

Patrolling Monocytes Control NK Cell Expression of Activating and Stimulatory Receptors to Curtail Lung Metastases.

The role of nonclassical, patrolling monocytes in lung tumor metastasis and their functional relationships with other immune cells remain poorly defined. Contributing to these gaps in knowledge is a lack of cellular specificity in commonly used approaches for depleting nonclassical monocytes. To circumvent these limitations and study the role of patrolling monocytes in melanoma metastasis to lungs, we generated C57BL/6J mice in which the Nr4a1 superenhancer E2 subdomain is ablated (E2 -/- mice). E2 -/- mice lack nonclassical patrolling monocytes but preserve classical monocyte and macrophage numbers and functions. Interestingly, NK cell recruitment and activation were impaired, and metastatic burden was increased in E2 -/-mice. E2 -/- mice displayed unchanged "educated" (CD11b+CD27+) and "terminally differentiated" (CD11b+CD27-) NK cell frequencies. These perturbations were accompanied by reduced expression of stimulatory receptor Ly49D on educated NK cells and increased expression of inhibitory receptor NKG2A/CD94 on terminally differentiated NK cells. Thus, our work demonstrates that patrolling monocytes play a critical role in preventing lung tumor metastasis via NK cell recruitment and activation.

Sex Differences in Coronary Artery Disease and Diabetes Revealed by scRNA-Seq and CITE-Seq of Human CD4+ T Cells.

Despite the decades-old knowledge that males and people with diabetes mellitus (DM) are at increased risk for coronary artery disease (CAD), the reasons for this association are only partially understood. Among the immune cells involved, recent evidence supports a critical role of T cells as drivers and modifiers of CAD. CD4+ T cells are commonly found in atherosclerotic plaques. We aimed to understand the relationship of CAD with sex and DM by single-cell RNA (scRNA-Seq) and antibody sequencing (CITE-Seq) of CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) of 61 men and women who underwent cardiac catheterization were interrogated by scRNA-Seq combined with 49 surface markers (CITE-Seq). CAD severity 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 sex and DM changed cell proportions and gene expression, we compared matched groups of men and women, as well as diabetic and non-diabetic subjects. We analyzed 41,782 single CD4+ T cell transcriptomes for sex differences in 16 women and 45 men with and without coronary artery disease and with and without DM. We identified 16 clusters in CD4+ T cells. The proportion of cells in CD4+ effector memory cluster 8 (CD4T8, CCR2+ Em) was significantly decreased in CAD+, especially among DM+ participants. This same cluster, CD4T8, was significantly decreased in female participants, along with two other CD4+ T cell clusters. 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. We conclude that (1) CAD and DM are clearly reflected in PBMC transcriptomes, and (2) significant differences exist between women and men and (3) between subjects with DM and non-DM.

Naive CD8+ T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity.

OBJECTIVE: Cardiovascular disease (CVD) remains a significant global health concern with a high degree of mortality. While CD4+ T cells have been extensively studied in CVD, the importance of CD8+ T cells in this disease, despite their abundance and increased activation in human atherosclerotic plaques, remains largely unknown. Thus, the objective of this study was to compare peripheral T-cell signatures between humans with a high (severe) risk of CVD (including myocardial infarction or stroke) and those with a low risk of CVD. Approach and Results: Using mass cytometry, we uncovered a naive CD8+ T (TN) cell population expressing CD95 (termed CD95+CD8+ stem cell memory T [CD8 TSCM] cells) that was enriched in patients with high compared with low CVD. This T-cell subset enrichment within individuals with high CVD was a relative increase and resulted from the loss of CD95lo cells within the TN compartment. We found that CD8 TSCM cells positively correlated with CVD risk in humans, while CD8+ TN cells were inversely correlated. Atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice also displayed respective 7- and 2-fold increases in CD8+ TSCM frequencies within the peripheral blood and aorta-draining paraaortic lymph nodes compared with C57BL/6J mice. CD8+ TSCM cells were 1.7-fold increased in aortas from western diet fed ApoE-/- mice compared with normal laboratory diet-fed ApoE-/- mice. Importantly, transfer of TSCM cells into immune-deficient Rag.Ldlr recipient mice that lacked T cells increased atherosclerosis, illustrating the importance of these cells in atherogenesis. CONCLUSIONS: CD8+ TSCM cells are increased in humans with high CVD. As these TSCM cells promote atherosclerosis, targeting them may attenuate atherosclerotic plaque progression.

Neuropilin-1 Expression on CD4 T Cells Is Atherogenic and Facilitates T Cell Migration to the Aorta in Atherosclerosis.

Neuropilin 1 (Nrp1) is a type I transmembrane protein that plays important roles in axonal guidance, neuronal development, and angiogenesis. Nrp1 also helps migrate thymus-derived regulatory T cells to vascular endothelial growth factor (VEGF)-producing tumors. However, little is known about the role of Nrp1 on CD4 T cells in atherosclerosis. In ApoE-/- mice fed a Western diet for 15 wk, we found a 2-fold increase in Nrp1+Foxp3- CD4 T cells in their spleens, periaortic lymph nodes, and aortas, compared with chow-fed mice. Nrp1+Foxp3- CD4 T cells had higher proliferation potential, expressed higher levels of the memory marker CD44, and produced more IFN-γ when compared with Nrp1- CD4 T cells. Treatment of CD4 T cells with oxLDL increased Nrp1 expression. Furthermore, atherosclerosis-susceptible mice selectively deficient for Nrp1 expression on T cells developed less atherosclerosis than their Nrp1-sufficient counterparts. Mechanistically, we found that CD4 T cells that express Nrp1 have an increased capacity to migrate to the aorta and periaortic lymph nodes compared to Nrp1- T cells, suggesting that the expression of Nrp1 facilitates the recruitment of CD4 T cells into the aorta where they can be pathogenic. Thus, we have identified a novel role of Nrp1 on CD4 T cells in atherosclerosis. These results suggest that manipulation of Nrp1 expression on T cells can affect the outcome of atherosclerosis and lower disease incidence.

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

RATIONALE: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood. OBJECTIVE: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis. METHODS AND RESULTS: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. CONCLUSIONS: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

Human Monocyte Heterogeneity as Revealed by High-Dimensional Mass Cytometry.

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 used 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). 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 atheroprotective role. Conclusions- Our data demonstrate 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.

Human Blood Monocyte Subsets: A New Gating Strategy Defined Using Cell Surface Markers Identified by Mass Cytometry.

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 use cytometry by time-of-flight mass cytometry to profile human monocytes using a panel of 36 cell surface markers. Using the dimensionality reduction approach visual interactive stochastic neighbor embedding (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 ≈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 3 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 use of this new gating scheme using conventional flow cytometry of peripheral blood mononuclear cells from subjects with cardiovascular disease. CONCLUSIONS: Using cytometry by time-of-flight 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.