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Background: CD (cluster of differentiation) 4 + T-cell responses to APOB (apolipoprotein B) are well characterized in atherosclerotic mice and detectable in humans. CD4 + T cells recognize antigenic peptides displayed on highly polymorphic HLA (human leukocyte antigen)-II. Immunogenicity of individual APOB peptides is largely unknown in humans. Only 1 HLA-II-restricted epitope was validated using the DRB1*07:01-APOB 3036 –3050 tetramer. We hypothesized that human APOB may contain discrete immunodominant CD4 + T-cell epitopes that trigger atherosclerosis-related autoimmune responses in donors with diverse HLA alleles. Methods: We selected 20 APOB-derived peptides (APOB 20 ) from an in silico screen and experimentally validated binding to the most commonly occurring human HLA-II alleles. We optimized a restimulation-based workflow to evaluate antigenicity of multiple candidate peptides in HLA-typed donors. This included activation-induced marker assay, intracellular cytokine staining, IFNγ (interferon gamma) enzyme–linked immunospot and cytometric bead array. High-throughput sequencing revealed TCR (T-cell receptor) clonalities of APOB-reactive CD4 + T cells. Results: Using stringent positive, negative, and crossover stimulation controls, we confirmed specificity of expansion-based protocols to detect CD4 + T cytokine responses to the APOB 20 pool. Ex vivo assessment of AIM + CD4 + T cells revealed a statistically significant autoimmune response to APOB 20 but not to a ubiquitously expressed negative control protein, actin. Resolution of CD4 + T responses to the level of individual peptides using IFNγ enzyme–linked immunospot led to the discovery of 6 immunodominant epitopes (APOB 6 ) that triggered robust CD4 + T activation in most donors. APOB 6 -specific responding CD4 + T cells were enriched in unique expanded TCR clonotypes and preferentially expressed memory markers. Cytometric bead array analysis detected APOB 6 -induced secretion of both proinflammatory and regulatory cytokines. In clinical samples from patients with angiographically verified coronary artery disease, APOB 6 stimulation induced higher activation and memory phenotypes and augmented secretion of proinflammatory cytokines TNF (tumor necrosis factor) and IFNγ, compared with patients with low coronary artery disease. Conclusions: Using 3 cohorts, each with ≈20 donors, we discovered and validated 6 immunodominant, HLA-II–restricted APOB epitopes. The immune response to these APOB epitopes correlated with coronary artery disease severity.

Background: B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgM OSE ) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers, leading to higher IgM OSE production and reduction in atherosclerotic plaque formation. Yet, the mechanism underlying this regulation remains unexplored. Methods: Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3 KO and Id3 WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3 -dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. Results: Through RNA sequencing, P62 was found to be enriched in Id3 KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-κB (nuclear factor kappa B), leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgM OSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings, P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgM OSE levels. Conclusions: This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgM OSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover, analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects, suggesting P62 as a new immunomodulatory target for treating atherosclerosis.

Objective:Neovascularization can maintain and even improve tissue perfusion in the setting of limb ischemia during peripheral artery disease. The molecular and cellular mechanisms mediating this process are incompletely understood. We investigate the potential role(s) for Id3 (inhibitor of differentiation 3) in regulating blood flow in a murine model of hindlimb ischemia (HLI).Approach and Results:HLI was modeled through femoral artery ligation and resection and blood flow recovery was quantified by laser Doppler perfusion imaging. Mice with global Id3 deletion had significantly impaired perfusion recovery at 14 and 21 days of HLI. Endothelial- or myeloid cell-specific deletion of Id3 revealed no effect on perfusion recovery while B-cell–specific knockout of Id3 (Id3BKO) revealed a significant attenuation of perfusion recovery. Flow cytometry revealed no differences in ischemia-induced T cells or myeloid cell numbers at 7 days of HLI, yet there was a significant increase in B-1b cells in Id3BKO. Consistent with these findings, ELISA (enzyme-linked immunoassay) demonstrated increases in skeletal muscle and plasma IgM. In vitro experiments demonstrated reduced proliferation and increased cell death when endothelial cells were treated with conditioned media from IgM-producing B-1b cells and tibialis anterior muscles in Id3BKOmice showed reduced density of total CD31+and αSMA+CD31+vessels.Conclusions:This study is the first to demonstrate a role for B-cell–specific Id3 in maintaining blood flow recovery during HLI. Results suggest a role for Id3 in promoting blood flow during HLI and limiting IgM-expressing B-1b cell expansion. These findings present new mechanisms to investigate in peripheral artery disease pathogenesis.

B cell–activating factor (BAFF), part of a tumor necrosis factor family of cytokines, was recently identified as a regulator of atherosclerosis; however, its role in aortic aneurysm has not been determined. Here, the study examined the effect of selective BAFF antagonism using an anti-BAFF antibody (blocks binding of BAFF to receptors BAFF receptor 3, transmembrane activator and CAML interactor, and B-cell maturation antigen) and mBaffR-mFc (blocks binding of BAFF to BAFF receptor 3) on a murine model of abdominal aortic aneurysm (AAA). In a prevention strategy, the antagonists were injected before the induction of AAA, and in an intervention strategy, the antagonists were injected after the induction of AAA. Both strategies attenuated the formation of AAA. In the intervention group, BAFF antagonism depleted most of the mature B-cell subsets in spleen and circulation, leading to enhanced resolution of inflammation in AAA as indicated by decreased infiltration of B cells and proinflammatory macrophages and a reduced number of apoptotic cells. In AAA tissues, B cells and macrophages were found in close contact. In vitro, B cells, irrespective of treatment with BAFF, impaired the efferocytosis activity of macrophages, suggesting a direct innate role of B cells on macrophage function. Altogether, BAFF antagonism affects survival of the mature B cells, promotes resolution of inflammation in the aorta, and attenuates the growth of AAA in mice.

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 diet–fed 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 diet–fed apolipoprotein-E knockout mice failed to uptake glucose and displayed impaired proliferation and activation, compared with CD4 T cells from standard laboratory diet–fed 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.

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.

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.

Immunoglobulin M (IgM) to oxidation specific epitopes (OSE) are inversely associated with atherosclerosis in mice and humans. The B-1b subtype of B-1 cells secrete IgM to OSE, and unlike B-1a cells, are capable of long-lasting IgM memory. What attributes make B-1b cells different than B-1a cells is unknown. Our objectives were to determine how B-1b cells produce more IgM compared to B-1a cells at homeostatic condition and to see the differences in the B-1a and B-1b cell distribution and IgM CDR-H3 sequences in mice with advanced atherosclerosis. Here,in-vivostudies demonstrated greater migration to spleen, splenic production of IgM and plasma IgM levels inApoE-/-Rag1-/-mice intraperitoneally injected with equal numbers of B-1b compared to B-1a cells. Bulk RNA seq analysis and flow cytometry of B-1a and B-1b cells identified CCR6 as a chemokine receptor more highly expressed on B-1b cells compared to B-1a. Knockout of CCR6 resulted in reduced B-1b cell migration to the spleen. Moreover, B-1b cell numbers were significantly higher in spleen of aged atheroscleroticApoE-/-mice compared to youngApoE-/-mice. Single cell sequencing results of IgHM in B-1a and B-1b cells from peritoneal cavity and spleen of atherosclerotic agedApoE-/-mice revealed significantly more N additions at the V-D and D-J junctions, greater diversity in V region usage and CDR-H3 sequences in B-1b compared to B-1a cells. In summary, B-1b cells demonstrated enhanced CCR6-mediated splenic migration, IgM production, and IgM repertoire diversification compared to B-1a cells. These findings suggest that potential strategies to selectively augment B-1b cell numbers and splenic trafficking could lead to increased and more diverse IgM targeting OSE to limit atherosclerosis.

Background: Detailed understanding of the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV)-2, the cause of coronavirus disease 2019 (CO­VID-19) has been hampered by a lack of quantitative antibody assays. Objective: The objective was to develop a quantitative assay for IgG to SARS-CoV-2 proteins that could be implemented in clinical and research laboratories. Methods: The biotin-streptavidin technique was used to conjugate SARS-CoV-2 spike receptor-binding domain (RBD) or nucleocapsid protein to the solid phase of the ImmunoCAP. Plasma and serum samples from patients hospitalized with COVID-19 (n = 60) and samples from donors banked before the emergence of COVID-19 (n = 109) were used in the assay. SARS-CoV-2 IgG levels were followed longitudinally in a subset of samples and were related to total IgG and IgG to reference antigens using an ImmunoCAP 250 platform. Results: At a cutoff of 2.5 μg/mL, the assay demonstrated sensitivity and specificity exceeding 95% for IgG to both SARS-CoV-2 proteins. Among 36 patients evaluated in a post-hospital follow-up clinic, median levels of IgG to spike-RBD and nucleocapsid were 34.7 μg/mL (IQR 18–52) and 24.5 μg/mL (IQR 9–59), respectively. Among 17 patients with longitudinal samples, there was a wide variation in the magnitude of IgG responses, but generally the response to spike-RBD and to nucleocapsid occurred in parallel, with peak levels approaching 100 μg/mL, or 1% of total IgG. Conclusions: We have described a quantitative assay to measure IgG to SARS-CoV-2 that could be used in clinical and research laboratories and implemented at scale. The assay can easily be adapted to measure IgG to mutated COVID-19 proteins, has good performance characteristics, and has a readout in standardized units.

BackgroundDespite 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.MethodsPeripheral 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.ResultsWe 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.ConclusionsWe 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.

Background: Atherosclerosis development in the aorta is region specific with the aortic arch region more prone to disease development than descending thoracic and abdominal aorta. Perivascular adipose tissue (PVAT) directly associates with the aorta, harbors high numbers of B cells, and regulates atherosclerosis development. Age-Associated B cells (ABCs: CD19 + CD11c + ) have recently been reported as a new subset of B cells with unique cell surface and transcriptional signatures. ABCs have been reported to be elevated in autoimmune diseases, where in, these cells have characteristics of auto-antibody producing memory B cells. However, the impact of ABCs in the PVAT near aortic arch and with atherosclerosis progression during aging has not be studied. Methods and Results: To study ABCs in atherosclerosis setting, we used 50- and 100- week old chow diet fed, ApoE KO mice. Flow cytometric analysis showed that there was no difference in frequency of total B cells in the PVAT near the aortic arch region between 50- and 100- week old mice. However, the frequency of ABCs was significantly higher in aortic arch PVAT in 100-week-old mice compared to 50-week-old mice. Additionally, these ABCs in aortic arch PVAT are CD44 + (activation marker) and most ABCs were actively proliferating cells (Ki67 + ), as compared to other CD11c - B cells in 100-week-old ApoE KO mice. Lesion area was measured following Sudan-IV enface staining of aorta and 100-week-old mice developed significantly more disease than the 50-week-old mice. Interestingly, we observed a significant correlation of atherosclerosis disease in aortic arch region with the frequency of ABCs in PVAT near aortic arch (p=0.001, R 2 =0.69). Conclusion: Results provide the first evidence that ABCs in the aortic arch PVAT are highly active and the percentage of these ABCs strongly associates with advanced atherosclerosis disease in aged animals.

Recent work by our group and others have shown individuals with IgE to the oligosaccharide allergen present in mammalian products (α-gal) have increased atheroma burden with increased necrosis and calcification compared to those without α -gal specific IgE. These data suggest α -gal specific IgE increase plaque severity and vulnerability, yet the mechanisms that promote production of IgE to α -gal are unknown. Previous work from our group utilized multi-omics single cell analysis of circulating PBMCs from subjects with coronary angiography at UVA and showed subjects with IgE α -gal sensitization had a higher frequency of CCR6hi switched memory (SWM) B cells and that the CCR6 ligand, CCL20, increased class switching to IgE. To determine mechanisms whereby CCR6 may mediated class switching to IgE, we enriched total B cells from healthy donor peripheral blood mononuclear cells and stimulated with 20ng/ml human IL-4 and 10μg/ml agnostic anti-human CD40 with or without 20ng/ml CCL20 for 3 days. After 3 days of treatment, cells were collected and stained with a 15-color panel and analyzed via flow cytometry. Cells were analyzed for surface expression of CD20 and CD3 to gate total B cells. Gated B cells were further separated into CD27+/IgM-/IgD- to define SWM B cell population. CCL20 treatment did not increase the total percent of SWM B cells; however, there was a 2-3-fold increase in CCR6+ SWM B cells compared to CCR6- SWM B cells. CCL20 treated CCR6+ SWM B cells have an average of 7% increase of phosphorylated mTOR in total SWM B cells compared to nonCCL20 treated CCR6+ SWM B cells. These results suggest CCL20 stimulation induces mTOR phosphorylation and activation. Thus, mTOR activation and downstream mediators may be necessary for α -gal induced B cell class switching. From these preliminary studies, we conclude that CCL20 stimulation increases the percent pmTOR+ SWM B cells. We will continue to investigate downstream mTOR mediators in regard to B cell class switching after CCL20 stimulation and how α-gal sensitization augments B cell IgE class switching and increases atherosclerosis with our novel α -gal-/- Apoe-/- mouse.

TET2 is an epigenetic regulator with an emerging role in regulating CVD severity. TET2 deficiency in mice (TET2 -/- ) was shown to increase atherosclerosis via upregulation of proinflammatory pathways in macrophages. While TET2 has been reported to regulate germinal center formation and class switch recombination in atherogenic B2 cells, its role in atheroprotective B1 cell subsets remains largely unexplored. To investigate the role of TET2 in B1 subsets, flow cytometry of cells from the peritoneal cavity (PEC), spleen and bone marrow of TET2 -/- mice and wildtype littermate controls (n = 14/group) was performed. Results demonstrated increased B1a (p = 0.0162) and B1b (p = 0.0056) cells in TET2 -/- mice in the PEC, their primary niche, but not in the spleen, while in the bone marrow only TET2 -/- B1a cells were increased (p = 0.0021). An enzyme linked immunosorbent assay using the plasma from these mice demonstrated a significant increase in total IgM in the TET2 -/- mice compared to the wildtype mice (p = 0.0287). In addition, sort purified PEC B1a, B1b and B2 cells from TET2 -/- mice and wildtype littermate controls were analyzed for methylation status and RNA expression (n = 24). RNAseq analysis of TET2 -/- PEC B1a, B1b, and B2 cells revealed significantly reduced chemokine receptor CCR6 expression in B1a (-4.88 fold change, 8.03E-08 padj.) and a trending reduction of CCR6 in B1b cells (-2.17 fold change, 0.3477 padj.) compared to wildtype. Further, methylation analysis showed significant hypermethylation of CCR6 in B1a and B1b TET2 -/- cells compared to wildtype. This was corroborated by gene pathway analysis of the RNAseq data which showed significant downregulation of chemotaxis and migration pathways in B1a and B1b TET2 -/- cells. We conclude that loss of TET2 increases hypermethylation of CCR6 and suppresses CCR6 RNA expression in B1 cells which may impair trafficking out of the PEC to the spleen, but not necessarily the bone marrow, leading to PEC accumulation of B1 subtypes and an increase in the total plasma level of atheroprotective IgM. Further study is needed to identify mechanisms of potential B1 cell-mediated atheroprotection in the case of TET2 loss.

Supplemental Digital Content is available in the text., Background: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. Methods: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. Results: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene–positive, extracellular matrix-remodeling, “pioneer” cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. Conclusions: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

The immune system plays an important role in obesity-induced adipose tissue inflammation and the resultant metabolic dysfunction, which can lead to hypertension, dyslipidemia, and insulin resistance and their downstream sequelae of type 2 diabetes mellitus and cardiovascular disease. While macrophages are the most abundant immune cell type in adipose tissue, other immune cells are also present, such as B cells, which play important roles in regulating adipose tissue inflammation. This brief review will overview B-cell subsets, describe their localization in various adipose depots and summarize our knowledge about the function of these B-cell subsets in regulating adipose tissue inflammation, obesity-induced metabolic dysfunction and atherosclerosis.

Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an in vivo model of obesity-associated dysmetabolism. Methods: Male leptin-deficient (ob/ob) mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells. Results: Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery. Conclusions: These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations in vivo. These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.

Murine B-1 cells produce IgMs that inactivate oxidation-specific epitopes and protect against atherosclerosis. Despite compelling data that IgM to MDA-LDL (IgM MDA-LDL ) is inversely associated with coronary artery disease (CAD) and cardiovascular (CV) events in human, the human B cell subtype that produces IgM MDA-LDL is unknown. Here, we utilized mass cytometry to identify the 11 circulating human B cell subtypes and discovered that only the frequency of CD20 + CD27 + IgM + cells was associated with IgM MDA-LDL levels. Notably, high expression of CD24, a GPI-anchored sialoglycoprotein, was associated with IgM MDA-LDL (r = 0.57, p = 0.002) and inversely associated with CAD severity (r = -0.58, p=0.001). Adoptive transfer (AT) of sorted CD20 + CD27 + IgM + CD24hi (B 27+IgM+CD24hi ) and CD24lo/- (B 27+IgM+CD24lo/- ) human B cells into humanized mice demonstrated that CD24 is critical for trafficking to the spleen and IgM production. Bulk RNAseq coupled with pathway analysis of B 27+IgM+CD24hi and B 27+IgM+CD24lo/- cells revealed enhanced BCR and CCR6 signaling in B 27+IgM+CD24hi . Imaging flow cytometry indicated colocalization between CD24 and CCR6 which was abrogated by CD24 blocking antibody (CD24mAb). Trans-well migration and internalization assays demonstrated that CD24 blockade increased CCL20-induced CCR6 internalization (p < 0.05, n =4), and reduced CCL20-induced migration of B 27+IgM+CD24hi cells (p < 0.05, n =4). CD24mAb treatment of B 27+IgM+CD24hi cells prior to AT into humanized mice also reduced the amount of surface CCR6 on B 27+IgM+CD24hi cells in vivo (p < 0.05, n = 4). Both CCR6 knockout and CD24 inhibition of B 27+IgM+CD24hi cells reduced migration to the spleen (p < 0.05, n = 4), and plasma level of human IgM (p = 0.057, n = 4). Lastly, single cell multi-omics sequencing of PBMCs from 60 CAD subjects revealed elevated CCR6 and IgM signaling in B 27+IgM+CD24hi cells in subjects with less severe CAD. Results provide the first evidence for a role for CD24 in regulating CCR6-mediated B cell homing to the spleen, IgM production and CAD in humans. Findings may have important implications for the new CD24 immunotherapy entering clinical trials and raise the possibility that augmenting CD24 and/or CCR6 on B 27+IgM+ cells may be an effective atheroprotective strategy.

Background: The co-stimulatory CD40-CD40L dyad is an important driver of atherosclerosis. CD40 exerts divergent, cell-specific roles, which differentially impacts atherogenesis. Objectives: We here investigate the role of the most prominent CD40-expressing cell type, the CD40 + B cell, in atherosclerosis. Methods: B cell subset specific CD40-expression of patients with mild and severe coronary artery disease (CAD) was determined by mass-cytometry and correlated to CAD severity. Underlying mechanisms were revealed using CD19-CD40 flfl -ApoE -/- (CD40 BKO ) and CD40 flfl -ApoE -/- (CD40 BWT ) mice. Results: Patients with severe CAD had similar CD40 expression levels on most B cell subsets but showed a profound decrease in CD40 on B1 cells. This was associated with increased atherosclerotic plaque burden and decreased plaque fibrosis. Likewise, CD40 BKO mice exhibited an increased plaque area and a more advanced stage of atherosclerosis. Absence of B cell CD40 caused a decrease in immunoglobulin (Ig) producing cells, including germinal center-, plasma-, but especially B1 cells, thereby reducing levels of (anti-ox/MDA-LDL) IgM and (anti-ox/MDA-LDL) IgG. Interestingly, transcriptomics analysis revealed that the absence of CD40 on B1b cells in particular, caused altered gene expression pathways related to lipid uptake ( Cd36, Ldlr ), cellular stress and metabolism ( Nr4a1, Hif1a ), and cell death ( Naip5/6, Top1 ). Indeed, in vitro analysis showed that B1b CD40KO cells took up excessive amounts of ac/oxLDL, exhibited defective BCR signaling, and were prone to apoptosis, especially in hyperlipidemic conditions. Finally, transfer of wild-type B1b cells into CD40 BKO mice prevented the increase in atherosclerosis. Conclusions: We here show that CD40 is a critical modulator of B1 cell protective function in hypercholesterolemia and atherosclerosis.

Atherosclerosis is associated with increased lipid peroxidation, leading to generation of multiple oxidation-specific epitopes (OSEs), contributing to the pathogenesis of atherosclerosis and its clinical manifestation. Oxidized cholesteryl esters (OxCEs) are a major class of OSEs found in human plasma and atherosclerotic tissue. To evaluate OxCEs as a candidate biomarker, we generated a novel mouse monoclonal Ab (mAb) specific to an OxCE modification of proteins. The mAb AG23 (IgG1) was raised in C57BL6 mice immunized with OxCE-modified keyhole limpet hemocyanin, and hybridomas were screened against OxCE-modified BSA. This method ensures mAb specificity to the OxCE modification, independent of a carrier protein. AG23 specifically stained human carotid artery atherosclerotic lesions. An ELISA method, with AG23 as a capture and either anti-apoAI or anti-apoB-100 as the detection Abs, was developed to assay apoAI and apoB-100 lipoproteins that have one or more OxCE epitopes. OxCE-apoA or OxCE-apoB did not correlate with the well-established oxidized phospholipid-apoB biomarker. In a cohort of subjects treated with atorvastatin, OxCE-apoA was significantly lower than in the placebo group, independent of the apoAI levels. These results suggest the potential diagnostic utility of a new biomarker assay to measure OxCE-modified lipoproteins in patients with CVD.

Supplemental Digital Content is available in the text., Objective: Few studies of population-based cohorts have investigated prospective associations of lymphoid and myeloid cell subsets in cardiovascular disease onset and progression. The purpose of this analysis was to determine associations of prespecified myeloid and lymphoid lineage cell subsets with common carotid artery intima-media thickness (IMT) progression. Approach and Results: We performed a prospective case-cohort study of 1195 participants from the Multi-Ethnic Study of Atherosclerosis who had peripheral blood mononuclear cells stored from the baseline examination. Key exposure variables were prespecified subsets of lymphoid and myeloid lineage immune cells, phenotyped by multicolor flow cytometry. The primary outcome was progression from baseline (Exam 1) to year 10 (Exam 5) in common carotid IMT. Higher proportions of nonclassical monocytes (CD14dimCD16++) were significantly associated with IMT progression over 10 years, but classical monocytes (CD14++CD16−), CD4+CD28− T cells, and T helper cells producing IL-17 (interleukin 17; T helper 17 cells) were not associated with significant changes in IMT over 10 years. There were significant interactions between monocyte subsets and sex with respect to IMT progression: in sex-stratified analyses, nonclassical monocytes were associated with significant IMT progression and classical monocytes were associated with significant IMT regression for men, whereas there were no significant associations of monocyte subsets with IMT change for women. Conclusions: Nonclassical monocytes were associated with progression of carotid IMT. There were significant sex differences in associations of monocyte subsets with IMT progression: for men, nonclassical monocytes were associated with IMT progression and classical monocytes were associated with regression, whereas these associations were null for women.

Objective: The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS isoenzymes (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and Results: Male Id3 −/− mice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected in Id3 −/− epididymal AT. HA accumulated in epididymal AT of high-fat diet–fed Id3 −/− mice and circulating levels of HA were elevated. Has2 mRNA expression was increased in epididymal AT of Id3 −/− mice. Luciferase promoter assays showed that Id3 suppressed Has2 promoter activity, while loss of Id3 stimulated Has2 promoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells of Id3 −/− mice, an effect sensitive to hyaluronidase. Conclusions: Our data demonstrate that loss of Id3 increases Has2 expression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.

TET2 is an evolutionarily conserved dioxygenase that catalyzes the conversion of 5-methyl-cytosine to 5- hydroxymethyl-cytosine, promotes DNA demethylation and regulates transcription. TET2 has been reported to regulate germinal center formation and class switch recombination in B2 B cells, yet its role in B1 B cell subsets remains largely unexplored. To investigate the role of TET2 in B1 subsets, flow cytometry of cells from the peritoneal cavity (PEC), spleen and bone marrow of TET2−/− mice and wildtype littermate controls (n = 14/group) was performed. In addition, sort purified PEC B1a, B1b and B2 cells were analyzed for methylation status (n = 24) and RNA expression (n = 24). Results demonstrated increased B1a (p = 0.0162) and B1b (p = 0.0056) cells in TET2−/− mice in the PEC, their primary niche, but not in the spleen, while in the bone marrow only TET2−/− B1a cells were increased (p = 0.0021). RNAseq analysis of PEC B1a, B1b, and B2 cells from TET2−/− and wildtype mice revealed reduced chemokine receptor CCR6 expression in B1a (−4.879068141 fold change, 1. 8.03E-08 padj.) and B1b cells (−2.17044776 fold change, 0.34771255 padj.) from the TET2−/− mice. Further, methylation analysis showed significant hypermethylation of CCR6 in B1a and B1b TET2−/− cells compared to wildtype B1a and B1b cells. This was corroborated by gene pathway analysis of the RNAseq data which showed significant downregulation of chemotaxis and migration pathways in B1a and B1b TET2−/− cells. We conclude that loss of TET2 increases hypermethylation of CCR6 and suppresses CCR6 RNA expression in B1 cells which may impair trafficking out of the PEC to the spleen, but not necessarily the bone marrow, leading to PEC accumulation of B1 subtypes. Supported by grants from the NIH: 1R01HL 136098-01 (McNamara, C PI), R01 HL141123 (McNamara, C PI), T32 HL007284 (Dennis, E)

Background: Atherosclerotic plaques in mice and humans contain natural killer (NK) cells. Data on the role of NK cells in atherosclerosis using different transgenic mice models is inconsistent. Some studies showed that NK cells augment atherosclerosis through their cytotoxic potential, while others reported no effect. Evidence in humans indicates that NK cells are atherogenic. Frequency of NK cells and expression of the activating NK cell receptors are associated with severe disease and symptomatic carotid atherosclerotic plaques in humans. Hypothesis: We tested if coronary artery disease (CAD) subjects with necrotic plaques have a higher frequency of the circulating NK cells. Methods: We performed mass cytometry on peripheral blood mononuclear cells from matched CAD subjects with low and high (n=9 each) necrotic plaque content as determined by intravascular ultrasound-virtual histology. Results: CAD subjects with high necrotic plaques have significantly higher atheroburden, stenosis, calcium, fatty plaque content, and lower plaque fibrosis. Interestingly, CAD subjects with high necrotic plaques exhibited a significantly higher frequency of the CD56 bright NK cells as compared to those with low necrotic plaques (4.618 ± 0.625 vs 2.481 ± 0.37; p=0.011). Moreover, frequency of CD25 + NK cells also trended to be higher in subjects with high necrotic plaques. The frequency of the cytotoxic CD56 dim NK cells did not differ between the two groups. Correlation analyses demonstrated a significant positive association of CD56 bright NK cells with atheroburden (r=0.43; p=0.04), stenosis (r=0.58; p=0.005), plaque necrotic (r=0.71; p=0.002), calcium contents (r=0.73; p=0.0001), and a negative association with the plaque fibrous content (r=0.71; p= 0.0003). CD25 + NK cells also showed similar trending associations with burden, stenosis and plaque features. Conclusions: Our data provides yet another evidence of the atherogenic role of NK cells in humans and indicates that the CD56 bright NK cells may contribute to the development of a vulnerable plaque. CD56 bright NK cells produce proinflammatory cytokines including IFN-γ and TNF-α and cytotoxic enzymes that may contribute to increased inflammation, cell activation, and apoptosis within the plaque.

BackgroundDetailed understanding of the immune response to SARS-CoV-2, the cause of coronavirus disease 2019 (COVID-19), has been hampered by a lack of quantitative antibody assays.ObjectiveTo develop a quantitative assay for IgG to SARS-CoV-2 proteins that could readily be implemented in clinical and research laboratories.MethodsThe biotin-streptavidin technique was used to conjugate SARS-CoV-2 spike receptor-binding-domain (RBD) or nucleocapsid protein to the solid-phase of the ImmunoCAP resin. Plasma and serum samples from patients with COVID-19 (n=51) and samples from donors banked prior to the emergence of COVID-19 (n=109) were used in the assay. SARS-CoV-2 IgG levels were followed longitudinally in a subset of samples and were related to total IgG and IgG to reference antigens using an ImmunoCAP 250 platform.ResultsPerformance characteristics demonstrated 100% sensitivity and 99% specificity at a cut-off level of 2.5 µg/mL for both SARS-CoV-2 proteins. Among 36 patients evaluated in a post-hospital follow-up clinic, median levels of IgG to spike-RBD and nucleocapsid were 34.7 µg/mL (IQR 18-52) and 24.5 µg/mL (IQR 9-59), respectively. Among 17 patients with longitudinal samples there was a wide variation in the magnitude of IgG responses, but generally the response to spike-RBD and to nucleocapsid occurred in parallel, with peak levels approaching 100 µg/mL, or 1% of total IgG.ConclusionsWe have described a quantitative assay to measure IgG to SARS-CoV-2 that could be used in clinical and research laboratories and implemented at scale. The assay can easily be adapted to measure IgG to novel antigens, has good performance characteristics and a read-out in standardized units.

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 in PBMCs. Among 31 participants in the WIHS Study, we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs and Louvain clustering, we identified 58 subsets among CD4 T, CD8 T, B, NK cells and monocytes. This resolution was superior to flow cytometry, mass cytometry or scRNA-sequencing without antibodies. Since the transcriptome was not needed for cell identification, combined protein and transcript scRNA-Seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportion. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease. In conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs.

Background/objectives The incidence of obesity continues to increase worldwide and while the underlying pathogenesis remains largely unknown, nutrient excess, manifested by “Westernization” of the diet and reduced physical activity have been proposed as key contributing factors. Western-style diets, in addition to higher caloric load, are characterized by excess of advanced glycation end products (AGEs), which have been linked to the pathophysiology of obesity and related cardiometabolic disorders. AGEs can be “trapped” in adipose tissue, even in the absence of diabetes, in part due to higher expression of the receptor for AGEs (RAGE) and/or decreased detoxification by the endogenous glyoxalase (GLO) system, where they may promote insulin resistance. It is unknown whether the expression levels of genes linked to the RAGE axis, including AGER (the gene encoding RAGE), Diaphanous 1 (DIAPH1), the cytoplasmic domain binding partner of RAGE that contributes to RAGE signaling, and GLO1 are differentially regulated by the degree of obesity and/or how these relate to inflammatory and adipocyte markers and their metabolic consequences. Subjects/methods We sought to answer this question by analyzing gene expression patterns of markers of the AGE/RAGE/DIAPH1 signaling axis in abdominal subcutaneous (SAT) and omental (OAT) adipose tissue from obese and morbidly obese subjects. Results In SAT, but not OAT, expression of AGER was significantly correlated with that of DIAPH1 (n = 16; $$\hat \beta = 0.719$$ β ̂ = 0.719 , [0.260, 1.177]; q = 0.008) and GLO1 (n = 16; $$\hat \beta = 0.773$$ β ̂ = 0.773 , [0.364, 1.182]; q = 0.004). Furthermore, in SAT, but not OAT, regression analyses revealed that the expression pattern of genes in the AGE/RAGE/DIAPH1 axis is strongly and positively associated with that of inflammatory and adipogenic markers. Remarkably, particularly in SAT, not OAT, the expression of AGER positively and significantly correlated with HOMA-IR (n = 14; $$\hat \beta = 0.794$$ β ̂ = 0.794 , [0.338, 1.249]; q = 0.018). Conclusions These observations suggest associations of the AGE/RAGE/DIAPH1 axis in the immunometabolic pathophysiology of obesity and insulin resistance, driven, at least in part, through expression and activity of this axis in SAT.

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

ObjectiveExamine if adding aerobic exercise to standard medical care (EX+SC) prior to bariatric surgery improves metabolic health in relation to surgical outcomes.MethodsFourteen bariatric patients (age: 42.3±2.5y, BMI: 45.1±2.5 kg/m2) met inclusion criteria and were match-paired to pre-operative SC (n = 7) or EX+SC (n = 7; walking 30min/d, 5d/wk, 65-85% HRpeak) for 30d. A 120min mixed meal tolerance test was performed pre- and post-intervention (~2d prior to surgery) to assess insulin sensitivity (Matsuda Index) and metabolic flexibility (indirect calorimetry). Aerobic fitness (VO2peak), body composition (BodPod), and adipokines (adiponectin, leptin) were also measured. Omental adipose tissue was collected during surgery to quantify gene expression of adiponectin and leptin, and operating time and length of hospital stay were recorded. ANOVA and Cohen's d effect size (ES) was used to test group differences.ResultsSC tended to increase percent body fat (P = 0.06) after the intervention compared to EX+SC. Although SC and EX+SC tended to raise insulin sensitivity (P = 0.11), EX+SC enhanced metabolic flexibility (P = 0.01, ES = 1.55), reduced total adiponectin (P = 0.01, ES = 1.54) with no change in HMW adiponectin and decreased the length of hospital stay (P = 0.05) compared to SC. Albeit not statistically significant, EX+SC increased VO2peak 2.9% compared to a 5.9% decrease with SC (P = 0.24, ES = 0.91). This increased fitness correlated to shorter operating time (r = -0.57, P = 0.03) and length of stay (r = -0.58, P = 0.03). Less omental total adiponectin (r = 0.52, P = 0.09) and leptin (r = 0.58, P = 0.05) expression correlated with shorter operating time, and low leptin expression was linked to shorter length of stay (r = 0.70, P = 0.01), and low leptin expression was linked to shorter length of stay (r = 0.70, P = 0.01).ConclusionAdding pre-operative aerobic exercise to standard care may improve surgical outcomes through a fitness and adipose tissue derived mechanism.

Supplemental Digital Content is available in the text., Objective— Emerging evidence suggests a link between coronary artery disease and type 2 immunity. We sought to test the hypothesis that IgE sensitization to the mammalian oligosaccharide galactose-α-1,3-galactose (α-Gal)—the target allergen of delayed anaphylaxis to red meat—is associated with coronary artery disease. Approach and Results— Total IgE and specific IgE to α-Gal were assayed on sera from 118 subjects who presented for cardiac catheterization and underwent intravascular ultrasound. IgE to α-Gal was detected in 26%, and atheroma burden was higher in sensitized subjects (P=0.02). Because α-Gal sensitization relates to an environmental exposure that could be a risk factor for early-onset coronary artery disease (ie, tick bites), we age stratified the cohort. In subjects ≤65 years of age, the strength of the association with atheroma burden was stronger (P

D-dimer has emerged as a biomarker of cardiovascular event risk, yet pathophysiological factors associated with plasma D-dimer levels in stable coronary artery disease (CAD) subjects are poorly understood. In 106 stable CAD subjects undergoing intravascular ultrasound with virtual histology (IVUS-VH), we measured D-dimer, lipoprotein(a) (Lp(a)), plasminogen, biomarkers reflecting oxidation-specific epitopes (OSE) such as oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB), OxPL on plasminogen (OxPL-PLG), and autoantibodies to phosphorylcholine-BSA [PC-BSA] and a malondialdehyde [MDA] mimotope. In univariate analysis, D-dimer was positively associated with Lp(a), OxPL-apoB, OxPL-PLG, and coronary artery calcium, and inversely associated with autoantibodies to OSE and plaque fibrosis. D-dimer levels > 500 ng/ml also showed positive association with plaque necrosis. After multivariate analysis, D-dimer remained significantly associated with Lp(a) and plaque calcium. While further studies are needed, results provide evidence that plasma D-dimer levels are associated with levels of OxPLs and IVUS-VH indices linked to plaque erosion and rupture.

Supplemental Digital Content is available in the text., Objective— Investigate the impact of modulating B cell FcγRIIb (Fcγ receptor IIb) expression on atherosclerosis. Approach and Results— Western diet–induced atherosclerosis was assessed in Ldlr−/− or Apoe−/− mice with B cell–specific overexpression of FcγRIIb or with an FcγRIIb promoter mutation that alters FcγRIIb expression in germinal center (GC) B cells. In males, overexpression of FcγRIIb on B cells severely reduced activated, class switched B cell responses, as indicated by reductions in GC B cells, plasma cells, and serum IgG but not IgM antibodies. Male mice overexpressing FcγRIIb developed less atherosclerosis, suggesting a pathogenic role for GC B cell IgG responses. In support of this hypothesis, male mice with a promoter polymorphism-driven reduction in FcγRIIb on GC B cells but not plasma cells have a converse phenotype of enhanced GC responses and IgG2c antibodies and enhanced atherosclerosis. IgG2c significantly enhanced TNF (tumor necrosis factor) secretion by CD11b+ CD11c+ cells expressing the high-affinity receptor FcγRIV. In females, overexpression of FcγRIIb on B cells not only reduced GC B cell responses but also substantially reduced B-1 cells and IgM antibodies, which translated into acceleration of atherosclerosis. Promoter-driven reduction in FcγRIIb did not alter GC B cell responses in females and, therefore, had no impact on atherosclerosis. Conclusions— B cell FcγRIIb differentially alters proatherogenic adaptive GC B cell and atheroprotective innate B-1 responses in male and female mice fed a western diet. Our results highlight the importance of a better understanding and ability to selectively target B cell responses in future immunotherapeutic approaches against human cardiovascular disease.

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.

Rationale: B-1 cell-derived natural IgM antibodies against oxidation-specific epitopes on low-density lipoprotein are anti-inflammatory and atheroprotective. Bone marrow (BM) B-1a cells contribute abundantly to IgM production, yet the unique repertoire of IgM antibodies generated by BM B-1a and the factors maintaining the BM B-1a population remain unexplored. CXCR4 (C-X-C motif chemokine receptor 4) has been implicated in human cardiovascular disease and B-cell homeostasis, yet the role of B-1 cell CXCR4 in regulating atheroprotective IgM levels and human cardiovascular disease is unknown. Objective: To characterize the BM B-1a IgM repertoire and to determine whether CXCR4 regulates B-1 production of atheroprotective IgM in mice and humans. Methods and Results: Single-cell sequencing demonstrated that BM B-1a cells from aged ApoE −/− mice with established atherosclerosis express a unique repertoire of IgM antibodies containing increased nontemplate-encoded nucleotide additions and a greater frequency of unique heavy chain complementarity determining region 3 sequences compared with peritoneal cavity B-1a cells. Some complementarity determining region 3 sequences were common to both compartments suggesting B-1a migration between compartments. Indeed, mature peritoneal cavity B-1a cells migrated to BM in a CXCR4-dependent manner. Furthermore, BM IgM production and plasma IgM levels were reduced in ApoE −/− mice with B-cell–specific knockout of CXCR4, and overexpression of CXCR4 on B-1a cells increased BM localization and plasma IgM against oxidation specific epitopes, including IgM specific for malondialdehyde-modified LDL (low-density lipoprotein). Finally, in a 50-subject human cohort, we find that CXCR4 expression on circulating human B-1 cells positively associates with plasma levels of IgM antibodies specific for malondialdehyde-modified LDL and inversely associates with human coronary artery plaque burden and necrosis. Conclusions: These data provide the first report of a unique BM B-1a cell IgM repertoire and identifies CXCR4 expression as a critical factor selectively governing BM B-1a localization and production of IgM against oxidation specific epitopes. That CXCR4 expression on human B-1 cells was greater in humans with low coronary artery plaque burden suggests a potential targeted approach for immune modulation to limit atherosclerosis.

Increasing brown adipose tissue abundance or activity or converting white into brown adipocytes holds promise for the treatment of type 2 diabetes and obesity. Several PPARγ agonists, including rosiglitazone, have been shown to robustly induce browning of white adipose tissue (WAT) in vitro but their effects in vivo are less pronounced, while PPARα agonists show modest browning effects both in vitro or in vivo. In the present study, we investigated whether PPARα and PPARγ dual agonism would have synergistic effects in inducing browning of WAT. We found that all tested dual PPARα/γ agonists robustly increased uncoupling protein 1 (Ucp1) expression in both mouse and human preadipocytes, with tesaglitazar giving the largest Ucp1 induction. Notably, tesaglitazar also strongly induced browning of WAT in vivo in both lean and obese mice at thermoneutrality, largely exceeding the increase in Ucp1 observed with rosiglitazone. Mechanistically, we found that selective PPARγ agonism was sufficient for the conversion of white into brown-like adipocytes in vitro, but dual PPARα/γ agonism was superior to selective PPARγ agonism at inducing white fat browning in vivo, at least in part via a PPARα-mediated increase in fibroblast growth factor 21 (FGF-21). In human preadipocytes in vitro, combined treatment with rosiglitazone and FGF-21 resulted in a robust and synergistic increase in UCP1 mRNA levels, superior to rosiglitazone- or FGF-21-monotreatments. Tesaglitazar-induced browning in obese mice was associated with increased energy expenditure, enhanced insulin sensitivity, reduced liver steatosis, and an overall improved metabolic profile compared to rosiglitazone and vehicle control groups. In conclusion, we found that dual PPARα/γ agonism robustly promotes browning of white fat in vivo, via synergistic action of FGF-21 and PPARγ agonism. Our findings identify a novel opportunity to develop compounds with robust browning of WAT, via dual modulation of PPARα and PPARγ. Disclosure T. Kroon: Employee; Self; AstraZeneca. M.J. Harms: Employee; Self; AstraZeneca. D. Nilsson: None. A. Lindblom: Employee; Self; AstraZeneca. P. Gennemark: Employee; Self; AstraZeneca. G. O'Mahony: Employee; Self; AstraZeneca. V. Osinski: None. C.A. McNamara: None. J. Boucher: Employee; Self; AstraZeneca.

Objectives: IgMs that inactivate oxidation specific epitopes (IgMOSE) on phospholipids such as low density lipoprotein (LDL) were shown to confer athero-protection. We developed a 24 antibodies mass cytometry panel (CyTOF) to identify the human B cell subtype producing IgM to malondialdehyde modified LDL (IgMMDA-LDL), a predominant IgMOSE in humans. We also utilized humanized mice model to characterize a role of CD24 mediated IgM production. Methods: Peripheral blood mononuclear cells from healthy donors and subjects with coronary artery disease (CAD) undergoing quantitative coronary angiography were used to perform high dimensional analysis using CyTOF, RNAseq and Abseq as well as adoptive transfer into humanized mice model. Results: CD20+CD27+IgM+ cells (B27+IgM+) spontaneously produced IgM and produced IgMMDA-LDL in response to MDA stimulation when injected into humanized mice; an effect significantly augmented in B27+IgM+ cells with high expression of CD24 (B27+IgM+CD24hi). CD24 expression also enhanced splenic and bone marrow trafficking of B27+IgM+ cells. Blocking CD24 with a mAb reduced CCR6 expression, increased CCL20-induced CCR6 internalization and impaired migration to the spleen leading to lower IgM production. Lastly, single cell protein and transcriptome sequencing of PBMCs from 60 CAD subjects revealed enhanced CCR6 and IgM signaling in B27+IgM+CD24hi cells in subjects with low compared to high CAD severity. Conclusions: Identification of IgMMDA-LDL-producing cells in humans has the potential to allow for the development of therapeutics aimed at cellular targeting that may allow for enhancing production of IgM to the many OSE produced during inflammatory states such as atherosclerosis.

B-1 cell derived IgM attenuates chronic inflammatory diseases like atherosclerosis. The spleen is a major lymphoid tissue where B-1 cells (both B-1a and B-1b) produce natural IgM. B-1 derived natural IgM has long been thought to contain restricted predominantly germline encoded V-D-J gene segments with low number of non-template encoded nucleotide (N)-additions, which are added at the junctions between V-D and D-J segments. However, differences in VDJ gene usage and N-addition frequency between peritoneal cavity (PerC) and spleen B-1 cells in aged atherosclerotic mice are unknown. B-1a and B-1b cells were single-cell sorted from PerC and spleen of 100-week-old, chow-fed ApoE−/− mice. Single cell sequencing of the immunoglobulin heavy chain variable region was performed. Splenic B-1 cells displayed increased diversity at the V-D and D-J junctions, as evidenced by increased average number of N-additions. Sequences containing >1 N-additions at both V-D and D-J junctions were 2% and 27% for PerC B-1a and B-1b sequences as compared to 44% and 44% of sequences in splenic B-1a and B-1b respectively. B-1 cells from PerC and spleen showed marked differences in VH, D and JH gene segment usage. Importantly, the most commonly expressed heavy chain complementarity determining region 3 (CDR-H3) amino acid sequence in spleen B-1a (AREVTTMYYFDY) and B-1b (AREDYYGSSYYFDY) cells was different from PerC B-1a (AGDYDGYWYFDV) and B-1b (AGDRDGYWYFDV) cells. Results provide clear evidence of a diversified IgM repertoire expressed by spleen B-1 cells compared to PerC B-1 cells during advanced atherosclerosis. The CDR-H3 sequences of splenic B-1 cells suggest underlying differences in antigen specificity which could further regulate disease progression.

Objective: IL-6 is implicated in the development of coronary artery disease (CAD), autoimmune (AI) disorders, and cytokine storm syndrome (CSS). IL-6 inhibitors are effective in treating AI disorders and are being tested for CAD and CSS. While some studies have reported on IL-6-induced STAT signaling in humans, a comprehensive map of IL-6 signaling in human immune cells is currently lacking. We developed a 32-antibody custom mass cytometry (CyTOF) panel to characterize IL-6 signaling across all major human immune cell subsets, and applied it to identify IL-6-induced immune signatures linked with unstable atherosclerotic plaque. Methods: Blood cells from healthy donors and CAD subjects undergoing virtual histology-intravascular ultrasound imaging were stimulated with IL-6, stained and ran in CyTOF. Unsupervised analytical tools (SPADE, UMAP, and Leiden clustering) were used to identify immune cell subsets and IL-6-induced intracellular phosphorylation status. Results: IL-6 induced STAT1 and STAT3 activation in CD4 and CD8 naïve T cell subsets and CD4 memory T subsets. Notably, we identified that IL-6 also activates STAT5 within the CD4 and CD8 naïve T subsets. IL-6 induced a much more robust activation of STAT1 as compared to STAT3 and STAT5. Other cell types such as CD14+ monocytes, and CD11c+ and CD123+ dendritic cells also showed IL-6-induced STAT activation. IL-6-induced phosphorylation of STAT1 and STAT3 in a novel PD-1+CD27−CD127lowCD4+ effector memory T cell subtype was associated with higher CAD burden and unstable plaque features. Conclusions: Findings are significant for mechanistic insights into IL-6-induced inflammation and may enable discovery of new approaches to reduce inflammation in CAD and other pathologies.

Thrombosis is a leading cause of morbidity and mortality. Detection of a prothrombotic state using biomarkers would be of great benefit to identify patients at risk of thrombosis that would benefit from thromboprophylaxis. Tissue factor (TF) is a highly procoagulant protein that under normal conditions is not present in the blood. However, increased levels of TF in the blood in the form of microparticles (MPs) (also called extracellular vesicles) are observed under various pathological conditions. In this review, we will discuss studies that have measured MP-TF activity in a variety of diseases using two similar FXa generation assay. One of the most robust signals for MP-TF activity (16-26 fold higher than healthy controls) is observed in pancreatic cancer patients with venous thromboembolism. In this case, the TF+ MPs appear to be derived from the cancer cells. Surprisingly, cirrhosis and acute liver injury are associated with 17-fold and 38-fold increases in MP-TF activity, respectively. Based on mouse models, we speculate that the TF+ MPs are derived from hepatocytes. More modest increases are observed in patients with urinary tract infections (6-fold) and in a human endotoxemia model (9-fold) where monocytes are the likely source of the TF+ MPs. Finally, there is no increase in MP-TF activity in the majority of cardiovascular disease patients. These studies indicate that MP-TF activity may be a useful biomarker to identify patients with particular diseases that have an increased risk of thrombosis.

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.

The development of new blood vessels is important during the progression of many diseases including cancer, obesity, and ischemia. While inhibiting blood vessel growth can prevent excess adiposity and tumor growth, it can also exacerbate symptoms of ischemia. Using flow cytometry to quantify total endothelial cells (ECs) in the gastrocnemius of mice who have undergone femoral artery resection (FAR), the number of ECs per gram of muscle increases approximately four-fold relative to uninjured gastrocnemius from the same mouse (442% increase, ± 208%) in a model of hindlimb ischemia. The helix-loop-helix (HLH) transcription factor Inhibitor of differentiation (ID3) promotes angiogenesis and whole tissue blood perfusion in models of cancer and obesity, but the potential role for ID3 in promoting ischemia-induced angiogenesis remains uninvestigated. Id3 mRNA levels in injured gastrocnemius are increased three days post-FAR relative to uninjured gastrocnemius (p < 0.05, n = 6). Furthermore, ID3 global knockout (KO) mice have attenuated perfusion recovery compared to wild type littermate controls at 14 days post-FAR (p < 0.05). Gastrocnemius muscles isolated from ID3 global KO mice 7 days post-FAR express higher levels of the cell cycle inhibitor p21 Cip1 mRNA than WT mice (p = 0.10, n = 3). Finally, flow cytometry analysis of cell subsets in a novel Id3 promoter-GFP reporter mouse line revealed that Id3 promoter activation is greater in CD31 + ECs than many other cell types (n = 10), suggesting ECs may be an important cell type in which ID3 is promoting pro-angiogenic effects. Together, these data support the hypothesis that ID3 promotes ischemia-induced angiogenesis by promoting EC proliferation and inhibiting cell cycle inhibitor p21 Cip1 . To investigate the role of ID3 in ECs during angiogenesis, an EC-specific, tamoxifen-inducible Id3 KO mouse (Id3 fl/fl Cdh5-Cre/ERT2) has been generated.

In adipose tissues of obese mice, macrophages accumulate in crown-like structures (CLS) formed around dying adipocytes. The CLS macrophages are thought to clear the dead adipocytes by exophagy, which involves exocytosis of lysosomes and digestion of apoptotic adipocytes. After digesting the plasma membrane and other cytosolic contents of the adipocyte, CLS macrophages come in contact with the large lipid droplet, however, it is unknown how macrophages response to such acellular adipocytes. We hypothesized that the acelular adipocytes in CLSs promote inflammation and metabolically activate the macrophages to promote clearance of the lipid. To mimic the in vivo scenario, we exposed cultured murine bone marrow-derived macrophages to lipid droplets isolated from the adipocytes of high-fat diet-induced obese C57BL/6 mice. In response to adipocyte lipid, macrophages accumulated lipid droplets, which were similar in size and number to lipid droplets of CLS macrophages in vivo . Acellular lipid exposure significantly increased TNF-α gene expression, which was suppressed by the CD36 inhibitor sulfo-N-succinimidyl oleate, supporting CD36-mediated inflammatory response. Moreover, lipid exposure significantly lowered metabolic activity of macrophages and impaired clearance of apoptotic bodies from 3T3-L1 adipocytes. Using specific inhibitors, unexpectedly we found that lipid droplet accumulation in macrophages was independent of CD36 activity or processes involved in exophagy such as exocytosis of lysosomes, extracellular lipase activity, lipolysis and phagocytosis. Interestingly, lipid droplet accumulation was dependent on acetyl-CoA carboxylase (ACC) as determined by use of ACC inhibitors soraphen A and TOFA, and siRNA knock-down of ACC in macrophages. Furthermore, confocal microscopy of whole mount adipose tissue revealed expression of ACC in CLS macrophages. Altogether, using a novel in vitro model we demonstrate that acelular adipocytes suppress metabolic activity, but induce inflammation and de novo lipogenesis-mediated lipid droplet formation in macrophages.

B1 cells exert protective effects in atherosclerosis through production of anti-inflammatory IgM antibodies recognizing oxidation-specific epitopes, such as MDA-LDL, present in diseased arteries. However, factors mediating B1 IgM production are currently unclear. We evaluated MDA-LDL binding and chemokine receptor expression on human B1 cells in a cohort of subjects undergoing intravascular ultrasound (IVUS) for coronary artery assessment. Results demonstrate that a subset of human B1 cells (~35%) is able to bind MDA-LDL. Moreover, expression of the chemokine receptor CXCR4 on circulating B1 cells associates with increased plasma levels of anti-MDA-LDL IgM antibodies (p=0.0009), and decreased plaque burden in coronary arteries (p=0.0002). Mice with B cell-specific loss of CXCR4 on the atherogenic ApoE -/- background (CXCR4 BKO ) demonstrate fewer B1a cells (n=6-8,pWT ). Furthermore, retroviral-mediated overexpression of CXCR4 on B1a cells in vivo is associated with increased B1a localization to the bone marrow (pWT or CXCR4 BKO B1a cells into lymphocyte-deficient Rag1 -/- ApoE -/- mice. After 16 weeks of Western diet feeding, recipients given CXCR4 BKO B1a cells demonstrate reduced plasma IgM levels (n=7,pWT B1a cells. Intriguingly, B1a transfer reduces plasma cholesterol levels in mice regardless of CXCR4 expression (n=7,pWT B1a cells have reduced aortic lesion area compared to PBS controls (n=7,pBKO B1a cells did not attain the same level of protection. Overall, these data suggest that CXCR4 is an important regulator of IgM production and B1a-mediated atheroprotection.

Marginal zone and follicular B cells together constitute the B2 cell population, which is known to promote cardiovascular diseases by secretion of pathogenic antibodies. However, it is not completely understood how B2 cells are activated. Here, we tested the hypothesis that B cell activating factor (BAFF) activates B2 cells and promote abdominal aortic aneurysm (AAA) formation. Since BAFF can either exist as a 3mer or multimerize to a highly active 60mer, we further examined if the 60mer is critical for B2 cell-mediated pathogenicity. Anti-BAFF antibody (Ab) Sandy-2 was injected to C57BL/6 male mice at 1 mg/kg once in every 14 days. AAA was induced by topical elastase model after 14 days of Sandy-2 injection. Native PAGE and ELISA methods were used to determine binding of Abs to recombinant BAFF 3mer and 60mer. For in vitro experiments, B cells were isolated from murine spleens. Activation of B cells was examined by Western blotting and RNA sequencing and by surface expression of CD23 and MHC II by flow cytometry. Metabolic reprogramming of B cells by BAFF was determined by extracellular flux analysis using a Seahorse XF24 Flux Analyzer. Sandy-2 bound to both 3mer and 60mer, resulting in suppressed AAA formation (n=8, pIn vitro , the 60mer, but not the 3mer, significantly activated both NF-kB1 and -kB2 signaling, and induced expression of B2 cell activation markers and anti-apoptotic genes in B cells. Inhibitors of NF-kB signaling decreased B cell activation in response to 60mer. The 60mer treatment significantly increased mitochondrial respiration and glycolysis in B cells, supporting an activated status. An antibody against multimerization site of BAFF (anti-multiBAFF) significantly suppressed B cell activation relative to a control Ab, in a neutrophil:B cell co-culture model. The effect of the anti-multiBAFF Ab on AAA formation is currently being tested in our laboratory. Altogether, our results suggest a critical role for BAFF 60mer in skewing B cells to an activated B2 cell phenotype, supporting a pathogenic role of B2 cells in AAA.

Background: Perivascular adipose tissue (PVAT) regulates artery physiology and pathology, such as promoting atherosclerosis development through local production of inflammatory cytokines. The phenotype of PVAT is region-specific and PVAT composed of brown adipose tissue (near the aortic arch and thoracic aorta) and white adipose tissue (near the abdominal aorta). B-1 cells limit adipose tissue inflammation and atherosclerosis through the production of IgM antibodies. PVAT harbors high numbers of B cells. Id3 is a basic helix-loop-helix protein and important for B cell development. B cell-specific Id3 deficiency (Id3 BKO ) increases B-1b cell numbers and provides atheroprotection. However, the location and regulation of IgM producing B-1 cells in the PVAT are unknown. Methods and Results: Flow cytometry analysis of PVAT of normal chow diet fed young ApoE -/- mice (n=5) demonstrated that the abundant CD19 + B cells (per gram fat) harbored in PVAT around aortic arch (2.7 + 0.69 x10 5 cells) compared to PVAT near the thoracic (0.9 + 0.29 x10 5 cells) and abdominal aorta (1.1 + 0.51 x10 5 cells). Interestingly, a large proportion (40%) of these B cells are belong to the CD19 hi B220 low B-1 subset in PVAT near aortic arch. CXCL13 is a chemokine, which is important for B-1 cell recruitment to omental fat. Real time-PCR data confirmed that high numbers of B-1 cell recruitment to PVAT near aortic arch is due to high expression of CXCL13 in the PVAT near the aortic arch compared to PVAT near thoracic aorta and abdominal aorta. Moreover, Flow cytometry and ELISPOT data in normal chow fed young ApoE -/- mice with Id3 BKO demonstrated that Id3 BKO increased B-1b cells (Id3 BKO : 1.2 + 0.16 x10 3 ; Id3 WT : 0.4 + 0.06 x10 3 ; p-value: BKO : 2.0 + 0.42 x10 3 ; Id3 WT : 0.78 + 0.23 x10 3 ; p-value: WT control mice. Conclusion: Results provide the first evidence that atheroprotective B-1 cell profile in PVAT is region-specific and identify Id3 and CXCL13 as regulators of aortic arch PVAT B-1b cell numbers and IgM production.

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., Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here Gaddis et al. show that Apolipoprotein AI prevents the conversion of Treg cells into pro-atherogenic T follicular helper cells, and thus regulates the immune response during atherogenesis.