Multidimensional profiling of human T cells reveals high CD38 expression, marking recent thymic emigrants and age-related naive T cell remodeling.

Thymic involution is a key factor in human immune aging, leading to reduced thymic output and a decline in recent thymic emigrant (RTE) naive T cells in circulation. Currently, the precise definition of human RTEs and their corresponding cell surface markers lacks clarity. Analysis of single-cell RNA-seq/ATAC-seq data distinguished RTEs by the expression of SOX4, IKZF2, and TOX and CD38 protein, whereby surface CD38hi expression universally identified CD8+ and CD4+ RTEs. We further determined the dynamics of RTEs and mature cells in a cohort of 158 individuals, including age-associated transcriptional reprogramming and shifts in cytokine production. Spectral cytometry profiling revealed two axes of aging common to naive CD8+ and CD4+ T cells: (1) a decrease in CD38++ cells (RTEs) and (2) an increase in CXCR3hi cells. Identification of RTEs enables direct assessment of thymic health. Furthermore, resolving the dynamics of naive T cell remodeling yields insight into vaccination and infection responsiveness throughout aging.

BAFF neutralization impairs the autoantibody-mediated clearance of dead adipocytes and aggravates obesity-induced insulin resistance.

B cell-activating factor (BAFF) is a critical TNF-family cytokine that regulates homeostasis and peripheral tolerance of B2 cells. BAFF overproduction promotes autoantibody generation and autoimmune diseases. During obesity, BAFF is predominantly produced by white adipose tissue (WAT), and IgG autoantibodies against adipocytes are identified in the WAT of obese humans. However, it remains to be determined if the autoantibodies formed during obesity affect WAT remodeling and systemic insulin resistance. Here, we show that IgG autoantibodies are generated in high-fat diet (HFD)-induced obese mice that bind to apoptotic adipocytes and promote their phagocytosis by macrophages. Next, using murine models of obesity in which the gonadal WAT undergoes remodeling, we found that BAFF neutralization depleted IgG autoantibodies, increased the number of dead adipocytes, and exacerbated WAT inflammation and insulin resistance. RNA sequencing of the stromal vascular fraction from the WAT revealed decreased expression of immunoglobulin light-chain and heavy-chain variable genes suggesting a decreased repertoire of B cells after BAFF neutralization. Further, the B cell activation and the phagocytosis pathways were impaired in the WAT of BAFF-neutralized mice. In vitro, plasma IgG fractions from BAFF-neutralized mice reduced the phagocytic clearance of apoptotic adipocytes. Altogether, our study suggests that IgG autoantibodies developed during obesity, at least in part, dampens exacerbated WAT inflammation and systemic insulin resistance.

Tipping the Scale: Atheroprotective IgM-Producing B Cells in Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease whose progression is fueled by proinflammatory moieties and limited by anti-inflammatory mediators. Whereas oxidative damage and the generation of oxidation-specific epitopes that act as damage-associated molecular patterns are highly inflammatory, IgM antibodies produced by B-1 and marginal zone B cells counteract unrestricted inflammation by neutralizing and encouraging clearance of these proinflammatory signals. In this review, we focus on describing the identities of IgM-producing B cells in both mice and humans, elaborating the mechanisms underlying IgM production, and discussing the potential strategies to augment the production of atheroprotective IgM. In addition, we will discuss promising therapeutic interventions in humans to help tip the scale toward augmentation of IgM production and to provide atheroprotection.

B-1 lymphocytes in adipose tissue as innate modulators of inflammation linked to cardiometabolic disease.

Fat is stored in distinct depots with unique features in both mice and humans and B cells reside in all adipose depots. We have shown that B cells modulate cardiometabolic disease through activities in two of these key adipose depots: visceral adipose tissue (VAT) and perivascular adipose tissue (PVAT). VAT refers to the adipose tissue surrounding organs, within the abdomen and thorax, and is comprised predominantly of white adipocytes. This depot has been implicated in mediating obesity-related dysmetabolism. PVAT refers to adipose tissue surrounding major arteries. It had long been thought to exist to provide protection and insulation for the vessel, yet recent work demonstrates an important role for PVAT in harboring immune cells, promoting their function and regulating the biology of the underlying vessel. The role of B-2 cells and adaptive immunity in adipose tissue biology has been nicely reviewed elsewhere. Given that, the predominance of B-1 cells in adipose tissue at homeostasis, and the emerging role of B-1 cells in a variety of disease states, we will focus this review on how B-1 cells function in VAT and PVAT depots to promote homeostasis and limit inflammation linked to cardiometabolic disease and factors that regulate this function.

Loss of TET2 increases B-1 cell number and IgM production while limiting CDR3 diversity.

Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of VH11 and VH12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM.

A protective role for B-1 cells and oxidation-specific epitope IgM in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a morbid fibrotic lung disease with limited treatment options. The pathophysiology of IPF remains poorly understood, and elucidation of the cellular and molecular mechanisms of IPF pathogenesis is key to the development of new therapeutics. B-1 cells are an innate B cell population which play an important role linking innate and adaptive immunity. B-1 cells spontaneously secrete natural IgM and prevent inflammation in several disease states. One class of these IgM recognize oxidation-specific epitopes (OSE), which have been shown to be generated in lung injury and to promote fibrosis. A main B-1 cell reservoir is the pleural space, adjacent to the typical distribution of fibrosis in IPF. In this study, we demonstrate that B-1 cells are recruited to the lung during injury where they secrete IgM to OSE (IgM OSE ). We also show that the pleural B-1 cell reservoir responds to lung injury through regulation of the chemokine receptor CXCR4. Mechanistically we show that the transcription factor Id3 is a novel negative regulator of CXCR4 expression. Using mice with B-cell specific Id3 deficiency, a model of increased B-1b cells, we demonstrate decreased bleomycin-induced fibrosis compared to littermate controls. Furthermore, we show that mice deficient in secretory IgM ( sIgM -/- ) have higher mortality in response to bleomycin-induced lung injury, which is partially mitigated through airway delivery of the IgM OSE E06. Additionally, we provide insight into potential mechanisms of IgM in attenuation of fibrosis through RNA sequencing and pathway analysis, highlighting complement activation and extracellular matrix deposition as key differentially regulated pathways.

Distinct Type 1 Immune Networks Underlie the Severity of Restrictive Lung Disease after COVID-19.

The variable etiology of persistent breathlessness after COVID-19 have confounded efforts to decipher the immunopathology of lung sequelae. Here, we analyzed hundreds of cellular and molecular features in the context of discrete pulmonary phenotypes to define the systemic immune landscape of post-COVID lung disease. Cluster analysis of lung physiology measures highlighted two phenotypes of restrictive lung disease that differed by their impaired diffusion and severity of fibrosis. Machine learning revealed marked CCR5+CD95+ CD8+ T-cell perturbations in mild-to-moderate lung disease, but attenuated T-cell responses hallmarked by elevated CXCL13 in more severe disease. Distinct sets of cells, mediators, and autoantibodies distinguished each restrictive phenotype, and differed from those of patients without significant lung involvement. These differences were reflected in divergent T-cell-based type 1 networks according to severity of lung disease. Our findings, which provide an immunological basis for active lung injury versus advanced disease after COVID-19, might offer new targets for treatment.

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.

Marginal zone B cells produce 'natural' atheroprotective IgM antibodies in a T cell-dependent manner.

AIMS: The adaptive immune response plays an important role in atherosclerosis. In response to a high-fat/high-cholesterol (HF/HC) diet, marginal zone B (MZB) cells activate an atheroprotective programme by regulating the differentiation and accumulation of 'poorly differentiated' T follicular helper (Tfh) cells. On the other hand, Tfh cells activate the germinal centre response, which promotes atherosclerosis through the production of class-switched high-affinity antibodies. We therefore investigated the direct role of Tfh cells and the role of IL18 in Tfh differentiation in atherosclerosis. METHODS AND RESULTS: We generated atherosclerotic mouse models with selective genetic deletion of Tfh cells, MZB cells, or IL18 signalling in Tfh cells. Surprisingly, mice lacking Tfh cells had increased atherosclerosis. Lack of Tfh not only reduced class-switched IgG antibodies against oxidation-specific epitopes (OSEs) but also reduced atheroprotective natural IgM-type anti-phosphorylcholine (PC) antibodies, despite no alteration of natural B1 cells. Moreover, the absence of Tfh cells was associated with an accumulation of MZB cells with substantially reduced ability to secrete antibodies. In the same manner, MZB cell deficiency in Ldlr-/- mice was associated with a significant decrease in atheroprotective IgM antibodies, including natural anti-PC IgM antibodies. In humans, we found a positive correlation between circulating MZB-like cells and anti-OSE IgM antibodies. Finally, we identified an important role for IL18 signalling in HF/HC diet-induced Tfh. CONCLUSION: Our findings reveal a previously unsuspected role of MZB cells in regulating atheroprotective 'natural' IgM antibody production in a Tfh-dependent manner, which could have important pathophysiological and therapeutic implications.

Single cell transcriptomics reveals recent CD8T cell receptor signaling in patients with coronary artery disease.

Coronary artery disease (CAD) is a major cause of death worldwide. The role of CD8+ T cells in CAD is unknown. Recent studies suggest a breakdown of tolerance in atherosclerosis, resulting in active T cell receptor (TCR) engagement with self-antigens. We hypothesized that TCR engagement would leave characteristic gene expression signatures. In a single cell RNA-sequencing analysis of CD8+ T cells from 30 patients with CAD and 30 controls we found significant enrichment of TCR signaling pathways in CAD+ subjects, suggesting recent TCR engagement. We also found significant enrichment of cytotoxic and exhaustion pathways in CAD cases compared to controls. Highly significant upregulation of TCR signaling in CAD indicates that CD8 T cells reactive to atherosclerosis antigens are prominent in the blood of CAD cases compared to controls.

Characterizing blood pressure trajectories in people living with HIV following antiretroviral therapy: A systematic review.

OBJECTIVES: The advent of antiretroviral therapy (ART) has reduced AIDS-related morbidity and mortality among people living with HIV (PLWH). Due to increased survival, PLWH have now been found to be at risk of chronic conditions related to ageing, such as cardiovascular disease (CVD). Hypertension is common in PLWH and is a major risk factor for the development of CVD. We conducted a systematic literature review to evaluate the research evidence on longitudinal blood pressure (BP) trajectories following ART initiation in PLWH. METHODS: We searched the following databases: PubMed, CINHAL, Scopus, and Web of Science (up to 15 March 2021) for peer-reviewed published studies that reported BP trajectories following ART initiation in PLWH. Three reviewers independently screened all studies by title and abstract. We included articles in English, published up to March 2021, that report office BP trajectories in PLWH initiating ART. A total of 10 publications met our inclusion criteria. Eight studies were prospective cohorts and two were retrospective. RESULTS: Nine out of 10 studies in the literature reported an increase in systolic BP (4.7-10.0 mmHg in studies with a follow-up range of 6 months to 8 years, and 3.0-4.7 mmHg/year in time-averaged studies). In addition, four out of 10 studies reported increases in diastolic BP (2.3-8.0 mmHg for a 6 month to 6.8-year follow-up range and 2.3 mmHg/year). CONCLUSION: Systolic BP consistently increases while diastolic BP changes are more heterogeneous following ART initiation in PLWH. However, the studies were highly variable with respect to population demographics, ART regimen and duration, and follow-up time. Nevertheless, given the risks of CVD complications, such as stroke, heart failure and myocardial infarction, associated with elevated BP, results highlight the importance of future research in this area. It will be important to better characterize BP trajectories over time, identify the most critical times for interventions to reduce BP, determine the long-term CVD consequences in PLWH with elevated BP, and understand how different ART regimens may or may not influence BP and CVD disease.

Human circulating CD24hi marginal zone B cells produce IgM targeting atherogenic antigens and confer protection from vascular disease.

IgMs that inactivate oxidation-specific epitopes (IgMOSE), which are secondary products of lipid peroxidization, protect against inflammatory diseases, including diet-induced atherosclerosis. However, the human B cell subtype that produces IgMOSE remains unknown. In this study, we used single-cell mass cytometry and adoptive transfer of B cell subtypes to NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice to identify B27+IgM+CD24hi cells as the major producers of IgMOSE in humans. Notably, these cells have characteristics of human circulatory marginal zone B (MZB) cells, which are known to be atheoroprotective IgM producers in mice. CD24 antibody treatment to reduce MZB cells and IgM in a hyperlipidemic humanized mouse model provides the evidence that MZB cells protect against vascular inflammation. Consistent with these findings, the frequency of B27+IgM+CD24hi cells (MZB) in patients inversely correlates with coronary artery disease severity.

Single-cell profiling of CD11c+ B cells in atherosclerosis.

Circulating CD11c+ B cells, a novel subset of activated B cells, have been linked to autoimmunity and shown to expand with age. Atherosclerosis is an age-associated disease that involves innate and adaptive immune responses to modified self-antigens. Yet, the expression of CD11c on specific B-cell subtypes and its link to atherosclerosis are poorly understood. In this study, we characterized the frequency of CD11c+ B cells in tissues in mice with aging. We observed an age-associated increase in CD11c+ B cells in the spleen and bone marrow of ApoE-/- mice, and this was associated with an increase in aortic plaque. In addition, we also utilized single-cell multi-omics profiling of 60 human subjects undergoing advanced imaging for coronary artery disease (CAD) to subtype CD11c+ B cells and determine their frequency in subjects with high and low severity of CAD. Using unsupervised clustering, we identified four distinct clusters of CD11c+ B cells, which include CD27 and IgD double negative 2 (DN2), age-associated (ABC), CD11c+ unswitched memory (USWM), and activated Naïve (aNav) B cells. We observed an increase in the frequency of both ABC B cells and DN2 B cells in patients with high CAD severity. Pathway analysis further demonstrated augmentation of autophagy, IFNg signaling, and TLR signaling in DN2 cells in high-severity CAD patients. On the other hand, an increase in the negative regulator of BCR signaling through CD72 was found in ABC cells in low-severity CAD patients. Through investigating scRNAseq of atheroma, these DN2 cells were also found to infiltrate human coronary atheroma.

BAFF antagonism via the BAFF receptor 3 binding site attenuates BAFF 60-mer-induced classical NF-κB signaling and metabolic reprogramming of B cells.

Human recombinant B cell activating factor (BAFF) is secreted as 3-mers, which can associate to form 60-mers in culture supernatants. However, the presence of BAFF multimers in humans is still debated and it is incompletely understood how BAFF multimers activate the B cells. Here, we demonstrate that BAFF can exist as 60-mers or higher order multimers in human plasma. In vitro, BAFF 60-mer strongly induced the transcriptome of B cells which was partly attenuated by antagonism using a soluble fragment of BAFF receptor 3. Furthermore, compared to BAFF 3-mer, BAFF 60-mer strongly induced a transient classical and prolonged alternate NF-κB signaling, glucose oxidation by both aerobic glycolysis and oxidative phosphorylation, and succinate utilization by mitochondria. BAFF antagonism selectively attenuated classical NF-κB signaling and glucose oxidation. Altogether, our results suggest critical roles of BAFF 60-mer and its BAFF receptor 3 binding site in hyperactivation of B cells.

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.

B-1b Cells Have Unique Functional Traits Compared to B-1a Cells at Homeostasis and in Aged Hyperlipidemic Mice With Atherosclerosis.

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-vivo studies demonstrated greater migration to spleen, splenic production of IgM and plasma IgM levels in ApoE-/-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 atherosclerotic ApoE-/- mice compared to young ApoE-/- mice. Single cell sequencing results of IgHM in B-1a and B-1b cells from peritoneal cavity and spleen of atherosclerotic aged ApoE-/- 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.

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.