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.

B-1 cells are a unique subset of B cells that are positively selected for expressing autoreactive BCRs. We isolated RNA from peritoneal (B-1a, B-1b, B-2) and splenic (B-1a, marginal zone, follicular) B cells from C57BL/6 mice and used 5'-RACE to amplify the IgH V region using massively parallel sequencing. By analyzing 379,000 functional transcripts, we demonstrate that B-1a cells use a distinct and restricted repertoire. All B-1 cell subsets, especially peritoneal B-1a cells, had a high proportion of sequences without N additions, suggesting predominantly prenatal development. Their transcripts differed markedly and uniquely contained V11 and V12 genes, which were rearranged only with a restricted selection of D and J genes, unlike other V genes. Compared to peritoneal B-1a, the peritoneal B-1b repertoire was larger, had little overlap with B-1a, and most sequences contained N additions. Similarly, the splenic B-1a repertoire differed from peritoneal B-1a sequences, having more unique sequences and more frequent N additions, suggesting influx of B-1a cells into the spleen from nonperitoneal sites. Two CDR3s, previously described as Abs to bromelain-treated RBCs, comprised 43% of peritoneal B-1a sequences. We show that a single-chain variable fragment designed after the most prevalent B-1a sequence bound oxidation-specific epitopes such as the phosphocholine of oxidized phospholipids. In summary, we provide the IgH V region library of six murine B cell subsets, including, to our knowledge for the first time, a comparison between B-1a and B-1b cells, and we highlight qualities of B-1 cell Abs that indicate unique selection processes.

Background: The blood-brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases' progression. This functional damage is one key event which is attempted to be mimicked in in vitro models. Recent studies showed the pivotal role of micro-environmental cells such as astrocytes for this barrier damage in mouse stroke in vitro models. The aim of this study was to evaluate the role of micro-environmental cells for the functional, paracellular breakdown in a human BBB cerebral ischemia in vitro model accompanied by a transcriptional analysis. Methods: Transwell models with human brain endothelial cell line hCMEC/D3 in mono-culture or co-culture with human primary astrocytes and pericytes or rat glioma cell line C6 were subjected to oxygen/glucose deprivation (OGD). Changes of transendothelial electrical resistance (TEER) and FITC-dextran 4000 permeability were recorded as measures for paracellular tightness. In addition, qPCR and high-throughput qPCR Barrier chips were applied to investigate the changes of the mRNA expression of 38 relevant, expressed barrier targets (tight junctions, ABC-transporters) by different treatments. Results: In contrast to the mono-culture, the co-cultivation with human primary astrocytes/pericytes or glioma C6 cells resulted in a significantly increased paracellular permeability after 5 h OGD. This indicated the pivotal role of micro-environmental cells for BBB breakdown in the human model. Hierarchical cluster analysis of qPCR data revealed differently, but also commonly regulated clustered targets dependent on medium exchange, serum reduction, hydrocortisone addition and co-cultivations. Conclusions: The co-cultivation with micro-environmental cells is necessary to achieve a functional breakdown of the BBB in the cerebral ischemia model within an in vivo relevant time window. Comprehensive studies by qPCR revealed that distinct expression clusters of barrier markers exist and that these are regulated by different treatments (even by growth medium change) indicating that controls for single cell culture manipulation steps are crucial to understand the observed effects properly.

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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,p<0.0001) and IgM antibody-secreting cells (n=6,p<0.01) in the bone marrow, and reduced plasma IgM levels (n=6-8,p<0.05), relative to littermate controls (CXCR4 WT ). Furthermore, retroviral-mediated overexpression of CXCR4 on B1a cells in vivo is associated with increased B1a localization to the bone marrow (p<0.01) and increased circulating levels of anti-MDA-LDL IgM antibodies (p<0.05). To determine the atheroprotective role of CXCR4 on the B1a cell subset, we adoptively transferred CXCR4 WT 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,p<0.001), and fewer donor B1a cells in the bone marrow and spleen (n=7,p<0.05) compared to recipients given CXCR4 WT B1a cells. Intriguingly, B1a transfer reduces plasma cholesterol levels in mice regardless of CXCR4 expression (n=7,p<0.05). However, CXCR4 further strengthens the atheroprotective ability of B1a cells, as recipients given CXCR4 WT B1a cells have reduced aortic lesion area compared to PBS controls (n=7,p<0.01) while recipients given CXCR4 BKO 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.