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B-1a cells provide immediate and essential protection from infection through production of natural Ig, which is germline-like due to minimal insertion of N region additions. We have previously demonstrated peritoneal B-1a cell-derived phosphorylcholine-specific and total IgM moves away from germline (as evidenced by an increase in N-additions) with age as a result of selection. In young mice, anti-phosphatidylcholine Abs, like anti-phosphorylcholine Abs, contain few N-additions, and have been shown to be essential in protection from bacterial sepsis. In this study, we demonstrate the germline-like status of phosphatidylcholine (PtC)-specific (PtC + ) peritoneal B-1a cell IgM does not change with age. In direct contrast, the splenic PtC + B-1a cell population does not preserve its IgM germline status in the aged mice. Furthermore, splenic PtC + B-1a cells displayed more diverse variable gene segments of the H chain (V H ) use in both the young and aged mice as compared with peritoneal PtC + B-1a cells. Whereas the peritoneal PtC + population increased V H 12 use with age, we observed differential use of V H 11, V H 12, and V H 2 between the peritoneal and splenic PtC + populations with age. These results suggest disparate selection pressures occur with age upon B-1a cells expressing different specificities in distinct locations. Overall, these results illuminate the need to further elucidate how B-1a cells are influenced over time in terms of production and selection, both of which contribute to the actual and available natural IgM repertoire with increasing age. Such studies would aid in the development of more effective vaccination and therapeutic strategies in the aged population., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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.

Streptococcus pneumoniae is the most common cause of pneumonia, which claims the lives of people over the age of 65 y seven times more frequently than those aged 5-49 y. B-1a cells provide immediate and essential protection from S. pneumoniae through production of natural Ig, which has minimal insertion of N-region additions added by the enzyme TdT. In experiments with SCID mice infected with S. pneumoniae, we found passive transfer of IgG-depleted serum from aged (18-24 mo old) mice had no effect whereas IgG-depleted serum from young (3 mo old) mice was protective. This suggests protective natural IgM changes with age. Using single cell PCR we found N-region addition, which is initially low in fetal-derived B-1a cell IgM developing in the absence of TdT, increased in 7- to 24-mo-old mice as compared with 3-mo-old mice. To determine the mechanism responsible for the age related change in B-1a cell IgM, we established a mixed chimera system in which mice were reconstituted with allotype-marked mature peritoneal B-1a cells and adult bone marrow cells. We demonstrated even in the presence of mature peritoneal B-1a cells, adult bone marrow contributed to the mature B-1a cell pool. More importantly, using this system we found over a 10-mo-period peritoneal B-1a cell IgM changed, showing the number of cells lacking N-region additions at both junctions fell from 49 to 29% of sequences. These results strongly suggest selection-induced skewing alters B-1a cell-derived natural Ab, which may in turn be responsible for the loss of natural IgM-mediated protection against pneumococcal infection., (Copyright © 2016 by The American Association of Immunologists, Inc.)

This study tested the hypothesis that B cells from salivary tissue are distinct in terms of proliferative capacity, immunoglobulin M secretion, repertoire, and autoantibody enrichment in Sjögren's syndrome. We sorted purified B cells from the spleen, cervical lymph nodes, and submandibular glands of a primary Sjögren's syndrome mouse model (Id3(-/-)). Enzyme-linked immunospot and proliferation assays were performed with stimulated B cells. We single-cell sorted B cells from the spleen, cervical lymph nodes, and submandibular gland tissue from Sjögren's syndrome mice and sequenced immunoglobulin M heavy-chain variable regions. Finally, autoantigen arrays were performed using immunoglobulin M derived from sera, cervical lymph nodes, spleens, and submandibular gland tissue of Id3(-/-) animals. Results suggest B cells from salivary tissue of Sjögren's syndrome mice are similar to those from secondary immune sites in terms of proliferative and secretory capacity. However, differences in repertoire usage, heavy chain complementarity-determining region 3 length, mutational frequency, and N region addition were observed among B cells derived from submandibular gland, cervical lymph node, and spleen tissue. Moreover, autoantigen array data show immunoglobulin M from salivary B cells have enriched specificity for Ro (Sjögren's syndrome A) and La (Sjögren's syndrome B). All together, these data suggest salivary B cells have unique repertoire characteristics that likely influence autoantigen binding and contribute to Sjögren's syndrome disease in a tissue-specific manner., (© Society for Leukocyte Biology.)

Peritoneal B1 cells are typified by spontaneous, constitutive secretion of IgM natural antibody, detected by ELISPOT assay, among other means. Recently, this key characteristic has been called into question, a reason for which we evaluated the integrity of IgM(+) ELISPOT spots. We found that fixed B1 cells fail to produce ELISPOT spots, that interference with Golgi function inhibits ELISPOT spot formation, and that B1 cell-derived immunoglobulin in supernatant samples is EndoH-resistant. These findings indicate that spots produced by B1 cells on ELISPOT assay reflect secretory IgM actively exported by viable B1 cells. Current paradigms propose that interferon response factor 4 (IRF4) is required for plasma cell differentiation and immunoglobulin secretion. However, we found that IgM secretion by peritoneal B1 cells is not altered in IRF4-null mice. In contrast, spontaneous IgM secretion by splenic B1 cells, which amounts to much more IgM secreted per cell, is dramatically reduced in the absence of IRF4. These results indicate that peritoneal B1 cells spontaneously secrete low levels of IgM via an IRF4-independent non-classical pathway, and, considering the low level of serum IgM in IRF-null mice, further suggest that accumulation of serum immunoglobulin depends on IRF4-dependent secretion by splenic B1 cells., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)