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Introduction: Natural antibody (NAb) derived from CD5+ B-1 cells maintains tissue homeostasis, controls inflammation, aids in establishing long-term protective responses against pathogens, and provides immediate protection from infection. CD5+ B-1 cell NAbs recognize evolutionarily fixed epitopes, such as phosphatidylcholine (PtC), found on bacteria and senescent red blood cells. Anti-PtC antibodies are essential in protection against bacterial sepsis. CD5+ B-1 cell-derived NAbs have a unique germline-like structure that lacks N-additions, a feature critical for providing protection against infection. Previously, we demonstrated the repertoire and germline status of PtC+CD5+ B-1 cell IgM obtained from male mice changes with age depending on the anatomical location of the B-1 cells. More recently, we demonstrated serum antibody from aged female mice maintains protection against pneumococcal infection, whereas serum antibody from male mice does not provide protection., Results: Here, we show that aged female mice have significantly more splenic PtC+CD5+ B-1 cells and more PtC specific serum IgM than aged male mice. Furthermore, we find both age and biological sex related repertoire differences when comparing B cell receptor (BCR) sequencing results of PtC+CD5+ B-1 cells. While BCR germline status of PtC+CD5+ B-1 cells from aged male and female mice is similar in the peritoneal cavity, it differs significantly in the spleen, where aged females retain germline configuration and aged males do not. Nucleic acid sensing toll-like receptors are critical in the maintenance of PtC+ B-1 cells; therefore, to begin to understand the mechanism of differences observed between the male and female PtC+CD5+ B-1 cell repertoire, we analyzed levels of cell-free nucleic acids and found increases in aged females., Conclusion: Our results suggest the antigenic milieu differs between aged males and females, leading to differential selection of antigen-specific B-1 cells over time. Further elucidation of how biological sex differences influence the maintenance of B-1 cells within the aging environment will be essential to understand sex and age-related disparities in the susceptibility to bacterial infection and will aid in the development of more effective vaccination and/or therapeutic strategies specific for males and females., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Webster, Tsuji, Clemente and Holodick.)

Natural Abs are primarily produced by B-1 cells and are essential for protection against Streptococcus pneumoniae The incidence and mortality rate for pneumococcal infection increases dramatically after age 65, disproportionately affecting males in both human and murine systems. To date, there is a significant gap in our understanding of the relationship among sex, aging, natural IgM efficacy, and the natural IgM repertoire. Our investigation demonstrates that the protective capacity of serum IgM against pneumococcal infection is maintained in IgM obtained from aged female mice but absent in IgM from aged male mice. To understand this difference in protective capacity, we examined serum Ig, discovering that the protective change was not associated with shifts in levels of phosphorylcholine (PC)- or pneumococcal capsular polysaccharide serotype 3-specific IgM. Interestingly, we observed that aged females have an increase in the total number of CD5 + B-1 cells, higher serum IL-5 levels, and a larger percentage of aged female CD5 + B-1 cells that express CD86 as compared with aged males. Furthermore, single-cell IgM repertoire analysis from peritoneal PC + , splenic PC + , and bone marrow CD5 + B-1 cell subsets demonstrated greater diversity with age and a higher level of germline status in female mice than previously observed in studies of aged male mice. Aged female CD5 + B-1 cells also expressed higher levels of transcripts associated with cell activity and self-renewal, such as Nanog and Hmga2 Taken together, these data indicate that females maintain a more diverse and active CD5 + B-1 cell pool and natural IgM repertoire, which has implications for sex-related susceptibility to infection and disease., (Copyright © 2022 by The American Association of Immunologists, Inc.)

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.

Bacterial sepsis is a serious life-threatening condition caused by an excessive immune response to infection. B-1 cells differ from conventional B-2 cells by their distinct phenotype and function. A subset of B-1 cells expressing CD5, known as B-1a cells, exhibits innate immune activity. Here we report that B-1a cells play a beneficial role in sepsis by mitigating exaggerated inflammation through a novel mechanism. Using a mouse model of bacterial sepsis, we found that the numbers of B-1a cells in various anatomical locations were significantly decreased. Adoptive transfer of B-1a cells into septic mice significantly attenuated systemic inflammation and improved survival, whereas B-1a cell-deficient CD19 -/- mice were more susceptible to infectious inflammation and mortality. We also demonstrated B-1a cells produced ample amounts of IL-10 which controlled excessive inflammation and the mice treated with IL-10-deficient B-1a cells were not protected against sepsis. Moreover, we identified a novel intracellular signaling molecule, cAMP-response element binding protein (CREB), which serves as a pivotal transcription factor for upregulating IL-10 production by B-1a cells in sepsis through its nuclear translocation and binding to putative responsive elements on IL-10 promoter. Thus, the benefit of B-1a cells in bacterial sepsis is mediated by CREB and the identification of CREB in B-1a cells reveals a potential avenue for treatment in bacterial sepsis., (Copyright © 2017 by The American Association of Immunologists, Inc.)

P3 is a murine, germline, IgM mAb that recognizes N -glycolylated gangliosides and other self-antigens. This antibody is able to induce an anti-idiotypic IgG response and B-T idiotypic cascade, even in the absence of any adjuvant or carrier protein. P3 mAb immunization induces the expression of activation markers in a significant percentage of B-1a cells in vivo. Interestingly, transfer of both B-1a and B-2 to BALB/Xid mice was required to recover anti-P3 IgG response in this model. In fact, P3 mAb activated B-2 cells, in vitro, inducing secretion of IFN-γ and IL-4, although this activation was not detected ex vivo. Interestingly, naïve CD8 + T cells increased the expression of activation markers and IFN-γ secretion in the presence of B-1a cells isolated from P3 mAb-immunized mice, even without in vitro restimulation. In contrast, B-2 cells were able to stimulate CD8 + T cells only if P3 was added in vitro. Using bioinformatics, a MHC class I-binding peptide from P3 V H region was identified. P3 mAb was able to induce a specific CTL response in vivo against cells presenting this peptide. Both humoral and CTL anti-idiotypic responses could be mechanisms to protect against the self-reactive antibody, contributing to keeping the tolerance to self-antigens.

The B-1 B cell population is an important bridge between innate and adaptive immunity primarily because B-1 cells produce natural Ab. Murine B-1 and B-2 cells arise from distinct progenitors; however, in humans, in part because it has been difficult to discriminate between them phenotypically, efforts to pinpoint the developmental origins of human B-1 and B-2 cells have lagged. To characterize progenitors of human B-1 and B-2 cells, we separated cord blood and bone marrow Lin - CD34 + hematopoietic stem cells into Lin - CD34 + CD38 lo and Lin - CD34 + CD38 hi populations. We found that transplanted Lin - CD34 + CD38 lo cells, but not Lin - CD34 + CD38 hi cells, generated a CD19 + B cell population after transfer into immunodeficient NOD.Cg-Prkdc scid Il2rg tm1wjl /SxJ neonates. The emergent CD19 + B cell population was found in spleen, bone marrow, and peritoneal cavity of humanized mice and included distinct populations displaying the B-1 or the B-2 cell phenotype. Engrafted splenic B-1 cells exhibited a mature phenotype, as evidenced by low-to-intermediate expression levels of CD24 and CD38. The engrafted B-1 cell population expressed a VH-DH-JH composition similar to cord blood B-1 cells, including frequent use of VH4-34 (8 versus 10%, respectively). Among patients with hematologic malignancies who underwent hematopoietic stem cell transplantation, B-1 cells were found in the circulation as early as 8 wk posttransplantation. Altogether, our data demonstrate that human B-1 and B-2 cells develop from a Lin - CD34 + CD38 lo stem cell population, and engrafted B-1 cells in humanized mice exhibit an Ig-usage pattern comparable to B-1 cells in cord blood., (Copyright © 2016 by The American Association of Immunologists, Inc.)

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.)

The investigation of immune senescence has uncovered many changes in B cell development, maintenance, and function with increasing age. However, most of these studies have focused on conventional B cell subsets in the spleen. The B-1 cell subset is an essential arm of the innate immune system, which in general has been understudied in terms of immune senescence. Here, we review what is currently known about B cells during aging and go on to describe why B-1 cell biology is an important component of the aging immune system in the context of diseases that most affect the aged population., (© 2015 New York Academy of Sciences.)

Natural Abs produced by B-1a cells are required for immediate protection against infection. The protective capacity of natural Abs is attributed to germline-like structure, which includes the relative absence of N-region addition. Previous studies have shown B-1a cell Ig from aged mice contains abundant nontemplated (N)-additions. B-1a cells have been shown to derive from a specific lineage-negative (Lin(-))CD45R(low/-)CD19(+) progenitor found both in fetal liver and adult bone marrow. In this study, we report identification of a fetal liver population characterized phenotypically as Lin(-)CD45R(-)CD19(-), which gives rise to IgM(+)IgD(low)CD45R(low)CD5(+)Mac-1(+)CD19(high)CD43(+)CD23(low) B-1a cells upon adoptive transfer to SCID recipients. These B-1a cells derived from the Lin(-)CD45R(-)CD19(-) fetal liver population produce natural Ab that binds pneumococcal Ags, but this Ig contains substantial N-addition despite initial absence of TdT. Furthermore, we show extensive N-addition is also present in B-1a cells derived from the Lin(-)CD45R(low/-)CD19(+) B-1 progenitor found in the bone marrow. Together these results demonstrate B-1a cell N-addition depends on the type of progenitor and the location of the progenitor during its development. These findings have implications for how regulation of different progenitors from fetal liver and bone marrow may play a role in the age-related increase in N-region addition by B-1a cells in normal animals.

B-1 cells play critical roles in defending against microbial invasion and in housekeeping removal of cellular debris. B-1 cells secrete natural antibody and manifest functions that influence T cell expansion and differentiation and in these and other ways differ from conventional B-2 cells. B-1 cells were originally studied in mice where they are easily distinguished from B-2 cells, but their identity in the human system remained poorly defined for many years. Recently, functional criteria for human B-1 cells were established on the basis of murine findings, and reverse engineering resulted in identification of the phenotypic profile, CD20(+)CD27(+)CD43(+)CD70(-), for B-1 cells found in both umbilical cord blood and adult peripheral blood. Human B-1 cells may contribute to multiple disease states through production of autoantibody and stimulation/modulation of T cell activity. Human B-1 cells could be a rich source of antibodies useful in treating diseases present in elderly populations where natural antibody protection may have eroded. Manipulation of human B-1 cell numbers and/or activity may be a new avenue for altering T cell function and treating immune dyscrasias., (© 2013 New York Academy of Sciences.)

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.)

The self-renewing capacity of B1 cells infers homeostatic regulation; however, previous work suggests the low level of N-region addition characterizing B1 cells early in life increases with age, which implies that the B1-cell population is not a closed system. To explore this, we evaluated N-region addition in CD5(+) B1 cells generated from adult BM. Adult BM cells were marked with GFP introduced by mouse stem cell virus transduction, and were then adoptively transferred into lethally irradiated recipients. Within 2-3 months, we found GFP-marked CD5(+) B cells in the peritoneal cavities of recipients, which we demonstrate here meet a variety of criteria for B1-cell traits including Mac-1 surface expression; annexin, elfin, and Pax-5 gene expression; mitogenic responsiveness to phorbol ester; and spontaneous immunoglobulin secretion. Notably, we found by single-cell PCR that this population of BM-derived CD5(+) B1 cells expressed immunoglobulin with abundant N-region addition (and little V(H)11/V(H)12 skewing), unlike CD5(+) B1 cells obtained from unmanipulated animals but reminiscent of B2 cells. Further, we confirmed that native CD5(+) B1 cells from older mice contain more N-region additions than native CD5(+) B1 cells from younger mice. These results suggest that adult BM progenitors contribute to the peritoneal CD5(+) B1-cell pool over time.