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Age-related deficits in the immune system have been associated with an increased incidence of infections, autoimmune diseases, and cancer. Human B cell populations change quantitatively and qualitatively in the elderly. However, the function of human B-1 cells, which play critical anti-microbial and housekeeping roles, have not been studied in the older age population. In the present work, we analyzed how the frequency, function and repertoire of human peripheral blood B-1 cells (CD19+CD20+CD27+CD38 CD43+) change with age. Our results show that not only the percentage of B-1 cells but also their ability to spontaneously secrete IgM decreased with age. Further, expression levels of the transcription factors XBP-1 and Blimp-1 were significantly lower, while PAX-5, characteristic of non-secreting B cells, was significantly higher, in healthy donors over 65 years (old) as compared to healthy donors between 20 and 45 years (young). To further characterize the B-1 cell population in older individuals, we performed single cell sequencing analysis of IgM heavy chains from healthy young and old donors. We found reduced repertoire diversity of IgM antibodies in B-1 cells from older donors as well as differences in usage of certain VH and DH specific genes, as compared to younger. Overall, our results show impairment of the human B-1 cell population with advancing age, which might impact the quality of life and onset of disease within the elderly population.

B-1 cells are a distinct lineage of tissue-resident B cells with crucial roles in innate immunity and tissue homeostasis. Mature B-1 cell pools are mostly maintained by self-renewal in their peripheral niches, in a process that is largely uncharacterized. Here, we investigated the role of the transcription repressor Bach2 in maintaining the B-1 cell pool. We found that B-1 cell numbers and antibody responses were dramatically reduced in adult mice bearing a B cell-specific Bach2 deletion, although the proportions of B-1 progenitors in early neonatal life were unaffected. Cells taken from the fetal liver or bone marrow of Bach2 -deleted mice were defective in reconstituting the B-1 cell pool in the peritonea of Rag2 -/- hosts, and peritoneal B-1 cell transplants from adult Bach2 -deleted mice failed to sustain their numbers in the host’s peritoneum. The mutant B-1 cells proliferated normally in vivo but were more apoptotic. They also expressed the reduced level of the self-renewal factor Bmi1. These results indicate that Bach2 deficiency does not affect the development of B-1 progenitors in fetal liver and bone marrow but impairs the self-renewal of mature B-1 cells in peripheral tissues, which is caused by increased apoptosis. Thus, this study suggests that a cell-autonomous function of Bach2 is crucial for maintaining a stable population size of B-1 cells in their peripheral niches.

B-1a cells constitutively secrete natural antibody that provides immediate protection against microbial pathogens and functions homeostatically to speed removal of apoptotic cell debris. Although B-1a cells are especially prominent in the peritoneal and pleural cavities, some B-1a cells reside in the spleen. A small subset of splenic B-1a cells in naïve, unimmunized mice express CD138, a recognized plasma cell antigen, whereas the bulk of splenic B-1a cells are CD138 negative. Splenic B-1a cells in toto have been shown to generate much more antibody per cell than peritoneal B-1a cells; however, specific functional information regarding CD138(+) splenic B-1a cells has been lacking. Here, we find a higher proportion of CD138(+) splenic B-1a cells spontaneously secrete more IgM as compared to CD138(-) B-1a cells. Moreover, IgM secreted by CD138(+) splenic B-1a cells is skewed with respect to N-region addition, and some aspects of VH and JH utilization, as compared to CD138(-) splenic B-1a cells and peritoneal B-1a cells. The small population of CD138(+) splenic B-1a cells is likely responsible for a substantial portion of natural IgM and differs from IgM produced by other B-1a cell subsets.

serotype ( ) 2 is an important bacterial pathogen in piglet nurseries and an emerging zoonotic agent without effective vaccines available. Immunoglobulin (Ig)M plays an essential role in host defense against . In mice, non-conventional B-1 cells are a major source of protective IgM against encapsulated bacterial pathogens, such as . Two IgM CD21 B-1-like cell subpopulations, distinguishable by CD11R1 expression, were described in pigs, but their properties and functions are poorly understood. This study aimed at a first characterization of the porcine early IgM B cell response against 2. We analyzed the same healthy pigs, naturally colonized by different serotypes, including 2, at four and eight weeks of age serologically and determined the frequency of different peripheral B cell subpopulations by flow cytometry. Furthermore, we isolated conventional IgM CD21 B-2 cells as well as non-conventional B-1-like cell subpopulations from peripheral blood of eight-weeks-old pigs to evaluate their potential of IgM secretion in response to innate and adaptive stimuli . Between the fourth and eighth week of life, a characteristic increase of 2-binding serum IgM antibodies, restricting bacterial growth, was observed. Moreover, we show for the first time that the significant increase of anti- serum IgM is associated with a relative increase of peripheral non-conventional IgM CD21 B-1-like cells , particularly of the IgM CD21 CD11R1 subpopulation. Noteworthy, sorted IgM CD21 CD11R1 B-1-like cells from eight-weeks-old pigs spontaneously secreted IgM . In addition, both non-conventional IgM CD21 B cell subpopulations, in contrast to conventional IgM CD21 B-2 cells, produced anti- IgM upon toll-like receptor (TLR) stimulation underlining their innate-like characteristics. We furthermore observed that both B-1-like subpopulations secrete 2-binding IgM upon stimulation with T cell-associated factors with highest amounts in IgM CD21 CD11R1 B-1-like cells even exceeding anti- IgM levels produced by B-2 cells. Porcine non-conventional B-1-like cells are a potent source of -binding IgM indicating a role in immunity during a critical phase of piglet rearing.

Only few studies have described the anti-tumor properties of natural antibodies (NAbs). In particular, natural IgM have been linked to cancer immunosurveillance due to its preferential binding to tumor-specific glycolipids and carbohydrate structures. Neu5GcGM3 ganglioside is a sialic acid–containing glycosphingolipid that has been considered an attractive target for cancer immunotherapy, since it is not naturally expressed in healthy human tissues and it is overexpressed in several tumors. Screening of immortalized mouse peritoneal-derived hybridomas showed that peritoneal B-1 cells contain anti-Neu5GcGM3 antibodies on its repertoire, establishing a link between B-1 cells, NAbs and anti-tumor immunity. Previously, we described the existence of naturally-occurring anti-Neu5GcGM3 antibodies with anti-tumor properties in healthy young humans. Interestingly, anti-Neu5GcGM3 antibodies level decreases with age and is almost absent in non-small cell lung cancer patients. Although anti-Neu5GcGM3 antibodies may be clinically relevant, the identity of the human B cells participating in this anti-tumor antibody response is unknown. In this work, we found an increased percentage of circulating human B-1 cells in healthy individuals with anti-Neu5GcGM3 IgM antibodies. Furthermore, anti-Neu5GcGM3 IgMs were generated predominantly by human B-1 cells and the antibodies secreted by these B-1 lymphocytes also recognized Neu5GcGM3-positive tumor cells. These data suggest a protective role for human B-1 cells against malignant transformation through the production of NAbs reactive to tumor-specific antigens such as Neu5GcGM3 ganglioside.

B-1 cells have intricate biology, with distinct function, phenotype and developmental origin from conventional B cells. They generate a B cell receptor with conserved germline characteristics and biased V(D)J recombination, allowing this innate-like lymphocyte to spontaneously produce self-reactive natural antibodies (NAbs) and become activated by immune stimuli in a T cell-independent manner. NAbs were suggested as “rheostats” for the chronic diseases in advanced age. In fact, age-dependent loss of function of NAbs has been associated with clinically-relevant diseases in the elderly, such as atherosclerosis and neurodegenerative disorders. Here, we analyzed comprehensively the ontogeny, phenotypic characteristics, functional properties and emerging roles of B-1 cells and NAbs in health and disease. Additionally, after navigating through the complexities of B-1 cell biology from development to aging, therapeutic opportunities in the field are discussed. Highlights • B-1 cell development is regulated by specific transcription factors and this innate-like B cell bypasses selection phases during lymphopoiesis to generate polyreactive BCRs. • B-1 cell-derived cytokines have immunoregulatory effects during homeostasis and diseases. • Natural antibodies act as “eat-me” signals for the phagocytosis of cellular debris and invading pathogens. • Impairment in natural antibody function is correlated with onset and progression of chronic diseases in the elderly.

Systemic lupus erythematosus is an autoimmune disease in which pathogenic autoantibodies targeting nucleic acid containing antigens promote inflammation and tissue damage. Recent reports suggest that deep B cell depletion will be a highly effective therapeutic strategy for lupus. However, elimination of all B cells confers susceptibility to infection. Thus, an approach which targets pathogenic B cells but spares protective ones would be ideal. The B-1 subset of B cells has been suggested to be either pathogenic or protective in lupus, depending on the study. We used several complementary approaches to define the contribution of B-1 cells to autoantibody production and immune cell activation in the Lyn-/- mouse model of lupus. We labeled activated B-1 cells to track their cellular and antibody progeny. Activated B-1 cells were also depleted or prevented from differentiating into plasma cells. B-1 cells contributed significantly to the accumulation of splenic plasma cells and total IgM characteristic of Lyn-/- mice in a manner at least partially independent of the transcription factor IRF4. Unlike T-bet+ B cells, they were not a major source of pathogenic lupus-associated autoantibodies. Rather, they limited both the production of IgG against other autoantigens and the activation of CD8+ T cells. These studies highlight a regulatory role for B-1 cells in shaping adaptive immune tolerance in the Lyn-/- lupus model.

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. 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. 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.

Similar to human chronic lymphocytic leukemia (CLL), the de novo New Zealand Black (NZB) mouse model has a genetically determined age-associated increase in malignant B-1 clones and decreased expression of microRNAs miR-15a and miR-16 in B-1 cells. In the present study, lentiviral vectors were employed in vivo to restore miR-15a/16, and both the short-term single injection and long-term multiple injection effects of this delivery were observed in NZB. Control lentivirus without the mir-15a/16 sequence was used for comparison. We found that in vivo lentiviral delivery of mir-15a/16 increased miR-15a/16 expression in cells that were transduced (detected by GFP expression) and in sera when compared with control lentivirus treatment. More importantly, mice treated with the miR-expressing lentivirus had decreased disease. The lentivirus had little systemic toxicity while preferentially targeting B-1 cells. Short-term effects on B-1 cells were direct effects, and only malignant B-1 cells transduced with miR-15a/16 lentivirus had decreased viability. In contrast, long-term studies suggested both direct and indirect effects resulting from miR-15a/16 lentivirus treatment. A decrease in B-1 cells was found in both the transduced and non-transduced populations. Our data support the potential use of systemic lentiviral delivery of miR-15a/16 to ameliorate disease manifestations of CLL.

B1 cells constitute a specialized subset of B cells, best characterized in mice, which is abundant in body cavities, including the peritoneal cavity. Through natural and antigen-induced antibody production, B1 cells participate in the early defense against bacteria. The G protein-coupled receptor 183 (GPR183), also known as Epstein-Barr virus-induced gene 2 (EBI2), is an oxysterol-activated chemotactic receptor that regulates migration of B cells. We investigated the role of GPR183 in B1 cells in the peritoneal cavity and omentum. B1 cells expressed GPR183 at the mRNA level and migrated towards the GPR183 ligand 7α,25-dihydroxycholesterol (7α,25-OHC). GPR183 knock-out (KO) mice had smaller omenta, but with normal numbers of B1 cells, whereas they had fewer B2 cells in the omentum and peritoneal cavity than wildtype (WT) mice. GPR183 was not responsible for B1 cell accumulation in the omentum in response to i.p. lipopolysaccharide (LPS)-injection, in spite of a massive increase in 7α,25-OHC levels. Lack of GPR183 also did not affect B1a- or B1b cell-specific antibody responses after vaccination. In conclusion, we found that GPR183 is non-essential for the accumulation and function of B1 cells in the omentum and peritoneal cavity, but that it influences the abundance of B2 cells in these compartments.