Sex Influences Age-Related Changes in Natural Antibodies and CD5+ B-1 Cells.

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.

Antigen Receptor Specificity and Cell Location Influence the Diversification and Selection of the B-1a Cell Pool with Age.

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 (VH) use in both the young and aged mice as compared with peritoneal PtC+ B-1a cells. Whereas the peritoneal PtC+ population increased VH12 use with age, we observed differential use of VH11, VH12, and VH2 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.

Diversification and CXCR4-Dependent Establishment of the Bone Marrow B-1a Cell Pool Governs Atheroprotective IgM Production Linked to Human Coronary Atherosclerosis.

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-1a Cells Protect Mice from Sepsis: Critical Role of CREB.

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.

B-1a cells protect mice from sepsis-induced acute lung injury.

BACKGROUND: Sepsis morbidity and mortality are aggravated by acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Mouse B-1a cells are a phenotypically and functionally unique sub-population of B cells, providing immediate protection against infection by releasing natural antibodies and immunomodulatory molecules. We hypothesize that B-1a cells ameliorate sepsis-induced ALI. METHODS: Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). PBS or B-1a cells were adoptively transferred into the septic mice intraperitoneally. After 20 h of CLP, lungs were harvested and assessed by PCR and ELISA for pro-inflammatory cytokines (IL-6, IL-1ß) and chemokine (MIP-2) expression, by histology for injury, by TUNEL and cleaved caspase-3 for apoptosis, and by myeloperoxidase (MPO) assay for neutrophil infiltration. RESULTS: We found that septic mice adoptively transferred with B-1a cells significantly decreased the mRNA and protein levels of IL-6, IL-1ß and MIP-2 in the lungs compared to PBS-treated mice. Mice treated with B-1a cells showed dramatic improvement in lung injury compared to PBS-treated mice after sepsis. We found apoptosis in the lungs was significantly inhibited in B-1a cell injected mice compared to PBS-treated mice after sepsis. B-1a cell treatment significantly down-regulated MPO levels in the lungs compared to PBS-treated mice in sepsis. The protective outcomes of B-1a cells in ALI was further confirmed by using B-1a cell deficient CD19-/- mice, which showed significant increase in the lung injury scores following sepsis as compared to WT mice. CONCLUSIONS: Our results demonstrate a novel therapeutic potential of B-1a cells to treat sepsis-induced ALI.

Age-Related Decline in Natural IgM Function: Diversification and Selection of the B-1a Cell Pool with Age.

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.

Human B-1 and B-2 B Cells Develop from Lin-CD34+CD38lo Stem Cells.

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+CD38lo and Lin-CD34+CD38hi populations. We found that transplanted Lin-CD34+CD38lo cells, but not Lin-CD34+CD38hi cells, generated a CD19+ B cell population after transfer into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1wjl/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+CD38lo stem cell population, and engrafted B-1 cells in humanized mice exhibit an Ig-usage pattern comparable to B-1 cells in cord blood.

B-1a cell diversity: nontemplated addition in B-1a cell Ig is determined by progenitor population and developmental location.

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.

Autoantigen can promote progression to a more aggressive TCL1 leukemia by selecting variants with enhanced B-cell receptor signaling.

(Auto)antigen engagement by the B-cell receptor (BCR) and possibly the sites where this occurs influence the outcome of chronic lymphocytic leukemia (CLL). To test if selection for autoreactivity leads to increased aggressiveness and if this selection plays out equally in primary and secondary tissues, we used T-cell leukemia (TCL)1 cells reactive with the autoantigen phosphatidylcholine (PtC). After repeated transfers of splenic lymphocytes from a single mouse with oligoclonal PtC-reactive cells, outgrowth of cells expressing a single IGHV-D-J rearrangement and superior PtC-binding and disease virulence occurred. In secondary tissues, increased PtC-binding correlated with enhanced BCR signaling and cell proliferation, whereas reduced signaling and division of cells from the same clone was documented in cells residing in the bone marrow, blood, and peritoneum, even though cells from the last site had highest surface membrane IgM density. Gene-expression analyses revealed reciprocal changes of genes involved in BCR-, CD40-, and PI3K-signaling between splenic and peritoneal cells. Our results suggest autoantigen-stimulated BCR signaling in secondary tissues promotes selection, expansion, and disease progression by activating pro-oncogenic signaling pathways, and that--outside secondary lymphoid tissues--clonal evolution is retarded by diminished BCR-signaling. This transferrable, antigenic-specific murine B-cell clone (TCL1-192) provides a platform to study the types and sites of antigen-BCR interactions and genetic alterations that result and may have relevance to patients.

B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells.

The production and release of small phospholipid membrane vesicles, or extracellular vesicles (EVs), is a trait of most prokaryotic and eukaryotic cells. EVs display heterogeneity in content, size, biogenesis, activity, and function. B cells uniquely express immunoglobulin and produce EVs; however, the relationship between these entities has not been clarified. Here, we used several methodologies to isolate large (11,000 × g) and small (110,000 × g) EVs and evaluate their IgM content, characteristics and activity. We found that B cells from multiple cell lines and primary B cells produce EVs that display monomeric IgM on the surface and contain encapsulated monomeric IgM, which is independent of secreted pentameric IgM. Our data indicate EV IgM can bind antigen specifically, and EV IgM can be incorporated intracellularly into secondary cells. These results suggest immunological activities different from secreted pentameric IgM that may constitute a separate and distinct antibody distribution system.

Modulation of microbiome diversity and cytokine expression is influenced in a sex-dependent manner during aging.

The microbiome and immune system have a unique interplay, which influences homeostasis within the organism. Both the microbiome and immune system play important roles in health and diseases of the aged including development of cancer, autoimmune disorders, and susceptibility to infection. Various groups have demonstrated divergent changes in the gut microbiota during aging, yet the compounding factor of biological sex within the context of aging remains incompletely understood, and little is known about the effect of housing location in the composition of gut microbiota in the context of both sex and age. To better understand the roles of sex, aging, and location in influencing the gut microbiome, we obtained normal healthy BALB/cByJ mice from a single source and aged male and female mice in two different geographical locations. The 16S rRNA was analyzed from fecal samples of these mice and cytokine levels were measured from serum.16S rRNA microbiome analysis indicated that both age and sex play a role in microbiome composition, whereas location plays a lesser role in the diversity present. Interestingly, microbiome changes occurred with alterations in serum expression of several different cytokines including IL-10 and IL-6, which were also both differentially regulated in context to sex and aging. We found both IL-10 and IL-6 play a role in the constitutive expression of pSTAT-3 in CD5+ B-1 cells, which are known to regulate the microbiome. Additionally, significant correlations were found between cytokine expression and significantly abundant microbes. Based on these results, we conclude aging mice undergo sex-associated alterations in the gut microbiome and have a distinct cytokine profile. Further, there is significant interplay between B-1 cells and the microbiome which is influenced by aging in a sex-dependent manner. Together, these results illustrate the complex interrelationship among sex, aging, immunity, housing location, and the gut microbiome.

Age-related changes in antigen-specific natural antibodies are influenced by sex.

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.

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.

Immunoglobulin secretion by B1 cells: differential intensity and IRF4-dependence of spontaneous IgM secretion by peritoneal and splenic B1 cells.

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.

Adult BM generates CD5+ B1 cells containing abundant N-region additions.

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.

Binding of LBP-1a to specific immunoglobulin switch regions in vivo correlates with specific repression of class switch recombination.

Upon stimulation of mature B cells, class switch recombination (CSR) can alter the specific immunoglobulin heavy chain constant region that is expressed. In a tissue culture cell line, we previously demonstrated that inhibition of late SV40 factor (LSF) family members enhanced IgM to IgA CSR. Here, isotype specificity of CSR regulation by LSF family members is addressed in primary mouse splenic B cells. First, we demonstrate that leader-binding protein-1a (LBP-1a) is the prevalent family member in B lymphocytes. Second, we demonstrate by ChIP that LBP-1a binds genomic sequences around mouse switch (S) regions in an isotype-specific manner, in accordance with computational predictions: binding is observed to Smu and Salpha, but not to the tested Sgamma1, regions. Importantly, binding of LBP-1a is tightly regulated, with occupancy at genomic S regions dramatically decreasing following LPS stimulation. Finally, the consequence of DNA-binding by LBP-1a is determined using bone marrow chimeric mice in which LSF/LBP-1 activity is inhibited in hematopoietic lineages. Upon in vitro stimulation of such primary B cells, CSR occurs with a higher efficiency to IgA, but not to IgG1. These results are supportive of a model whereby LBP-1a represses CSR in an isotype-specific manner via direct interaction with S regions involved in the recombination.

Defining Natural Antibodies.

The traditional definition of natural antibodies (NAbs) states that these antibodies are present prior to the body encountering cognate antigen, providing a first line of defense against infection thereby, allowing time for a specific antibody response to be mounted. The literature has a seemingly common definition of NAbs; however, as our knowledge of antibodies and B cells is refined, re-evaluation of the common definition of Nabs may be required. Defining Nabs becomes important as the function of NAb production is used to define B cell subsets (1) and as these important molecules are shown to play numerous roles in the immune system (Figure 1). Herein, we aim to briefly summarize our current knowledge of NAbs in the context of initiating a discussion within the field of how such an important and multifaceted group of molecules should be defined.

CD25 + B-1a Cells Express Aicda.

B-1a cells are innate-like B-lymphocytes producing natural antibodies. Activation-induced cytidine deaminase (AID), a product of the Aicda gene, plays a central role in class-switch recombination and somatic hypermutation in B cells. Although a role for Aicda in B-1a cells has been suggested on the basis of experiments with knock out (KO) mice, whether B-1a cells express Aicda, and if so, which B-1a cell subpopulation expresses Aicda, remains unknown. Here, we demonstrate that B-1 cells express Aicda, but at a level below that expressed by germinal center (GC) B cells. We previously reported that B-1a cells can be subdivided based on CD25 expression. We show here that B-1a cell Aicda expression is concentrated in the CD25+ B-1a cell subpopulation. These results suggest the possibility that previous studies of memory B cells identified on the basis of Aicda expression may have inadvertently included an unknown number of CD25+ B-1a cells. Although B-1a cells develop normally in the absence of Aicda, a competitive reconstitution assay reveals enhanced vigor for AID KO B-1a cell bone marrow (BM) progenitors, as compared with wild-type BM B-1 cell progenitors. These results suggest that AID inhibits the development of B-1a cells from BM B-1 cell progenitors in a competitive environment.

B-CD8+ T Cell Interactions in the Anti-Idiotypic Response against a Self-Antibody.

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 VH 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.

Expansion of B-1a Cells with Germline Heavy Chain Sequence in Lupus Mice.

B6.Sle1.Sle2.Sle3 (B6.TC) lupus-prone mice carrying the NZB allele of Cdkn2c, encoding for the cyclin-dependent kinase inhibitor P18(INK4), accumulate B-1a cells due to a higher rate of proliferative self-renewal. However, it is unclear whether this affects primarily early-appearing B-1a cells of fetal origin or later-appearing B-1a cells that emerge from bone marrow. B-1a cells are the major source of natural autoantibodies, and it has been shown that their protective nature is associated with a germline-like sequence, which is characterized by few N-nucleotide insertions and a repertoire skewed toward rearrangements predominated during fetal life, VH11 and VH12. To determine the nature of B-1a cells expanded in B6.TC mice, we amplified immunoglobulin genes by PCR from single cells in mice. Sequencing showed a significantly higher proportion of B-1a cell antibodies that display fewer N-additions in B6.TC mice than in B6 control mice. Following this lower number of N-insertions within the CDR-H3 region, the B6.TC B-1a cells display shorter CDR-H3 length than B6 B-1a cells. The absence of N-additions is a surrogate for fetal origin, as TdT expression starts after birth in mice. Therefore, our results suggest that the B-1a cell population is not only expanded in autoimmune B6.TC mice but also qualitatively different with the majority of cells from fetal origin. Accordingly, our sequencing results also demonstrated the overuse of VH11 and VH12 in autoimmune B6.TC mice as compared to B6 controls. These results suggest that the development of lupus autoantibodies in these mice is coupled with skewing of the B-1a cell repertoire and possible retention of protective natural antibodies.