TLR Engagement Induces an Alternate Pathway for BCR Signaling that Results in PKCδ Phosphorylation.

Recently, we reported that preexposure of B cells to IL-4 induced an alternate, signalosome-independent BCR signaling pathway leading to protein kinase C (PKC)δ phosphorylation (pTyr311), which occurs in the membrane compartment. This is considered to represent a form of receptor crosstalk and signal integration. Unlike the classical BCR signaling pathway, Lyn kinase is indispensable for BCR-induced downstream events in the alternate pathway. Our previous report that alternate BCR signaling leading to ERK phosphorylation is triggered by LPS and PAM3CSK4 (much like IL-4) raises the possibility that other signaling outcomes such as PKCδ phosphorylation might be similarly affected. To explore the range of mediators capable of producing an alternate pathway for BCR signaling, we examined PKCδ translocation and phosphorylation in LPS- and PAM3CSK4-treated B cells stimulated by anti-Ig. We found that LPS and PAM3CSK4 alter the signaling pathway used by the BCR to produce PKCδ phosphorylation. As with IL-4, elements of the signalosome are not needed for PKCδ phosphorylation when BCR triggering occurs after LPS and PAM3CSK4. However, with LPS and PAM3CSK4, anti-Ig-induced phosphorylation of PKCδ takes place in the cytosol, in contrast to the IL-4-induced alternate pathway, wherein PKCδ phosphorylation occurs in the membrane. Furthermore, the BCR signaling pathway induced by LPS and PAM3CSK4 differs from that induced by IL-4 by not requiring Lyn. Thus, an alternate, signalosome-independent BCR signaling pathway for PKCδ phosphorylation is induced by TLR agonists but differs in important ways from the alternate pathway induced by IL-4.

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.

Human VH4-34 antibodies derived from B1 cells are more frequently autoreactive than VH4-34 antibodies derived from memory cells.

Human B1 cells produce natural antibodies characterized by overutilization of heavy chain variable region VH4-34 in comparison to other B cell populations. VH4-34-containing antibodies have been reported to be autoreactive and to be associated with lupus and other autoimmune dyscrasias. However, it has been unclear to what extent VH4-34 antibodies manifest autoreactivity in B1 cells or other B cell populations-in other words, are VH4-34 containing antibodies autoreactive wherever found, or mainly within the B1 cell population? To address this issue we sort purified single human B1 and memory B cells and then amplified, sequenced, cloned and expressed VH4-34-containing antibodies from 76 individual B cells. Each of these antibodies was tested for autoreactivity by HEp-2 IFA and autoantigen ELISA. Antibodies were scored as autoreactive if positive by either assay. We found VH4-34 antibodies rescued from B1 cells were much more frequently autoreactive (14/48) than VH4-34 antibodies rescued from memory B cells (2/28). Among B1 cell antibodies, 4 were HEp-2+, 6 were dsDNA+ and 4 were positive for both. Considering only HEp-2+ antibodies, again these were found more frequently among B1 cell VH4-34 antibodies (8/48) than memory B cell VH4-34 antibodies (1/28). We found autoreactivity was associated with greater CDR3 length, as expected; however, we found no association between autoreactivity and a previously described FR1 "hydrophobic patch". Our results indicate that autoreactive VH4-34-containing antibodies tend to reside within the human B1 cell population.

Human B-1 cells are important contributors to the naturally-occurring IgM pool against the tumor-associated ganglioside Neu5GcGM3.

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.

Small Heat Shock Proteins Collaborate with FAIM to Prevent Accumulation of Misfolded Protein Aggregates.

Cells and tissues are continuously subject to environmental insults, such as heat shock and oxidative stress, which cause the accumulation of cytotoxic, aggregated proteins. We previously found that Fas Apoptosis Inhibitory Molecule (FAIM) protects cells from stress-induced cell death by preventing abnormal generation of protein aggregates similar to the effect of small heat shock proteins (HSPs). Protein aggregates are often associated with neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we sought to determine how FAIM protein dynamics change during cellular stress and how FAIM prevents the formation of amyloid-ß aggregates/fibrils, one of the pathological hallmarks of AD. Here, we found that the majority of FAIM protein shifts to the detergent-insoluble fraction in response to cellular stress. A similar shift to the insoluble fraction was also observed in small heat shock protein (sHSP) family molecules, such as HSP27, after stress. We further demonstrate that FAIM is recruited to sHSP-containing complexes after cellular stress induction. These data suggest that FAIM might prevent protein aggregation in concert with sHSPs. In fact, we observed the additional effect of FAIM and HSP27 on the prevention of protein aggregates using an in vitro amyloid-ß aggregation model system. Our work provides new insights into the interrelationships among FAIM, sHSPs, and amyloid-ß aggregation.

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.

Microparticle immunocapture assay for quantitation of protein multimer amount and size.

Cellular stress and toxicity are often associated with the formation of protein multimers, or aggregates. Numerous degenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, prion-propagated disease, amyotrophic lateral sclerosis, cardiac amyloidosis, and diabetes, are characterized by aggregated protein deposits. Current methods are limited in the ability to assess multimer size along with multimer quantitation and to incorporate one or more ancillary traits, including target specificity, operative simplicity, and process speed. Here, we report development of a microparticle immunocapture assay that combines the advantages inherent to a monoclonal antibody:protein interaction with highly quantitative flow cytometry analysis. Using established reagents to build our platform, and aggregation-prone amyloid beta 1-42 peptide (Aß42) and alpha-synuclein to demonstrate proof of principle, our results indicate that this assay is a highly adaptable method to measure multimer size and quantity at the same time in a technically streamlined workflow applicable to laboratory and clinical samples.

Skewing of the antibody repertoire in cerebrospinal fluid B cells from healthy controls and patients with schizophrenia.

Autoantibodies play a role in the etiology of some neuropsychiatric disorders. To address the possibility that B cells and their antibodies may be involved in the pathophysiology of schizophrenia, we examined B cells in cerebrospinal fluid (CSF) and peripheral blood (PB) of 4 schizophrenic patients (SP) and 4 healthy control (HC) volunteers by analyzing immunoglobulin VH gene usage. All CSF samples contained measurable levels of B cells. We found for both SP and HC, CSF B cells represented a select subset of, and were not the same as, B cells in PB. Moreover, we found statistically significant differences in antibodies generated by CSF B cells in SP compared to CSF B cells in HC. Although binding characteristics of CSF SP-associated B cell antibodies is unknown, the study number is small, and pathophysiology has not been established, these results suggest the value of focusing further study on the distinctly separate population of CSF B cells in SP.

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.

Fas Apoptosis Inhibitory Molecule Blocks and Dissolves Pathological Amyloid-ß Species.

A number of neurodegenerative diseases are associated with the accumulation of misfolded proteins, including Alzheimer's disease (AD). In AD, misfolded proteins such as tau and amyloid-ß (Aß) form pathological insoluble deposits. It is hypothesized that molecules capable of dissolving such protein aggregates might reverse disease progression and improve the lives of afflicted AD patients. Here we report new functions of the highly conserved mammalian protein, Fas Apoptosis Inhibitory Molecule (FAIM). We found that FAIM-deficient Neuro 2A cells accumulate Aß oligomers/fibrils. We further found that recombinant human FAIM prevents the generation of pathologic Aß oligomers and fibrils in a cell-free system, suggesting that FAIM functions without any additional cellular components. More importantly, recombinant human FAIM disaggregates and solubilizes established Aß fibrils. Our results identify a previously unknown, completely novel candidate for understanding and treating irremediable, irreversible, and unrelenting neurodegenerative diseases.

Signal Integration by Translocation and Phosphorylation of PKCδ in the B Cell Alternate Pathway.

B cell signaling for activation via the BCR occurs as an isolated event only in vitro; in real life, BCR signaling takes place within a complex milieu that involves interactions with agents that trigger additional receptors. Chief among these is IL-4. We have shown that BCR signaling is reprogrammed by IL-4 receptor engagement and that this reprogramming involves creation of a new, signalosome-independent, Lyn-dependent alternate signaling pathway in B cells isolated from BALB/cByJ mice. A unique aspect of the alternate pathway is protein kinase Cδ (PKCδ) phosphorylation. In dissecting this pathway, we unexpectedly found that Lyn is associated with IL-4Rα, that IL-4 induces Lyn activation, and that Lyn immunoprecipitated from IL-4-treated B cells capably phosphorylates PKCδ in a cell-free system. However, PKCδ phosphorylation does not occur in the absence of BCR triggering in vivo. This raised the question of why IL-4 alone failed to produce PKCδ phosphorylation. We considered the possibility that Lyn and PKCδ may be spatially separated. As expected, before any treatment, Lyn is located primarily in the membrane fraction, whereas PKCδ is located mainly in the cytosol fraction. However, when anti-Ig follows IL-4 treatment, PKCδ is found in the membrane fraction and phosphorylated. This translocation of PKCδ to the membrane fraction is not affected by loss of Lyn, although PKCδ phosphorylation requires Lyn. Thus, PKCδ phosphorylation through the alternate pathway represents the result of signal integration, whereby neither IL-4 nor anti-Ig working alone produces this outcome, but together they achieve this result by Lyn activation (IL-4) and PKCδ translocation (IL-4 followed by anti-Ig).

A new murine Rpl5 (uL18) mutation provides a unique model of variably penetrant Diamond-Blackfan anemia.

Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins are found in most individuals with Diamond-Blackfan anemia (DBA), whereas somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human patients with DBA. In this study, we characterized a novel ENU mouse mutant (Skax23m1Jus) with growth and skeletal defects, cardiac malformations, and increased mortality. After genetic mapping and whole-exome sequencing, we identified an intronic Rpl5 mutation, which segregated with all affected mice. This mutation was associated with decreased ribosome generation, consistent with Rpl5 haploinsufficiency. Rpl5Skax23-Jus/+ animals had a profound delay in erythroid maturation and increased mortality at embryonic day (E) 12.5, which improved by E14.5. Surviving mutant animals had macrocytic anemia at birth, as well as evidence of ventricular septal defect (VSD). Surviving adult and aged mice exhibited no hematopoietic defect or VSD. We propose that this novel Rpl5Skax23-Jus/+ mutant mouse will be useful in studying the factors influencing the variable penetrance that is observed in DBA.

The Alternate Pathway for BCR Signaling Induced by IL-4 Requires Lyn Tyrosine Kinase.

BCR signaling triggers a cascade of intracellular mediators that eventuates in transcription factor activation. Signaling is proximally mediated by Src family tyrosine kinases, the most abundant being Lyn. Key mediators are grouped together as the signalosome, and failure of any single member of this group leads to failure of signaling via this classical pathway. Recent work has revealed an alternate pathway for BCR signaling, in which signalosome elements are bypassed for downstream events such as ERK and PKCδ phosphorylation. This pathway is created by B cell treatment with IL-4 prior to BCR triggering. After IL-4 treatment, the alternate pathway for pERK operates in parallel with the classical pathway for pERK, whereas PKCδ phosphorylation is specific to the alternate pathway. Remarkably, Lyn is not required for B cell activation via the classical pathway; however, Lyn is indispensable and irreplaceable for B cell activation via the alternate pathway. Thus, Lyn operates at a branch point that determines the nature of the B cell response to BCR activation. The mechanism underlying the absolute dependence of alternate pathway signaling on Lyn is unknown. Here, our current understanding of receptor crosstalk between IL-4R and BCR is summarized along with several possible mechanisms for the role of Lyn in alternate pathway signaling. Further dissection of alternate pathway signaling and the role of Lyn is likely to provide important information relating to normal B cell responses, malignant B cell expansion, and generic principles relating to receptor interactions and crosstalk.

The Use of B-Cell Polysome Profiling to Validate Novel RPL5 (uL18) and RPL26 (uL24) Variants in Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome usually caused by heterozygous variants in ribosomal proteins (RP) and which leads to severe anemia. Genetic studies in DBA rely primarily on multigene panels that often result in variants of unknown significance. Our objective was to optimize polysome profiling to functionally validate new large subunit RP variants. We determined the optimal experimental conditions for B-cell polysome profiles then performed this analysis on 2 children with DBA and novel missense RPL5 (uL18) and RPL26 (uL24) variants of unknown significance. Both patients had reduced 60S and 80S fractions when compared with an unaffected parent consistent with a large ribosomal subunit defect. Polysome profiling using primary B-cells is an adjunctive tool that can assist in validation of large subunit RP variants of uncertain significance. Further studies are necessary to validate this method in patients with known DBA mutations, small RP subunit variants, and silent carriers.

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.

FAIM Is a Non-redundant Defender of Cellular Viability in the Face of Heat and Oxidative Stress and Interferes With Accumulation of Stress-Induced Protein Aggregates.

A key element of cellular homeostasis lies in the way in which misfolded and dysfunctional proteins are handled. Cellular pathways that include proteasomal destruction and autophagic disposal are components of normal proteostasis. Here we report a novel molecule that plays a non-redundant role in maintaining homeostasis, Fas Apoptosis Inhibitory Molecule (FAIM). FAIM is highly conserved throughout evolution and bears no homology to any other protein. We found that FAIM counteracts heat and oxidative stress-induced loss of cell viability. FAIM is recruited to ubiquitinated proteins induced by cellular stress and the levels of stress-induced protein aggregates are much greater in FAIM-deficient cell lines. Primary fibroblasts from FAIM-deficient mice showed the same proteostasis deficits as cell lines. Administration of a mediator of oxidative stress to FAIM-deficient animals induced more ubiquitinated protein aggregates and more organ damage as compared to wild type mice. These results identify a completely new actor that protects cells against stress-induced loss of viability by preventing protein aggregation.

FAIM Opposes Aggregation of Mutant SOD1 That Typifies Some Forms of Familial Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative illness that is unremittingly fatal and for which no effective treatment exists. All forms of ALS are characterized by protein aggregation. In familial forms of ALS, specific and heritable aggregation-prone proteins have been identified, such as mutant superoxide dismutase (SOD1). It has been suggested that a factor capable of preventing mutant SOD1 protein aggregation and/or disassembling mutant SOD1 protein aggregates would ameliorate SOD1-associated forms of familial ALS. Here we identify Fas Apoptosis Inhibitory Molecule (FAIM), a highly evolutionarily conserved 20 kDa protein, as an agent with this activity. We show FAIM counteracts intracellular accumulation of mutant SOD1 protein aggregates, which is increased in the absence of FAIM, as determined by pulse-shape analysis and filter trap assays. In a cell-free system, FAIM inhibits aggregation of mutant SOD1, and further disassembles and solubilizes established mutant SOD1 protein aggregates, as determined by thioflavin T (ThT), filter trap, and sedimentation assays. In sum, we report here a previously unknown activity of FAIM that opposes ALS disease-related protein aggregation and promotes proteostasis of an aggregation-prone ALS protein.

Impairment of PD-L2 positive B1a cells enhances susceptibility to sepsis in RasGRP1-deficient mice.

RasGRP1 is a key molecule that mediates antigen-initiated signaling for activation of the RAS-MAPK pathway in lymphocytes. Patients with aberrant RasGRP1 expression experience lymphocyte dysfunction and are afflicted with recurrent microbial infections. Yet, the underlying mechanism that accounts for microbial infection remains unknown. We previously reported that B1a cells are heterogeneous with respect to PD-L2 expression and that RasGRP1 deficiency preferentially impairs PD-L2+ B1a cell development. In the present study, we show that PD-L2+ B1a cells exhibit increased capacity for differentiation to CD138+ plasma cells that secrete natural IgM antibody, as well as IL-10 and GM-CSF, in response to TLR stimulation. In keeping with this, we show here that RasGRP1-deficent mice are much more susceptible to septic infection triggered by cecalligation and puncture than wild type mice, and that reconstitution of RasGRP1-deficient mice with wild type PD-L2+ B1a cells greatly rescues RasGRP1-deficient mice from sepsis. Thus, this study indicates a mechanism for the association of RasGRP1 deficiency with predispostion to infection in the loss of a particular B1a subpopulation.

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.

Human B-1 Cells and B-1 Cell Antibodies Change With Advancing Age.

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+CD38low/intCD43+) 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.