Neonatal Exposure to Commensal-Bacteria-Derived Antigens Directs Polysaccharide-Specific B-1 B Cell Repertoire Development.

B-1 B cells derive from a developmental program distinct from that of conventional B cells, through B cell receptor (BCR)-dependent positive selection of fetally derived precursors. Here, we used direct labeling of B cells reactive with the N-acetyl-D-glucosamine (GlcNAc)-containing Lancefield group A carbohydrate of Streptococcus pyogenes to study the effects of bacterial antigens on the emergent B-1 B cell clonal repertoire. The number, phenotype, and BCR clonotypes of GlcNAc-reactive B-1 B cells were modulated by neonatal exposure to heat-killed S. pyogenes bacteria. GlcNAc-reactive B-1 clonotypes and serum antibodies were reduced in germ-free mice compared with conventionally raised mice. Colonization of germ-free mice with a conventional microbiota promoted GlcNAc-reactive B-1 B cell development and concomitantly elicited clonally related IgA+ plasma cells in the small intestine. Thus, exposure to microbial antigens in early life determines the clonality of the mature B-1 B cell repertoire and ensuing antibody responses, with implications for vaccination approaches and schedules.

Mast cells crosstalk with B cells in the gut and sustain IgA response in the inflamed intestine.

B lymphocytes are among the cell types whose effector functions are modulated by mast cells (MCs). The B/MC crosstalk emerged in several pathological settings, notably the colon of inflammatory bowel disease (IBD) patients is a privileged site in which MCs and IgA+ cells physically interact. Herein, by inducing conditional depletion of MCs in red MC and basophil (RMB) mice, we show that MCs control B cell distribution in the gut and IgA serum levels. Moreover, in dextran sulfate sodium (DSS)-treated RMB mice, the presence of MCs is fundamental for the enlargement of the IgA+ population in the bowel and the increase of systemic IgA production. Since both conventional B-2 and peritoneal-derived B cells populate the intestine and communicate with MCs in physiological conditions and during inflammation, we further explored this interplay through the use of co-cultures. We show that MCs finely regulate different aspects of splenic B cell biology while peritoneal B cells are unresponsive to the supporting effects provided by MCs. Interestingly, peritoneal B cells induce a pro-inflammatory skewing in MCs, characterized by increased ST2 and TNF-α expression. Altogether, this study uncovers the versatility of the B/MC liaison and highlights key aspects for the resolution of intestinal inflammation.

Spleen supports a pool of innate-like B cells in white adipose tissue that protects against obesity-associated insulin resistance.

Lipid accumulation in obesity triggers a low-grade inflammation that results from an imbalance between pro- and anti-inflammatory components of the immune system and acts as the major underlying mechanism for the development of obesity-associated diseases, notably insulin resistance and type 2 diabetes. Innate-like B cells are a subgroup of B cells that respond to innate signals and modulate inflammatory responses through production of immunomodulatory mediators such as the anti-inflammatory cytokine IL-10. In this study, we examined innate-like B cells in visceral white adipose tissue (VAT) and the relationship of these cells with their counterparts in the peritoneal cavity and spleen during diet-induced obesity (DIO) in mice. We show that a considerable number of innate-like B cells bearing a surface phenotype distinct from the recently identified "adipose natural regulatory B cells" populate VAT of lean animals, and that spleen represents a source for the recruitment of these cells in VAT during DIO. However, demand for these cells in the expanding VAT outpaces their recruitment during DIO, and the obese environment in VAT further impairs their function. We further show that removal of splenic precursors of innate-like B cells through splenectomy exacerbates, whereas supplementation of these cells via adoptive transfer ameliorates, DIO-associated insulin resistance. Additional adoptive transfer experiments pointed toward a dominant role of IL-10 in mediating the protective effects of innate-like B cells against DIO-induced insulin resistance. These findings identify spleen-supplied innate-like B cells in VAT as previously unrecognized players and therapeutic targets for obesity-associated diseases.

Homeostatic role of B-1 cells in tissue immunity.

To date, studies of tissue-resident immunity have mainly focused on innate immune cells and T cells, with limited data on B cells. B-1 B cells are a unique subset of B cells with innate-like properties, enriched in murine pleural and peritoneal cavities and distinct from conventional B-2 cells in their ontogeny, phenotype and function. Here we discuss how B-1 cells represent exemplar tissue-resident immune cells, summarizing the evidence for their long-term persistence & self-renewal within tissues, differential transcriptional programming shaped by organ-specific environmental cues, as well as their tissue-homeostatic functions. Finally, we review the emerging data supporting the presence and homeostatic role of B-1 cells across non-lymphoid organs (NLOs) both in mouse and human.

The impact of Escherichia coli contamination products present in recombinant African horse sickness virus serotype 4 proteins on the innate and humoral immune responses.

Transcriptome analysis was used to characterise the in vitro primary and secondary immune responses induced in horse peripheral blood mononuclear cells (PBMC) stimulated for 24 h with the individual recombinant proteins of a virulent AHSV serotype 4 (AHSV4) field isolate (rAHSV4 proteins) that were previously expressed in Escherichia coli (E. coli). The results showed that the E. coli contamination products greatly affected the innate and humoral immune response transcripts. Hence, the impact of E. coli contamination products present in the individual rAHSV4 proteins on the translational immune response was determined. The combined amplification effects of synergistic pattern recognition receptors (PRRs), TNF-α and IL-1ß signalling induced potent pro-inflammatory responses that were too overwhelming for the anti-inflammatory cytokines and regulators to control. In addition to inducing robust B cell and antibody-mediated responses, lipopolysaccharide (LPS) activation of the innate-like B cells and subsequent polyreactive (natural) antibody responses could potentially contribute to endotoxin tolerance.

Dysfunction of CD27+IgD+ B cells correlates with aggravated systemic lupus erythematosus.

OBJECTIVE: The apoptotic signaling pathway is obviously disordered in systemic lupus erythematosus (SLE). Natural IgM (nIgM) is important in clearing apoptotic cells and preventing them from triggering deleterious autoimmunity. B-1 and innate-like B (ILBs) cells are the main nIgM producers. Human CD27+IgD+ B cells (un-switched memory B cells) are considered ILBs. However, their functional properties in SLE remain undefined. METHODS: Peripheral blood sample of 50 SLE patients and 50 healthy controls were collected, and twelve SLE patients were assessed in a follow-up study. The amount of CD27+IgD+ B cell in each population was analyzed by flow cytometry. The IgM and IL-10 levels of CD27+IgD+ B cell were assessed by ELISPOT and qRT-PCR, respectively. SPSS 17.0 (SPSS, USA) was employed for data analysis. P < 0.05 indicated statistical significance. RESULT: The amounts of CD27+IgD+ B cell were significantly decreased in SLE patients than healthy control (P < 0.01). CD27+IgD+ B cell amounts were positively correlated with WBC (r = 0.337, P = 0.017), platelet count (r = 0.396, P = 0.004), and serum C3 levels (r = 0.415, P = 0.003) and negatively correlated with serum creatinine levels (r = - 0.285, P = 0.045), SLEDAI(r = - 0.724, P = 0.000), and anti-dsDNA(r = - 0.477, P = 0.000). The IgM and IL-10 levels of CD27+IgD+ B in active SLE were decreased than healthy control (P < 0.001). Moreover, CD27+IgD+ B cells are increased in SLE cases after treatment than before treatment (P < 0.001). CONCLUSION: The amounts of CD27+IgD+ B cell were significantly decreased in SLE patients compared with the healthy population, and CD27+IgD+ B cell was verified to be correlated with clinical and immunological features in SLE patients. CD27+IgD+ B cells had impaired function associated with IgM and IL-10 production in active SLE. Moreover, the amounts of CD27+IgD+ B cells were recovered to the normal level in SLE cases with treatment-related disease remission. Key Points • CD27+IgD+ B cell amounts are significantly decreased in SLE patients than healthy control. • CD27+IgD+ B cells are functionally impaired in producing natural antibody-like IgM and IL-10 in SLE patients. • CD27+IgD+ B cell amounts are correlated with clinical and immunological features in SLE.

Role of Programmed Cell Death Protein-1 and Lymphocyte Specific Protein Tyrosine Kinase in the Aryl Hydrocarbon Receptor- Mediated Impairment of the IgM Response in Human CD5+ Innate-Like B Cells.

Innate-like B cells (ILBs) are a heterogeneous population B cells which participate in innate and adaptive immune responses. This diverse subset of B cells is characterized by the expression of CD5 and has been shown to secrete high levels of immunoglobulin M (IgM) in the absence of infection or vaccination. Further, CD5+ ILBs have been shown to express high basal levels of lymphocyte specific protein tyrosine kinase (LCK) and programmed cell death protein-1 (PD-1), which are particularly sensitive to stimulation by interferon gamma (IFNγ). Previous studies have demonstrated that activation of the aryl hydrocarbon receptor (AHR), a cytosolic ligand-activated transcription factor, results in suppressed IgM responses and is dependent on LCK. A recent study showed that CD5+ ILBs are particularly sensitive to AHR activation as evidenced by a significant suppression of the IgM response compared to CD5- B cells, which were refractory. Therefore, the objective of this study was to further investigate the role of LCK and PD-1 signaling in AHR-mediated suppression of CD5+ ILBs. In addition, studies were conducted to establish whether IFNγ alters the levels of LCK and PD-1 in CD5+ ILBs. We found that AHR activation led to a significant upregulation of total LCK and PD-1 proteins in CD5+ ILBs, which correlated with suppression of IgM. Interestingly, treatment with recombinant IFNγ reduced LCK protein levels and reversed AHR-mediated IgM suppression in CD5+ ILBs in a similar manner as LCK inhibitors. Collectively, these results support a critical role for LCK and PD-1 in AHR-mediated suppression of the IgM response in human CD5+ ILBs.

Identification of a Sensitive Human Immunological Target of Aryl Hydrocarbon Receptor Activation: CD5+ Innate-Like B Cells.

Xenobiotic-mediated activation of the aryl hydrocarbon receptor (AHR) is immunotoxic in a number of immune cell types, with the B cell being a well-established sensitive target. Recent advances have provided evidence that the B cell repertoire is a heterogeneous population, with subpopulations exhibiting vastly different cellular and functional phenotypes. Recent work from our laboratory identified the T cell specific kinase lck as being differentially regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent activator of AHR. While LCK is primarily expressed in T cells, a subset of CD5+ B cells also express LCK. CD5 positivity describes a broad class of B lymphocytes termed innate-like B cells (ILBs) that are critical mediators of innate immunity through constitutive secretion of polyvalent natural immunoglobulin M (IgM). We hypothesized that CD5+ ILBs may be sensitive to AHR-mediated immunotoxicity. Indeed, when CD5+ B cells were isolated from the CD19+ pool and treated with TCDD, they showed increased suppression of the CD40 ligand-induced IgM response compared to CD5- B cells. Further, characterization of the CD5+ population indicated increased basal expression of AHR, AHR repressor (AHRR), and cytochrome p450 family 1 member a1 (CYP1A1). Indeed the levels of AHR-mediated suppression of the IgM response from individual donors strongly correlated with the percentage of the B cell pool that was CD5+, suggesting that CD5+ B cells are more sensitive to AHR-mediated impairment. Together these data highlight the sensitive nature of CD5+ ILBs to AHR activation and provide insight into mechanisms associated with AHR activation in human B cells.

Purification and Immune Phenotyping of B-1 Cells from Body Cavities of Mice.

B-1 cells are fetal-origin B lymphocytes with unique developmental and functional characteristics that can generate natural, polyreactive antibodies with important functions in tissue homeostasis and immune defense. While B-1 cell frequencies in bone marrow and secondary lymphoid tissues are low, relative high frequencies exist within peritoneal and pleural cavities of mice, including both CD5+ and CD5- B-1 cells. These cells represent B-1 reservoirs that, when activated, migrate to lymphoid tissues to secrete antibodies and/or cytokines. Here, we outline efficient methods for the extraction and magnetic isolation of CD5+ B-1 cells from the peritoneal and pleural cavities as well as the separation and phenotypic characterization of CD5+ and CD5- B-1 cells by flow cytometry.

Glycan Reactive Natural Antibodies and Viral Immunity.

Adaptive antibody responses provide a crucial means of host defense against viral infections by mediating the neutralization and killing infectious pathogens. At the forefront of humoral defense against viruses lie a subset of innate-like serum antibodies known as natural antibodies (NAbs). NAbs serve multifaceted functions in host defense and play an essential role in early immune responses against viruses. However, there remain many unanswered questions with regard to both the breadth of viral antigens recognized by NAbs, and how B cell ontology and individual antigenic histories intersect to control the development and function of antiviral human NAbs. In the following article we briefly review the current understanding of the functions and source of NAbs in the immune repertoire, their role during antiviral immune responses, the factors influencing the maturation of the NAb repertoire, and finally, the gaps and future research needed to advance our understanding of innate-like B cell biology for the purpose of harnessing NAbs for host defense against viral infections.

Fc Receptor-Like 6 (FCRL6) Discloses Progenitor B Cell Heterogeneity That Correlates With Pre-BCR Dependent and Independent Pathways of Natural Antibody Selection.

B-1a cells produce "natural" antibodies (Abs) to neutralize pathogens and clear neo self-antigens, but the fundamental selection mechanisms that shape their polyreactive repertoires are poorly understood. Here, we identified a B cell progenitor subset defined by Fc receptor-like 6 (FCRL6) expression, harboring innate-like defense, migration, and differentiation properties conducive for natural Ab generation. Compared to FCRL6- pro B cells, the repressed mitotic, DNA damage repair, and signaling activity of FCRL6+ progenitors, yielded VH repertoires with biased distal Ighv segment accessibility, constrained diversity, and hydrophobic and charged CDR-H3 sequences. Beyond nascent autoreactivity, VH11 productivity, which predominates phosphatidylcholine-specific B-1a B cell receptors (BCRs), was higher for FCRL6+ cells as was pre-BCR formation, which was required for Myc induction and VH11, but not VH12, B-1a development. Thus, FCRL6 revealed unexpected heterogeneity in the developmental origins, regulation, and selection of natural Abs at the pre-BCR checkpoint with implications for autoimmunity and lymphoproliferative disorders.

Impaired CD27+IgD+ B Cells With Altered Gene Signature in Rheumatoid Arthritis.

Natural antibodies, particularly natural IgM, are proved to play indispensable roles in the immune defenses against common infections. More recently, the protective roles of these natural IgM were also recognized in autoimmune diseases. They are mainly produced by B-1 and innate-like B cells (ILBs). Human CD19+CD27+IgD+ B cells, also termed as un-switched memory B cells, were proposed to be a kind of ILBs. However, functional features and characteristics of these cells in rheumatoid arthritis (RA) remained poorly understood. In this study, we found that human CD27+IgD+ B cells could produce natural antibody-like IgM. Under RA circumstance, the frequencies of these cells were significantly decreased. Moreover, the IgM-producing capacities of these cells were also dampened. Interestingly, the BCR repertoire of these cells was altered in RA, demonstrating decreased diversity with preferential usage alteration from VH3-23D to VH1-8. Single cell sequencing further revealed the proinflammatory biased features of these cells in RA. These CD27+IgD+ B cells were negatively correlated with RA patient disease activities and clinical manifestations. After effective therapy with disease remission in RA, these cells could be recovered. Taken together, these results have revealed that CD27+IgD+ B cells were impaired in RA with dysfunctional features, which might contribute to the disease perpetuation.

Marginal-Zone B-Cells Are Main Producers of IgM in Humans, and Are Reduced in Patients With Autoimmune Vasculitis.

In mice, B1 and marginal zone (MZ) B-cells play an important role in prevention of autoimmunity through production of regulatory cytokines and natural antibodies. There is limited knowledge about the human counterparts of these cells. We therefore investigated functions of MZ-like B-cells and the frequency of circulating MZ-like and B1-like B-cells in healthy controls (HC), as well as in patients with autoimmune vasculitis to learn more about the role of these cells in autoimmune disease. After stimulation with CpG oligodeoxynucleotides (ODN) of class B in vitro, MZ-like B-cells were the main producers of IgM whereas switched memory B-cells primarily produced IgG and IgA. TNF and IL-10 were produced by both MZ-like and switched memory B-cells. Neither antibody nor TNF/IL-10 production by the B-cell subsets differed between patients and HC. Patients with autoimmune vasculitis, irrespective of disease activity, had lower percentage and absolute numbers of circulating MZ-like B-cells, and lower absolute numbers of B1-like B-cells. The percentage of B1-like B-cells was reduced during active disease. These findings remained significant when the analysis was confined to active treatment-naïve patients (disease onset).Our results suggest that human innate-like B-cells might have a physiological role in prevention of autoimmunity.