Publisher Correction: Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten-eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.

B cell signaling and fate decision.

Antigen receptors on the surface of B lymphocytes trigger adaptive immune responses after encountering their cognate antigens but also control a series of antigen-independent checkpoints during B cell development. These physiological processes are regulated by the expression and function of cell surface receptors, intracellular signaling molecules, and transcription factors. The function of these proteins can be altered by a dynamic array of post-translational modifications, using two interconnected mechanisms. These modifications can directly induce an altered conformational state in the protein target of the modification itself. In addition, they can create new binding sites for other protein partners, thereby contributing to where and when such multiple protein assemblies are activated within cells. As a new type of post-transcriptional regulator, microRNAs have emerged to influence the development and function of B cells by affecting the expression of target mRNAs.

Silencing and activating anergic B cells.

Despite the existence of central tolerance mechanisms, including clonal deletion and receptor editing to eliminate self-reactive B cells, moderately self-reactive cells still survive in the periphery (about 20% of peripheral B cells). These cells normally exist in a functionally silenced state called anergy; thus, anergy has been thought to contribute to tolerance by active-silencing of potentially dangerous B cells. However, a positive rationale for the existence of these anergic B cells has recently been suggested by discoveries that broadly neutralizing antibodies for HIV and influenza virus possess poly- and/or auto-reactivity. Given the conundrum of generating inherent holes in the immune repertoire, retaining weakly self-reactive BCRs on anergic B cells could allow these antibodies to serve as an effective defense against pathogens, particularly in the case of pathogens that mimic forbidden self-epitopes to evade the host immune system. Thus, anergic B cells should be brought into a silenced or activated state, depending on their contexts. Here, we review recent progress in our understanding of how the anergic B cell state is controlled in B cell-intrinsic and B cell-extrinsic ways.

Humoral responses are enhanced by facilitating B cell viability by Fcrl5 overexpression in B cells.

B cell initial activity is regulated through a balance of activation and suppression mediated by regulatory molecules expressed in B cells; however, the molecular mechanisms underlying this process remain incompletely understood. In this study, we investigated the function of the Fc receptor-like (Fcrl) family molecule Fcrl5, which is constitutively expressed on naïve B cells, in humoral immune responses. Our study demonstrated that B cell-specific overexpression of Fcrl5 enhanced antibody (Ab) production in both T cell-independent type 1 (TI1) and T cell-dependent (TD) responses. Additionally, it promoted effector B cell formation under competitive conditions in TD responses. Mechanistically, in vitro ligation of Fcrl5 by agonistic Abs reduced cell death and enhanced proliferation in lipopolysaccharide (LPS)-stimulated B cells. In the presence of anti-CD40 Abs and IL-5, the Fcrl5 ligation not only suppressed cell death but also enhanced differentiation into plasma cells. These findings reveal a novel role of Fcrl5 in promoting humoral immune responses by enhancing B cell viability and plasma cell differentiation.

Regulation mechanisms of CARMA1-Bcl10-MALT1 complex assembly inferred from the analysis of TRAF6-deficient cells.

The CARMA1-Bcl10-MALT1 (CBM) signalosome is a crucial module of NF-κB activation in B cell receptor (BCR) signaling. Biophysical studies have shown that the E3 ubiquitin ligase TRAF6 cooperatively modifies the CBM signalosome; however, the specific details regarding how TRAF6 is involved in BCR signal-induced CBM formation remain unclear. In this study, we aimed to reveal the influences of TRAF6 on CBM formation and TAK1 and IKK activities using DT40 B cells which lack all the exons of TRAF6. In TRAF6-null cells we found: (i) attenuation of TAK1 activity and abolishment of IKK activity and (ii) sustained binding of CARMA1 to Bcl10. To account for the molecular mechanism causing these dynamics, we performed a mathematical model analysis. The mathematical model analysis showed that the regulation of IKK activation by TRAF6 can reproduce TAK1 and IKK activities in TRAF6 null cells, and that the TRAF6 related signal-dependent inhibitor suppresses CARMA1 binding to Bcl10 in wild-type cells. These results suggest that TRAF6 contributes to the positive regulation of IKK activation via TAK1, alongside the negative signal-dependent regulation of CARMA1 binding to Bcl10.

Special AT-Rich Sequence-Binding Protein 1 Supports Survival and Maturation of Naive B Cells Stimulated by B Cell Receptors.

Epigenetic mechanisms underpin the elaborate activities of essential transcription factors in lymphocyte development. Special AT-rich sequence-binding protein 1 (SATB1) is a chromatin remodeler that orchestrates the spatial and temporal actions of transcription factors. Previous studies have revealed the significance of SATB1 in T cell lineage. However, whether and how SATB1 controls B cell lineage development is yet to be clarified. In this study, we show that SATB1 is an important factor during splenic B cell maturation. By analyzing SATB1/Tomato reporter mice, we determined the dynamic fluctuation of SATB1 expression in the B cell lineage. Although SATB1 expression decreased to minimal levels during B cell differentiation in the bone marrow, it resurged markedly in naive B cells in the spleen. The expression was dramatically downregulated upon Ag-induced activation. Splenic naive B cells were subdivided into two categories, namely SATB1high and SATB1-/low, according to their SATB1 expression levels. SATB1high naive B cells were less susceptible to death and greater proliferative than were SATB1-/low cells during incubation with an anti-IgM Ab. Additionally, SATB1high cells tended to induce the expression of MHC class II, CD86, and CD83. Accordingly, naive B cells from B lineage-specific SATB1 conditional knockout mice were more susceptible to apoptosis than that in the control group upon anti-IgM Ab stimulation in vitro. Furthermore, conditional knockout mice were less capable of producing Ag-specific B cells after immunization. Collectively, our findings suggest that SATB1 expression increases in naive B cells and plays an important role in their survival and maturation.

MHC class II inhibits the generation of IL-17A+ Vγ6 γδ T cells in the thymus at perinatal stage.

Vγ6+ γδ T cells develop in the thymus at the perinatal stage and are exclusive IL-17A producers among γδ T cells. The loss of MHC class II led to the expansion of IL-17A+ Vγ6+ γδ T cells in the thymus. Thus, MHC class II in the thymus inhibits the generation of IL-17A+ Vγ6+ γδ T cells.

Pyruvate enhances oral tolerance via GPR31.

CX3CR1high myeloid cells in the small intestine mediate the induction of oral tolerance by driving regulatory T (Treg) cells. Bacterial metabolites, e.g. pyruvate and lactate, induce a dendrite extension of CX3CR1high myeloid cells into the intestinal lumen via GPR31. However, it remains unclear whether the pyruvate-GPR31 axis is involved in the induction of oral tolerance. Here, we show that pyruvate enhances oral tolerance in a GPR31-dependent manner. In ovalbumin (OVA)-fed Gpr31-deficient mice, an OVA-induced delayed-type hypersensitivity response was substantially induced, demonstrating the defective induction of oral tolerance in Gpr31-deficient mice. The percentage of RORγt+ Treg cells in the small intestine was reduced in Gpr31-deficient mice. In pyruvate-treated wild-type mice, a low dose of OVA efficiently induced oral tolerance. IL-10 production from intestinal CX3CR1high myeloid cells was increased by OVA ingestion in wild-type mice, but not in Gpr31-deficient mice. CX3CR1high myeloid cell-specific IL-10-deficient mice showed a defective induction of oral tolerance to OVA and a decreased accumulation of OVA-specific Treg cells in the small intestine. These findings demonstrate that pyruvate enhances oral tolerance through a GPR31-dependent effect on intestinal CX3CR1high myeloid cells.

Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation.

B cells can suppress autoimmunity by secreting interleukin-10 (IL-10). Although subpopulations of splenic B lineage cells are reported to express IL-10 in vitro, the identity of IL-10-producing B cells with regulatory function in vivo remains unknown. By using IL-10 reporter mice, we found that plasmablasts in the draining lymph nodes (dLNs), but not splenic B lineage cells, predominantly expressed IL-10 during experimental autoimmune encephalomyelitis (EAE). These plasmablasts were generated only during EAE inflammation. Mice lacking plasmablasts by genetic ablation of the transcription factors Blimp1 or IRF4 in B lineage cells developed an exacerbated EAE. Furthermore, IRF4 positively regulated IL-10 production that can inhibit dendritic cell functions to generate pathogenic T cells. Our data demonstrate that plasmablasts in the dLNs serve as IL-10 producers to limit autoimmune inflammation and emphasize the importance of plasmablasts as IL-10-producing regulatory B cells.

Quiescent B Cells Acquire Sensitivity to Cell Cycle Arresting Agents by B Cell Receptor Stimulation.

For the treatment of autoimmune diseases, depletion of B cells specific for auto-antigens is important because they will be a source of plasmablasts/plasma cells to produce autoantibodies. However, because some types of B cells like naïve B cells and memory B cells are at quiescent phase, they are insensitive to anticancer drugs which exert cytotoxicity by arresting the cell cycle. Here we show that B cell receptor (BCR) stimulation increases the sensitivity of anticancer drugs by promoting the proliferation of quiescent B cells. The BCR stimulation to primary naïve B cells enhanced sensitivity to several anticancer drugs which arrest the cell cycle through different mechanisms. The present results indicated that combination of the BCR stimulation and anticancer drugs is a promising strategy for the antigen-specific depletion of pathogenic quiescent B cells.

Heterogeneous subsets of B-lineage regulatory cells (Breg cells).

B cells represent a key cellular component of humoral immunity. Besides antigen presentation and antibody production, B cells also play a role in immune regulation and induction of tolerance through several mechanisms. Our understanding of B-lineage cells with regulatory ability has been revolutionized by the delineation of heterogeneous subsets of these cells. Specific environmental signals may further determine the polarization and function of B-lineage regulatory cells. With the availability of new genetic, molecular and pharmacological tools, considerable advances have been made toward our understanding of the surface phenotype, developmental processes and functions of these cells. These exciting discoveries, some of which are still controversial, also raise many new questions, which makes the inhibitory function of B cells a rapidly growing field in immunopathology. Here we review highlights of the regulatory activity of B cells and the recent advances in the function and phenotype of these B-cell subsets in healthy and diseased states.

Essential function for the calcium sensor STIM1 in mast cell activation and anaphylactic responses.

Mast cells have key functions as effectors of immunoglobulin E-mediated allergic inflammatory diseases. Allergen stimulation induces Ca2+ influx and elicits the secretion of inflammatory mediators from mast cells. Here we show that the Ca2+-binding endoplasmic reticulum protein STIM1 is critical to mast cell function. STIM1-deficient fetal liver-derived mast cells had impaired Ca2+ influx mediated by the high-affinity immunoglobulin E receptor FcepsilonRI and activation of the transcription factors NF-kappaB and NFAT. Mast cells lacking STIM1 also had much less degranulation and cytokine production after FcepsilonRI stimulation. In addition, alterations in STIM1 expression affected the sensitivity of immunoglobulin E-mediated immediate-phase anaphylactic responses in vivo. Thus, STIM1 is key in promoting the Ca2+ influx that is essential for FcepsilonRI-mediated mast cell activation and anaphylaxis.

The calcium sensors STIM1 and STIM2 control B cell regulatory function through interleukin-10 production.

A chief Ca(2+) entry pathway in immune cells is store-operated Ca(2+) (SOC) influx, which is triggered by depletion of Ca(2+) from the endoplasmic reticulum (ER). However, its physiological role in B cells remains elusive. Here, we show that ER calcium sensors STIM1- and STIM2-induced SOC influx is critical for B cell regulatory function. B cell-specific deletion of STIM1 and STIM2 in mice caused a profound defect in B cell receptor (BCR)-induced SOC influx and proliferation. However, B cell development and antibody responses were unaffected. Remarkably, B cells lacking both STIM proteins failed to produce the anti-inflammatory cytokine IL-10 because of defective activation of nuclear factor of activated T cells (NFAT) after BCR stimulation. This resulted in exacerbation of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Our data establish STIM-dependent SOC influx as a key signal for B cell regulatory function required to limit autoimmunity.

B Cell Receptor Signaling.

Signaling through the B cell receptor (BCR) plays a critical role at multiple checkpoints of B cell biology. BCR acts as a gatekeeper of the progression of their early development in bone marrow (BM). It is also essential in triggering mechanisms such as clonal deletion and receptor editing to eliminate autoreactive B cells. In the periphery, it most importantly functions as a receptor that recognizes various extracellular antigens in response to bacterial and viral infections for conferring host defense. The recognition of antigens by BCR is the first step to receive T cell help for the functional differentiation of naive B cells toward plasma cells, germinal center (GC) B cells and memory B cells. In addition, similar to the role of BCR in the early stages of B cell development, BCR signaling plays a crucial role in the prevention of dysregulated activation of autoreactive B cells which can induce autoimmunity in the secondary lymphoid organs. Thus, since BCR is essential for the proper elicitation of immune responses by B cells, signaling through the BCR is tightly controlled by the intracellular positive and negative regulators. In this chapter, the mechanisms of activation and repression of BCR signaling are reviewed on the basis of the recent findings.

Role of Calcium Signaling in B Cell Activation and Biology.

Increase in intracellular levels of calcium ions (Ca2+) is one of the key triggering signals for the development of B cell response to the antigen. The diverse Ca2+ signals finely controlled by multiple factors participate in the regulation of gene expression, B cell development, and effector functions. B cell receptor (BCR)-initiated Ca2+ mobilization is sourced from two pathways: one is the release of Ca2+ from the intracellular stores, endoplasmic reticulum (ER), and other is the prolonged influx of extracellular Ca2+ induced by depleting the stores via store-operated calcium entry (SOCE) and calcium release-activated calcium (CRAC) channels. The identification of stromal interaction molecule 1(STIM1), the ER Ca2+ sensor, and Orai1, a key subunit of the CRAC channel pore, has now provided the tools to understand the mode of Ca2+ influx regulation and physiological relevance. Herein, we discuss our current understanding of the molecular mechanisms underlying BCR-triggered Ca2+ signaling as well as its contribution to the B cell biological processes and diseases.

UDP-Induced Phagocytosis and ATP-Stimulated Chemotactic Migration Are Impaired in STIM1-/- Microglia In Vitro and In Vivo.

STIM1 is the only currently known intracellular calcium sensor that functions as the calcium influx regulator controlling immune cell activation. STIM1 function in immune cell calcium signalling has been studied extensively; however, its role in microglia, innate immune cells in brain, has not been fully understood. Here, we report that STIM1-/- murine microglia lost store-operated calcium influx and displayed aberrant immunological functions. Microglial functions regulated by chronic and global [Ca2+]i changes were reduced significantly, including cytokine releases and opsonin-dependent phagocytosis. More dramatically, cellular functions governed by Ca2+ regulation in local microdomains at the cell periphery, such as UDP-induced phagocytosis and ATP-stimulated chemotactic migration, were severely reduced in STIM1-/- microglia. Interestingly, UDP-induced Orai1 mobilization to the peripheral region was greatly attenuated in STIM1-/- microglia. Their chemotactic migration defect was reproduced in vivo in embryonic brain; the aggregated number of STIM1-/- microglia in LPS- (lipopolysaccharide-) injected lesions was much smaller than that in wild-type microglia. Furthermore, the neuron phagoptosis activities of activated microglia were significantly diminished in the STIM1-/- microglia. These in vitro and in vivo results suggest that STIM1-mediated store-operated calcium entry is important for the regulation of global [Ca2+]i changes which differentiates into active immune state of microglia, but it is more crucial for the regulation of local [Ca2+] microdomains which mediates the acute motility of murine microglia.

STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense.

The stromal-interacting molecule 1 (STIM1) is a potent sensor of intracellular calcium, which in turn regulates entry of external calcium through plasma membrane channels to affect immune cell activation. Although the contribution of STIM1 to calcium signaling in lymphocytes has been well studied, the role of this protein in neutrophil-mediated inflammation and host defense is unknown. We report that STIM1-deficient murine neutrophils show loss of store-operated calcium entry (SOCE) in response to both soluble ligands that activate G-proteins as well as Fcγ-receptor or integrin ligation that activates tyrosine kinase signaling. This results in modest defects in phagocytosis and degranulation responses but a profound block in superoxide production by the phagocyte oxidase. We trace the primary intracellular target of calcium to be protein kinase C isoforms α and ß (PKCα and PKCß), which in turn phosphorylate subunits of the oxidase leading to superoxide production. In vivo the loss of SOCE in stim1(-/-) chimeric mice results in marked susceptibility to bacterial infections but also protection from tissue injury in hepatic ischemia/reperfusion injury. These results demonstrate the critical role of STIM1-mediated SOCE and define major protein targets of calcium signaling in neutrophil activation during inflammatory disease.

Signals controlling the development and activity of regulatory B-lineage cells.

The fundamental concepts surrounding B cells with inhibitory function (regulatory B cells) are now being established. In the context of autoimmune and inflammatory animal models, B cells play an immunomodulatory role via IL-10 production and contribute to limitation of the pathogenesis. Recent studies have notably identified the human counterparts of these cells, which have been suggested to be relevant to the pathophysiology of disease. Clear criteria to identify these cell subsets and the key molecular mechanisms underlying their physiological features are required for understanding the big picture of regulatory B cells. Plasmablasts have recently been identified as a major IL-10-producing regulatory B-cell subset and Ca(2+) signaling has furthermore been found to contribute to B-cell IL-10 expression. In this review, the signaling components controlling IL-10-dependent B-cell regulatory function and the development of IL-10-competent/-producing B cells and plasmablasts are discussed.