Search

B cells play a crucial role in recognizing external antigens through their surface B cell receptor (BCR), essential for generating protective antibodies and immune memory. Regulation of B cell immune activation is closely linked to various upper respiratory tract diseases. Adenoid hypertrophy (ATH), a common childhood condition, is characterized by lymphoid follicular hyperplasia, yet its exact mechanisms remain elusive. This review consolidates research on BCR trophic signaling that sustains B cell viability in resting state. It clarifies the regulatory mechanisms governing B cell immune activation and the establishment of rapid, effective immune memory, with a specific focus on early molecular activation events and the pivotal role of mIgG-tail in memory antibody responses. Dysregulation of B cell activation processes can disrupt immune balance, potentially precipitating disease. This synthesis explores the connection between regulation of B cell activation and ATH pathogenesis, examining the potential impacts of signaling pathway dysregulation or mutations on ATH. The review aims to enhance understanding of the disease mechanisms underlying ATH, with the goal of identifying new diagnostic and therapeutic targets for this condition.

Simple Summary: The B cell receptor (BCR) is a membrane-bound protein complex that is required for the normal development of B cells. BCR signalling is also involved in the pathogenesis of B cell cancers. While there is substantial literature on genomic analyses of the BCR, there are limited proteomic studies of receptor structure and its interactions with neighbouring proteins. This is partly due to the location of the BCR in the surface-membrane lipid environment that has limited the ability to enrich the complex for proteomic analysis. Here, we report an enrichment technique that can be used for mass spectrometry analyses of the BCR from live B cells. B cells are central to the adaptive immune response and provide long-lasting immunity after infection. B cell activation is mediated by the surface membrane-bound B cell receptor (BCR) following recognition of a specific antigen. The BCR has been challenging to analyse using mass spectrometry (MS) due to the difficulty of isolating and enriching this membrane-bound protein complex. There are approximately 120,000 BCRs on the B cell surface; however, depending on the B cell activation state, there may be hundreds-of-millions to billions of proteins in a B cell. Consequently, advanced proteomic techniques such as MS workflows that use purified proteins to yield structural and protein-interaction information have not been published for the BCR complex. This paper describes a method for enriching the BCR complex that is MS-compatible. The method involves a Protein G pull down on agarose beads using an intermediary antibody to each of the BCR complex subcomponents (CD79a, CD79b, and membrane immunoglobulin). The enrichment process is shown to pull down the entire BCR complex and has the advantage of being readily compatible with further proteomic study including MS analysis. Using intermediary antibodies has the potential to enrich all isotypes of the BCR, unlike previous methods described in the literature that use protein G-coated beads to directly pull down the membrane IgG (mIgG) but cannot be used for other mIg isotypes. [ABSTRACT FROM AUTHOR]

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen‐specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long‐term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen‐specific peripheral IgG+ memory B cells into antibody‐secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture‐derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG‐Fc fucosylation to be regulated at the single‐clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture‐derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies. [ABSTRACT FROM AUTHOR]

Abstract Background Neuromyelitis optica spectrum disorder (NMOSD) stands out among CNS inflammatory demyelinating diseases (CIDDs) due to its unique disease characteristics, including severe clinical attacks with extensive lesions and its association with systemic autoimmune diseases. We aimed to investigate whether characteristics of B cell receptors (BCRs) differ between NMOSD and other CIDDs using high-throughput sequencing. Methods From a prospective cohort, we recruited patients with CIDDs and categorized them based on the presence and type of autoantibodies: NMOSD with anti-aquaporin-4 antibodies, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) with anti-myelin oligodendrocyte glycoprotein antibodies, double-seronegative demyelinating disease (DSN), and healthy controls (HCs). The BCR features, including isotype class, clonality, somatic hypermutation (SHM), and the third complementarity-determining region (CDR3) length, were analyzed and compared among the different disease groups. Results Blood samples from 33 patients with CIDDs (13 NMOSD, 12 MOGAD, and 8 DSN) and 34 HCs were investigated for BCR sequencing. Patients with NMOSD tended to have more activated BCR features compare to the other disease groups. They showed a lower proportion of unswitched isotypes (IgM and IgD) and a higher proportion of switched isotypes (IgG), increased clonality of BCRs, higher rates of SHM, and shorter lengths of CDR3. Notably, advanced age was identified as a clinical factor associated with these activated BCR features, including increased levels of clonality and SHM rates in the NMOSD group. Conversely, no such clinical factors were found to be associated with activated BCR features in the other CIDD groups. Conclusions NMOSD patients, among those with CIDDs, displayed the most pronounced B cell activation, characterized by higher levels of isotype class switching, clonality, SHM rates, and shorter CDR3 lengths. These findings suggest that B cell-mediated humoral immune responses and characteristics in NMOSD patients are distinct from those observed in the other CIDDs, including MOGAD. Age was identified as a clinical factor associated with BCR activation specifically in NMOSD, implying the significance of persistent B cell activation attributed to anti-aquaporin-4 antibodies, even in the absence of clinical relapses throughout an individual’s lifetime.

Phylogenetic trees can be built from B cell receptor (BCR) sequences and used to study somatic hypermutation and selection. The unique biology of B cells has led to the development of B cell-specific phylogenetic methods. Single-cell sequencing can improve tree building with paired heavy and light chain sequences and (potentially) with associated transcriptomic information. Phylogenetic trees can be used to study B cell migration, differentiation, and evolution over time, but more work is needed to study these processes in a model-based manner. Phylogenetic trees built from B cell receptor (BCR) sequences can trace the history of mutation and selection in B cell clones. Advances in single-cell sequencing can significantly improve all aspects of B cell tree building. Further, there is potential for new methods using single-cell data to drive exciting advances in tracking cellular migration, differentiation, and evolution over time. The widespread availability of single-cell RNA sequencing (scRNA-seq) has led to the development of new methods for understanding immune responses. Single-cell transcriptome data can now be paired with B cell receptor (BCR) sequences. However, RNA from BCRs cannot be analyzed like most other genes because BCRs are genetically diverse within individuals. In humans, BCRs are shaped through recombination followed by mutation and selection for antigen binding. As these processes co-occur with cell division, B cells can be studied using phylogenetic trees representing the mutations within a clone. B cell trees can link experimental timepoints, tissues, or cellular subtypes. Here, we review the current state and potential of how B cell phylogenetics can be combined with single-cell data to understand immune responses. [ABSTRACT FROM AUTHOR]

Background: Neuromyelitis optica spectrum disorder (NMOSD) stands out among CNS inflammatory demyelinating diseases (CIDDs) due to its unique disease characteristics, including severe clinical attacks with extensive lesions and its association with systemic autoimmune diseases. We aimed to investigate whether characteristics of B cell receptors (BCRs) differ between NMOSD and other CIDDs using high-throughput sequencing. Methods: From a prospective cohort, we recruited patients with CIDDs and categorized them based on the presence and type of autoantibodies: NMOSD with anti-aquaporin-4 antibodies, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) with anti-myelin oligodendrocyte glycoprotein antibodies, double-seronegative demyelinating disease (DSN), and healthy controls (HCs). The BCR features, including isotype class, clonality, somatic hypermutation (SHM), and the third complementarity-determining region (CDR3) length, were analyzed and compared among the different disease groups. Results: Blood samples from 33 patients with CIDDs (13 NMOSD, 12 MOGAD, and 8 DSN) and 34 HCs were investigated for BCR sequencing. Patients with NMOSD tended to have more activated BCR features compare to the other disease groups. They showed a lower proportion of unswitched isotypes (IgM and IgD) and a higher proportion of switched isotypes (IgG), increased clonality of BCRs, higher rates of SHM, and shorter lengths of CDR3. Notably, advanced age was identified as a clinical factor associated with these activated BCR features, including increased levels of clonality and SHM rates in the NMOSD group. Conversely, no such clinical factors were found to be associated with activated BCR features in the other CIDD groups. Conclusions: NMOSD patients, among those with CIDDs, displayed the most pronounced B cell activation, characterized by higher levels of isotype class switching, clonality, SHM rates, and shorter CDR3 lengths. These findings suggest that B cell-mediated humoral immune responses and characteristics in NMOSD patients are distinct from those observed in the other CIDDs, including MOGAD. Age was identified as a clinical factor associated with BCR activation specifically in NMOSD, implying the significance of persistent B cell activation attributed to anti-aquaporin-4 antibodies, even in the absence of clinical relapses throughout an individual's lifetime. [ABSTRACT FROM AUTHOR]

Simple Summary: The treatment of B cell malignancies was transformed by the development of a monoclonal antibody—rituximab—that targets CD20, a protein expressed on the surface of B cells. However, some types of B cell malignancies do not express CD20 or can reduce the expression of the protein to escape therapy. Consequently, there is a need to develop tailored therapies against other targets expressed by B cells. The structures of key B cell proteins, the B cell receptor, and its co-receptors CD22, CD19 and CD81, have recently been solved and provide the opportunity to guide development of new antibodies and other therapies targeted at malignant B cells. Here, we review high-resolution protein structures of the BCR, CD22, CD19 and CD81 molecules, treatments that have been developed against these targets and discuss structural features that will enable the design of novel antibodies. B cells are central to the adaptive immune response, providing long lasting immunity after infection. B cell activation is mediated by a cell surface B cell receptor (BCR) following recognition of an antigen. BCR signaling is modulated by several co-receptors including CD22 and a complex that contains CD19 and CD81. Aberrant signaling through the BCR and co-receptors promotes the pathogenesis of several B cell malignancies and autoimmune diseases. Treatment of these diseases has been revolutionized by the development of monoclonal antibodies that bind to B cell surface antigens, including the BCR and its co-receptors. However, malignant B cells can escape targeting by several mechanisms and until recently, rational design of antibodies has been limited by the lack of high-resolution structures of the BCR and its co-receptors. Herein we review recently determined cryo-electron microscopy (cryo-EM) and crystal structures of the BCR, CD22, CD19 and CD81 molecules. These structures provide further understanding of the mechanisms of current antibody therapies and provide scaffolds for development of engineered antibodies for treatment of B cell malignancies and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Introduction: Early development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known. Methods: To identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects. Results: We found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing. Discussion: Together, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development. [ABSTRACT FROM AUTHOR]

IntroductionEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known.MethodsTo identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects.ResultsWe found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing.DiscussionTogether, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

B cell maturation and immunoglobulin (Ig) repertoire selection are governed by expression of a functional B cell receptor (BCR). Naïve B cells co-express their BCR as IgM and IgD isotype. However, the role of the additionally expressed IgD on naïve B cells is not known. Here we assessed the impact of IgD on naïve B cell maturation and Ig repertoire selection in 8 individuals from3 different families with heterozygous loss-of-function or loss-of expression mutations in IGHD. Although naïve B cells from these individuals expressed IgM on their surface, the IGHD variant in heterozygous state entailed a chimeric situation by allelic exclusion with almost half of the naïve B cell population lacking surface IgD expression. Flow cytometric analyses revealed a distinct phenotype of IgD-negative naïve B cells with decreased expression of CD19, CD20 and CD21 as well as lower BAFF-R and integrin-b7 expression. IgD-negative B cells were less responsive in vitro after engaging the IgM-BCR, TLR7/9 or CD40 pathway. Additionally, a selective disadvantage of IgD-negative B cells within the T2 transitional and mature naïve B cell compartment as well as reduced frequencies of IgMlo/- B cells within the mature naïve B cell compartment lacking IgD were evident. RNA-Ig-seq of bulk sorted B cell populations showed an altered selection of distinct VH segments in the IgD-negativemature naïve B cell population. We conclude that IgD expression on human naïve B cells is redundant for generation of naïve B cells in general, but further shapes the naive B cell compartment starting from T2 transitional B cells. Our observations suggest an unexpected role of IgD expression to be critical for selection of distinct Ig VH segments into the pre-immune Ig repertoire and for the survival of IgMlo/- naïve B cells known to be enriched in poly-/autoreactive B cell clones. [ABSTRACT FROM AUTHOR]

Aberrant B cell signaling plays a critical in role in various systemic and organ-specific autoimmune diseases. This is supported by genetic evidence by many functional studies in B cells from patients or specific animal models and by the observed efficacy of small-molecule inhibitors. In this review, we first discuss key signal transduction pathways downstream of the B cell receptor (BCR) that ensure that autoreactive B cells are removed from the repertoire or functionally silenced. We provide an overview of aberrant BCR signaling that is associated with inappropriate B cell repertoire selection and activation or survival of peripheral B cell populations and plasma cells, finally leading to autoantibody formation. Next to BCR signaling, abnormalities in other signal transduction pathways have been implicated in autoimmune disease. These include reduced activity of several phosphates that are downstream of co-inhibitory receptors on B cells and increased levels of BAFF and APRIL, which support survival of B cells and plasma cells. Importantly, pathogenic synergy of the BCR and Toll-like receptors (TLR), which can be activated by endogenous ligands, such as self-nucleic acids, has been shown to enhance autoimmunity. Finally, we will briefly discuss therapeutic strategies for autoimmune disease based on interfering with signal transduction in B cells. [ABSTRACT FROM AUTHOR]

B cell maturation and immunoglobulin (Ig) repertoire selection are governed by expression of a functional B cell receptor (BCR). Naïve B cells co-express their BCR as IgM and IgD isotype. However, the role of the additionally expressed IgD on naïve B cells is not known. Here we assessed the impact of IgD on naïve B cell maturation and Ig repertoire selection in 8 individuals from 3 different families with heterozygous loss-of-function or loss-of expression mutations in IGHD. Although naïve B cells from these individuals expressed IgM on their surface, the IGHD variant in heterozygous state entailed a chimeric situation by allelic exclusion with almost half of the naïve B cell population lacking surface IgD expression. Flow cytometric analyses revealed a distinct phenotype of IgD-negative naïve B cells with decreased expression of CD19, CD20 and CD21 as well as lower BAFF-R and integrin-β7 expression. IgD-negative B cells were less responsive in vitro after engaging the IgM-BCR, TLR7/9 or CD40 pathway. Additionally, a selective disadvantage of IgD-negative B cells within the T2 transitional and mature naïve B cell compartment as well as reduced frequencies of IgMlo/- B cells within the mature naïve B cell compartment lacking IgD were evident. RNA-Ig-seq of bulk sorted B cell populations showed an altered selection of distinct VH segments in the IgD-negative mature naïve B cell population. We conclude that IgD expression on human naïve B cells is redundant for generation of naïve B cells in general, but further shapes the naive B cell compartment starting from T2 transitional B cells. Our observations suggest an unexpected role of IgD expression to be critical for selection of distinct Ig VH segments into the pre-immune Ig repertoire and for the survival of IgMlo/- naïve B cells known to be enriched in poly-/autoreactive B cell clones.

Although birds have been used historically as a model animal for immunological research, resulting in remarkable achievements, immune cell development in birds themselves has yet to be fully elucidated. In this study, we firstly generated an immunodeficient chicken model using a CRISPR/Cas9-mediated recombination activating gene 1 (RAG1) knockout, to investigate avian-specific immune cell development. Unlike previously reported immunoglobulin (Ig) heavy chain knockout chickens, the proportion and development of B cells in both RAG1+/- and RAG1-/- embryos were significantly impaired during B cell proliferation (embryonic day 16 to 18). Our findings indicate that, this is likely due to disordered B cell receptor (BCR)-mediated signaling and interaction of CXC motif chemokine receptor (CXCR4) with CXCL12, resulting from disrupted Ig V(D)J recombination at the embryonic stage. Histological analysis after hatching showed that, unlike wild-type (WT) and RAG1+/- chickens, lymphatic organs in 3-week old RAG1-/- chickens were severely damaged. Furthermore, relative to WT chickens, RAG1+/- and RAG1-/- birds had reduced serum Igs, fewer mature CD4+ and CD8+ T lymphocytes. Furthermore, BCRmediated B cell activation in RAG1+/- chickens was insufficient, leading to decreased expression of the activation-induced deaminase (AID) gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of RAG1 in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of RAG1 during immune cell development. This model will provide ongoing insights for understanding chicken immune system development and applied in the fields of immunology and biomedical science. [ABSTRACT FROM AUTHOR]

We previously reported that monoclonal mouse immunoglobulin (Ig) A, OA-4, attenuates sensitization in mice by suppressing B cell activation. Here, it is demonstrated for the first time that mouse IgA inhibits mouse B cell activation in vitro under natural conditions (i.e. in the absence of chemical, physical, and genetic modifications of IgA and B cells). Mouse splenocytes were stimulated with anti-B cell receptor (BCR) antibody or lipopolysaccharide (LPS) in the presence or absence of OA-4. Splenic B cell proliferation and the activation of several intracellular signaling molecules were measured. Anti-BCR antibody-induced proliferation was markedly inhibited by OA-4 or the commercially available mouse IgA S107, whereas LPS-induced proliferation was weakly attenuated by a high concentration of OA-4. Moreover, OA-4 markedly decreased the anti-BCR antibody-induced phosphorylation of p44/42 mitogen-activated protein kinase (ERK) and CD22 and decreased phosphorylated phospholipase (PLC) γ2 and intracellular Ca2+ levels moderately, whereas protein kinase B (Akt) phosphorylation was not affected by OA-4. The MAPK/ERK kinase-ERK and phosphoinositide 3-kinase-Akt pathways were found to play a role in the proliferation of splenocytes induced by anti-BCR antibody based on experiments with their inhibitors. In contrast to that in splenic B cells, ERK phosphorylation induced by anti-BCR antibody in A20 cells was not inhibited by OA-4. The modulatory effects of IgA were different among the cell types and signaling pathways. IgA is a potential immunoregulatory drug utilizing new mechanisms that affect splenic B cells but not A20 lymphomas. [ABSTRACT FROM AUTHOR]

The poor efficacy of seasonal influenza virus vaccines is often attributed to pre-existing immunity interfering with the persistence and maturation of vaccine-induced B cell responses. We previously showed that a subset of vaccine-induced B cell lineages are recruited into germinal centers (GCs) following vaccination, suggesting that affinity maturation of these lineages against vaccine antigens can occur. However, it remains to be determined whether seasonal influenza vaccination stimulates additional evolution of vaccine-specific lineages, and previous work has found no significant increase in somatic hypermutation among influenza-binding lineages sampled from the blood following seasonal vaccination in humans. Here, we investigate this issue using a phylogenetic test of measurable immunoglobulin sequence evolution. We first validate this test through simulations and survey measurable evolution across multiple conditions. We find significant heterogeneity in measurable B cell evolution across conditions, with enrichment in primary response conditions such as HIV infection and early childhood development. We then show that measurable evolution following influenza vaccination is highly compartmentalized: while lineages in the blood are rarely measurably evolving following influenza vaccination, lineages containing GC B cells are frequently measurably evolving. Many of these lineages appear to derive from memory B cells. We conclude from these findings that seasonal influenza virus vaccination can stimulate additional evolution of responding B cell lineages, and imply that the poor efficacy of seasonal influenza vaccination is not due to a complete inhibition of vaccine-specific B cell evolution.

Aberrant B cell signaling plays a critical in role in various systemic and organ-specific autoimmune diseases. This is supported by genetic evidence by many functional studies in B cells from patients or specific animal models and by the observed efficacy of small-molecule inhibitors. In this review, we first discuss key signal transduction pathways downstream of the B cell receptor (BCR) that ensure that autoreactive B cells are removed from the repertoire or functionally silenced. We provide an overview of aberrant BCR signaling that is associated with inappropriate B cell repertoire selection and activation or survival of peripheral B cell populations and plasma cells, finally leading to autoantibody formation. Next to BCR signaling, abnormalities in other signal transduction pathways have been implicated in autoimmune disease. These include reduced activity of several phosphates that are downstream of co-inhibitory receptors on B cells and increased levels of BAFF and APRIL, which support survival of B cells and plasma cells. Importantly, pathogenic synergy of the BCR and Toll-like receptors (TLR), which can be activated by endogenous ligands, such as self-nucleic acids, has been shown to enhance autoimmunity. Finally, we will briefly discuss therapeutic strategies for autoimmune disease based on interfering with signal transduction in B cells.

Signaling by the B cell antigen receptor (BCR) initiates actin remodeling. The assembly of branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, allowing B cells to form membrane protrusions that can scan the surface of antigen-presenting cells (APCs). The resulting Arp2/3 complex-dependent actin retrograde flow promotes the centripetal movement and progressive coalescence of BCR microclusters, which amplifies BCR signaling. Glia maturation factor γ (GMFγ) is an actin disassembly-protein that releases Arp2/3 complex-nucleated actin filaments from actin networks. By doing so, GMFγ could either oppose the actions of the Arp2/3 complex or support Arp2/3 complex-nucleated actin polymerization by contributing to the recycling of actin monomers and Arp2/3 complexes. We now show that reducing the levels of GMFγ in human B cell lines via transfection with a specific siRNA impairs the ability of B cells to spread on antigen-coated surfaces, decreases the velocity of actin retrograde flow, diminishes the coalescence of BCR microclusters into a central cluster at the B cell-APC contact site, and decreases APC-induced BCR signaling. These effects of depleting GMFγ are similar to what occurs when the Arp2/3 complex is inhibited. This suggests that GMFγ cooperates with the Arp2/3 complex to support BCR-induced actin remodeling and amplify BCR signaling at the immune synapse.

Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in synapses of primary mouse B cells and human B cell lines forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction.

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.

The gap junction (GJ) protein connexin 43 (Cx43) is both necessary and sufficient for B cell receptor (BCR)-mediated cell spreading. To address how Cx43 mediates this effect, we blocked its function genetically, by expressing mutants of Cx43, and pharmacologically, by using chemical inhibitors. While various point mutations of Cx43 inhibited B cell spreading, treatment with channel blocking drugs did not, suggesting that this response was independent of channel function. The critical region of Cx43 appears to be the cytoplasmic carboxyl-terminal (CT) domain, which has previously been shown to be important for B cell spreading. Consistent with this, mutations of either tyrosine 247 or 265 found in the CT were sufficient to inhibit spreading. Thus Cx43 may influence B cell spreading by mechanisms requiring protein binding to, or modification of, these sites in the CT tail.

Germinal centers (GCs) are sites of B cell clonal expansion, diversification, and antibody affinity selection. This process is limited and directed by T follicular helper cells that provide helper signals to B cells that endocytose, process, and present cognate antigens in proportion to their B cell receptor (BCR) affinity. Under this model, the BCR functions as an endocytic receptor for antigen capture. How signaling through the BCR contributes to selection is not well understood. To investigate the role of BCR signaling in GC selection, we developed a tracker for antigen binding and presentation and a Bruton's tyrosine kinase drug-resistant-mutant mouse model. We showed that BCR signaling per se is necessary for the survival and priming of light zone B cells to receive T cell help. Our findings provide insight into how high-affinity antibodies are selected within GCs and are fundamental to our understanding of adaptive immunity and vaccine development. [Display omitted] • Developed a tracker of in vivo antigen binding and presentation • Generated a Bruton's tyrosine kinase drug-resistant mouse model • BCR signaling protects LZ B cells from apoptosis • BCR signaling primes and synergizes with T cell help The role that B cell receptor (BCR) signaling plays in selection within germinal centers (GCs) is not well understood. Here, Chen et al. demonstrate that BCR signaling is necessary for the survival and priming of light zone (LZ) B cells and that these signals synergize with T cell help to enhance positive selection of GC B cells. [ABSTRACT FROM AUTHOR]

B cells form an essential part of the adaptive immune system by producing specific antibodies that can neutralize toxins and target infected or malignant cells for destruction. During B cell activation, a fundamental role is played by a specialized intercellular structure called the immunological synapse ( IS). The IS serves as a platform for B cell recognition of foreign, often pathogenic, antigens on the surface of antigen-presenting cells ( APC). This recognition is elicited by highly specific B cell receptors ( BCR) that subsequently trigger carefully orchestrated intracellular signaling cascades that lead to cell activation. Furthermore, antigen internalization, essential for full B cell activation and differentiation into antibody producing effector cells or memory cells, occurs in the IS. Recent developments especially in various imaging-based methods have considerably advanced our understanding of the molecular control of B cell activation. Interestingly, the cellular cytoskeleton is emerging as a key player at several stages of B cell activation, including the initiation of receptor signaling. Here, we discuss the functions and molecular mechanisms of the IS and highlight the multifaceted role of the actin cytoskeleton in several aspects of B cell activation. [ABSTRACT FROM AUTHOR]

Abstract: The actin cytoskeleton is a dynamic cellular network known for its function in cell morphology and motility. Recent studies using high resolution and real time imaging techniques have revealed that actin plays a critical role in signal transduction, primarily by modulating the dynamics and organization of membrane-associated receptors and signaling molecules. This review summarizes what we have learned so far about a regulatory niche of the actin cytoskeleton in the signal transduction of the B cell receptor (BCR). The activation of the BCR is initiated and regulated by a close coordination between the dynamics of surface BCRs and the cortical actin network. The actin cytoskeleton is involved in regulating the signaling threshold of the BCR to antigenic stimulation, the kinetics and amplification of BCR signaling activities, and the timing and kinetics of signaling downregulation. Actin exerts its regulatory function by controlling the kinetics, magnitude, subcellular location, and nature of BCR clustering and BCR signaling complex formation at every stage of signaling. The cortical actin network is remodeled by initial detachment from the plasma membrane, disassembly and subsequent reassembly into new actin structures in response to antigenic stimulation. Signaling responsive actin regulators translate BCR stimulatory and inhibitory signals into a series of actin remodeling events, which enhance signaling activation and down-regulation by modulating the lateral mobility and spatial organization of surface BCR. The mechanistic understanding of actin-mediated signaling regulation in B cells will help us explore B cell-specific manipulations of the actin cytoskeleton as treatments for B cell-mediated autoimmunity and B cell cancer. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé. [Copyright &y& Elsevier]

Abstract: Autoantibodies clearly contribute to tissue inflammation in systemic lupus erythematosus. In order to therapeutically target B cells making pathogenic autoantibodies, it is necessary to identify their phenotype. It is also important to understand the defects in B cell repertoire selection that permit pathogenic autoreactive B cells to enter the immunocompetent B cell repertoire. We present the data that both marginal zone and follicular B cells can produce pathogenic autoantibodies. Moreover, we discuss how B cell survival and maturation are regulated centrally prior to antigen activation and in the periphery after antigen activation to form the repertoire that generates the spectrum of circulating antibodies. [Copyright &y& Elsevier]

Age related changes in the structure and function of the immune system, collectively termed immunosenescence, result in poor responses to infections, increased susceptibility to cancers and increased incidence of autoimmune diseases. The humoral immune response, maintained by the B cell compartment, has a key role in an effective immune system—not only in producing high affinity antibodies that are crucial for vaccination strategies, but in assisting other components of the immune system in their function. Hence an understanding of B cell immunosenescence in particular is vital in designing strategies to combat the effects of age on immune function. Numerous studies have been undertaken using small animal models in order to understand immunosenescence, and these have contributed greatly to our understanding of the events that underpin impaired immune responses. However, there are key differences between the human and the mouse and a clear understanding of these differences is required when extrapolating from one species to the other. In this article we present an overview of B cell development and summarise current data on age-related B cell changes, at both the population level and at the individual mechanistic level. Areas of similarity and difference between human and mouse models are highlighted. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are involved in the innate recognition of foreign material and their activation leads to both innate and adaptive immune responses directed against invading pathogens. TLR9 is intracellularly expressed in the endo-lysosomal compartments of specialized immune cells. TLR9 is activated in response to DNA, in particular DNA containing unmethylated CpG motifs that are more prevalent in microbial than mammalian DNA. By detecting foreign DNA signatures TLR9 can sense the presence of certain viruses or bacteria inside the cell and mount an immune response. However, under certain conditions, TLR9 can also recognize self-DNA and this may promote immune pathologies with uncontrolled chronic inflammation. The autoimmune disease systemic lupus erythematosis (SLE) is characterized by the presence of immune stimulatory complexes containing autoantibodies against endogenous DNA and DNA- and RNA-associated proteins. Recent evidence indicates that the autoimmune response to these complexes involves TLR9 and the related single-stranded RNA-responsive TLRs 7 and 8, and therefore some breakdown in the normal ability of these TLRs to distinguish self and foreign DNA. Evidence suggests that immune cells use several mechanisms to discriminate between stimulatory and nonstimulatory DNA; however, it appears that TLR9 itself binds rather indiscriminately to a broad range of DNAs. We therefore propose that there is an additional recognition step by which TLR9 senses differences in the structures of bound DNA. [ABSTRACT FROM AUTHOR]