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To evaluate the expression of recombinant activating gene (RAG) and B cell receptor (BCR) gene rearrangements in mature peripheral B lymphocytes in Takayasu arteritis (TA) to explore the possible mechanism of humoral immune response in TA. Ten patients with TA and 10 age- and sex-matched healthy volunteers (control group) from Beijing Shijitan Hospital, Capital Medical University and Peking Union Medical College Hospital, between 2022 and 2023, were included in this study. The mRNA of the RAG was measured using real-time quantitative PCR (RT-PCR). Western blotting was used to detect RAG protein expression levels. NGS technology was used to detect BCR gene rearrangement. The mRNA expression level of RAG1 and RAG2 in peripheral mature B lymphocytes in patients with TA was significantly higher than in the control group (RAG1 5.56 ± 1.71 vs. 1.94 ± 0.86, p < 0.05; RAG2 5.26 ± 1.59 vs. 1.65 ± 0.64, p < 0.05), respectively. The protein expression level of the RAG1 and the RAG2 in peripheral mature B lymphocytes in patients with TA was significantly higher than in the healthy control group (RAG1 4.33 ± 1.58 vs. 1.52 ± 0.59, p < 0.001; RAG2 4.67 ± 1.88 vs. 1.59 ± 0.56, p < 0.001). The number of peripheral B lymphocyte BCR clonotypes in the group of patients with TA was significantly higher than in the normal control group (1574 ± 317.7 vs. 801.3 ± 202.1, p < 0.05). The abundance of IGHV clones in patients with TA was higher than in the normal control group (31.185% vs. 13.449%), which was positively correlated with the expression levels of RAG1 and RAG2 (correlation coefficient r = 1.00, p < 0.001), respectively. High expression of the RAG gene coexists with secondary BCR gene rearrangement in mature peripheral B lymphocytes in patients with TA, providing important clues regarding the potential humoral response in TA; however, further studies with larger samples are needed., (© 2024 John Wiley & Sons Ltd.)

In people living with HIV (PLWH), the susceptibility to malignancies is notably augmented, with lymphoma emerging as a predominant malignancy. Even in the antiretroviral therapy (ART) era, aggressive B-cell lymphoma stands out as a paramount concern. Yet, the pathogenesis of HIV related lymphoma (HRL) largely remains an enigma. Recent insights underscore the pivotal role of the dysregulated B cell receptor (BCR) signaling cascade, evidencing its oncogenic potential across a spectrum of lymphomas. Intricate interplays between HIV and BCR structural-functional integrity have been identified in PLWH. In this review, we elucidated the mechanism by which the BCR signaling pathway is involved in HRL, mainly including the following aspects: HIV can reshape BCR structure by modulating of activation-induced cytidine deaminase (AID) and recombination-activating gene (RAG) dynamics; HIV can act as a chronic antigen to activate the BCR signaling pathway, such as upregulating PI3K and MAPK signaling pathway and reducing the expression of CD300a; HIV co-infection with other oncogenic viruses may also influence tumor formation mediated by the BCR signaling pathway. This review aims to elucidate the intricate regulation of the BCR signaling pathway by HIV in B cell lymphoma, providing a novel perspective on the pathogenesis of lymphoma in HIV-affected environments., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Multiple sclerosis (MS) is a chronic, multifactorial, inflammatory and demyelinating disease of the central nervous system (CNS), which involves an autoimmune response against components of the myelin sheaths. Anti-B cell therapies have been proven to be successful in reducing relapses. Therefore, the study of B cells in both phases of the disease (relapse and remission) is of great importance. Here, we analyzed peripheral blood-cell BCR repertoire from 11 MS patients during a relapse phase and during remission, 6 patients with other inflammatory neurological diseases (OIND) and 10 healthy subjects (HCs), using next generation sequencing. In addition, immunoglobulins G, M, A and D were quantified in the serum of patients and controls, using ELISA. BCR repertoire of relapsing MS patients showed lower diversity, as well as a higher rate of somatic hypermutation compared to the other study groups. Within this group, the highest percentage of shared clonotypes was observed. IGHV4-32 gene was identified as a potential differential biomarker between MS and OIND, as well as IGL3-21 gene as a potential MS biomarker. On the other hand, an elevation of IgG and IgD was found in the serum of MS patients during remission, and the serum IgG was also elevated in MS patients during relapse. In conclusion, these results show the important role of B cells in the pathogenesis of the MS relapses and a new panorama on the analysis of the peripheral blood BCR repertoire to obtain diagnostic tools for MS. Furthermore, this work highlights the need of studies in diverse populations, since results reported in Caucasian populations may not coincide with the immunological course of MS patients in other latitudes, due to differences in genetic background and environmental exposures., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024. Published by Elsevier Inc.)

Background: Tuberculosis (TB) is a leading cause of death worldwide from a single infectious agent. In China the most common extra-pulmonary TB (EPTB) is pleural tuberculosis (PLTB). An important clinical feature of PLTB is that the lymphocytes associated with TB will accumulate in the pleural fluid. The adaptive immune repertoires play important roles in Mycobacterium tuberculosis (Mtb) infection., Methods: In this study, 10 PLTB patients were enrolled, and their Peripheral Blood Mononuclear Cells(PBMCs) and Pleural Effusion Mononuclear Cells(PEMCs) were collected. After T cells were purified from PBMCs and PEMCs, high-throughput immunosequencing of the TCRβ chain (TRB), TCRγ chain(TRG), and B cell receptor(BCR) immunoglobulin heavy chain (IGH) were conducted on these samples., Results: The TRB, TRG, and BCR IGH repertoires were characterized between the pleural effusion and blood in PLTB patients, and the shared clones were analyzed and collected. The binding activity of antibodies in plasma and pleural effusion to Mtb antigens was tested which indicates that different antibodies responses to Mtb antigens in plasma and pleural effusion in PLTB patients. Moreover, GLIPH2 was used to identify the specificity groups of TRB clusters and Mtb-specific TRB sequences were analyzed and collected by VJ mapping., Conclusion: We characterize the adaptive immune repertoires and identify the shared clones and Mtb-specific clones in pleural effusion and blood in PLTB patients which can give important clues for TB diagnosis, treatment, and vaccine development., Competing Interests: Authors YD, LD and TS were employed by company Hangzhou ImmuQuad Biotechnologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Du, Deng, Deng, Du, Xing, Tao, Li, Xie, Zhang, Sun and Li.)

When mature B cells are activated by antigens, the selection of these activated B cells takes place particularly during T cell-dependent immune responses in which an improved antibody repertoire is generated through somatic hypermutation in germinal centers (GCs). In this process the importance of antigen presentation by GC B cells, and subsequent T follicular helper (Tfh) cell help in positive selection of GC B cells, has been well appreciated. By contrast, the role of B cell receptor (BCR) signaling per se remains unclear. Strong experimental support for the involvement of BCR signaling in GC B cell selection has now been provided. Interestingly, these studies suggest that several checkpoints operating through the BCR ensure affinity maturation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The molecular landscape of chronic lymphocytic leukemia (CLL) has been extensively characterized, and various potent prognostic biomarkers were discovered. The genetic composition of the B-cell receptor (BCR) immunoglobulin (IG) was shown to be especially powerful for discerning indolent from aggressive disease at diagnosis. Classification based on the IG heavy chain variable gene (IGHV) somatic hypermutation status is routinely applied. Additionally, BCR IGH stereotypy has been implicated to improve risk stratification, through characterization of subsets with consistent clinical profiles. Despite these advances, it remains challenging to predict when CLL progresses to requiring first-line therapy, thus emphasizing the need for further refinement of prognostic indicators. Signaling pathways downstream of the BCR are essential in CLL pathogenesis, and dysregulated components within these pathways impact disease progression. Considering not only genomics but the entirety of factors shaping BCR signaling activity, this review offers insights in the disease for better prognostic assessment of CLL.

Glucocorticoids have been used for decades to treat lymphomas without an established mechanism of action. Using functional genomic, proteomic, and chemical screens, we discover that glucocorticoids inhibit oncogenic signaling by the B cell receptor (BCR), a recurrent feature of aggressive B cell malignancies, including diffuse large B cell lymphoma and Burkitt lymphoma. Glucocorticoids induce the glucocorticoid receptor (GR) to directly transactivate genes encoding negative regulators of BCR stability (LAPTM5; KLHL14) and the PI3 kinase pathway (INPP5D; DDIT4). GR directly represses transcription of CSK, a kinase that limits the activity of BCR-proximal Src-family kinases. CSK inhibition attenuates the constitutive BCR signaling of lymphomas by hyperactivating Src-family kinases, triggering their ubiquitination and degradation. With the knowledge that glucocorticoids disable oncogenic BCR signaling, they can now be deployed rationally to treat BCR-dependent aggressive lymphomas and used to construct mechanistically sound combination regimens with inhibitors of BTK, PI3 kinase, BCL2, and CSK., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

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., Competing Interests: Declaration of interests S.H.K. receives consulting fees from Peraton. K.B.H. receives consulting fees from Prellis Biologics., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Next-generation sequencing has the potential to uncover the complex nature of B cell immunity by revealing the full complexity of B cell receptor (BCR) repertoires in health and disease. However, there are drawbacks which can compromise the validity of the repertoire analysis caused by quantitative bias and accumulation of sequencing errors during the library preparation and sequencing. Here, we provide an optimized protocol designed to minimize bias for reproducible and accurate preparation of human BCR repertoire libraries for high-throughput sequencing., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Advances in high-throughput sequencing technologies have facilitated the large-scale characterization of B cell receptor (BCR) repertoires. However, the vast amount and high diversity of the BCR sequences pose challenges for efficient and biologically meaningful analysis. Here, we introduce fastBCR, an efficient computational approach for inferring B cell clonal families from massive BCR heavy chain sequences. We demonstrate that fastBCR substantially reduces the running time while ensuring high accuracy on simulated datasets with diverse numbers of B cell lineages and varying mutation rates. We apply fastBCR to real BCR sequencing data from peripheral blood samples of COVID-19 patients, showing that the inferred clonal families display disease-associated features, as well as corresponding antigen-binding specificity and affinity. Overall, our results demonstrate the advantages of fastBCR for analyzing BCR repertoire data, which will facilitate the identification of disease-associated antibodies and improve our understanding of the B cell immune response., Competing Interests: Declaration of interests X.H. is a shareholder of GV20 Oncotherapy and the Chief Information Officer of its subsidiary, GV20 Therapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Since the pioneering works of Berg and Purcell, discriminating between diffusion followed by binding has played a central role in understanding cell signaling. B cell receptors (BCR) and antibodies (Ab) challenge that simplified view as binding to the antigen follows after a chain of diffusion and rotations, including whole molecule rotation and independent tilts and twists of their Fab arms due to their Y-shaped structure and flexibility. In this paper, we combine analytical calculations with Brownian simulations to derive the first-passage times due to these three rotations positioning the Fab paratopes at a proper distance and orientation required for antigen binding. Our results indicate that when measuring Ab-Ag effective kinetic binding rates, using experimental methods in which the analyte is in solution only gives values proportional to the intrinsic binding rates, [Formula: see text], and [Formula: see text], for values of [Formula: see text] up to [Formula: see text]. Beyond that, a plateau of the effective 3D on rate between [Formula: see text] and [Formula: see text] is attained. Additionally, for BCR-Ag interactions, the effective 2D on and off binding rates can only be inferred from the corresponding effective 3D on and off rates for values of effective 3D on rates lower than [Formula: see text]. This is highly relevant when trying to relate BCR-antigen-binding strength and B cell response, especially during germinal center reactions. Therefore, there is a pressing need to reexamine our current understanding of the BCR-antigen kinetic rates in germinal centers using the latest experimental assays for BCR-Ag interactions.

The humoral immune response is dependent on B cell activation and differentiation, which is typically triggered by the formation of immunological synapses at the interface between B cells and the antigen presenting surfaces. However, due to the highly dynamic and transient feature of immunological synapses, it has been difficult to capture and investigate the molecular events that occur within them. The planar lipids bilayer (PLB) supported antigen presenting surface combined with high-resolution high-speed total internal reflection fluorescence microscope (TIRFM) live cell imaging system has been proved to be a powerful tool that allows us to visualize the dynamic events in immunological synapse. In addition, the phospholipid phosphatidylinositol-(4,5)-biphosphate (PIP2) plays a unique role in B cell activation, and it is difficult to investigate the synaptic dynamics of PIP2 molecules. Hence, we describe here the general procedures for the utilization of a PLB based antigen presenting system combining TIRFM based imaging methods to visualize the spatial-temporal co-distribution of PIP2 and BCR microcluster within the B cell immunological synapse., Competing Interests: Disclosure The authors declare no competing financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The B cell receptor (BCR) interacts with foreign antigens to mediate B cell activation and secretion of antibodies. B cell activation begins with initiation of signaling pathways, such as NFAT, NF-κB, and MAPK, and endocytosis of the BCR-antigen complex. Many studies have investigated the signaling pathways associated with BCR activation, and this work has led to significant advances in drug therapies to treat cancer and autoimmune diseases that are linked to aberrant BCR signaling. Less is known, however, about the mechanisms of BCR endocytosis and the role endocytosis plays in B cell pathogenesis. This chapter will review key characteristics of the BCR, including a review of signaling pathways, and endocytic mechanisms associated with the activated BCR. We will also review recent studies investigating the role of BCR endocytosis disease pathogenesis., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The B cell and T cell antigen receptors (BCR and TCR) share a common architecture in which variable dimeric antigen-binding modules assemble with invariant dimeric signaling modules to form functional receptor complexes. In the TCR, a highly conserved T cell receptor αβ (TCRαβ) transmembrane (TM) interface forms a rigid structure around which its three dimeric signaling modules assemble through well-characterized polar interactions. Noting that the key features stabilizing this TCRαβ TM interface also appear with high evolutionary conservation in the TM sequences of the membrane immunoglobulin (mIg) heavy chains that form the BCR's homodimeric antigen-binding module, we asked whether the BCR contained an analogous TM structure. Using an unbiased biochemical and computational modeling approach, we found that the mouse IgM BCR forms a core TM structure that is remarkably similar to that of the TCR. This structure is reinforced by a network of interhelical hydrogen bonds, and our model is nearly identical to the arrangement observed in the just-released cryo-electron microscopy (cryo-EM) structures of intact human BCRs. Our biochemical analysis shows that the integrity of this TM structure is vital for stable assembly with the BCR signaling module CD79AB in the B cell endoplasmic reticulum, and molecular dynamics simulations indicate that BCRs of all five isotypes can form comparable structures. These results demonstrate that, despite their many differences in composition, complexity, and ligand type, TCRs and BCRs rely on a common core TM structure that has been shaped by evolution for optimal receptor assembly and stability in the cell membrane.

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.

Although B cells and T cells are integral players of the adaptive immune system and act in co-dependent ways to orchestrate immune responses, existing methods to study the immune repertoire have largely focused on separate analyses of B cell receptor (BCR) and T cell receptor (TCR) repertoires. Based on our hypothesis that the shared history of immune exposures and the shared cellular machinery for recombination result in similarities between BCR and TCR repertoires in an individual, we examine any commonalities and interrelationships between BCR and TCR repertoires. We find that the BCR and TCR repertoires have covarying clonal architecture and diversity, and that the pattern of correlations appears to be altered in immune-mediated diseases. Furthermore, hierarchical clustering of public B and T cell clonotypes in both health and disease based on correlation of clonal proportion revealed distinct clusters of B and T cell clonotypes that exhibit increased sequence similarity, share motifs, and have distinct amino acid characteristics. Our findings point to common principles governing memory formation, recombination, and clonal expansion to antigens in B and T cells within an individual. A significant proportion of public BCR and TCR repertoire can be clustered into nonoverlapping and correlated clusters, suggesting a novel way of grouping B and T cell clonotypes., Competing Interests: BH is the CTO of Genealogy Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financal relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 Hong, Shin, Hong, Lee and Han.)

Germinal centers (GCs) are essential for antibody affinity maturation. GC B cells have a unique repertoire of cell surface glycans compared with naive B cells, yet functional roles for changes in glycosylation in the GC have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a downregulation in ligands for CD22, an inhibitory co-receptor of the B cell receptor. To test a functional role for downregulation of CD22 ligands in the GC, we generate a mouse model that maintains CD22 ligands on GC B cells. With this model, we demonstrate that glycan remodeling plays a critical role in the maintenance of B cells in the GC. Sustained expression of CD22 ligands induces higher levels of apoptosis in GC B cells, reduces memory B cell and plasma cell output, and delays affinity maturation of antibodies. These defects are CD22 dependent, demonstrating that downregulation of CD22 ligands on B cells plays a critical function in the GC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The B cell receptor (BCR) repertoire is highly diverse among individuals. Poor similarity of the spectrum among inbred baseline mice may limit the ability to discriminate true signals from those involving specific experimental factors. The repertoire similarity of the baseline status lacks intensive measurements., Results: We measured the repertoire similarity of IgH in blood and spleen samples from untreated BALB/c and C57BL/6J mice to investigate the baseline status of the two inbred strains. The antibody pool was stratified by the isotype of IgA, IgG and IgM. Between individuals, the results showed better convergence of CDR3 and clonal lineage profiles in IgM than in IgA and IgG, and better robustness of somatic mutation networks in IgM than in IgA and IgG. It also showed that the CDR3 clonotypes and clonal lineages shared better in the spleen samples than in the blood samples. The animal batch differences were detected in CDR3 evenness, mutated clonotype proportions, and maximal network degrees. A cut-off of 95% identity in the CDR3 nucleotide sequences was suitable for clonal lineage establishment., Conclusions: Our findings reveal a natural landscape of BCR repertoire similarities between baseline mice and provide a solid reference for designing studies of mouse BCR repertoires., (© 2022. The Author(s).)

A comprehensive study of the B cell response against SARS-CoV-2 could be significant for understanding the immune response and developing therapeutical antibodies and vaccines. To define the dynamics and characteristics of the antibody repertoire following SARS-CoV-2 infection, we analyzed the mRNA transcripts of immunoglobulin heavy chain (IgH) repertoires of 24 peripheral blood samples collected between 3 and 111 days after symptom onset from 10 COVID-19 patients. Massive clonal expansion of naive B cells with limited somatic hypermutation (SHM) was observed in the second week after symptom onset. The proportion of low-SHM IgG clones strongly correlated with spike-specific IgG antibody titers, highlighting the significant activation of naive B cells in response to a novel virus infection. The antibody isotype switching landscape showed a transient IgA surge in the first week after symptom onset, followed by a sustained IgG elevation that lasted for at least 3 months. SARS-CoV-2 infection elicited poly-germ line reactive antibody responses. Interestingly, 17 different IGHV germ line genes recombined with IGHJ6 showed significant clonal expansion. By comparing the IgH repertoires that we sequenced with the 774 reported SARS-CoV-2-reactive monoclonal antibodies (MAbs), 13 shared spike-specific IgH clusters were found. These shared spike-specific IgH clusters are derived from the same lineage of several recently published neutralizing MAbs, including CC12.1, CC12.3, C102, REGN10977, and 4A8. Furthermore, identical spike-specific IgH sequences were found in different COVID-19 patients, suggesting a highly convergent antibody response to SARS-CoV-2. Our analysis based on sequencing antibody repertoires from different individuals revealed key signatures of the systemic B cell response induced by SARS-CoV-2 infection. IMPORTANCE Although the canonical delineation of serum antibody responses following SARS-CoV-2 infection has been well established, the dynamics of antibody repertoire at the mRNA transcriptional level has not been well understood, especially the correlation between serum antibody titers and the antibody mRNA transcripts. In this study, we analyzed the IgH transcripts and characterized the B cell clonal expansion and differentiation, isotype switching, and somatic hypermutation in COVID-19 patients. This study provided insights at the repertoire level for the B cell response after SARS-CoV-2 infection.

Context: We previously reported that monoclonal mouse immunoglobulin (Ig) A, OA-4, attenuates sensitization in mice by suppressing B cell activation., Objective: 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)., Materials and Methods: 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., Results: 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 Ca 2+ 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., Conclusion: IgA is a potential immunoregulatory drug utilizing new mechanisms that affect splenic B cells but not A20 lymphomas.

The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or 'public' antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site. As proof of concept, we deployed a library of RNA bacteriophage VLPs displaying random peptides to identify a multivalent antigen that selectively triggered germline BCRs using the human V H gene IGVH1-2*02. This VLP selectively primed IGHV1-2*02 BCRs that were present within a highly diversified germline antibody repertoire within humanized mice. Our approach thus provides methodology to generate antigens that engage specific BCR configurations of interest, in the absence of structure-based information., Competing Interests: BC and DP have an equity stake in Flagship Laboratories 72. RMB, DR and NL were employed by Bristol Myers Squibb. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ronsard, Yousif, Peabody, Okonkwo, Devant, Mogus, Barnes, Rohrer, Lonberg, Peabody, Chackerian and Lingwood.)

Mature B cells express B cell antigen receptor (BCR), toll-like receptors (TLR) and TNF family receptors including CD40 and B-cell activating factor receptor (BAFFR). These receptors transduce cellular signals to govern the physiological and pathological processes in B cells including B cell development and differentiation, survival, proliferation, and antibody-mediated immune responses as well as autoimmune diseases and B cell lymphomagenesis. Effective antibody-mediated immune responses require class switch recombination (CSR), a somatic DNA recombination event occurring at the immunoglobulin heavy chain ( Igh ) gene locus. Mature B cells initially express IgM as their BCR, and CSR enables the B cells to switch from expressing IgM to expressing different classes of antibodies including IgG, IgA or IgE that exhibit distinct effector functions. Here, we briefly review recent findings about how the signaling crosstalk of the BCR with TLRs, CD40 and BAFFR regulates CSR, antibody-mediate immune responses, and B cell anergy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen and Wang.)

The functions of bone marrow plasma cells (BMPC) beyond antibody production are not fully elucidated and distinct subsets of BMPC suggest potential different functions. Phenotypic differences were identified for human BMPC depending on CD19 expression. Since CD19 is a co-stimulatory molecule of the B-cell-receptor (BCR), and IgA + and IgM + BMPC express the BCR on their surface, we here studied whether CD19 expression affects cellular responses, such as BCR signaling and the expression of checkpoint molecules. We analyzed 132 BM samples from individuals undergoing routine total hip arthroplasty. We found that both CD19 + and CD19 - BMPC expressed BCR signaling molecules. Notably, the BCR-associated kinase spleen tyrosine kinase (SYK) including pSYK was higher expressed in CD19 + BMPC compared to CD19 - BMPC. BCR stimulation also resulted in increased kinase phosphorylation downstream of the BCR while expression of CD19 remained stable afterwards. Interestingly, the BCR response was restricted to IgA + BMPC independently of CD19 expression. With regard to the expression of checkpoint molecules, CD19 - BMPC expressed higher levels of co-inhibitory molecule programmed cell death protein-1 (PD-1) than CD19 + BMPC. IgA + BMPC characteristically upregulated PD-1 upon BCR stimulation in contrast to other PC subsets and inhibition of the kinase SYK abrogated PD-1 upregulation. In contrast, expression of PD-1 ligand, B and T lymphocyte attenuator (BTLA) and CD28 did not change upon BCR activation of IgA + BMPC. Here, we identify a distinct characteristic of IgA + BMPC that is independent of the phenotypic heterogeneity of the subsets according to their CD19 expression. The data suggest that IgA + BMPC underlie different regulatory principles and/or exert distinct regulatory functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wiedemann, Lettau, Wirries, Jungmann, Salhab, Gasparoni, Mei, Perka, Walter, Radbruch, Lino and Dörner.)

B-cell receptor (BCR) knock-in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one-step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD-GT8 60mer, a germline-targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class-switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

High-resolution confocal imaging has provided new insights in the process of receptor-mediated endocytosis in variety of cell types. We describe here the protocol for investigating B cell receptor (BCR)-mediated internalization of membrane bound antigens using confocal microscopy. We describe the method to prepare plasma membrane sheets (PMS) in a small area, bind fluorescently tagged antigens to the PMS and activate B cells on the PMS. We also describe the method for analyzing antigen internalization using confocal microscopy and computational image analysis. This protocol is useful for the study of antigen internalization by B cells and can be applied for studying receptor-mediated endocytosis in other cells as well. The setup we describe here is especially useful for studying rare cell types when the number of cells available is limiting.

Epstein-Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation., Competing Interests: Competing interest statement: F.C. is a co-founder of enGene Statistics GmbH. T.O. reported grants from Merck KGaA, and Gilead, and personal fees from Merck KGaA, Roche, Kronos Bio, all outside the submitted work.

Severe COVID-19 is associated with profound lymphopenia and an elevated neutrophil to lymphocyte ratio. We applied a novel dimer avoidance multiplexed polymerase chain reaction next-generation sequencing assay to analyze T (TCR) and B cell receptor (BCR) repertoires. Surprisingly, TCR repertoires were markedly diminished during the early onset of severe disease but recovered during the convalescent stage. Monitoring TCR repertoires could serve as an indicative biomarker to predict disease progression and recovery. Panoramic concurrent assessment of BCR repertoires demonstrated isotype switching and a transient but dramatic early IgA expansion. Dominant B cell clonal expansion with decreased diversity occurred following recovery from infection. Profound changes in T cell homeostasis raise critical questions about the early events in COVID-19 infection and demonstrate that immune repertoire analysis is a promising method for evaluating emergent host immunity to SARS-CoV-2 viral infection, with great implications for assessing vaccination and other immunological therapies., (Copyright © 2020 Niu, Li, Li, Pan, Wang, Feng, Mo, Yan, Ye, Luo, Qu, Weber, Byrne-Steele, Wang, Yu, Li, Myers, Lotze, Zhong, Han and Chen.)

The MC38 (derived from carcinogen‑induced colon adenocarcinoma) tumor model is sensitive to anti‑programmed cell death‑1 (anti-PD‑1) treatment. However, there is no comprehensive description of the T and B cell receptor (TCR, BCR) repertoires of the MC38 tumor model following anti‑PD‑1 treatment, an improved understanding of which is highly important in the development of anti‑PD‑1 immunotherapy. The present study analyzed the TCR and BCR repertoires of three types of tissue, including tumor, spleen and tumor draining lymph node (DLN) from 20 MC38 syngeneic mice receiving murine anti‑PD‑1 (mDX400) treatment or mouse immunoglobulin G1 (mIgG1) control treatment. To obtain enough tissues for high‑throughput sequencing, samples were collected on day 8 after the start of initial treatment. The usage frequencies of seven TCR β chain (TRB) V genes and one TRBJ gene were significantly different between mDX400‑ and mIgG1‑group tumors. TCR repertoire diversity was significantly lower in mDX400‑group tumors compared with mIgG1‑group tumors, with the top 10 most frequent TCR clonotypes notably expanded in mDX400‑group tumors. In addition, the proportion of high‑frequency TCR clonotypes from mDX400‑group tumors that were also present both in the DLN and spleen was significantly higher than that in mIgG1‑group tumors. Among the highly expanded TCR clonotypes, one TCR clonotype was consistently expanded in >50% of the mDX400‑group tumors compared with mIgG1‑group tumors. Similarly, one BCR clonal family was highly expanded in >50% of mDX400‑group tumor samples. The consistently expanded TCR and BCR clones were co‑expanded in 29% of mDX400‑group tumors. Moreover, mutation rates of immunoglobulin heavy chain sequences in the spleen within complementarity determining region 2 and framework region 3 were significantly higher in the mDX400 group than in the mIgG1 group. The findings of this study may contribute to an improved understanding of the molecular mechanisms of anti‑PD‑1 treatment.

The KLHL14 gene acquires frequent inactivating mutations in mature B cell malignancies, especially in the MYD88 L265P , CD79B mutant (MCD) genetic subtype of diffuse large B cell lymphoma (DLBCL), which relies on B cell receptor (BCR) signaling for survival. However, the pathogenic role of KLHL14 in DLBCL and its molecular function are largely unknown. Here, we report that KLHL14 is in close proximity to the BCR in the endoplasmic reticulum of MCD cell line models and promotes the turnover of immature glycoforms of BCR subunits, reducing total cellular BCR levels. Loss of KLHL14 confers relative resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib and promotes assembly of the MYD88-TLR9-BCR (My-T-BCR) supercomplex, which initiates prosurvival NF-κB activation. Consequently, KLHL14 inactivation allows MCD cells to maintain NF-κB signaling in the presence of ibrutinib. These findings reinforce the central role of My-T-BCR-dependent NF-κB signaling in MCD DLBCL and suggest that the genetic status of KLHL14 should be considered in clinical trials testing inhibitors of BTK and BCR signaling mediators in DLBCL., Competing Interests: The authors declare no competing interest.

In terms of diagnosing and treating diseases, our adaptive immune system is the "best doctor." It carries out these tasks with unmatched precision, with the help of both T and B cell receptors, our most diverse set of genes, distinguishing one individual from another. It does this by generating autologous extraordinary diversity in the receptors, ranging from 10 15 to 10 25 for each chain of the rearranged receptors. By combining multiplex PCR and next-generation sequencing (NGS), we have developed high throughput methods to study adaptive immunity. The adaptome is the sum-total of expressed T and B cell receptor genes in a sample, composed of seven chains, including the alpha/beta and gamma/delta chains for T cells, and heavy/lambda or kappa chains for B cells. Immune repertoire is the sum-total of the individual clonotypes within one chain, including individual complementarity-determining regions (CDR) 3 sequences. In order to reflect the breadth and depth of the true adaptome, the following criteria assessing any method needs to be ascertained: (1) Methods need to be inclusive and quantitative; (2) Analysis should consider what questions need to be addressed and whether bulk or single cell sequencing provide the best tools for assessing the underlying biology and addressing important questions; (3) Measures of clonal diversity are key to understand the underlying structure and providence of the repertoire; and (4) Convergent evolution may allow a surprising degree of homologous or identical CDR3s associated with individual disease entities, creating hope for novel diagnostics and/or disease burden assessments. Integrating studies of the peripheral blood, lymph nodes, and tumor allows dynamic interrogation of the alterations occurring with age, treatment, and response to emergent and established therapies.

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.

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., Competing Interests: SR, LW, DM, MW, SC, PT No competing interests declared, (© 2019, Roper et al.)

To efficiently initiate activation responses against rare ligands in the microenvironment, lymphocytes employ sophisticated mechanisms involving signaling amplification. Recently, a signaling amplification mechanism initiated from phosphatidylinositol (PI) 4, 5-biphosphate [PI(4,5)P2] hydrolysis and synthesis for sustained B cell activation has been reported. Antigen and B cell receptor (BCR) recognition triggered the prompt reduction of PI(4,5)P2 density within the BCR microclusters, which led to the positive feedback for the synthesis of PI(4,5)P2 outside of the BCR microclusters. At single molecule level, the diffusion of PI(4,5)P2 was slow, allowing for the maintenance of a PI(4,5)P2 density gradient between the inside and outside of the BCR microclusters and the persistent supply of PI(4,5)P2 from outside to inside of the BCR microclusters. Here, we review studies that have contributed to uncovering the molecular mechanisms of PI(4,5)P2-derived signaling amplification model. Based on these studies, we proposed a "gasoline engine model" in which the activation of B cell signaling inside the microclusters is similar to the working principle of burning gasoline within the engine chamber of a gasoline engine. We also discuss the evidences showing the potential universality of this model and future prospects., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In cancer biology, the functional interpretation of genomic alterations is critical to achieve the promise of genomic profiling in the clinic. For chronic lymphocytic leukemia (CLL), a heterogeneous disease of B-lymphocytes maturing under constitutive B cell receptor (BCR) stimulation, the functional role of diverse clonal mutations remains largely unknown. Here, we demonstrate that alterations in BCR signaling dynamics underlie the progression of B cells toward malignancy. We reveal emergent dynamic features-bimodality, hypersensitivity, and hysteresis-in the BCR signaling pathway of primary CLL B cells. These signaling abnormalities in CLL quantitatively derive from BCR clustering and constitutive signaling with positive feedback reinforcement, as demonstrated through single-cell analysis of phospho-responses, computational modeling, and super-resolution imaging. Such dysregulated signaling segregates CLL patients by disease severity and clinical presentation. These findings provide a quantitative framework and methodology to assess complex and heterogeneous leukemia pathology and to inform therapeutic strategies in parallel with genomic profiling., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

B lymphocytes use B cell receptors (BCRs) to recognize antigens. It is still not clear how BCR transduces antigen-specific physical signals upon binding across cell membrane for the conversion to chemical signals, triggering downstream signaling cascades. It is hypothesized that through a series of conformational changes within BCR, antigen engagement in the extracellular domain of BCR is transduced to its intracellular domain. By combining site-specific labeling methodology and FRET-based assay, we monitored conformational changes in the extracellular domains within BCR upon antigen engagement. Conformational changes within heavy chain of membrane-bound immunoglobulin (mIg), as well as conformational changes in the spatial relationship between mIg and Igβ were observed. These conformational changes were correlated with the strength of BCR activation and were distinct in IgM- and IgG-BCR. These findings provide molecular mechanisms to explain the fundamental aspects of BCR activation and a framework to investigate ligand-induced molecular events in immune receptors., Competing Interests: ZS, SL, XL, ZW, YM, CC, WL No competing interests declared, (© 2019, Shen et al.)

The complement system and Toll-like receptors (TLRs) are essential contributors of innate immunity. Separate activation of these systems has been shown to play a role in initiating and shaping the adaptive immune response, however the modulation of various B cell functions by the simultaneous involvement of these two systems has not yet been uncovered. We demonstrate here that occupancy of complement receptor type 1 (CR1, CD35) by its natural, complement component C3-derived ligand significantly and dose dependently reduces the TLR9-induced expression of activation markers, cytokine production, proliferation, and antibody production by human B cells, but has no effect on the TLR7-induced functions. The synergistic response to the simultaneous engagement of either TLR9 or TLR7 along with the BCR however, is significantly inhibited by CR1 occupancy. Our findings imply that both under physiological and pathological conditions, when complement- and TLR-activating microbial and damage products are present in the B cell environment, the cooperation between CR1 and TLR7 or TLR9 provides additional levels of the regulation of human B cell functions.

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., Competing Interests: MB, KC, JS, LA, RC, DS, MD, XW, JP, DC, JH, MG No competing interests declared

Epstein-Barr virus (EBV) is a widespread human virus that establishes latent infection, potentially leading to tumors, hematological disorders, and other severe diseases. EBV infections are associated with diverse symptoms and affect various organs; therefore, early diagnosis and treatment are crucial. B cell receptor (BCR) repertoires of B cell surface immunoglobulins have been widely studied for their association with various infectious diseases. However, the specific genetic changes that modulate the BCR repertoires after an EBV infection are still poorly understood. In this study, we employed high-throughput sequencing (HTS) to investigate the diversity of BCR repertoires in an EBV-transformed lymphoblastic cell line (LCL). Compared with the noninfected control B cell line, the LCL exhibited a decrease in overall BCR diversity but displayed an increase in the expansion of some dominant rearrangements such as IGHV4-31/IGHJ4, IGHV4-59/IGHJ4, IGHV5-51/IGHJ3, and IGHV3-74/IGHJ3. A higher frequency of occurrence of these rearrangement types was confirmed in patients with EBV infection. Interestingly, the IGHV3-74 rearrangement was only detected in EBV-infected children, suggesting that our experimental observations were not coincidental. In addition, we identified a highly dominant consensus motif, CAR(xRx)YGSG(xYx)FD, in complementarity-determining region 3 (CDR3) sequences of the heavy chain in the LCL. Our findings demonstrated the utility of HTS technology for studying the variations in signature motifs of the BCR repertoires after EBV infection. We propose that the analysis of BCR repertoire sequences represents a promising method for diagnosing early EBV infections and developing novel antibody- and vaccine-based therapies against such infections.

B cell linker (BLNK) is a key linker protein of B cell receptor (BCR) signaling pathway. BLNK participates in the regulation of PLC-γactivity and the activation of Ras pathway through its typical structure and interaction network with other proteins, and is thus widely involved in the regulation of B cell proliferation, differentiation, apoptosis and signal transduction. Furthermore, it is closely related to anaphylactic diseases, multiple sclerosis, chromosomal aneuploidy, aneuglobulinemia, B lymphocytic leukemia and lymphoma. Herein we review the structure and biological function of BLNK and its role in B cell-related diseases. BLNK can cooperate with a series of effective proteins to activate BCR signaling pathway, thereby regulating the development, maturation and function of B cells. The functional mutation of BLNK can destroy the homeostasis of B cells and affect the development and maturation of B cells, which leads to the occurrence of B cell related diseases. A comprehensive understanding of the biological functions of BLNK not only provides insights into the pathogenesis of B cell-related diseases, but also inspires new ideas and helps to find breakthroughs for the treatment of these diseases with BLNK as the therapeutic target.

Adaptive lymphocytes express a panel of immunoreceptors on the cell surface. Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids forming microdomains in the plasma membrane can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. We have shown that negatively charged acidic phospholipids can interact with positively charged juxtamembrane polybasic regions of immunoreceptors, such as TCR-CD3, CD28 and IgG-BCR, to regulate protein structure and function. Furthermore, we pay our attention to protein transmembrane domains. We show that a membrane-snorkeling Lys residue in integrin αLβ2 regulates transmembrane heterodimer formation and integrin adhesion through ionic interplay with acidic phospholipids and calcium ions (Ca 2+ ) in T cells, thus providing a new mechanism of integrin activation. Here, we review our recent progress showcasing the importance of both juxtamembrane and intramembrane ionic protein-lipid interactions., (© 2019 Elsevier Inc. All rights reserved.)

Antigen-dependent engagement of germinal center (GC) B cell receptors (BCRs) promotes antigen internalization and presentation for follicular helper T cells. However, whether BCR signaling is critical or synergistic with T cell help for GC B cell selection or differentiation is unclear. Here, by adapting an experimental approach that enables independent delivery of BCR-crosslinking antigen or T cell help to GC B cells in vivo, we showed that T cell help was sufficient to induce GC B cell expansion and plasmablast formation. However, although BCR crosslinking could not by itself promote GC B cell selection or differentiation, it could synergize with T cell help to enhance the GC and plasmablast responses when T cell help was limiting. These findings indicate that GC B cells can integrate variable inputs from T cell help and BCR signaling in vivo for an optimal process of selection and differentiation, critical for potent long-term humoral immunity., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The adaptation of high-throughput sequencing to the B cell receptor and T cell receptor has made it possible to characterize the adaptive immune receptor repertoire (AIRR) at unprecedented depth. These AIRR sequencing (AIRR-seq) studies offer tremendous potential to increase the understanding of adaptive immune responses in vaccinology, infectious disease, autoimmunity, and cancer. The increasingly wide application of AIRR-seq is leading to a critical mass of studies being deposited in the public domain, offering the possibility of novel scientific insights through secondary analyses and meta-analyses. However, effective sharing of these large-scale data remains a challenge. The AIRR community has proposed minimal information about adaptive immune receptor repertoire (MiAIRR), a standard for reporting AIRR-seq studies. The MiAIRR standard has been operationalized using the National Center for Biotechnology Information (NCBI) repositories. Submissions of AIRR-seq data to the NCBI repositories typically use a combination of web-based and flat-file templates and include only a minimal amount of terminology validation. As a result, AIRR-seq studies at the NCBI are often described using inconsistent terminologies, limiting scientists' ability to access, find, interoperate, and reuse the data sets. In order to improve metadata quality and ease submission of AIRR-seq studies to the NCBI, we have leveraged the software framework developed by the Center for Expanded Data Annotation and Retrieval (CEDAR), which develops technologies involving the use of data standards and ontologies to improve metadata quality. The resulting CEDAR-AIRR (CAIRR) pipeline enables data submitters to: (i) create web-based templates whose entries are controlled by ontology terms, (ii) generate and validate metadata, and (iii) submit the ontology-linked metadata and sequence files (FASTQ) to the NCBI BioProject, BioSample, and Sequence Read Archive databases. Overall, CAIRR provides a web-based metadata submission interface that supports compliance with the MiAIRR standard. This pipeline is available at http://cairr.miairr.org, and will facilitate the NCBI submission process and improve the metadata quality of AIRR-seq studies.

Immunoglobulin A nephropathy (IgAN) is a type of glomerular disorder associated with immune dysregulation, and understanding B‑/T‑cell receptors (BCRs/TCRs) may be valuable for the development of specific immunotherapeutic interventions. In the present study, B and T cells were isolated from IgAN patients and healthy controls, and the composition of the BCR/TCR complementarity‑determining region (CDR)3 was analyzed by multiplex polymerase chain reaction, high‑throughput sequencing and bioinformatics. The present results revealed that the BCR/TCR CDR3 clones were expressed at very low frequencies, and the composition of clone types in patients with IgAN was skewed; the majority of clones were unique, and only 12 BCR and 228 TCR CDR3 clones were public ones, of which 16 were expressed at a significantly higher frequency in patients with IgAN (P<0.001). There were also certain conserved amino acid residues between unique clones or groups, and the residues GMDV, EQY and EQF were recurring only in the IgAN group. In addition, some VDJ gene recombinations indicated great variation between groups, including 4 high‑frequency VDJ gene recombinations in the IgAN patients (P<0.001). Immune repertoires provide novel information, and conserved BCR/TCR CDR3 clones and VDJ gene recombinations with great variation may be potential therapeutic targets for IgAN patients.

The role of microenvironment-mediated biophysical forces in human lymphomas remains elusive. Diffuse large B cell lymphomas (DLBCLs) are heterogeneous tumors, which originate from highly proliferative germinal center B cells. These tumors, their associated neo-vessels, and lymphatics presumably expose cells to particular fluid flow and survival signals. Here, we show that fluid flow enhances proliferation and modulates response of DLBCLs to specific therapeutic agents. Fluid flow upregulates surface expression of B cell receptors (BCRs) and integrin receptors in subsets of ABC-DLBCLs with either CD79A/B mutations or WT BCRs, similar to what is observed with xenografted human tumors in mice. Fluid flow differentially upregulates signaling targets, such as SYK and p70S6K, in ABC-DLBCLs. By selective knockdown of CD79B and inhibition of signaling targets, we provide mechanistic insights into how fluid flow mechanomodulates BCRs and integrins in ABC-DLBCLs. These findings redefine microenvironment factors that regulate lymphoma-drug interactions and will be critical for testing targeted therapies., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Naive B cells co-express two BCR isotypes, IgM and IgD, with identical antigen-binding domains but distinct constant regions. IgM but not IgD is downregulated on autoreactive B cells. Because these isotypes are presumed to be redundant, it is unknown how this could impose tolerance. We introduced the Nur77-eGFP reporter of BCR signaling into mice that express each BCR isotype alone. Despite signaling strongly in vitro, IgD is less sensitive than IgM to endogenous antigen in vivo and developmental fate decisions are skewed accordingly. IgD-only Lyn -/- B cells cannot generate autoantibodies and short-lived plasma cells (SLPCs) in vivo, a fate thought to be driven by intense BCR signaling induced by endogenous antigens. Similarly, IgD-only B cells generate normal germinal center, but impaired IgG1 + SLPC responses to T-dependent immunization. We propose a role for IgD in maintaining the quiescence of autoreactive B cells and restricting their differentiation into autoantibody secreting cells., Competing Interests: MN, JM, AS, LG, FB, JZ No competing interests declared, (© 2018, Noviski et al.)

Positive selection of germinal center (GC) B cells is driven by B cell receptor (BCR) affinity and requires help from follicular T helper cells. The transcription factors c-Myc and Foxo1 are critical for GC B cell selection and survival. However, how different affinity-related signaling events control these transcription factors in a manner that links to selection is unknown. Here we showed that GC B cells reprogram CD40 and BCR signaling to transduce via NF-κB and Foxo1, respectively, whereas naive B cells propagate both signals downstream of either receptor. Although either BCR or CD40 ligation induced c-Myc in naive B cells, both signals were required to highly induce c-Myc, a critical mediator of GC B cell survival and cell cycle reentry. Thus, GC B cells rewire their signaling to enhance selection stringency via a requirement for both antigen receptor- and T cell-mediated signals to induce mediators of positive selection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Approximately one third of Diffuse Large B cell Lymphomas (DLBCL) are refractory or relapse. Novel therapeutic approaches under scrutiny include inhibitors of B-cell receptor (BCR) signaling. Protein kinase CK2 propels survival, proliferation and stress response in solid and hematologic malignancies and promotes a "non-oncogene addiction" phenotype. Whether this kinase regulates BCR signaling, being a suitable pharmacological target in DLBCL, is unknown., Objective: The objective was to establish if CK2 controls DLBCL cell survival and the BCR signaling, to check if the combination of CK2 inhibitor CX-4945 and BCR blockers Ibrutinib and Fostamatinib is more effectively cytotoxic for DLBCL cells than the single agents and to survey the changes in signaling molecules downstream BCR upon CK2 inhibition., Method: A panel of GC and ABC DLBCL cells was treated with CX-4945 and Fostamatinib or Ibrutinib. BCR signaling was assayed by intracellular Ca++ measurement and looking at the phosphorylation of signaling molecules. The effects on cell survival were assessed by flow cytometry, western blot and MTT assays., Results: CK2 inhibition with CX-4945 causes DLBCL cell death. CX-4945 impaired AKT phosphorylation and intracellular Ca++ mobilization upon BCR engagement. The CK2 inhibitor acted synergistically with either the SYK inhibitor Fostamatinib or the BTK inhibitor Ibrutinib in inducing DLBCL cell death. CX-4945 was equally effective in GC and ABC DLBCL subtypes as well as in "double hit" DLBCL cell lines., Conclusion: These findings suggest a role for CK2 downstream of the BCR in controlling survival pathways crucial for cell growth of different DLBCL subtypes. Also, the use of CX-4945 in combination with BCR signaling blockers could represent a novel rational therapeutic approach in the DLBCL., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Antibody responses are initiated by the binding of antigens to cell surface expressed B cell receptors (BCRs) that trigger signaling cascades resulting in the activation of B cells. However, it has been difficult to study these cascades due to their fast, dynamic, and transient nature. Using a conventional antigen-presenting system, such as planar lipid bilayers (PLBs), the initial events during B cell activation have been difficult to be captured. Here, we describe the general procedures for the utilization of a photoactivatable antigen-presenting system in the combination with a high speed live cell imaging method to investigate the early activation events in the same B cells in response to antigen stimulation.

The production of antibodies requires the expansion and selection of high-affinity B cell clones. This process is initiated by antigen uptake through the B cell receptor (BCR), which recognizes and binds antigen displayed on the surface of an antigen-presenting cell (APC). To acquire the antigen, B cells use myosin contractility to physically pull BCR-antigen clusters from the APC membrane. These mechanical forces influence association and dissociation rates of BCR-antigen bonds, resulting in affinity-dependent acquisition of antigen by B cells. Mechanical regulation of B cell antigen acquisition from APCs remains poorly understood, although the recent development of DNA-based force sensors has enabled the measurement of mechanical forces generated in B cell-APC contacts. In this chapter, we describe a protocol to design, synthesize, and purify DNA-based force sensors to measure B cell antigen extraction forces using fluorescence microscopy.