Notch2 signaling governs activated B cells to form memory B cells.

Memory B cells (MBCs) are essential for humoral immunological memory and can emerge during both the pre-germinal center (GC) and GC phases. However, the transcription regulators governing MBC development remain poorly understood. Here, we report that the transcription regulator Notch2 is highly expressed in MBCs and their precursors at the pre-GC stage and required for MBC development without influencing the fate of GC and plasma cells. Mechanistically, Notch2 signaling promotes the expression of complement receptor CD21 and augments B cell receptor (BCR) signaling. Reciprocally, BCR activation up-regulates Notch2 surface expression in activated B cells via a translation-dependent mechanism. Intriguingly, Notch2 is dispensable for GC-derived MBC formation. In summary, our findings establish Notch2 as a pivotal transcription regulator orchestrating MBC development through the reciprocal enforcement of BCR signaling during the pre-GC phase and suggest that the generation of GC-independent and -dependent MBCs is governed by distinct transcriptional mechanisms.

Ultrasound-Guided Fine Needle Biopsy of Human Axillary Lymph Nodes to Assess B Cell Responses to Vaccination.

Ultrasound-guided fine needle biopsy, also known as fine needle aspiration, of human axillary lymph nodes is a safe and effective procedure to assess the immune response within the lymph nodes following vaccination. Once acquired, lymph node cells can be characterized via flow cytometric immunophenotyping and/or single-cell RNA sequencing for gene expression and T and B cell receptors. Analysis of the immune cells from the lymph nodes enables the investigation of T and B cells that may interact at this site. These interactions may lead to germinal center formation and expansion, critical for the generation of effective immunity to vaccination. Directly studying the dynamic processes and interaction of the key cells has been challenging in humans due to the anatomically protected location of these cells. Here, we describe the methods involved in ultrasound-guided fine needle biopsy of human axillary lymph nodes in response to vaccination and subsequent analyses of the B cell populations.

Human B Cell Receptor Repertoire Sequencing.

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.

B Cell Receptor Transgenic Mice as Tools to Study Memory B Cells.

B cell receptor (BCR) transgenic mice allow the control of the initial target (antigen) specificity of naïve B cells and to investigate their properties following activation. Here, I describe how BCR transgenic B cells can be used in combination with adoptive cell transfer and immunization models to study memory B cell formation and reactivation.

E41K Mutation Activates Bruton's Tyrosine Kinase by Stabilizing an Inositol Hexakisphosphate dependent Invisible Dimer.

Bruton's tyrosine kinase (BTK) regulates diverse cellular signaling of the innate and adaptive immune system in response to microbial pathogens. Downregulation or constitutive activation of BTK is reported in patients with autoimmune diseases or various B-cell leukemias. BTK is a multidomain protein tyrosine kinase that adopts an Src-like autoinhibited conformation maintained by the interaction between the kinase and PH-TH domains. The PH-TH domain plays a central role in regulating BTK function. BTK is activated by binding to PIP3 at the plasma membrane upon stimulation by the B-cell receptor (BCR). The PIP3 binding allows dimerization of the PH-TH domain and subsequent transphosphorylation of the activation loop. Alternatively, a recent study shows that the multivalent T-cell-independent (TI) antigen induces BCR response by activating BTK independently of PIP3 binding. It was proposed that a transiently stable IP6-dependent PH-TH dimer may activate BTK during BCR activation by the TI antigens. However, no IP6-dependent PH-TH dimer has been identified yet. Here, we investigated a constitutively active PH-TH mutant (E41K) to determine if the elusive IP6-dependent PH-TH dimer exists. We showed that the constitutively active E41K mutation activates BTK by stabilizing the IP6-dependent PH-TH dimer. We observed that a downregulating mutation in the PH-TH domain (R28H) linked to X-linked agammaglobulinemia impairs BTK activation at the membrane and in the cytosol by preventing PH-TH dimerization. We conclude that the IP6 dynamically remodels the BTK active fraction between the membrane and cytoplasm. Stimulating with IP6 increases the cytosolic fraction of the activated BTK.

Leveraging altered lipid metabolism in treating B cell malignancies.

B cell malignancies, comprising over 80 heterogeneous blood cancers, pose significant prognostic challenges due to intricate oncogenic signaling. Emerging evidence emphasizes the pivotal role of disrupted lipid metabolism in the development of these malignancies. Variations in lipid species, such as phospholipids, cholesterol, sphingolipids, and fatty acids, are widespread across B cell malignancies, contributing to uncontrolled cell proliferation and survival. Phospholipids play a crucial role in initial signaling cascades leading to B cell activation and malignant transformation through constitutive B cell receptor (BCR) signaling. Dysregulated cholesterol and sphingolipid homeostasis support lipid raft integrity, crucial for propagating oncogenic signals. Sphingolipids impact malignant B cell stemness, proliferation, and survival, while glycosphingolipids in lipid rafts modulate BCR activation. Additionally, cancer cells enhance fatty acid-related processes to meet heightened metabolic demands. In obese individuals, the obesity-derived lipids and adipokines surrounding adipocytes rewire lipid metabolism in malignant B cells, evading cytotoxic therapies. Genetic drivers such as MYC translocations also intrinsically alter lipid metabolism in malignant B cells. In summary, intrinsic and extrinsic factors converge to reprogram lipid metabolism, fostering aggressive phenotypes in B cell malignancies. Therefore, targeting altered lipid metabolism has translational potential for improving risk stratification and clinical management of diverse B cell malignancy subtypes.

Method for B Cell Receptor Enrichment in Malignant 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.

Neutralizing antibody responses against contemporary and future influenza A(H3N2) viruses in paradoxical clades elicited by repeated and single vaccinations.

As one of the most effective measures to prevent seasonal influenza viruses, annual influenza vaccination is globally recommended. Nevertheless, evidence regarding the impact of repeated vaccination to contemporary and future influenza has been inconclusive. A total of 100 subjects singly or repeatedly immunized with influenza vaccines including 3C.2a1 or 3C.3a1 A(H3N2) during 2018-2019 and 2019-2020 influenza season were recruited. We investigated neutralization antibody by microneutralization assay using four antigenically distinct A(H3N2) viruses circulating from 2018 to 2023, and tracked the dynamics of B cell receptor (BCR) repertoire for consecutive vaccinations. We found that vaccination elicited cross-reactive antibody responses against future emerging strains. Broader neutralizing antibodies to A(H3N2) viruses and more diverse BCR repertoires were observed in the repeated vaccination. Meanwhile, a higher frequency of BCR sequences shared among the repeated-vaccinated individuals with consistently boosting antibody response was found than those with a reduced antibody response. Our findings suggest that repeated seasonal vaccination could broaden the breadth of antibody responses, which may improve vaccine protection against future emerging viruses.

Multi-omic Analysis of Human B-cell Activation Reveals a Key Lysosomal BCAT1 Role in mTOR Hyperactivation by B-cell receptor and TLR9.

B-lymphocytes play major adaptive immune roles, producing antibody and driving T-cell responses. However, how immunometabolism networks support B-cell activation and differentiation in response to distinct receptor stimuli remains incompletely understood. To gain insights, we systematically investigated acute primary human B-cell transcriptional, translational and metabolomic responses to B-cell receptor (BCR), Toll-like receptor 9 (TLR9), CD40-ligand (CD40L), interleukin-4 (IL4) or combinations thereof. T-independent BCR/TLR9 co-stimulation, which drives malignant and autoimmune B-cell states, jointly induced PD-L1 plasma membrane expression, supported by NAD metabolism and oxidative phosphorylation. BCR/TLR9 also highly induced the transaminase BCAT1, which localized to lysosomal membranes to support branched chain amino acid synthesis and mTORC1 hyperactivation. BCAT1 inhibition blunted BCR/TLR9, but not CD40L/IL4-triggered B-cell proliferation, IL10 expression and BCR/TLR pathway-driven lymphoma xenograft outgrowth. These results provide a valuable resource, reveal receptor-mediated immunometabolism remodeling to support key B-cell phenotypes including PD-L1 checkpoint signaling, and identify BCAT1 as a novel B-cell therapeutic target.

Altered Expression of B Cell Receptor Signaling Pathway Genes in Peripheral Blood of Patients with Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease and has adverse implications. The exact mechanism of its pathogenesis is not fully understood and remains to be elucidated. In the current study we aimed to identify key genes that can serve as potential biomarkers and therapeutic targets for MS and shed light on pathogenesis mechanisms involved in MS. We analyzed a gene expression dataset (GES21942) and found 266 differentially expressed genes (DEGs) including 183 upregulated and 83 downregulated genes in MS patients compared to controls. Then we conducted pathway enrichment on DEGs and selected the top enriched pathway i.e., B cell receptor signaling pathway, and 5 genes of this pathway (CR2, BLK, BLNK, RASGRP3, and KRAS) for further investigation in our clinical samples. We recruited 50 MS patients and 50 controls and assessed the expression of selected genes in the circulation of patients versus controls. Expression of CR2, BLK, BLNK, and RASGRP3 were significantly higher in MS cases compared with controls. There was no significant difference in expression of KRAS between patients and controls. All of the selected genes with differential expression had noticeable diagnostic power and CR2 was the most robust gene in differentiating MS cases from controls. Additionally, a combination of genes resulted in enhanced diagnostic power. Collectively our results suggest that the B cell receptor signaling pathway and the selected genes from this pathway may be implicated in the pathogenesis of MS and each of these genes can be considered as potential diagnostic biomarkers and therapeutic targets.

Single cells and TRUST4 reveal immunological features of the HFRS transcriptome.

The etiology of hemorrhagic fever with renal syndrome (HFRS) is significantly impacted by a variety of immune cells. Nevertheless, the existing techniques for sequencing peripheral blood T cell receptor (TCR) or B cell receptor (BCR) libraries in HFRS are constrained by both limitations and high costs. In this investigation, we utilized the computational tool TRUST4 to generate TCR and BCR libraries utilizing comprehensive RNA-seq data from peripheral blood specimens of HFRS patients. This facilitated the examination of clonality and diversity within immune libraries linked to the condition. Despite previous research on immune cell function, the underlying mechanisms remain intricate, and differential gene expression across immune cell types and cell-to-cell interactions within immune cell clusters have not been thoroughly explored. To address this gap, we performed clustering analysis on 11 cell subsets derived from raw single-cell RNA-seq data, elucidating characteristic changes in cell subset proportions under disease conditions. Additionally, we utilized CellChat, a tool for cell-cell communication analysis, to investigate the impact of MIF family, CD70 family, and GALECTIN family cytokines-known to be involved in cell communication-on immune cell subsets. Furthermore, hdWGCNA analysis identified core genes implicated in HFRS pathogenesis within T cells and B cells. Trajectory analysis revealed that most cell subsets were in a developmental stage, with high expression of transcription factors such as NFKB and JUN in Effector CD8+ T cells, as well as in Naive CD4+ T cells and Naive B cells. Our findings provide a comprehensive understanding of the dynamic changes in immune cells during HFRS pathogenesis, identifying specific V genes and J genes in TCR and BCR that contribute to advancing our knowledge of HFRS. These insights offer potential implications for the diagnosis and treatment of this autoimmune disease.

Comprehensive analysis of juvenile idiopathic arthritis patients' immune characteristics based on bulk and single-cell sequencing data.

Background: The pathogenesis of juvenile idiopathic arthritis (JIA) is strongly influenced by an impaired immune system. However, the molecular mechanisms underlying its development and progression have not been elucidated. In this study, the computational methods TRUST4 were used to construct a T-cell receptor (TCR) and B-cell receptor (BCR) repertoire from the peripheral blood of JIA patients via bulk RNA-seq data, after which the clonality and diversity of the immune repertoire were analyzed. Results: Our findings revealed significant differences in the frequency of clonotypes between the JIA and healthy control groups in terms of the TCR and BCR repertoires. This work identified specific V genes and J genes in TCRs and BCRs that could be used to expand our understanding of JIA. After single-cell RNA analysis, the relative percentages of CD14 monocytes were significantly greater in the JIA group. Cell-cell communication analysis revealed the significant role of the MIF signaling pathway in JIA. Conclusion: In conclusion, this work describes the immune features of both the TCR and BCR repertoires under JIA conditions and provides novel insight into immunotherapy for JIA.

Targeting the B cell receptor signaling pathway in chronic lymphocytic leukemia.

Aberrant signal transduction through the B cell receptor (BCR) plays a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). BCR-dependent signaling is necessary for the growth and survival of neoplastic cells, making inhibition of down-stream pathways a logical therapeutic strategy. Indeed, selective inhibitors against Bruton's tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) have been shown to induce high rates of response in CLL and other B cell lymphomas. In particular, the development of BTK inhibitors revolutionized the treatment approach to CLL, demonstrating long-term efficacy. While BTK inhibitors are widely used for multiple lines of treatment, PI3K inhibitors are much less commonly utilized, mainly due to toxicities. CLL remains an incurable disease and effective treatment options after relapse or development of TKI resistance are greatly needed. This review provides an overview of BCR signaling, a summary of the current therapeutic landscape, and a discussion of the ongoing trials targeting BCR-associated kinases.

Molecular targets of glucocorticoids that elucidate their therapeutic efficacy in aggressive lymphomas.

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.

Single-cell 5' RNA sequencing of camelid peripheral B cells provides insights into cellular basis of heavy-chain antibody production.

Camelids produce both conventional tetrameric antibodies (Abs) and dimeric heavy-chain antibodies (HCAbs). Although B cells that generate these two types of Abs exhibit distinct B cell receptors (BCRs), whether these two B cell populations differ in their phenotypes and developmental processes remains unclear. Here, we performed single-cell 5' RNA profiling of peripheral blood mononuclear cell samples from Bactrian camels before and after immunization. We characterized the functional subtypes and differentiation trajectories of circulating B cells in camels, and reconstructed single-cell BCR sequences. We found that in contrast to humans, the proportion of T-bet+ B cells was high among camelid peripheral B cells. Several marker genes of human B cell subtypes, including CD27 and IGHD, were expressed at low levels in the corresponding camel B cell subtypes. Camelid B cells expressing variable genes of HACbs (VHH) were widely present in various functional subtypes and showed highly overlapping differentiation trajectories with B cells expressing variable genes of conventional Abs (VH). After immunization, the transcriptional changes in VHH+ and VH+ B cells were largely consistent. Through structure modeling, we identified a variety of scaffold types among the reconstructed VHH sequences. Our study provides insights into the cellular context of HCAb production in camels and lays the foundation for developing single-B cell-based camelid single-domain Ab screening.

Identification of B cell subsets based on antigen receptor sequences using deep learning.

B cell receptors (BCRs) denote antigen specificity, while corresponding cell subsets indicate B cell functionality. Since each B cell uniquely encodes this combination, physical isolation and subsequent processing of individual B cells become indispensable to identify both attributes. However, this approach accompanies high costs and inevitable information loss, hindering high-throughput investigation of B cell populations. Here, we present BCR-SORT, a deep learning model that predicts cell subsets from their corresponding BCR sequences by leveraging B cell activation and maturation signatures encoded within BCR sequences. Subsequently, BCR-SORT is demonstrated to improve reconstruction of BCR phylogenetic trees, and reproduce results consistent with those verified using physical isolation-based methods or prior knowledge. Notably, when applied to BCR sequences from COVID-19 vaccine recipients, it revealed inter-individual heterogeneity of evolutionary trajectories towards Omicron-binding memory B cells. Overall, BCR-SORT offers great potential to improve our understanding of B cell responses.

B cell receptor signaling proteins as biomarkers for progression of CLL requiring first-line therapy.

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.

Loss of TET2 increases B-1 cell number and IgM production while limiting CDR3 diversity.

Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of VH11 and VH12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM.

Hyper-N-glycosylated SEL1L3 as auto-antigenic B-cell receptor target of primary vitreoretinal lymphomas.

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress into primary central nervous system lymphoma (PCNSL). To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed B-cell receptors (BCR) from PVRL patients and tested for binding against human auto-antigens. SEL1L3, a protein with multiple glycosylation sites, was identified as the BCR target in 3/20 PVRL cases. SEL1L3 induces proliferation and BCR pathway activation in aggressive lymphoma cell lines. Moreover, SEL1L3 conjugated to a toxin killed exclusively lymphoma cells with respective BCR-reactivity. Western Blot analysis indicates the occurrence of hyper-N-glycosylation of SEL1L3 at aa 527 in PVRL patients with SEL1L3-reactive BCRs. The BCR of a PVRL patient with serum antibodies against SEL1L3 was cloned from a vitreous body biopsy at diagnosis and of a systemic manifestation at relapse. VH4-04*07 was used in both lymphoma manifestations with highly conserved CDR3 regions. Both BCRs showed binding to SEL1L3, suggesting continued dependence of lymphoma cells on antigen stimulation. These results indicate an important role of antigenic stimulation by post-translationally modified auto-antigens in the genesis of PVRL. They also provide the basis for a new treatment approach targeting unique lymphoma BCRs with ultimate specificity.

Factors Governing B Cell Recognition of Autoantigen and Function in Type 1 Diabetes.

Islet autoantibodies predict type 1 diabetes (T1D) but can be transient in murine and human T1D and are not thought to be directly pathogenic. Rather, these autoantibodies signal B cell activity as antigen-presenting cells (APCs) that present islet autoantigen to diabetogenic T cells to promote T1D pathogenesis. Disrupting B cell APC function prevents T1D in mouse models and has shown promise in clinical trials. Autoantigen-specific B cells thus hold potential as sophisticated T1D biomarkers and therapeutic targets. B cell receptor (BCR) somatic hypermutation is a mechanism by which B cells increase affinity for islet autoantigen. High-affinity B and T cell responses are selected in protective immune responses, but immune tolerance mechanisms are known to censor highly autoreactive clones in autoimmunity, including T1D. Thus, different selection rules often apply to autoimmune disease settings (as opposed to protective host immunity), where different autoantigen affinity ceilings are tolerated based on variations in host genetics and environment. This review will explore what is currently known regarding B cell signaling, selection, and interaction with T cells to promote T1D pathogenesis.