Peripheral apoptosis and limited clonal deletion during physiologic murine B lymphocyte development.

Self-reactive and polyreactive B cells generated during B cell development are silenced by either apoptosis, clonal deletion, receptor editing or anergy to avoid autoimmunity. The specific contribution of apoptosis to normal B cell development and self-tolerance is incompletely understood. Here, we quantify self-reactivity, polyreactivity and apoptosis during physiologic B lymphocyte development. Self-reactivity and polyreactivity are most abundant in early immature B cells and diminish significantly during maturation within the bone marrow. Minimal apoptosis still occurs at this site, however B cell receptors cloned from apoptotic B cells show comparable self-reactivity to that of viable cells. Apoptosis increases dramatically only following immature B cells leaving the bone marrow sinusoids, but above 90% of cloned apoptotic transitional B cells are not self-reactive/polyreactive. Our data suggests that an apoptosis-independent mechanism, such as receptor editing, removes most self-reactive B cells in the bone marrow. Mechanistically, lack of survival signaling rather than clonal deletion appears to be the underpinning cause of apoptosis in most transitional B cells in the periphery.

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.

Revealing molecular and cellular heterogeneity in hypopharyngeal carcinogenesis through single-cell RNA and TCR/BCR sequencing.

Introduction: Hypopharyngeal squamous cell carcinoma (HSCC) is one of the malignant tumors with the worst prognosis in head and neck cancers. The transformation from normal tissue through low-grade and high-grade intraepithelial neoplasia to cancerous tissue in HSCC is typically viewed as a progressive pathological sequence typical of tumorigenesis. Nonetheless, the alterations in diverse cell clusters within the tissue microenvironment (TME) throughout tumorigenesis and their impact on the development of HSCC are yet to be fully understood. Methods: We employed single-cell RNA sequencing and TCR/BCR sequencing to sequence 60,854 cells from nine tissue samples representing different stages during the progression of HSCC. This allowed us to construct dynamic transcriptomic maps of cells in diverse TME across various disease stages, and experimentally validated the key molecules within it. Results: We delineated the heterogeneity among tumor cells, immune cells (including T cells, B cells, and myeloid cells), and stromal cells (such as fibroblasts and endothelial cells) during the tumorigenesis of HSCC. We uncovered the alterations in function and state of distinct cell clusters at different stages of tumor development and identified specific clusters closely associated with the tumorigenesis of HSCC. Consequently, we discovered molecules like MAGEA3 and MMP3, pivotal for the diagnosis and treatment of HSCC. Discussion: Our research sheds light on the dynamic alterations within the TME during the tumorigenesis of HSCC, which will help to understand its mechanism of canceration, identify early diagnostic markers, and discover new therapeutic targets.

Predictability of B cell clonal persistence and immunosurveillance in breast cancer.

B cells and T cells are important components of the adaptive immune system and mediate anticancer immunity. The T cell landscape in cancer is well characterized, but the contribution of B cells to anticancer immunosurveillance is less well explored. Here we show an integrative analysis of the B cell and T cell receptor repertoire from individuals with metastatic breast cancer and individuals with early breast cancer during neoadjuvant therapy. Using immune receptor, RNA and whole-exome sequencing, we show that both B cell and T cell responses seem to coevolve with the metastatic cancer genomes and mirror tumor mutational and neoantigen architecture. B cell clones associated with metastatic immunosurveillance and temporal persistence were more expanded and distinct from site-specific clones. B cell clonal immunosurveillance and temporal persistence are predictable from the clonal structure, with higher-centrality B cell antigen receptors more likely to be detected across multiple metastases or across time. This predictability was generalizable across other immune-mediated disorders. This work lays a foundation for prioritizing antibody sequences for therapeutic targeting in cancer.

A blueprint for tumor-infiltrating B cells across human cancers.

B lymphocytes are essential mediators of humoral immunity and play multiple roles in human cancer. To decode the functions of tumor-infiltrating B cells, we generated a B cell blueprint encompassing single-cell transcriptome, B cell-receptor repertoire, and chromatin accessibility data across 20 different cancer types (477 samples, 269 patients). B cells harbored extraordinary heterogeneity and comprised 15 subsets, which could be grouped into two independent developmental paths (extrafollicular versus germinal center). Tumor types grouped into the extrafollicular pathway were linked with worse clinical outcomes and resistance to immunotherapy. The dysfunctional extrafollicular program was associated with glutamine-derived metabolites through epigenetic-metabolic cross-talk, which promoted a T cell-driven immunosuppressive program. These data suggest an intratumor B cell balance between extrafollicular and germinal-center responses and suggest that humoral immunity could possibly be harnessed for B cell-targeting immunotherapy.

BAP31 Plays an Essential Role in Mouse B Cell Development via Regulation of BCR Signaling.

B cell receptor-associated protein 31 (BAP31) is a transmembrane protein that is widely expressed and primarily located in the endoplasmic reticulum (ER). B cells play a crucial role in the immune system, and BAP31 significantly contributes to the functions of various immune cells. However, the specific role of BAP31 in B lymphocytes development remains unknown. In this study, we utilized a mouse model with BAP31 deleted from B cells to investigate its effects. Our findings reveal a block in early B cell development in the bone marrow and a significant decrease in the number of B cells in peripheral lymphoid organs taken from BAP31 B cell conditional knockout (BAP31-BCKO) mice. B cell receptor (BCR) signaling is crucial for the normal development and differentiation of B lymphocytes. BAP31, an endoplasmic reticulum membrane protein, directly regulates the BCR signaling pathway and was shown to be significantly positively correlated with B cell activation and proliferation. These findings establish BAP31 as a crucial regulator of early B cell development.

Single-cell BCR and transcriptome analysis reveals peripheral immune signatures in patients with thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is the most prevalent orbital disease in adults caused by an autoimmune disorder, which can lead to disfigurement and vision impairment. Developing effective treatments for this condition presents challenges due to our limited understanding of its underlying immune aberrations. In this study, we profiled the immune components in the peripheral blood of patients with TAO as well as healthy individuals, utilizing single-cell RNA sequencing and B-cell receptor repertoires (BCR) analysis. We observed a significant reduction in the proportions of regulatory B cells (Bregs) and type 2 conventional dendritic cells (DCs) in patients with TAO during the active phase. Conversely, there was a significant increase in the proportion of type 1 DCs. Further analysis of cell differentiation trajectory revealed potential impairment in the transition of B cells towards Breg phenotype during the active phase of TAO. Besides, the activation process of TAO appeared to involve inflammation and immune dysfunction, as indicated by the dynamic changes in the activities of key regulators. The abnormalities in the peripheral immune system, such as the reduced capacity of Bregs to suppress inflammation, were primarily driven by the enhanced interaction among Breg, DCs, and monocytes (i.e., CD22-PTPRC and BTLA-TNFRSF14). Collectively, our findings offer a comprehensive insight into the molecular regulation and cellular reconfiguration during the active phase of TAO at the single-cell level, in order to explore the pathogenesis of TAO and provide new ideas for the future treatment of TAO.

mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

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.

Inferring B Cell Phylogenies from Paired H and L Chain BCR Sequences with Dowser.

Abs are vital to human immune responses and are composed of genetically variable H and L chains. These structures are initially expressed as BCRs. BCR diversity is shaped through somatic hypermutation and selection during immune responses. This evolutionary process produces B cell clones, cells that descend from a common ancestor but differ by mutations. Phylogenetic trees inferred from BCR sequences can reconstruct the history of mutations within a clone. Until recently, BCR sequencing technologies separated H and L chains, but advancements in single-cell sequencing now pair H and L chains from individual cells. However, it is unclear how these separate genes should be combined to infer B cell phylogenies. In this study, we investigated strategies for using paired H and L chain sequences to build phylogenetic trees. We found that incorporating L chains significantly improved tree accuracy and reproducibility across all methods tested. This improvement was greater than the difference between tree-building methods and persisted even when mixing bulk and single-cell sequencing data. However, we also found that many phylogenetic methods estimated significantly biased branch lengths when some L chains were missing, such as when mixing single-cell and bulk BCR data. This bias was eliminated using maximum likelihood methods with separate branch lengths for H and L chain gene partitions. Thus, we recommend using maximum likelihood methods with separate H and L chain partitions, especially when mixing data types. We implemented these methods in the R package Dowser: https://dowser.readthedocs.io.

Single-cell immune repertoire analysis.

Single-cell T cell and B cell antigen receptor-sequencing data analysis can potentially perform in-depth assessments of adaptive immune cells that inform on understanding immune cell development to tracking clonal expansion in disease and therapy. However, it has been extremely challenging to analyze and interpret T cells and B cells and their adaptive immune receptor repertoires at the single-cell level due to not only the complexity of the data but also the underlying biology. In this Review, we delve into the computational breakthroughs that have transformed the analysis of single-cell T cell and B cell antigen receptor-sequencing data.

[Biological characteristics and clinical significance of stereotyped B-cell receptor in chronic lymphocytic leukemia].

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western adults, although the incidence of CLL is relatively low in Asian populations. However, with the aging population, the incidence of CLL is increasing in China. The interaction between CLL cells and the microenvironment plays a crucial role in the recognition of antigens by the B-cell receptor immunoglobulin (BCR IG). The mutational status of the immunoglobulin heavy variable region (IGHV) is a classical prognostic marker for CLL. Over 40% of CLL patients exhibit biased usage of IGHV and highly similar amino acid sequences in the heavy complementarity-determining region 3 (HCDR3), known as the BCR stereotypy. Different subgroups of stereotyped BCR exhibit distinct biological and clinical features. Among them, subset #2 with mutated IGHV and poor prognosis, as well as the subset #8 with a high risk of Richter transformation, have been recommended by the European Research Initiative on CLL to be included in clinical reports on IGHV mutational status. This review summarizes the definition, distribution, biological characteristics, and clinical significance of clonality patterns of the BCR in CLL.

Sensitive B-cell receptor repertoire analysis shows repopulation correlates with clinical response to rituximab in rheumatoid arthritis.

BACKGROUND: Although B-cell depleting therapy in rheumatoid arthritis (RA) is clearly effective, response is variable and does not correlate with B cell depletion itself. METHODS: The B-cell receptor (BCR) repertoire was prospectively analyzed in peripheral blood samples of twenty-eight RA patients undergoing rituximab therapy. Timepoints of achieved BCR-depletion and -repopulation were defined based on the percentage of unmutated BCRs in the repertoire. The predictive value of early BCR-depletion (within one-month post-treatment) and early BCR-repopulation (within 6 months post-treatment) on clinical response was assessed. RESULTS: We observed changes in the peripheral blood BCR repertoire after rituximab treatment, i.e., increased clonal expansion, decreased clonal diversification and increased mutation load which persisted up to 12 months after treatment, but started to revert at month 6. Early BCR depletion was not associated with early clinical response but late depleters did show early response. Patients with early repopulation with unmutated BCRs showed a significant decrease in disease activity in the interval 6 to 12 months. Development of anti-drug antibodies non-significantly correlated with more BCR repopulation. CONCLUSION: Our findings indicate that rather than BCR-depletion it is repopulation with unmutated BCRs, possibly from naïve B cells, which induces remission. This suggests that (pre-existing) differences in B-cell turnover between patients explain the interindividual differences in early clinical effect.

Envelope protein-specific B cell receptors direct lentiviral vector tropism in vivo.

While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.

B cell repertoire sequencing of HIV-1 pediatric elite-neutralizers identifies multiple broadly neutralizing antibody clonotypes.

Introduction: A limited subset of HIV-1 infected adult individuals typically after at least 2-3 years of chronic infection, develop broadly neutralizing antibodies (bnAbs), suggesting that highly conserved neutralizing epitopes on the HIV-1 envelope glycoprotein are difficult for B cell receptors to effectively target, during natural infection. Recent studies have shown the evolution of bnAbs in HIV-1 infected infants. Methods: We used bulk BCR sequencing (BCR-seq) to profile the B cell receptors from longitudinal samples (3 time points) collected from a rare pair of antiretroviralnaïve, HIV-1 infected pediatric monozygotic twins (AIIMS_329 and AIIMS_330) who displayed elite plasma neutralizing activity against HIV-1. Results: BCR-seq of both twins revealed convergent antibody characteristics including V-gene use, CDRH3 lengths and somatic hypermutation (SHM). Further, antibody clonotypes with genetic features similar to highly potent bnAbs isolated from adults showed ongoing development in donor AIIMS_330 but not in AIIMS_329, corroborating our earlier findings based on plasma bnAbs responses. An increase in SHM was observed in sequences of the IgA isotype from AIIMS_330. Discussion: This study suggests that children living with chronic HIV-1 can develop clonotypes of HIV-1 bnAbs against multiple envelope epitopes similar to those isolated from adults, highlighting that such B cells could be steered to elicit bnAbs responses through vaccines aimed to induce bnAbs against HIV-1 in a broad range of people including children.

scRNA+TCR+BCR-seq revealed the proportions and gene expression patterns of dual receptor T and B lymphocytes in NPC and NLH.

While the relationship between single receptor lymphocytes and cancer has been deeply researched, the origin and biological roles of dual receptor lymphocytes in tumor microenvironment (TME) remain largely unknown. And since nasopharyngeal carcinoma (NPC) is a type of cancer closely associated with immune infiltration, studying the TME of NPC holds particular significance. Utilizing single-cell RNA sequencing combined with T cell receptor (TCR) and B cell receptor (BCR) sequencing (scRNA + TCR + BCR-seq), we analyzed data from 7 patients with NPC and 3 patients with nasopharyngeal lymphatic hyperplasia (NLH). In our research, it was firstly found that the presence of dual receptor lymphocytes in both the TME of NPC and the inflammatory environment of NLH. We also confirmed their clonal expansion, suggesting their potential involvement in the immune response. Subsequently, we further discovered the lineage and the pairing characteristics. It was found that the dual receptor lymphocytes in NPC and NLH mainly originate from memory cells, and the predominant pairing type for dual TCR was ß+α1+α2 and dual BCR was heavy+κ+λ. By further analyzing their gene expression, we compared the function of dual receptor cells with single receptor cells in the context of both NPC and NLH. This groundbreaking research has enhanced our comprehension of the features of dual-receptor cells and has contributed to a better understanding of the TME in NPC. By comparing with NLH, it illuminates part of the alterations in the process of malignant transformation in NPC. These findings present the potential to acquire improved diagnostic markers and treatment modalities.

B cell receptor repertoire analysis in primary Sjogren's syndrome salivary glands identifies repertoire features associated with clinical activity.

BACKGROUND: Primary Sjogren's syndrome (pSS) is a complex autoimmune disease featuring damage to salivary and lacrimal glands, with the possibility of manifestations across multiple organs. Antibody-producing B cells have long been appreciated to play a significant role in pSS pathogenesis, with a number of autoreactive antibody species having been identified to be elevated in pSS patients. While several studies have attempted to characterize the BCR repertoires of peripheral blood B cells in pSS patients, much remains unknown about the repertoire characteristics of gland-infiltrating B cells. METHODS: Through paired scRNAseq and scBCRseq, we profiled the BCR repertoires of both infiltrating and circulating B cells in a small cohort of patients. We further utilize receptor reconstruction analyses to further investigate repertoire characteristics in a wider cohort of pSS patients previously profiled through RNAseq. RESULTS: Via integrated BCR and transcriptome analysis of B cell clones, we generate a trajectory progression pattern for infiltrated memory B cells in pSS. We observe significant differences in BCR repertoires between the peripheral blood and labial gland B cells of pSS patients in terms of relative expansion, isotype usage, and BCR clustering. We further observe significant decreases in IgA2 isotype usage among pSS patient labial and parotid gland B cells these analyses relative to controls as well as a positive correlation between kappa/lambda light chain usage and clinical disease activity. CONCLUSIONS: Through BCR repertoire analysis of pSS patient salivary glands, we identify a number of novel repertoire characteristics that may serve as useful indicators of clinical disease and disease activity. By collecting these BCR repertoires into an accessible database, we hope to also enable comparative analysis of patient repertoires in pSS and potentially other autoimmune disorders.

Mutation-specific CAR T cells as precision therapy for IGLV3-21R110 expressing high-risk chronic lymphocytic leukemia.

The concept of precision cell therapy targeting tumor-specific mutations is appealing but requires surface-exposed neoepitopes, which is a rarity in cancer. B cell receptors (BCR) of mature lymphoid malignancies are exceptional in that they harbor tumor-specific-stereotyped sequences in the form of point mutations that drive self-engagement of the BCR and autologous signaling. Here, we use a BCR light chain neoepitope defined by a characteristic point mutation (IGLV3-21R110) for selective targeting of a poor-risk subset of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR) T cells. We develop murine and humanized CAR constructs expressed in T cells from healthy donors and CLL patients that eradicate IGLV3-21R110 expressing cell lines and primary CLL cells, but neither cells expressing the non-pathogenic IGLV3-21G110 light chain nor polyclonal healthy B cells. In vivo experiments confirm epitope-selective cytolysis in xenograft models in female mice using engrafted IGLV3-21R110 expressing cell lines or primary CLL cells. We further demonstrate in two humanized mouse models lack of cytotoxicity towards human B cells. These data provide the basis for advanced approaches of resistance-preventive and biomarker-guided cellular targeting of functionally relevant lymphoma driver mutations sparing normal B cells.

Computational Analysis of B-Cell Receptor (BCR) Immune Repertoires with Abalign.

The widespread application of high-throughput sequencing technology has generated massive sequences of B-cell receptor (BCR) immune repertoires. Computational analysis of these data has gained significant attention due to the increasing importance of immunotherapy and precision medicine. It not only reveals the diversity and dynamic changes in immune responses, contributing to the study of associated diseases, but also provides valuable information for immunodiagnostics and drug development. Recently, we introduced a BCR-specific multiple sequence alignment (MSA) method along with a comprehensive platform software called Abalign, which stands out as an excellent choice for analyzing BCR immune repertoires due to its unique high-throughput processing capability. It offers ultra-fast MSA functionality and a wide range of analytical features, including BCR/antibody extraction, clonal grouping, lineage tree construction, mutation profiling, diversity statistics, VJ gene assignment, antibody humanization, and more. Importantly, users can perform these analyses using the graphical user interface without any programming skills or scripts. In this article, we present a series of protocols that integrate Abalign's analysis modules into a cohesive workflow. This step-by-step workflow provides detailed instructions for software installation, data preparation, and comprehensive analysis of BCR immune repertoires. This workflow facilitates the efficient acquisition of comprehensive results in profiling BCR immune repertoires, offering insights into the impacts of infectious diseases, allergies, autoimmune disorders, tumor immunology, and antibody drugs. Abalign is freely available at http://cao.labshare.cn/abalign/. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Resource preparation Basic Protocol 2: Analyzing BCR immune repertoires Support Protocol 1: Aiding antibody humanization Support Protocol 2: Constructing B-cell lineage trees Alternate Protocol: Running with Linux command line Basic Protocol 3: Comparing BCR immune repertoires.