Unravelling B cell heterogeneity: insights into flow cytometry-gated B cells from single-cell multi-omics data.

Introduction: B cells play a pivotal role in adaptive immunity which has been extensively characterised primarily via flow cytometry-based gating strategies. This study addresses the discrepancies between flow cytometry-defined B cell subsets and their high-confidence molecular signatures using single-cell multi-omics approaches. Methods: By analysing multi-omics single-cell data from healthy individuals and patients across diseases, we characterised the level and nature of cellular contamination within standard flow cytometric-based gating, resolved some of the ambiguities in the literature surrounding unconventional B cell subsets, and demonstrated the variable effects of flow cytometric-based gating cellular heterogeneity across diseases. Results: We showed that flow cytometric-defined B cell populations are heterogenous, and the composition varies significantly between disease states thus affecting the implications of functional studies performed on these populations. Importantly, this paper draws caution on findings about B cell selection and function of flow cytometric-sorted populations, and their roles in disease. As a solution, we developed a simple tool to identify additional markers that can be used to increase the purity of flow-cytometric gated immune cell populations based on multi-omics data (AlliGateR). Here, we demonstrate that additional non-linear CD20, CD21 and CD24 gating can increase the purity of both naïve and memory populations. Discussion: These findings underscore the need to reconsider B cell subset definitions within the literature and propose leveraging single-cell multi-omics data for refined characterisation. We show that single-cell multi-omics technologies represent a powerful tool to bridge the gap between surface marker-based annotations and the intricate molecular characteristics of B cell subsets.

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.

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination.

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies.

Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM.

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.

Analysis of T cell receptor clonotypes in tumor microenvironment identifies shared cancer-type-specific signatures.

Despite the conventional view that a truly random V(D)J recombination process should generate a highly diverse immune repertoire, emerging reports suggest that there is a certain bias toward the generation of shared/public immune receptor chains. These studies were performed in viral diseases where public T cell receptors (TCR) appear to confer better protective responses. Selective pressures generating common TCR clonotypes are currently not well understood, but it is believed that they confer a growth advantage. As very little is known about public TCR clonotypes in cancer, here we set out to determine the extent of shared TCR clonotypes in the intra-tumor microenvironments of virus- and non-virus-driven head and neck cancers using TCR sequencing. We report that tumor-infiltrating T cell clonotypes were indeed shared across individuals with the same cancer type, where the majority of shared sequences were specific to the cancer type (i.e., viral versus non-viral). These shared clonotypes were not particularly enriched in EBV-associated nasopharynx cancer but, in both cancers, exhibited distinct characteristics, namely shorter CDR3 lengths, restricted V- and J-gene usages, and also demonstrated convergent V(D)J recombination. Many of these shared TCRs were expressed in patients with a shared HLA background. Pattern recognition of CDR3 amino acid sequences revealed strong convergence to specific pattern motifs, and these motifs were uniquely found to each cancer type. This suggests that they may be enriched for specificity to common antigens found in the tumor microenvironment of different cancers. The identification of shared TCRs in infiltrating tumor T cells not only adds to our understanding of the tumor-adaptive immune recognition but could also serve as disease-specific biomarkers and guide the development of future immunotherapies.

Research Recommendations Following the Discovery of Pain Sensitizing IgG Autoantibodies in Fibromyalgia Syndrome.

BACKGROUND: Fibromyalgia syndrome (FMS) is the most common chronic widespread pain condition in rheumatology. Until recently, no clear pathophysiological mechanism for fibromyalgia had been established, resulting in management challenges. Recent research has indicated that serum immunoglobulin Gs (IgGs) may play a role in FMS. We undertook a research prioritisation exercise to identify the most pertinent research approaches that may lead to clinically implementable outputs. METHODS: Research priority setting was conducted in five phases: situation analysis; design; expert group consultation; interim recommendations; consultation and revision. A dialogue model was used, and an international multi-stakeholder expert group was invited. Clinical, patient, industry, funder, and scientific expertise was represented throughout. Recommendation-consensus was determined via a voluntary closed eSurvey. Reporting guideline for priority setting of health research were employed to support implementation and maximise impact. RESULTS: Arising from the expert group consultation (n = 29 participants), 39 interim recommendations were defined. A response rate of 81.5% was achieved in the consensus survey. Six recommendations were identified as high priority- and 15 as medium level priority. The recommendations range from aspects of fibromyalgia features that should be considered in future autoantibody research, to specific immunological investigations, suggestions for trial design in FMS, and therapeutic interventions that should be assessed in trials. CONCLUSIONS: By applying the principles of strategic priority setting we directed research towards that which is implementable, thereby expediating the benefit to the FMS patient population. These recommendations are intended for patients, international professionals and grant-giving bodies concerned with research into causes and management of patients with fibromyalgia syndrome.

Author Correction: Ultrasensitive amplicon barcoding for next-generation sequencing facilitating sequence error and amplification-bias correction.

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

Dynamic regulation of hypoxia-inducible factor-1α activity is essential for normal B cell development.

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell-activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.

The B cell immunobiology that underlies CNS autoantibody-mediated diseases.

A rapidly expanding and clinically distinct group of CNS diseases are caused by pathogenic autoantibodies that target neuroglial surface proteins. Despite immunotherapy, patients with these neuroglial surface autoantibody (NSAb)-mediated diseases often experience clinical relapse, high rates of long-term morbidity and adverse effects from the available medications. Fundamentally, the autoantigen-specific B cell lineage leads to production of the pathogenic autoantibodies. These autoantigen-specific B cells have been consistently identified in the circulation of patients with NSAb-mediated diseases, accompanied by high serum levels of autoantigen-specific antibodies. Early evidence suggests that these cells evade well-characterized B cell tolerance checkpoints. Nearer to the site of pathology, cerebrospinal fluid from patients with NSAb-mediated diseases contains high levels of autoantigen-specific B cells that are likely to account for the intrathecal synthesis of these autoantibodies. The characteristics of their immunoglobulin genes offer insights into the underlying immunobiology. In this Review, we summarize the emerging knowledge of B cells across the NSAb-mediated diseases. We review the evidence for the relative contributions of germinal centres and long-lived plasma cells as sources of autoantibodies, discuss data that indicate migration of B cells into the CNS and summarize insights into the underlying B cell pathogenesis that are provided by therapeutic effects.

Ultrasensitive amplicon barcoding for next-generation sequencing facilitating sequence error and amplification-bias correction.

The ability to accurately characterize DNA variant proportions using PCR amplification is key to many genetic studies, including studying tumor heterogeneity, 16S microbiome, viral and immune receptor sequencing. We develop a novel generalizable ultrasensitive amplicon barcoding approach that significantly reduces the inflation/deflation of DNA variant proportions due to PCR amplification biases and sequencing errors. This method was applied to immune receptor sequencing, where it significantly improves the quality and estimation of diversity of the resulting library.

Deep Sequencing of B Cell Receptor Repertoires From COVID-19 Patients Reveals Strong Convergent Immune Signatures.

Deep sequencing of B cell receptor (BCR) heavy chains from a cohort of 31 COVID-19 patients from the UK reveals a stereotypical naive immune response to SARS-CoV-2 which is consistent across patients. Clonal expansion of the B cell population is also observed and may be the result of memory bystander effects. There was a strong convergent sequence signature across patients, and we identified 1,254 clonotypes convergent between at least four of the COVID-19 patients, but not present in healthy controls or individuals following seasonal influenza vaccination. A subset of the convergent clonotypes were homologous to known SARS and SARS-CoV-2 spike protein neutralizing antibodies. Convergence was also demonstrated across wide geographies by comparison of data sets between patients from UK, USA, and China, further validating the disease association and consistency of the stereotypical immune response even at the sequence level. These convergent clonotypes provide a resource to identify potential therapeutic and prophylactic antibodies and demonstrate the potential of BCR profiling as a tool to help understand patient responses.

Genetic modification of primary human B cells to model high-grade lymphoma.

Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models.

Combined Influence of B-Cell Receptor Rearrangement and Somatic Hypermutation on B-Cell Class-Switch Fate in Health and in Chronic Lymphocytic Leukemia.

A diverse B-cell receptor (BCR) repertoire is required to bind a wide range of antigens. BCRs are generated through genetic recombination and can be diversified through somatic hypermutation (SHM) or class-switch recombination (CSR). Patterns of repertoire diversity can vary substantially between different health conditions. We use isotype-resolved BCR sequencing to compare B-cell evolution and class-switch fate in healthy individuals and in patients with chronic lymphocytic leukemia (CLL). We show that the patterns of SHM and CSR in B-cells from healthy individuals are distinct from CLL. We identify distinct properties of clonal expansion that lead to the generation of antibodies of different classes in healthy, malignant, and non-malignant CLL BCR repertoires. We further demonstrate that BCR diversity is affected by relationships between antibody variable and constant regions leading to isotype-specific signatures of variable gene usage. This study provides powerful insights into the mechanisms underlying the evolution of the adaptive immune responses in health and their aberration during disease.

Antibody repertoire analysis in polygenic autoimmune diseases.

High-throughput sequencing of the DNA/RNA encoding antibody heavy- and light-chains is rapidly transforming the field of adaptive immunity. It can address key questions, including: (i) how the B-cell repertoire differs in health and disease; and (ii) if it does differ, the point(s) in B-cell development at which this occurs. The advent of technologies, such as whole-genome sequencing, offers the chance to link abnormalities in the B-cell antibody repertoire to specific genomic variants and polymorphisms. Here, we discuss the current research using B-cell antibody repertoire sequencing in three polygenic autoimmune diseases where there is good evidence for a pathological role for B-cells, namely systemic lupus erythematosus, multiple sclerosis and rheumatoid arthritis. These autoimmune diseases exhibit significantly skewed B-cell receptor repertoires compared with healthy controls. Interestingly, some common repertoire defects are shared between diseases, such as elevated IGHV4-34 gene usage. B-cell clones have effectively been characterized and tracked between different tissues and blood in autoimmune disease. It has been hypothesized that these differences may signify differences in B-cell tolerance; however, the mechanisms and implications of these defects are not clear.

Dynamic variation of CD5 surface expression levels within individual chronic lymphocytic leukemia clones.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonally derived mature CD5high B cells; however, the cellular origin of CLL is still unknown. Patients with CLL also harbor variable numbers of CD5low B cells, but the clonal relationship of these cells to the bulk disease is unknown and can have important implications for monitoring, treating, and understanding the biology of CLL. Here, we use B-cell receptors (BCRs) as molecular barcodes to first show by single-cell BCR sequencing that the great majority of CD5low B cells in the blood of CLL patients are clonally related to CD5high CLL B cells. We investigate whether CD5 state switching was likely to occur continuously as a common event or as a rare event in CLL by tracking somatic BCR mutations in bulk CLL B cells and using them to reconstruct the phylogenetic relationships and evolutionary history of the CLL in four patients. Using statistical methods, we show that there is no parsimonious route from a single or low number of CD5low switch events to the CD5high population, but rather, large-scale and/or dynamic switching between these CD5 states is the most likely explanation. The overlapping BCR repertoires between CD5high and CD5low cells from CLL patient peripheral blood reveal that CLL exists in a continuum of CD5 expression. The major proportion of CD5low B cells in patients are leukemic, thus identifying CD5low B cells as an important component of CLL, with implications for CLL pathogenesis, clinical monitoring, and the development of anti-CD5-directed therapies.

Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods.

BACKGROUND: Deep-sequencing methods are rapidly developing in the field of B-cell receptor (BCR) and T-cell receptor (TCR) diversity. These promise to revolutionise our understanding of adaptive immune dynamics, identify novel antibodies, and allow monitoring of minimal residual disease. However, different methods for BCR and TCR enrichment and amplification have been proposed. Here we perform the first systematic comparison between different methods of enrichment, amplification and sequencing for generating BCR and TCR repertoires using large sample numbers. RESULTS: Resampling from the same RNA or cDNA pool results in highly correlated and reproducible repertoires, but resampling low frequency clones leads to stochastic variance. Repertoires generated by different sequencing methods (454 Roche and Illumina MiSeq) and amplification methods (multiplex PCR, 5' Rapid amplification of cDNA ends (5'RACE), and RNA-capture) are highly correlated, and resulting IgHV gene frequencies between the different methods were not significantly different. Read length has an impact on captured repertoire structure, and ultimately full-length BCR sequences are most informative for repertoire analysis as diversity outside of the CDR is very useful for phylogenetic analysis. Additionally, we show RNA-based BCR repertoires are more informative than using DNA. CONCLUSIONS: Repertoires generated by different sequencing and amplification methods are consistent, but we show that read lengths, depths and error profiles should be considered in experimental design, and multiple sampling approaches could be employed to minimise stochastic sampling variation. This detailed investigation of immune repertoire sequencing methods is essential for informing basic and clinical research.

Network properties derived from deep sequencing of human B-cell receptor repertoires delineate B-cell populations.

The adaptive immune response selectively expands B- and T-cell clones following antigen recognition by B- and T-cell receptors (BCR and TCR), respectively. Next-generation sequencing is a powerful tool for dissecting the BCR and TCR populations at high resolution, but robust computational analyses are required to interpret such sequencing. Here, we develop a novel computational approach for BCR repertoire analysis using established next-generation sequencing methods coupled with network construction and population analysis. BCR sequences organize into networks based on sequence diversity, with differences in network connectivity clearly distinguishing between diverse repertoires of healthy individuals and clonally expanded repertoires from individuals with chronic lymphocytic leukemia (CLL) and other clonal blood disorders. Network population measures defined by the Gini Index and cluster sizes quantify the BCR clonality status and are robust to sampling and sequencing depths. BCR network analysis therefore allows the direct and quantifiable comparison of BCR repertoires between samples and intra-individual population changes between temporal or spatially separated samples and over the course of therapy.