Autoimmune diseases are characterized by serum autoantibodies, some of which are pathogenic, causing severe manifestations and organ injury. However, autoantibodies of the same antigenic reactivity ...are also present in the serum of asymptomatic people years before they develop any clinical signs of autoimmunity. Autoantibodies can arise during multiple stages of B cell development, and various genetic and environmental factors drive their production. However, what drives the development of pathogenic autoantibodies is poorly understood. Advances in single‐cell technology have enabled the deep analysis of rare B cell clones producing pathogenic autoantibodies responsible for vasculitis in patients with primary Sjögren's syndrome complicated by mixed cryoglobulinaemia. These findings demonstrated a cascade of genetic events involving stereotypic immunoglobulin V(D)J recombination and transforming somatic mutations in lymphoma genes and V(D)J regions that disrupted antibody quality control mechanisms and decreased autoantibody solubility. Most studies consider V(D)J mutations that enhance autoantibody affinity to drive pathology; however, V(D)J mutations that increase autoantibody propensity to form insoluble complexes could be a major contributor to autoantibody pathogenicity. Defining the molecular characteristics of pathogenic autoantibodies and failed tolerance checkpoints driving their formation will improve prognostication, enabling early treatment to prevent escalating organ damage and B cell malignancy.
Clonal anergy is an enigmatic self-tolerance mechanism because no apparent purpose is served by retaining functionally silenced B cells bearing autoantibodies. Human autoantibodies with IGHV4-34*01 ...heavy chains bind to poly-N-acetyllactosamine carbohydrates (I/i antigen) on erythrocytes and B lymphocytes, cause cold agglutinin disease, and are carried by 5% of naive B cells that are anergic. We analyzed the specificity of three IGHV4-34*01 IgG antibodies isolated from healthy donors immunized against foreign rhesus D alloantigen or vaccinia virus. Each IgG was expressed and analyzed either in a hypermutated immune state or after reverting each antibody to its unmutated preimmune ancestor. In each case, the preimmune ancestor IgG bound intensely to normal human B cells bearing I/i antigen. Self-reactivity was removed by a single somatic mutation that paradoxically decreased binding to the foreign immunogen, whereas other mutations conferred increased foreign binding. These data demonstrate the existence of a mechanism for mutation away from self-reactivity in humans. Because 2.5% of switched memory B cells use IGHV4-34*01 and >43% of these have mutations that remove I/i binding, clonal redemption of anergic cells appears efficient during physiological human antibody responses.
The adaptive immune system is tasked with producing antibodies that recognize a wide scope of potential pathogens, including those never before encountered, and concurrently avoiding formation of ...antibodies binding host tissues. The diverse repertoire of antibodies produced by V(D)J recombination inevitably includes autoantibodies that bind to self‐antigens, estimated to be as much as 70% of nascent antibodies on immature B cells. Early theoretical models of tolerance hypothesized that such self‐reactive clones could not possibly be allowed to survive and mature. However from the first direct view of the fate of nascent B cells carrying a self‐binding antibody it was clear that many “forbidden clones” circulate to secondary lymphoid tissues, where they adopt an IgMlow IgD+ cell surface phenotype and are prevented from secreting autoantibodies by a series of tolerance checkpoints referred to as “clonal anergy.” Since anergic B cells can be reactivated to secrete pathogenic autoantibodies in certain settings, the advantage of controlling self‐reactive antibodies by clonal anergy has until recently remained enigmatic. Here we review this topic and recent advances showing that anergic B cells are recruited into the germinal center to mutate away from self‐reactivity, undergoing “clonal redemption” into cells making antibodies with exquisite specificity for foreign immunogens.
Antibodies have the specificity to differentiate foreign antigens that mimic self antigens, but it remains unclear how such specificity is acquired. In a mouse model, we generated B cells displaying ...an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis detected rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity. Crystal structures revealed that these mutations exploited subtle topological differences to achieve 5000-fold preferential binding to foreign over self epitopes. Resolution of antigenic mimicry drove the optimal affinity maturation trajectory, highlighting the value of retaining self-reactive clones as substrates for protective antibody responses.
Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared ...mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.
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•Single-cell omics reveal rare cells making a common pathogenic human autoantibody•Antibody mutations cause pathogenicity by phase transition to insoluble aggregates•Lymphoma driver mutations are present in cells making pathogenic autoantibodies•Driver mutations dysregulate NF-κB signaling, cell cycle, and antibody mutation
Integrated proteomics and single-cell analysis reveal rogue cell clones with lymphoma driver mutations producing damaging antibodies with the propensity for phase transition into insoluble antibody-autoantigen complexes.
Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define ...the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire.
Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial ...methylation encroachment across the 5′ or 3′ CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer.
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•Promoter CpG islands display asymmetric border methylation encroachment in cancer•5hmC is enriched in normal cells at CpG island shores prone to methylation spread•H3K4me1 patterns at CpG island borders are associated with the mode of encroachment•H3K4me1 disruption results in DNA methylation alterations at island borders
Skvortsova et al. identify promoter CpG islands that display partial methylation encroachment across one or both borders, which is often associated with gene suppression, and show that the pattern of H3K4me1 at CpG island borders in normal cells predicts patterns of cancer CpG island hypermethylation.
Abstract
Motivation
The B-cell receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. The vast repertoire and adaptive ...variation of BCR sequences due to V(D)J recombination and somatic hypermutation necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing presents the opportunity for simultaneous capture of paired BCR heavy and light chains and the transcriptomic signature.
Results
We developed VDJPuzzle, a novel bioinformatic tool that reconstructs productive, full-length B-cell receptor sequences of both heavy and light chains and extract somatic mutations on the VDJ region. VDJPuzzle successfully reconstructed BCRs from 100% (n=117) human and 96.5% (n=200) murine B cells. The reconstructed BCRs were successfully validated with single-cell Sanger sequencing.
Availability and implementation
VDJPuzzle is available at https://bitbucket.org/kirbyvisp/vdjpuzzle2.
Supplementary information
Supplementary data are available at Bioinformatics online.
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