Highlight of 2023: CAR T cells driving precision therapy for autoimmune disease.

CAR T cell therapy is showing remarkable results in autoimmune disease with treatment-refractory patients showing durable drug-free remission. Here, we highlight five key papers from 2023 that are driving the development of CAR T cells to improve precision, safety, efficacy and accessibility for the treatment of autoantibody-associated autoimmune diseases.

Seronegative Sjögren Syndrome: A Forgotten Entity?

As a highly varied disease, Sjögren syndrome (SS; also termed Sjögren disease) is estimated to affect 0.01% to 0.72% of the population, with an overwhelming bias toward the female gender.1 SS causes a significant burden to the quality of life of patients and inhibits their function.

Isolated anti-Ro52 identifies a severe subset of Sjögren's syndrome patients.

Introduction: Serum autoantibodies targeting the SSA/Ro proteins are a key component of the classification criteria for the diagnosis of Sjögren's syndrome (SS). Most patients' serum reacts with both Ro60 and Ro52 proteins. Here we compare the molecular and clinical characteristics of patients diagnosed with SS with anti-Ro52 in the presence or absence of anti-Ro60/La autoantibodies. Methods: A cross-sectional study was performed. Patients in the SS biobank at Westmead Hospital (Sydney, Australia) that were positive for anti-Ro52 were included and stratified based on the absence (isolated) or presence (combined) of anti-Ro60/La, measured by line immunoassay. We examined clinical associations and the serological and molecular characteristics of anti-Ro52 using ELISA and mass spectrometry in serological groups. Results: A total of 123 SS patients were included for study. SS patients with isolated anti-Ro52 (12%) identified a severe serological subset characterised by higher disease activity, vasculitis, pulmonary involvement, rheumatoid factor (RhF) and cryoglobulinaemia. Serum antibodies reacting with Ro52 in the isolated anti-Ro52 subset displayed less isotype switching, less immunoglobulin variable region subfamily usage and a lower degree of somatic hypermutation than the combined anti-Ro52 subset. Conclusions: In our cohort of SS patients, isolated anti-Ro52 represents a severe subset of SS, and is associated with the presence of cryoglobulinaemia. We therefore provide clinical relevance to the stratification of SS patients by their sero-reactivities. It is possible that the autoantibody patterns may be immunological epiphenomena of the underlying disease process, and further work is required to unearth the mechanisms of the differential clinical phenotypes.

Structural and functional characterization of capsid binding by anti-AAV9 monoclonal antibodies from infants after SMA gene therapy.

Success in the treatment of infants with spinal muscular atrophy (SMA) underscores the potential of vectors based on adeno-associated virus (AAV). However, a major obstacle to the full realization of this potential is pre-existing natural and therapy-induced anti-capsid humoral immunity. Structure-guided capsid engineering is one possible approach to surmounting this challenge but necessitates an understanding of capsid-antibody interactions at high molecular resolution. Currently, only mouse-derived monoclonal antibodies (mAbs) are available to structurally map these interactions, which presupposes that mouse and human-derived antibodies are functionally equivalent. In this study, we have characterized the polyclonal antibody responses of infants following AAV9-mediated gene therapy for SMA and recovered 35 anti-capsid mAbs from the abundance of switched-memory B (smB) cells present in these infants. For 21 of these mAbs, seven from each of three infants, we have undertaken functional and structural analysis measuring neutralization, affinities, and binding patterns by cryoelectron microscopy (cryo-EM). Four distinct patterns were observed akin to those reported for mouse-derived mAbs, but with early evidence of differing binding pattern preference and underlying molecular interactions. This is the first human and largest series of anti-capsid mAbs to have been comprehensively characterized and will prove to be powerful tools for basic discovery and applied purposes.

Phases and Natural History of Sjögren's Disease: A New Model for an Old Disease?

Sjögren's disease (SjD) is an archetypal and heterogenous autoimmune disorder that is characterized by exocrine glandular dysfunction. A proportion of patients develop severe extraglandular manifestations, such as cryoglobulinemia, and have an increased risk of lymphoma, both of which can adversely affect quality of life and occasionally mortality. As with most autoimmune disorders, the pathogenesis is poorly understood and difficult to predict, and, frustratingly, there is a lack of targeted therapies to cure this disease. We review the disease manifestations of SjD and propose a staged model for understanding the evolution of pathology. In longitudinal studies, most patients remain relatively stable in terms of their laboratory and clinical parameters. However, in the setting of various risk factors, a proportion of patients develop severe symptoms and/or lymphoma. We discuss potential underlying mechanisms for disease progression and the strengths and limitations of using a staged model to correlate the pathogenesis and spectrum of manifestations in SjD. Ultimately, understanding how and why some patients remain relatively stable, whereas others progress and develop florid systemic disease and a fraction develop lymphoma, is key to developing preventative and therapeutic treatments.

STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological NKG2Dhi CD8+ T cell dysregulation and accumulation.

The association between cancer and autoimmune disease is unexplained, exemplified by T cell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ T cell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ T cell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subset also expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ T cells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ T cell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Genetic and structural basis of the human anti-α-galactosyl antibody response.

Humans lack the capacity to produce the Galα1-3Galß1-4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.

Anti-Ro52/TRIM21 serological subsets identify differential clinical and laboratory parameters.

INTRODUCTION: Anti-Ro52/tripartite motif-containing protein 21 (TRIM21) IgG is one of the most common autoantibodies found in systemic autoimmune diseases and is typically found in conjunction with anti-Ro60 and/or anti-La. A retrospective, cross-sectional study was undertaken to examine the clinical and laboratory features of two serological subsets: patients with anti-Ro52/TRIM21 autoantibodies in the absence of anti-Ro60 and anti-La (isolated anti-Ro52/TRIM21) and patients with anti-Ro52/TRIM21 in the presence of anti-Ro60 and/or anti-La. METHODS: Over a 12-month period, patients tested positive for anti-Ro52/TRIM21 via line immunoassay (LIA) at the Westmead Hospital (Australia) immunopathology laboratory were included. The presence of anti-Ro60 and/or anti-La via same LIA was noted. Associated laboratory and medical records were perused to extract demographic, laboratory, and clinical information. RESULTS: There were 346 patients within the study period, and 39.9% of the patients positive for anti-Ro52/TRIM21 lacked anti-Ro60/anti-La autoantibodies. Isolated anti-Ro52/TRIM21 patients tend to be older, have lower anti-Ro52/TRIM21 titres, have lower rheumatoid factors, and have lower proportions of neutropaenia compared to patients who were positive for anti-Ro52/TRIM21 and anti-Ro60/La. This occurred independent to diagnoses of Sjögren's syndrome or systemic lupus erythematosus. Coexisting neurological syndromes, pulmonary pathologies, and malignancies were more prevalent in the isolated anti-Ro52/TRIM21 subset. CONCLUSIONS: Patients with isolated anti-Ro52/TRIM21 tend to have distinct and important clinical and laboratory associations. It is unclear if these patients evolve or remain a stable subset and how they originate immunologically. Longitudinal and prospective studies are required to ascertain the overall predictive and prognostic value of this stratification. Key Points • Anti-Ro52/TRIM21 is an autoantibody found in autoimmunity and non-immunological conditions. • Sixty percent of anti-Ro52/TRIM21 patients are positive for anti-Ro60. • Isolated anti-Ro52/TRIM21 has reduced anti-Ro52/TRIM21 and rheumatoid factor titres. • Isolated anti-Ro52/TRIM21 is associated with anaemia and malignancies.

Transforming mutations in the development of pathogenic B cell clones and autoantibodies.

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.

Uncontrolled CD21low age-associated and B1 B cell accumulation caused by failure of an EGR2/3 tolerance checkpoint.

CD21low age-associated or atypical memory B cells are autoantibody enriched and poised for plasma cell differentiation. These cells overaccumulate in chronic infections, autoimmune disease, and immunodeficiency, posing the question of what checkpoints normally oppose their accumulation. Here, we reveal a critical role for paralogous calcium-NFAT-regulated transcription factors EGR2 and EGR3 that are induced in self-reactive B cells. CD21low and B1 B cells lacking EGR2 and EGR3 accumulate and circulate in young mice in numbers 10- to 20-fold greater than normal and overexpress a large set of EGR2 ChIP-seq target genes, including known drivers of plasma cell differentiation. Most follicular B cells constitutively express Egr2 proportionally to surface IgM downregulation by self-antigens, and EGR2/3 deficiency abolishes this cardinal feature of B cell anergy. These results explain the cardinal features of B cell anergy, define a key transcriptional checkpoint repressing CD21low B cell formation, and inform how NFATC1 or EGR2 mutations promote B1 cell-derived chronic lymphocytic leukemias.

Anti-Ro60 and anti-Ro52/TRIM21: Two distinct autoantibodies in systemic autoimmune diseases.

As iconic and important diagnostic autoantibodies, anti-Ro60 and anti-Ro52/tri-partite motif-containing 21 (TRIM21) make a common appearance in a number of systemic autoimmune disorders such as systemic lupus erythematosus (SLE). These autoantibodies often co-exist together; yet despite their close relationship, there is no evidence that they are physically linked and probably reflect a convergence of separate processes of failed immunological tolerance. Confusingly, they are sometimes classed together as the "SSA" or "Ro" autoantibody system without clear distinction between the two. In this Short Communication, we discuss the diagnostic merits for separate detection and reporting of these two autoantibodies, and discuss avenues for future research. Indeed, further insight into their fascinating origins and pathogenic roles in autoimmunity will surely shed light on how we can prevent and treat devastating autoimmune disorders.

Human transitional and IgMlow mature naïve B cells preserve permissive B-cell receptors.

The level of immunoglobulin M (IgM) displayed on the surface of peripheral blood B cells exhibits a broad dynamic range and has been associated with both development and selection. To determine whether IgM surface expression associates with distinct immunoglobulin heavy-chain (IGH) repertoire properties, we performed deep IgM sequencing of peripheral blood transitional and mature naïve B cells in the upper and lower quartiles of surface IgM expression for 12 healthy donors. Mature naïve B cells within the lowest quartile for surface IgM expression displayed more diverse IGH features including increased complementarity-determining region 3 length, IGHJ6 segment usage and aromatic amino acids compared with mature naïve B cells with high surface IgM. There were no differences between IGH repertoires for transitional B cells with high or low surface IgM. These findings suggest that a selection checkpoint during progression of transitional to mature naïve B cells reduces the breadth of the IGH repertoire among high surface IgM B cells but that diversity is preserved in B cells expressing low levels of surface IgM.

When B cells break bad: development of pathogenic B cells in Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is often considered a B cell-mediated disease, yet the precise role of B cells in the pathogenesis is not fully understood. This is exemplified by the failure of multiple clinical trials directed at B cell depletion or inhibition. To date, most prognostic markers for severe disease outcomes are autoantibodies, but the underlying mechanisms by which B cells drive diverse disease presentations in pSS likely extend beyond autoantibody production. Here we outline an expanded role of B cells in disease pathogenesis drawing on examples from animal models of SS, and from other autoimmune diseases that share similar clinical or immunological abnormalities. We focus on recent findings from the detailed analysis of pathogenic B cells in patients with pSS to propose strategies for patient stratification to improve clinical trial outcomes. We conclude that an integrated cellular, molecular and genetic analysis of patients with pSS will reveal the underlying pathogenic mechanisms and guide precision medicine.

Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

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.

Activated PI3Kδ breaches multiple B cell tolerance checkpoints and causes autoantibody production.

Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity.

Clonal redemption and clonal anergy as mechanisms to balance B cell tolerance and immunity.

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.

DNA Hypermethylation Encroachment at CpG Island Borders in Cancer Is Predisposed by H3K4 Monomethylation Patterns.

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.

Sequencing and Affinity Determination of Antigen-Specific B Lymphocytes from Peripheral Blood.

Here we describe methods for screening human blood to isolate peripheral blood mononuclear cells (PBMCs) capable of binding fluorescently labeled antigen, as well as methods for the amplification and sequencing of B cell receptor (BCR) heavy and light chain genes. Detailed protocols are provided for transient mammalian expression in a hexahistidine-tagged Fab format, purification by immobilized metal affinity chromatography (IMAC), and affinity determination by BioLayer interferometry (BLI).

B-cell receptor reconstruction from single-cell RNA-seq with VDJPuzzle.

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.

Molecular Profiling and Clonal Tracking of Secreted Rheumatoid Factors in Primary Sjögren's Syndrome.

OBJECTIVE: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking. METHODS: Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. RESULTS: Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig VH ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig VH 4-34 IgM-RF decreased following immunosuppression and remission of MC. CONCLUSION: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.