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.

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.

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.

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.

Targeting downstream transcription factors and epigenetic modifications following Toll-like receptor 7/8 ligation to forestall tissue injury in anti-Ro60 associated heart block.

Based on the consistent demonstration of fibrosis of the atrioventricular node surrounded by macrophages and multinucleated giant cells in anti-Ro antibody exposed fetuses dying with heart block, this study focuses on macrophage signaling stimulated by ssRNA associated with the Ro60 protein and the impact of antagonizing innate cell drivers such as TLR7/8. Transcriptome and epigenetic modifications which affect transcription factors, NF-κB and STAT1, were selected to evaluate the phenotype of macrophages in which TLR7/8 was ligated following treatment with either anti-Ro60/Ro60/hY3 RNA immune complexes or transfection with hY3. Based on microarray, TNF and IL6 were among the most highly upregulated genes in both stimulated conditions, each of which was significantly inhibited by preincubation with hydroxychloroquine (HCQ). In contrast, following stimulation of macrophages with either TNF-α or IFN-α, which do not signal through TLR, the resultant gene expression was refractory to HCQ. Ligation of TLR7/8 resulted in increased histone methylation as measured by increased H3K4me2, a requirement for binding of NF-κB at certain promoters, specifically the kB1 region in the TNF promoter (ChIP-qPCR), which was significantly decreased by HCQ. In summary, these results support that the HCQ-sensitive phenotype of hY3 stimulated macrophages reflects the bifurcation of TLR downstream signals involving NF-κB and STAT 1 pathways and for the former dimethylation of H3K4. Accordingly, HCQ may act more as a preventive measure in downregulating the initial production of IFN-α or TNF-α and not affect the resultant autocoid stimulation reflected in TNF-α and IFN-α responsive genes. The beneficial scope of antimalarials in the prevention of organ damage, inclusive of heart block in an anti-Ro offspring or more broadly SLE, may include in part, a mechanism targeting TLR-dependent epigenetic modification.

A central role of plasmin in cardiac injury initiated by fetal exposure to maternal anti-Ro autoantibodies.

OBJECTIVE: Cardiac neonatal lupus (cardiac-NL), initiated by surface binding of anti-Ro60 autoantibodies to apoptotic cardiocytes during development, activates the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. Subsequent accumulation of apoptotic cells and plasmin generation facilitates increased binding of anti-Ro60 by disrupting and cleaving circulating ß2-glycoprotein I (ß2GPI) thereby eliminating its protective effect. The association of soluble levels of components of the uPA/uPAR system with cardiac-NL was examined. METHODS: Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies. RESULTS: uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 ± 0.1 vs 1.9 ± 0.05 ng/ml (P < 0.0001), 6.6 ± 0.3 vs 2.1 ± 0.2 ng/ml (P < 0.0001) and 435 ± 34 vs 220 ± 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 ± 0.1 vs 1.9 ± 0.05 ng/ml; P = 0.0086, 6.2 ± 1.4 vs 2.2 ± 0.7 ng/ml; P = 0.147 and 412 ± 61 vs 260 ± 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region. CONCLUSION: Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.

Complement receptor 3 influences toll-like receptor 7/8-dependent inflammation: implications for autoimmune diseases characterized by antibody reactivity to ribonucleoproteins.

Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFα in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFα secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFα release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.

Pathogenicity and proteomic signatures of autoantibodies to Ro and La.

Ro/SSA and La/SSB comprise a linked set of autoantigens that are clinically important members of the extractable nuclear antigen family and key translational biomarkers for lupus and primary Sjögren's syndrome. Autoantibodies directed against the Ro60 and La polypeptide components of the Ro/La ribonucleoprotein complex, and the structurally unrelated Ro52 protein, mediate tissue damage in the neonatal lupus syndrome, a model of passively acquired autoimmunity in humans in which the most serious manifestation is congenital heart block (CHB). Recent studies have concentrated on two distinct pathogenic mechanisms by which maternal anti-Ro/La autoantibodies can cause CHB: by forming immune complexes with apoptotic cells in developing fetal heart; and/or by acting as functional autoantibodies that cross-react with and inhibit calcium channels. Although the precise role of the individual autoantibodies is yet to be settled, maternal anti-Ro60 and anti-Ro52 remain the most likely culprits. This article will discuss the molecular pathways that culminate in the development of CHB, including the recent discovery of ß2 glycoprotein I as a protective factor, and present a proteomic approach based on direct mass spectrometric sequencing, which may give a more representative snapshot of the idiotype repertoire of these autoantibodies than genomic-based technologies.

A novel role of endothelin-1 in linking Toll-like receptor 7-mediated inflammation to fibrosis in congenital heart block.

Autoimmune associated congenital heart block (CHB) may result from pathogenic cross-talk between inflammatory and profibrosing pathways. Incubation of macrophages with immune complexes (IC) composed of Ro60, a target of the pathologic maternal autoantibodies necessary for CHB, hY3 ssRNA, and affinity-purified anti-Ro60 antibody induces the Toll-like receptor 7 (TLR7)-dependent generation of supernatants that provoke a fibrosing phenotype in human fetal cardiac fibroblasts. We show herein that these cells are a major source of TGFß and that endothelin-1 (ET-1) is one of the key components responsible for the profibrosing effects generated by stimulated macrophages. Supernatants from macrophages incubated with IC induced the fibroblast secretion of TGFß, which was inhibited by treating the macrophages with an antagonist of TLR7. Under the same conditions, the induced fibroblast secretion of TGFß was decreased by inhibitors of the ET-1 receptors ETa or ETb or by an anti-ET-1 antibody but not by an isotype control. Exogenous ET-1 induced a profibrosing phenotype, whereas fibroblasts transfected with either ETa or ETb siRNA were unresponsive to the profibrosing effects of the IC-generated macrophage supernatants. Immunohistochemistry of the hearts from two fetuses dying with CHB revealed the presence of ET-1-producing mononuclear cells in the septal region in areas of calcification and fibrosis. In conclusion, these data support a novel role of ET-1 in linking TLR7 inflammatory signaling to subsequent fibrosis and provide new insight in considering therapeutics for CHB.

Ro 60 functions as a receptor for beta(2)-glycoprotein I on apoptotic cells.

OBJECTIVE: The autoantigens 60-kd Ro/SSA (Ro 60) and beta(2)-glycoprotein I (beta(2)GPI) are both displayed on the surface membrane of apoptotic cells. Epitope-spreading experiments have suggested that these autoantigens may be present as a complex on the apoptotic cell surface. This study was undertaken to investigate whether beta(2)GPI interacts with Ro 60 on apoptotic cells and alters the binding of anti-Ro 60 IgG. METHODS: The interaction between soluble recombinant Ro 60 fragments and beta(2)GPI was investigated in vitro by direct and saturation binding assays using native human beta(2)GPI and recombinant domain deletion mutants. Binding of beta(2)GPI to early and late apoptotic cells was assessed by multiparameter flow cytometry, and specificity of binding was determined by competitive inhibition with soluble recombinant Ro 60 and anti-Ro 60 IgG. RESULTS: The Ro 60 fragment expressing a surface-exposed epitope (apotope) bound with high affinity (K(d) = approximately 15 nM) to domain V of beta(2)GPI in vitro. Beta(2)-glycoprotein I bound to the surface of apoptotic cells in a dose-dependent manner and was blocked by the Ro 60 apotope fragment. In reciprocal competitive inhibition studies, beta(2)GPI blocked the binding of anti-Ro 60 autoantibodies to apoptotic cells in a dose-dependent manner, and anti-Ro 60 IgG inhibited the binding of beta(2)GPI. Moreover, beta(2)GPI showed a 2-fold increase in binding to apoptotic cells that overexpress Ro 60 on the surface. CONCLUSION: These results demonstrate that Ro 60 functions as a novel receptor for beta(2)GPI on the surface of apoptotic cells. The formation of Ro 60-beta(2)GPI complexes may protect against anti-Ro 60 autoantibody-mediated tissue injury.