Objectives Systemic sclerosis (SSc) is a deleterious disease. Its clinical management is complicated by strong interpatient heterogeneity. Progressive organ fibrosis is linked to autoantibodies against topoisomerase 1 (TOP1). Here, we hypothesised that the interaction between anti-TOP1 autoantibodies (ATAs) and TOP1 could influence SSc pathogenesis. TOP1 is a DNA-binding enzyme. Therefore, we studied the effects and functional consequences of TOP1-DNA binding on ATA recognition. Methods ATA monoclonal antibodies (ATA mAbs) and ATA-expressing B cell lines were generated from patient-derived TOP1-reactive B cells. Reactivity of monoclonal and polyclonal ATAs towards TOP1 and TOP1-DNA cleavage complexes (TOP1cc) was determined by enzyme-linked immunosorbent assay and mass photometry. Immunostimulatory properties of ATA mAbs in complex with TOP1/TOP1cc were assessed on monocytic THP-1 cells and primary monocytes. The stimulatory effects of TOP1-DNA complexes were tested on ATA-expressing B cells. Results TOP1-DNA binding differentially affected TOP1 recognition by ATA mAbs. A subset of ATA mAbs showed enhanced binding to TOP1cc, whereas others recognised TOP1 and TOP1cc to a similar extent. ATAs with enhanced TOP1cc recognition were observed in ATA+ patients and correlated with ATA levels and interstitial lung disease. These ‘TOP1cc-enhanced ATAs’ affected the enzymatic function of TOP1 and, in complex with TOP1cc, increased proinflammatory cytokine production by THP-1 cells and primary monocytes. B cells expressing ‘TOP1cc-enhanced ATAs’ responded strongly to stimulation with TOP1-DNA complexes, whereas additive effects were not observed for B cells expressing ‘regular’ ATAs. Conclusions The differential recognition of TOP1 upon DNA binding by ATAs demonstrates heterogeneity in the ATA B cell response, possibly impacting disease-relevant processes in severe SSc.

Abstract Objectives Transformative observations demonstrate unprecedented success of B cell-depleting interventions in many human autoimmune diseases, calling for a deeper understanding of the triggers leading to B cell-mediated autoimmunity and its perpetuation in human disease. Here, we investigated whether the autoreactive B cell response targeting human topoisomerase 1 (TOP1), a hallmark of systemic sclerosis, could cross-react with TOP1 of microbial origin. Methods Homologies between human and microbial TOP1 were analyzed using Foldseek. TOP1-reactive monoclonal antibodies from patient-derived, human TOP1-reactive B cell receptors were generated and assessed for reactivity against human TOP1 and TOP1 from a prototypic yeast, Saccharomyces cerevisiae ( S. cerevisiae ). Reactivity of polyclonal serum IgG from anti-TOP1 autoantibody (ATA) + , anti-centromere autoantibody (ACA) + SSc patients and healthy donors (HDs) was tested. Finally, B cell lines were generated expressing human ATA to study B cell activation upon antigenic stimulation. Results Structural homologues of human TOP1 were found in many microbes, particularly in fungi. Taking TOP1 from S. cerevisiae as a prototype, microbial TOP1 was recognized by polyclonal patient IgG and by several monoclonal ATAs. Importantly, S. cerevisiae TOP1 also activated B cells expressing a patient-derived, human TOP1-reactive B cell receptor. Patients affected by interstitial lung disease most frequently showed recognition of microbial TOP1. Conclusions These findings identify fungi as potential drivers of immune dysregulation in human autoimmunity, specifically in SSc, highlighting microbial antigen cross-reactive cells as important therapeutic targets. Moreover, these data provide first functional evidence for a breach of B cell tolerance against human TOP1 triggered by cross-reactivity to fungal TOP1.

OBJECTIVES:Although B cell activating factor (BAFF) serum levels are reported to correlate with the extent of skin fibrosis and markers of systemic inflammation in SSc, their relationship with BAFF-receptor (BAFF-R) expression levels and their potential as biomarker for active disease is not investigated so far. This study was undertaken to investigate BAFF/BAFF-R dynamics in SSc and how they relate to autoantibody subtype and disease activity. METHODS:Peripheral blood mononuclear cells of 69 SSc patients (ACA+: n = 37; ATA+: n = 32) and 10 age- and sex-matched controls were stained for BAFF-R followed by flow cytometry analyses. Serum BAFF and autoantibody levels were measured by ELISA. RESULTS:BAFF levels were significantly elevated in SSc patients compared with the controls. This was more pronounced in clinically active than clinically stable patients, and in ATA+ compared with the controls and ACA+ SSc patients. Concurrently, BAFF-R expression was reduced on total and on several B cell subsets. BAFF-R expression correlated inversely to serum BAFF levels. In addition, BAFF levels correlated with ESR and CRP in ATA+ SSc patients. Of note, we observed a moderate correlation between BAFF and ATA-IgG, but not between BAFF and ACA-IgG. CONCLUSIONS:Our study indicates that elevated BAFF levels, coupled with a reduction in BAFF-R expression, are significant characteristics in ATA+ SSc and of clinically active patients regardless of autoantibody status. Furthermore, BAFF levels show correlations with markers of systemic inflammation and autoantibody levels, specifically in ATA+ SSc. These findings might indicate distinct regulation of B cell responses in ATA+ and ACA+ SSc, particularly emphasizing their relevance in the context of clinically active SSc.

OBJECTIVES:The presence of immunoglobulin A (IgA) autoantibodies has been described in many autoimmune diseases, and some of its characteristics, such as IgA dimerization, are considered a sign of a mucosal origin. However, limited information is available about the (patho)physiological conditions leading to the development of monomeric vs dimeric (autoreactive) IgA in humans. Therefore, we investigated IgA dimerization in rheumatic autoimmune diseases with a possible mucosal origin, as well as after vaccination. METHODS:Plasma of patients with rheumatic disease (rheumatoid arthritis [RA], systemic sclerosis [SSc], anti-neutrophil cytoplasmic antibody associated vasculitis [AAV], systemic lupus erythematosus [SLE]), SARS-CoV-2 vaccinated healthy individuals, and healthy controls was used for size exclusion chromatography (SEC). Enzyme-linked immunosorbent assays were performed on SEC fractions to determine the size of IgA. Results were confirmed using western blot and tandem mass spectrometry. RESULTS:The proportion of dimeric IgA was increased for autoantibodies compared to total IgA. This was most evident in RA, AAV, and SLE (dimeric autoreactive IgA ≈ 60%-80% vs total IgA ≈ 20%, SLE ≈ 60% total IgA), but not in SSc. Intramuscular vaccination against SARS-CoV-2 also led to an increased proportion of dimeric anti-spike IgA shortly after vaccination, irrespective of previous mucosal exposure. CONCLUSIONS:These findings indicate that dimeric (autoreactive) IgA responses are associated with newly emerging antigen-specific immune activation, where production temporarily shifts to dimeric IgA. Mucosal triggering is not necessarily always involved in these IgA immune responses. These findings provide key insights into the circumstances for IgA dimerization and suggest that dimeric IgA could serve as a marker for immunological disease activity in autoimmunity.

Objectives Systemic sclerosis (SSc) is a deleterious disease. Its clinical management is complicated by strong interpatient heterogeneity. Progressive organ fibrosis is linked to autoantibodies against topoisomerase 1 (TOP1). Here, we hypothesized that the molecular interaction between anti-TOP1 autoantibodies (ATAs) and TOP1 could be a relevant determinant of SSc pathogenesis. DNA binding by TOP1 might affect its interaction with ATAs. Therefore, we studied the effect of DNA binding by TOP1 on ATA recognition. Methods ATA monoclonal antibodies (ATA mAbs) were generated from patient-derived TOP1-reactive B cells. Reactivity of ATA mAbs and antibodies in patient plasma towards TOP1 and TOP1-DNA cleavage complexes (TOP1cc) was determined by ELISA and mass photometry. Immunostimulatory properties of ATA mAbs in complex with TOP1/TOP1cc were assessed by stimulation of monocytic THP-1 cells. Results DNA binding by TOP1 differentially affected the recognition of TOP1 by ATA mAbs. A subset of mAbs showed enhanced binding to TOP1cc, whereas others recognized TOP1 and TOP1cc to a similar extent. ATAs with enhanced TOP1cc recognition were observed in plasma of ATA+ SSc patients and correlated with ATA levels and interstitial lung disease. These ‘TOP1cc-enhanced ATAs’ variably affected the enzymatic function of TOP1 and circulated predominantly as IgG1 and IgM. The interaction of ‘TOP1cc-enhanced ATA mAbs’ with TOP1cc strongly increased IL-8 production by THP-1 cells. Conclusions The differential recognition of TOP1 upon DNA binding by ATAs demonstrates heterogeneity in the ATA B cell response, possibly impacting on disease-relevant processes in severe SSc. ### Competing Interest Statement The authors have declared no competing interest. ReumaNederland, LLP5 Innovative Health Initiative, https://ror.org/04afqts81, 777357 Health Holland, https://ror.org/056cwr036, LSHM18055-5GF, LSHM22042-SFG European Research Council, https://ror.org/0472cxd90, AdG2019-884796 Dutch Research Council, https://ror.org/04jsz6e67, 09150172010067

OBJECTIVE:Systemic sclerosis (SSc) is a rare but severe autoimmune disease characterized by immune dysregulation, fibrosis, and vasculopathy. Although previous studies have highlighted the presence of functional autoantibodies targeting the angiotensin II receptor type 1 (AT1) and endothelin-1 type A receptor (ETAR), leading to autoantibody-mediated receptor stimulation and subsequent activation of endothelial cells (ECs), a comprehensive understanding of the direct interaction between these autoantibodies and their receptors is currently lacking. Moreover, existing data confirming the presence of these autoantibodies in SSc often rely on similar methodologies and assays. Our aim was to replicate previous findings and to investigate the functional effects of IgG derived from patients with SSc (SSc IgG) on AT1 and ETAR signaling, the downstream EC response, and the presence of AT1-binding autoantibodies in circulation. METHODS:Quantitative polymerase chain reaction and cytokine enzyme-linked immunosorbent assay, alongside a real-time cell analyzer, were used to assess receptor-specific functional characteristics of purified SSc IgG (n = 18). Additionally, a novel protein capture assay using solubilized epitope-tagged AT1 was developed to detect AT1-binding autoantibodies in plasma samples from patients with SSc (n = 28) and healthy donors (n = 14). RESULTS:No evidence for EC activation in an AT1- or ETAR-dependent manner was revealed. Furthermore, stimulation with SSc IgG did not induce receptor activation or alter G protein-coupled receptor signaling on agonist stimulation in a model with receptor overexpression. Lastly, no AT1-binding autoantibodies were detected in plasma samples from patients with SSc when using epitope-tagged solubilized AT1. CONCLUSION:Overall, our study did not provide evidence to support the presence of AT1- or ETAR-activating autoantibodies in purified SSc IgG or AT1-binding autoantibodies in the circulation of patients with SSc.

Background: Rheumatoid arthritis (RA) is a prevalent autoimmune disease characterized by B-cells that produce autoantibodies against post translationally modified proteins, such as anti-citrullinated protein antibodies (ACPA), anti-acetylated protein antibodies (AAPA) and anti-carbamylated protein antibodies (a-CarP), together called anti-modified protein antibodies (AMPA). What initially leads to the breach in tolerance and eventually causes rheumatoid arthritis is still unknown, but there are indications, such as changes in the fecal microbiome composition of RA patients, that this tolerance breach takes place in the intestines. Objectives: Since antibody responses in the intestines are dominated by IgA, particularly in dimeric form, we set out to characterize the AMPA IgA response in detail. Methods: Sera from ACPA-positive RA, ACPA-negative RA and healthy donors were fractionated using size exclusion chromatography, separating proteins on size. Next, all fractions were tested by ELISA for ACPA IgA, AAPA IgA and anti-CarP IgA, as well as total IgM, IgA and IgG. Additionally, AMPA were purified from serum to determine their size by IgA western blot and to investigate the IgA tailpiece by tandem mass spectrometry (MS/MS). Lastly, monoclonal IgA was produced with both tailpiece variants, after which its ability to polymerize was analyzed. Results: Intriguingly, of all AMPA IgA, ~70% was found to be polymeric, while total IgA contained only ~20% polymeric IgA. MS/MS analysis furthermore showed a difference in the AMPA IgA tailpiece: in the dimeric fraction ~60% had the complete tailpiece, while in the monomeric fraction this was ~20%. Recombinant IgA with such a complete tailpiece formed up to ~90% polymers, while this was ~40% in IgA with a truncated tailpiece. Conclusion: These data indicate that AMPA IgA responses are differently regulated compared to the total IgA antibody response and that the tailpiece might be related to a difference in polymer formation. Moreover, the high abundance of polymeric AMPA IgA suggests a mucosal origin. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Anouk G. van Mourik: None declared, Sanne Reijm: None declared, Alice Bacon: None declared, Myrthe A.M van Delft: None declared, Nivine Levarht: None declared, Rayman Tjokrodirijo: None declared, Peter van Veelen: None declared, Leendert A Trouw Research funding from INOVA diagnostics, part of Werfen, Rene E.M. Toes Research funding from INOVA diagnostics, part of Werfen, Karin A. van Schie: None declared, Diane van der Woude Galapagos, Galapagos.

Objectives: Troponin I has been suggested as a more specific diagnostic biomarker for myocardial involvement in systemic sclerosis than the frequently used troponin T. The aim of this study is to evaluate the additive value of troponin I to detect myocardial involvement in systemic sclerosis. To this end, we evaluated the association between troponin I levels and myocardial involvement in systemic sclerosis patients. Methods: A cross-sectional observational study was performed, including 20 healthy controls and four groups of each 20 systemic sclerosis patients from the Leiden Combined Care in Systemic Sclerosis cohort: (1) patients with myocardial involvement, (2) patients with myositis, (3) patients with elevated troponin T and creatine kinase levels but without organ involvement, and (4) patients without any signs of organ involvement. Troponin I levels were measured using enzyme-linked immunosorbent assay. Troponin I levels were compared between the different groups using the Mann–Whitney U and Kruskal–Wallis tests. Results: The mean age of the 80 included patients was 56 years; 61% of the study population was female. Troponin I levels were not significantly different between patients with and without myocardial involvement (2.7 (0.5–15.3) vs 1.2 (0.1–6.6) ng/L; p = 0.117). Systemic sclerosis patients were more often positive for troponin I than healthy controls (70.0% vs 30.0%; p = 0.001). Conclusion: Elevated troponin I was not of additional value to diagnose myocardial involvement in systemic sclerosis patients.

Background To investigate the presence of different isotypes of anti-carbamylated protein (CarP) antibodies in systemic sclerosis (SSc) patients and its association with skin involvement. Methods Sera of 194 SSc patients from the Leiden CCISS cohort, fulfilling ACR/EULAR 2013 criteria and a clinical diagnosis of SSc, 83 patients with other connective tissue diseases/Raynaud’s Phenomenon, 24 rheumatoid arthritis patients and 98 age and sex-matched healthy controls were tested for the presence of anti-CarP IgG, IgA and IgM, determined by ELISA. Clinical characteristics, that were evaluated in SSc patients, included age, anti-topoisomerase antibodies (ATA), anti-centromere antibodies (ACA) and modified Rodnan Skin Score (mRSS). Results The SSc patients were 55 (SD:13) years and 155 (80%) were female. Forty-four (23%) patients tested positive for ATA, and 80 (42%) ACA. The median mRSS was 2 (range: 0; 47). Prevalence of anti-CarP IgG was higher in SSc patients than in healthy controls (8% vs 3%, p = 0.007. Prevalence of anti-CarP IgA and IgM and levels of anti-CarP isotypes were comparable between SSc patients and healthy controls. Fifteen (8%) SSc patients tested positive for anti-CarP IgG, 16 (8%) for anti-CarP IgA, and 36 (19%) for anti-CarP IgM. There were no significant correlations between age and levels of anti-CarP isotypes. No correlation between anti-CarP IgG levels and mRSS was found ( r = 0.141, p = 0.049), nor for anti-CarP IgM and IgA levels. Anti-CarP IgA levels were higher in ATA compared to ACA positive SSc patients (ATA: 616 aU/ml [359; 1103]; ACA: 424 aU/ml [300; 673], p = 0.015). Conclusion SSc patients can test positive for Anti-CarP IgG, IgA and IgM. We do not observe a relevant clinical association between anti-CarP antibody response and skin involvement in SSc.

BACKGROUND:Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms.METHODS:Polyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation.RESULTS:Pentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG.CONCLUSIONS:We provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation.

OBJECTIVE:Biomarkers that are associated with future progression to rheumatoid arthritis (RA) and joint destruction have been discovered previously in patients with arthralgia. The present study examined these RA biomarkers in inflammatory bowel disease (IBD) patients with arthropathies.PATIENTS AND METHODS:Sera from 155 IBD patients with and 99 IBD patients without arthropathies were analyzed for immunoglobulin (Ig) M rheumatoid factor (RF), IgA-RF, anti-cyclic citrullinated peptide 2, anti-cyclic citrullinated peptide 3.1, and anti-carbamylated protein antibody positivity using enzyme-linked immunosorbent assays. The prevalence of the autoantibodies in the IBD patients was compared with the prevalence in RA patients.RESULTS:No differences were found in biomarker positivity between IBD patients with and without arthropathies. Significantly more biomarker positivity (P<0.001) was observed in RA patients compared with IBD patients with arthropathies. Also, smoking turned out to be significantly associated with positivity for IgM-RF or IgA-RF.CONCLUSION:Our findings suggest that there is no apparent clinical value in the detection of RA biomarkers in serum of IBD patients to help identify arthropathies.

Evolution of serum cytokine profile after hematopoietic stem cell transplantation in systemic sclerosis patients

Introduction: Although the role of platelets in rheumatoid arthritis (RA) is relatively unexplored, recent studies point towards a contribution of platelets in arthritis.We set out to determine platelet phenotype in RA and studied whether this could be influenced by the presence of anti-citrullinated protein antibodies (ACPA).Methods: Platelets from healthy controls were incubated in the presence of plasma of patients with RA or age-and sex-matched healthy controls and plasma from ACPA neg or ACPA pos patients or in the presence of plate-bound ACPA.Characteristics of platelets isolated from patients with RA were correlated to disease activity.Results: Platelets isolated from healthy controls displayed markers of platelet activation in the presence of plasma derived from RA patients, as determined by P-selectin expression, formation of aggregates and secretion of soluble CD40 ligand (sCD40L).Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres.In accordance with these findings, enhanced platelet activation was observed after incubation with ACPA pos plasma versus ACPA neg plasma.Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation.In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level.Conclusions: We show for the first time that ACPA can mediate an FcγRIIa-dependent activation of platelets.As ACPA can be detected several years before RA disease onset and activated platelets contribute to vascular permeability, these data implicate a possible role for ACPA-mediated activation of platelets in arthritis onset.

INTRODUCTION:Anti-carbamylated protein (anti-CarP) antibodies have been described in rheumatoid arthritis (RA) and arthralgia patients at risk of developing RA. To what extent these autoantibodies are specific for RA is unknown. Therefore, we investigated the diagnostic performance of the presence of anti-CarP antibodies for RA in a setting of early arthritis.METHODS:Anti-CarP antibodies were detected using carbamylated fetal calf serum as substrate. Anti-CCP2 antibodies were measured using enzyme-linked immunosorbent assay and immunoglobulin M (IgM) rheumatoid factor (RF) as part of routine care. Sera were derived from patients in the Leiden Early Arthritis Clinic cohort obtained at inclusion. Test characteristics were determined using the fulfillment of the 2010 RA criteria after 1 year as outcome.RESULTS:In total 2086 early arthritis patients were studied regarding the presence of anti-CarP antibodies. We observed that the sensitivity and specificity of the presence of anti-CarP antibodies for RA were 44 % and 89 %, respectively. As a reference, sensitivity and specificity of the presence of anti-CCP2 antibodies were 54 % and 96 %, respectively, and of IgM-RF 59 % and 91 %. Patients harboring anti-CarP antibodies not classified as RA were mainly diagnosed with undifferentiated arthritis and less frequently reactive arthritis and psoriatic arthritis.CONCLUSION:Anti-CarP antibodies are predominantly present in RA but can also be detected in other forms of arthritis.

OBJECTIVE:The presence of anti-citrullinated protein antibodies (ACPA) and IgM-rheumatoid factor (IgM-RF) years before the clinical diagnosis of rheumatoid arthritis (RA) suggests they are possibly involved in the pathogenic process underlying RA. In this study, we analysed whether anti-carbamylated protein (anti-CarP) antibodies, a novel autoantibody system against carbamylated proteins, can also be detected in healthy individuals before they developed RA. METHODS:Multiple sera from asymptomatic blood donors prior to the onset of their RA symptoms and sera from age-matched and sex-matched controls were tested for the presence of antibodies directed against carbamylated-fetal calf serum (Ca-FCS), carbamylated-fibrinogen (Ca-Fib), cyclic citrullinated-peptide 2 and IgM-RF. RESULTS:Anti-Ca-FCS and anti-Ca-Fib antibodies were each present in 27% and 38% of the last serum samples of blood donors prior to the diagnosis of RA. Both anti-Ca-FCS and anti-Ca-Fib antibodies could be detected many years before the onset of RA. Anti-CarP antibodies as well as ACPA are, on average, detected earlier than IgM-RF. CONCLUSIONS:In addition to ACPA and IgM-RF, also the newly identified anti-CarP antibodies appear many years before the diagnosis of RA.