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Objectives: To investigate how individual rheumatoid arthritis (RA) autoantibodies associate with individual signs and symptoms at the time of RA diagnosis. Methods: IgA, IgG, IgM rheumatoid factor (RF), antibodies against cyclic citrullinated peptide version 2 (anti-CCP2) and 16 individual antibodies against citrullinated protein (ACPA) reactivities were analysed centrally in baseline sera from 1600 patients with RA classified according to the 1987 American College of Rheumatology (ACR) criteria. These results were related to C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), number of swollen and tender joints (SJC and TJC), 28-joint disease activity scores (DAS28 and DAS28CRP), global disease activity evaluated by the patients and Health Assessment Questionnaire, all obtained at baseline. Results: Individually, all autoantibodies except immunoglobulin G (IgG) RF associated with low SJC and TJC and with high ESR. In IgM RF-negative patients, ACPA associated strictly with low number of swollen and tender joints. This association persisted in multiple regression and stratified analyses where IgM and IgA RF instead associated with inflammation expressed as ESR. Among subjects without any ACPA peptide reactivity, there was no association between RF isotypes and ESR. The effect of RF on ESR increased with the number of ACPA reactivities, especially for IgM RF. In patients fulfilling the 1987 ACR criteria without taking RF into account, associations between IgM RF and high ESR, as well as between ACPA and low joint counts, remained. Conclusion: Whereas ACPA associate with low counts of affected joints in early RA, RF associates with elevated measures of systemic inflammation in an ACPA-dependent manner. This latter finding corroborates in vitro models of ACPA and RF in immune complex-induced inflammation. These phenotypic associations are independent of classification criteria.

Background: Why the adaptive immune system turns against citrullinated antigens in rheumatoid arthritis (RA) and whether anti-citrullinated protein antibodies (ACPAs) contribute to pathogenesis are questions that have triggered intense research, but still are not fully answered. Neutrophils may be crucial in this context, both as sources of citrullinated antigens and also as targets of ACPAs. To better understand how ACPAs and neutrophils contribute to RA, we studied the reactivity of a broad spectrum of RA patient-derived ACPA clones to activated or resting neutrophils, and we also compared neutrophil binding using polyclonal ACPAs from different patients. Methods: Neutrophils were activated by Ca2+ ionophore, PMA, nigericin, zymosan or IL-8, and ACPA binding was studied using flow cytometry and confocal microscopy. The roles of PAD2 and PAD4 were studied using PAD-deficient mice or the PAD4 inhibitor BMS-P5. Results: ACPAs broadly targeted NET-like structures, but did not bind to intact cells or influence NETosis. We observed high clonal diversity in ACPA binding to neutrophil-derived antigens. PAD2 was dispensable, but most ACPA clones required PAD4 for neutrophil binding. Using ACPA preparations from different patients, we observed high patient-to-patient variability in targeting neutrophil-derived antigens and similarly in another cellular effect of ACPAs, the stimulation of osteoclast differentiation. Conclusions: Neutrophils can be important sources of citrullinated antigens under conditions that lead to PAD4 activation, NETosis and the extrusion of intracellular material. A substantial clonal diversity in targeting neutrophils and a high variability among individuals in neutrophil binding and osteoclast stimulation suggest that ACPAs may influence RA-related symptoms with high patient-to-patient variability.

A majority of circulating IgG is produced by plasma cells residing in the bone marrow (BM). Long-lived BM plasma cells constitute our humoral immune memory and are essential for infection-specific immunity. They may also provide a reservoir of potentially pathogenic autoantibodies, including rheumatoid arthritis (RA)-associated anti-citrullinated protein autoantibodies (ACPA). Here we investigated paired human BM plasma cell and peripheral blood (PB) B-cell repertoires in seropositive RA, four ACPA+ RA patients and one ACPA- using two different single-cell approaches, flow cytometry sorting, and transcriptomics, followed by recombinant antibody generation. Immunoglobulin (Ig) analysis of >900 paired heavy-light chains from BM plasma cells identified by either surface CD138 expression or transcriptome profiles (including gene expression of MZB1, JCHAIN and XBP1) demonstrated differences in IgG/A repertoires and N-linked glycosylation between patients. For three patients, we identified clonotypes shared between BM plasma cells and PB memory B cells. Notably, four individuals displayed plasma cells with identical heavy chains but different light chains, which may indicate receptor revision or clonal convergence. ACPA-producing BM plasma cells were identified in two ACPA+ patients. Three of 44 recombinantly expressed monoclonal antibodies from ACPA+ RA BM plasma cells were CCP2+, specifically binding to citrullinated peptides. Out of these, two clones reacted with citrullinated histone-4 and activated neutrophils. In conclusion, single-cell investigation of B-cell repertoires in RA bone marrow provided new understanding of human plasma cells clonal relationships and demonstrated pathogenically relevant disease-associated autoantibody expression in long-lived plasma cells.

Objectives: To elucidate antibody responses after the second and third dose of COVID-19 vaccine in patients with inflammatory rheumatic diseases (IRD) treated with biologic/targeted disease modifying anti-rheumatic drugs (b/ts DMARDs). Methods: Antibody levels to antigens representing spike full length protein and spike S1 were measured before vaccination, 2–12 weeks after the second dose, before and after the third dose using multiplex bead-based serology assay. Positive antibody response was defined as antibody levels over cut off (seropositivity) in seronegative individuals or ≥ 4-fold increase in antibodies in individuals seropositive for both spike proteins. Results: Patients (n = 414) receiving b/ts DMARDs (283 had arthritis, 75 systemic vasculitis and 56 other autoimmune diseases) and controls (n = 61) from five Swedish regions participated. Treatments groups were: rituximab (n = 145); abatacept (n = 22); Interleukin 6 receptor inhibitors [IL6i (n = 79)]; JAnus Kinase Inhibitors [JAKi (n = 58)], Tumour Necrosis Factor inhibitor [TNFi (n = 68)] and Interleukin12/23/17 inhibitors [IL12/23/17i (n = 42)]. Percentage of patients with positive antibody response after two doses was significantly lower in rituximab (33,8%) and abatacept (40,9%) (p < 0,001) but not in IL12/23/17i, TNFi or JAKi groups compared to controls (80,3%). Higher age, rituximab treatment and shorter time between last rituximab course and vaccination predicted impaired antibody response. Antibody levels collected 21–40 weeks after second dose decreased significantly (IL6i: p = 0,02; other groups: p < 0,001) compared to levels at 2–12 week but most participants remained seropositive. Proportion of patients with positive antibody response increased after third dose but was still significantly lower in rituximab (p < 0,001). Conclusions: Older individuals and patients on maintenance rituximab have an impaired response after two doses of COVID-19 vaccine which improves if the time between l

Objectives To assess the association between venous thromboembolic (VTE) events and autoantibodies, following patients from RA diagnosis, measuring occurrence, levels and collective load of different autoantibodies against post-translational protein modifications, in particular recognizing citrullination (e.g. citrullinated fibrinogen) and RF by isotype. Methods A cohort of 2814 patients with newly diagnosed RA were followed for incident VTE through register linkages. Sera from RA diagnosis were centrally analysed for antibodies to second generation cyclic citrullinated peptides (anti-CCP2), 20 anti-citrullinated protein antibody (ACPA) fine-specificities, antibodies to additional protein modifications (carbamylation and acetylation) and RF by isotype. Association between baseline serology status and future VTE was analysed using Cox regression adjusted for age, sex and calendar period of RA diagnosis, overall and stratified by anti-CCP2 and RF positivity. Results During a median 16 years of follow-up, 213 first-ever VTE events were registered (5.0/1000 person-years). IgG anti-CCP2 (present in 65% of cohort) associated with VTE (hazard ratio [HR] = 1.33, 95% CI: 1.00, 1.78), in a dose-response manner. The risk of VTE increased with number of ACPA fine-specificities. IgM RF, but no other RF isotypes, associated with VTE (HR = 1.38, 95% CI: 1.04, 1.82). The associations were independent from smoking and HLA-DRB1 shared epitope alleles. None of the carbamylated or acetylated antibody reactivities associated with VTE. Conclusion Anti-CCP2, load of ACPA fine-specificities and IgM RF at RA diagnosis are associated with an increased risk of future VTE in RA. Antibodies to citrullinated fibrinogen did not differ substantially from other ACPA fine-specificities. Autoreactivity to other post-translational modifications was not associated with VTE risk., Funding Agencies|Swedish Research Council; NordForsk; Vinnova; Region Stockholm/Karolinska Institutet Funds (ALF); Swedish Heart Lung Foundation

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium Porphyromonas gingivalis to citrullinate proteins, it has been suggested that production of anti-citrullinated protein antibodies (ACPA), which are present in a majority of RA patients, may be triggered in the gum mucosa. To address this hypothesis, we investigated the antibody response to a citrullinated P. gingivalis peptide in relation to the autoimmune ACPA response in early RA, and examined citrulline-reactivity in monoclonal antibodies derived from human gingival B cells. Antibodies to a citrullinated peptide derived from P. gingivalis (denoted CPP3) and human citrullinated peptides were analyzed by multiplex array in 2,807 RA patients and 372 controls; associations with RA risk factors and clinical features were examined. B cells from inflamed gingival tissue were single-cell sorted, and immunoglobulin (Ig) genes were amplified, sequenced, cloned and expressed (n=63) as recombinant monoclonal antibodies, and assayed for citrulline-reactivities by enzyme-linked immunosorbent assay. Additionally, affinity-purified polyclonal anti-cyclic-citrullinated peptide (CCP2) IgG, and monoclonal antibodies derived from RA blood and synovial fluid B cells (n=175), were screened for CPP3-reactivity. Elevated anti-CPP3 antibody levels were detected in RA (11%), mainly CCP2+ RA, compared to controls (2%), p<0.0001, with a significant association to HLA-DRB1 shared epitope alleles, smoking and baseline pain, but with low correlation to autoimmune ACPA fine-specificities. Monoclonal antibodies derived from gingival B cells showed cross-reactivity between P. gingivalis CPP3 and human citrullinated peptides, and a CPP3+/CCP2+ clone, derived from an RA blood memory B cell, was identified. Our data support the possibility that immunity to P. gingivalis derived citrullinated antigens, triggered in the inflamed gum mucosa, may contribute to the

QC 20230113

Objectives Pulmonary manifestations in RA are common comorbidities, but the underlying mechanisms are largely unknown. The added value of a multiplex of ACPA and genetic risk markers was evaluated for the development of pulmonary fibrosis (PF) in an inception cohort. Methods A total of 1184 patients with early RA were consecutively included and followed prospectively from the index date until death or 31 December 2016. The presence of 21 ACPA fine specificities was analysed using a custom-made microarray chip (Thermo Fisher Scientific, Uppsala, Sweden). Three SNPs, previously found related to PF were evaluated, rs2609255 (FAM13A), rs111521887 (TOLLIP) and rs35705950 (MUC5B). ACPA and genetic data were available for 841 RA patients, of whom 50 developed radiologically defined PF. Results In unadjusted analyses, 11 ACPA specificities were associated with PF development. In multiple variable analyses, six ACPA specificities were associated with increased risk of PF: vimentin (Vim)60-75, fibrinogen (Fib)beta 62-78 (72), Fib alpha 621-635, Bla26, collagen (C)II359-369 and F4-CIT-R (P < 0.01 to P < 0.05). The number of ACPA specificities was also related to PF development (P < 0.05 crude and adjusted models). In multiple variable models respectively adjusted for each of the SNPs, the number of ACPA specificities (P < 0.05 in all models), anti-Vim60-75 (P < 0.05, in all models), anti-Fib beta 62-78 (72) (P < 0.001 to P < 0.05), anti-CII359-369 (P < 0.05 in all models) and anti-F4-CIT-R AQ4 (P < 0.01 to P < 0.05), anti-Fib alpha 621-635 (P < 0.05 in one) and anti-Bla26 (P < 0.05 in two) were significantly associated with PF development. Conclusion The development of PF in an inception cohort of RA patients was associated with both presence of certain ACPA and the number of ACPA specificities and risk genes.

Objectives To assess the association between venous thromboembolic (VTE) events and autoantibodies, following patients from RA diagnosis, measuring occurrence, levels and collective load of different autoantibodies against post-translational protein modifications, in particular recognizing citrullination (e.g. citrullinated fibrinogen) and RF by isotype. Methods A cohort of 2814 patients with newly diagnosed RA were followed for incident VTE through register linkages. Sera from RA diagnosis were centrally analysed for antibodies to second generation cyclic citrullinated peptides (anti-CCP2), 20 anti-citrullinated protein antibody (ACPA) fine-specificities, antibodies to additional protein modifications (carbamylation and acetylation) and RF by isotype. Association between baseline serology status and future VTE was analysed using Cox regression adjusted for age, sex and calendar period of RA diagnosis, overall and stratified by anti-CCP2 and RF positivity. Results During a median 16 years of follow-up, 213 first-ever VTE events were registered (5.0/1000 person-years). IgG anti-CCP2 (present in 65% of cohort) associated with VTE (hazard ratio [HR] = 1.33, 95% CI: 1.00, 1.78), in a dose-response manner. The risk of VTE increased with number of ACPA fine-specificities. IgM RF, but no other RF isotypes, associated with VTE (HR = 1.38, 95% CI: 1.04, 1.82). The associations were independent from smoking and HLA-DRB1 shared epitope alleles. None of the carbamylated or acetylated antibody reactivities associated with VTE. Conclusion Anti-CCP2, load of ACPA fine-specificities and IgM RF at RA diagnosis are associated with an increased risk of future VTE in RA. Antibodies to citrullinated fibrinogen did not differ substantially from other ACPA fine-specificities. Autoreactivity to other post-translational modifications was not associated with VTE risk., Funding Agencies|Swedish Research Council; NordForsk; Vinnova; Region Stockholm/Karolinska Institutet Funds (ALF); Swedish Heart Lung Foundation

QC 20220315

Seropositive rheumatoid arthritis (RA) is characterized by the presence of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) with different fine-specificities. Yet, other serum anti-modified protein autoantibodies (AMPA), e.g. anti-carbamylated (Carb), -acetylated (KAc), and malondialdehyde acetaldehyde (MAA) modified protein antibodies, have been described. In this comprehensive study, we analyze 30 different IgG and IgA AMPA reactivities to Cit, Carb, KAc, and MAA antigens detected by ELISA and autoantigen arrays in N=1985 newly diagnosed RA patients. Association with patient characteristics such as smoking and disease activity were explored. Carb and KAc reactivities by different assays were primarily seen in patients also positive for anti-citrulline reactivity. Modified vimentin (mod-Vim) peptides were used for direct comparison of different AMPA reactivities, revealing that IgA AMPA recognizing mod-Vim was mainly detected in subsets of patients with high IgG anti-Cit-Vim levels and a history of smoking. IgG reactivity to acetylation was mainly detected in a subset of patients with Cit and Carb reactivity. Anti-acetylated histone reactivity was RA-specific and associated with high anti-CCP2 IgG levels, multiple ACPA fine-specificities, and smoking status. This reactivity was also found to be present in CCP2+ RA-risk individuals without arthritis. Our data further demonstrate that IgG autoreactivity to MAA was increased in RA compared to controls with highest levels in CCP2+ RA, but was not RA-specific, and showed low correlation with other AMPA. Anti-MAA was instead associated with disease activity and was not significantly increased in CCP2+ individuals at risk of RA. Notably, RA patients could be subdivided into four different subsets based on their AMPA IgG and IgA reactivity profiles. Our serology results were complemented by screening of monoclonal antibodies derived from single B cells from RA patients for the same antigens as th

Objective To investigate the relationship between anti–citrullinated protein antibodies (ACPAs), specific ACPA subspecificities, rheumatoid factor (RF) isotypes, and incident cardiovascular (CV) events in patients with rheumatoid arthritis (RA). Methods Serum samples from Swedish patients with new-onset RA (diagnosed within 1 year of symptom onset between 1996 and 2009) were centrally typed for anti–cyclic citrullinated peptide 2 (anti-CCP2) antibodies, 20 ACPA subspecificities, and RF isotypes. Patients were followed up longitudinally in nationwide registers to monitor the occurrence of acute coronary syndrome (ACS), stroke, CV-related death, and major adverse CV events (MACE). The association between each serologic marker and CV outcome, and the impact of adjustment for the Disease Activity Score in 28 joints (DAS28), smoking status, and income at baseline, were assessed using Cox proportional hazards models. In addition, associations of serologic markers with all-cause mortality were explored. Results In total, 2,814 patients with RA were included in the study. The median follow-up was 13 years, during which the CV end points of ACS, stroke, or CV-related death were reported to occur in 375 patients. Occurrence and/or levels of anti-CCP2 were associated with risk of incident ACS (hazard ratio [HR] 1.46, 95% confidence interval [95% CI] 1.03–2.06), stroke (HR 1.47, 95% CI 1.03–2.10), CV-related death (P = 0.024 for association with anti-CCP2 levels), and MACE (HR 1.34, 95% CI 1.06–1.70). Similarly, an association with the number of ACPA subspecificities was observed; however, this could not be attributed to any individual or group of ACPA subspecificities. Presence of IgM-RF was associated with all CV end points except ACS, and IgA-RF was exclusively associated with CV-related death. Adjustment for smoking status, income, and DAS28 scores decreased most of the HRs, whereas IgA-RF remained associated with CV-related death (HR 1.61, 95% CI 1.05–2.48). All of the ass

Objective. Anti-citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigens that are targeted by ACPAs remains a matter of debate. This study utilized patient-derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA. Methods. Twelve human monoclonal ACPAs (positive by the second-generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head-to-head comparison of their reactivities was performed. For profiling, we used a complementary DNA-based protein array (Engine GmbH) and 3 peptide-screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histonederived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). Results. The fine-specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone-distinct consensus binding motifs (e.g., Cit-Gly, Gly-Cit, or Arg-Cit-Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA-binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated-lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for Cit peptides. Conclusion. ACPAs and anti-modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically r, QC 20201201