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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 last rituximab course and vaccination extends and also after an additional vaccine dose. Rituximab patients should be prioritized for booster vaccine doses. TNFi, JAKi and IL12/23/17i does not diminished humoral response to primary and an additional vaccination.

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)β62–78 (72), Fibα621–635, Bla26, collagen (C)II359–369 and F4-CIT-R (P 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.

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium

Objective Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). Aside from autoantibody production, the function of autoantigen-specific B cells remains poorly understood in the context of this disease. This study set out to elucidate autoantigen-specific B cell functions through the isolation and immortalization of unique citrullinated protein/peptide (CP)-reactive B cell clones from RA patients. Methods B cell clones from either the blood or synovial fluid of cyclic citrullinated peptide 2 (CCP2) antibody-positive RA patients were immortalized by genetic reprogramming with Bcl-6 and Bcl-xL. Enzyme-linked immunosorbent assay and flow cytometry were used to identify CCP2-reactive clones and to further characterize surface marker and cytokine expression as well as B cell receptor signaling competence. Global gene expression profiles were interrogated by RNA sequencing. Results Three unique CP-reactive memory B cell clones were generated from the blood or synovial fluid of 2 RA patients. CP-reactive memory B cells did not appear to be broadly cross-reactive, but rather had a fairly restricted epitope recognition profile. These clones were able to secrete both pro- and antiinflammatory cytokines and had a unique surface profile of costimulatory molecules and receptors, including CD40 and C5a receptor type 1, when compared to non-CP-reactive clones from the same patient. In addition, CP-reactive clones bound citrullinated protein, but not native protein, and could mobilize calcium in response to antigen binding. Conclusion CP-reactive memory B cells comprise a rare, seemingly oligoclonal population with restricted epitope specificity and distinct phenotypic and molecular characteristics suggestive of antigen-presenting cells. Cloning by genetic reprogramming opens new avenues to study the function of autoreactive memory B cells, especially in terms of antigen processing, presentation, and subsequent T cell polarization.

ObjectivesWe explored the effects of B-cell directed therapy in subjects at risk of developing autoantibodypositive rheumatoid arthritis (RA), who never experienced inflammatory arthritis before, and explored biomarkers predictive of arthritis development.MethodsIndividuals positive for both anti-citrullinated peptide antibodies and rheumatoid factor but without arthritis were included in a randomised, double-blind, placebo-controlled study to receive a single infusion of 1000 mg rituximab or placebo.ResultsEighty-one individuals received treatment and were followed up for a mean of 29.0 (0–54) months, during which 30/81 (37%) individuals developed arthritis. The observed risk of developing arthritis in the placebo-treated group was 40%, which was decreased by 55% (HR 0.45, 95% CI 0.154 to 1.322) in the rituximab-treated group at 12 months. Rituximab treatment caused a delay in arthritis development of 12 months compared with placebo treatment at the point when 25% of the subjects had developed arthritis (pConclusionsA single infusion of 1000 mg rituximab significantly delays the development of arthritis in subjects at risk of developing RA, providing evidence for the pathogenetic role of B cells in the earliest, prearthritis stage of autoantibody positive RA.

Objective: Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. Methods: IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. Results: Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. Conclusion: These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.

Objective Anti-citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen-specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity. Methods Blood samples were obtained from patients in a University of Minnesota cohort with ACPA-positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α-enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in RA patients. Citrullinated peptide tetramer-bound B cells were subjected to flow cytometric cell sorting and single-cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality. Results Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V-region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline-dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen-specific mAb with cross-reactive binding profiles also promoted arthritis in mice. Conclusion Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline-multispecific B cell clades with pathogenic potential.

Publisher's version (útgefin grein), 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 assessed serologic makers were associated with all-cause mortality. Conclusion: RF isotypes and ACPAs are associated with future CV events in patients with RA. ACPA levels and number of subspecificities seem more important than the occurrence of particular subspecificities, and these associations were not explained by a history of ever smoking., Karolinska institutet (strategic research area epidemiology), The nordic research council (nordforsk), The rheumatology research foundation (foreum), Stiftelsen för Strategisk Forskning, Vetenskapsrådet, Stockholms Läns Landsting, The swedish heart lung foundation

Background: Porphyromonas gingivalis (P.g) is unique among pathogens due to its ability to generate citrullinated proteins in an inflammatory milieu, potentially mediating the loss of immune tolerance, the production of anticitrullinated protein antibodies (ACPAs), and subsequently the development of rheumatoid arthritis (RA). Based on this hypothesis, we set out to investigate whether P.g is linked to ACPAs in a well-characterized German population. Participants and methods: A total of 600 participants (292 women and 308 men with a mean age of 67 years) of the European Prospective Investigation into Cancer and Nutrition-Potsdam study were selected in 2013, and paired saliva and serum samples were collected. Salivary P.g DNA and serum anticyclic citrullinated peptide (anti-CCP2) levels were quantified by real-time polymerase chain reaction and anti-CCP2 enzyme-linked immunosorbent assay, respectively. In selected participants, additional ACPA fine-specificities were also analysed on a custom-made multiplex peptide array. Results: Among participants with C-reactive protein greater than 3.0 mg/l, a one-unit increase in P.g DNA was associated with an almost twofold increase in anti-CCP2 levels. Moreover, participants with high P.g DNA had on average approximately 2.8-times higher anti-CCP2 levels when compared with participants with low P.g DNA, (Holm-adjusted p value = 0.01). Furthermore, citrullinated epitopes on α-enolase and vimentin were common ACPA reactivities among participants who also had high P.g DNA and elevated C-reactive protein. Conclusions: Our study suggests that in specific subgroups of individuals with systemic inflammation, higher salivary P.g DNA is associated with elevated serum ACPA. These data support a role for P.g in the development of anticitrulline immunity.

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 histone-derived 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 citrullinated 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 recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti-carbamylated and anti-citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.

Background: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), anti-acetylated (KAc), and anti-malondialdehyde acetaldehyde (MAA) modified protein antibodies, have been described. By using RA patient single-cell derived monoclonal antibodies we have previously shown that individual ACPA clones recognize small distinct citrulline-containing epitopes giving them extensive multireactivity when these epitopes are found in many peptides and proteins. Moreover, certain CCP2+ multireactive ACPA clones bind also to cabamylated and acetylated autoantigens [1].Objectives:To provide a comprehensive evaluation of serum IgG and IgA autoreactivity to different post-translational modifications in RA.Methods:We analyzed 30 different IgG and IgA AMPA reactivities to modified antigens by ELISA and autoantigen arrays, in N=1985 newly diagnosed RA patients and population controls. The study utilized both previously established (i.e IgG and IgA CCP2; IgG ACPA fine-specificities; IgG anti-Carb fibrinogen and Carb FCS; IgG and IgA Cit/Carb/KAc/Orn(Ac)-vimentin), and novel assays (e.g. IgG anti-MAA and IgG anti-acetylated histones). Association with patient characteristics such as smoking and disease activity were explored. The newly developed assays were also evaluated in SLE disease controls and CCP2+ RA-risk individuals without arthritis.Results:Carb and KAc reactivities by different assays were primarily seen in patients also positive for 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 acetylation reactivity was mainly detected in a subset of patients with Cit and Carb reactivity. Anti-acetylated histone 2B reactivity was RA-specific and associated with high anti-CCP2 IgG levels, multiple ACPA fine-specificities, and smoking. 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.Conclusion:We conclude that autoantibodies exhibiting different patterns of ACPA fine-specificities as well as Carb and KAc reactivity are present in RA and may be derived from multireactive B-cell clones. Anti-Carb and anti-KAc could be considered reactivities within the “Cit-umbrella” similar to ACPA fine-specificities, while MAA is distinctly different.References:[1]Sahlström P, Hansson M, Steen J, Amara K, Titcombe PJ, Forsström B, Stålesen R, Israelsson L, Piccoli L, Lundberg K, Klareskog L, Mueller DL, Catrina AI, Skriner K, Malmström V, Grönwall C. Different Hierarchies of Anti-Modified Protein Autoantibody Reactivities in Rheumatoid Arthritis. Arthritis Rheumatol. 2020 Oct;72(10):1643-1657. PMID: 32501655Caroline Grönwall: None declared, Lisa Liljefors: None declared, Holger Bang Employee of: Employee at ORGENTEC Diagnostika GmbH, Aase Hensvold: None declared, Monika Hansson: None declared, Linda Mathsson-Alm Employee of: Employee at Thermo Fisher Scientific, Lena Israelsson: None declared, Anna Svärd: None declared, Cyril CLAVEL: None declared, Elisabet Svenungsson: None declared, Iva Gunnarsson: None declared, Guy Serre: None declared, Saedis Saevarsdottir: None declared, Alf Kastbom: None declared, Lars Alfredsson: None declared, Vivianne Malmström: None declared, Johan Rönnelid: None declared, Anca Catrina: None declared, Karin Lundberg: None declared, Lars Klareskog: None declared

Background:The presence of anti-citrullinated protein antibodies (ACPAs) is a hallmark of rheumatoid arthritis (RA) that precede the development of the disease by years and is used for its clinical diagnosis. However, there are RA subjects that test negative for ACPA and thus the early diagnosis on these patients may be delayed. Furthermore, the presence or absence of ACPA in RA supports the hypothesis that on these two subsets of patients underlie different pathogenesis and clinical outcomes.Objectives:In this work, we searched for serum autoantibodies useful to assist the early diagnosis of ACPA-seronegative RA and its management.Methods:We profiled the serum autoantibody repertoire of 80 ACPA-seronegative and 80 ACPA-seropositive RA subjects from the Swedish population-based Epidemiological Investigation of RA (EIRA) cohort. A suspension bead array platform built on protein fragments within Human Protein Atlas and selected from an initial untargeted screening using arrays containing 2660 total antigens was employed to identify IgG and IgA serum autoantibodies. A validation phase on antigen suspension bead arrays was carried out on another set of samples from EIRA containing 386 ACPA-seropositive, 358 ACPA-seronegative and 372 randomly selected control subjects of the same age and sex. A sample-specific threshold based on 20 times the median absolute deviation plus the median of all signals was selected to determine the reactivity of samples. The Wilcoxon rank sum test and Fisher’s test were applied for the comparison of autoantibody levels and reactivity frequencies between the groups.Results:Our data revealed four antigens associated with the ACPA status (Table 1). Testis-specific Y-encoded-like protein 4 (TSPYL4) showed significantly higher IgG reactivity frequency in ACPA-seronegative subjects compared to ACPA-seropositive (8% vs. 3%; PTable 1.Scheme of the different phases of the study, the features within each phase and the results. The reactivity to four antigens allows to distinguish ACPA-seronegative (ACPA-), ACPA seropositive (ACPA+) and controls.PhasesUntargeteddiscoveryTargeteddiscoveryTargetedvalidationNumber of samples80 ACPA-80 ACPA-358 ACPA-372 Controls80 ACPA+80 ACPA+386 ACPA+Antigen arrayplatformPlanararraysSuspensionbead array 1Suspensionbead array 2Number of antigens26606227Number of candidatebiomarkers6227 4 (TSPYL4,MAP2K6,ANOS1,MURC)Conclusion:Upon further validation in other early RA sample cohorts, our data suggest the measurement of these four autoantibodies may be useful for the early diagnosis of ACPA-seronegative RA and give insight into the pathogenesis of the different RA subsets.Characters from table content including title and footnotes:Disclosure of Interests:None declared

Objective Antibodies to the citrullinated protein antigens (ACPAs) are important in the diagnosis and pathogenesis of rheumatoid arthritis (RA). However, the prevalence of ACPAs with different fine specificities in different populations is unclear. This study sought to examine the fine specificity of the antibody responses toward citrullinated proteins in RA patients from Malaysia, an area where genetic and environmental determinants of RA are different from those in more frequently studied cohorts of Caucasian subjects. Methods A multiplex analytic microarray system was used to analyze the occurrence of antibodies to 10 different citrullinated peptides (filaggrin [fil307–324], vimentin [Vim2–17, Vim60–75], fibrinogen [Fibα563–583, Fibα580–600, Fibβ36–52, Fibβ62–81a, Fibβ62–81b], enolase [Eno5–21], and type II collagen [CitCII355–378]) in serum samples from 4,089 RA patients (1,231 Malaysian and 2,858 Swedish) and 827 healthy control subjects (249 Malaysian and 578 Swedish). The positive reaction threshold for each peptide was set separately for each population based on a specificity of 98%. Results Distinct differences in the frequencies of 5 ACPA fine specificities (Vim60–75, Vim2–17, Fibβ62–81b, Eno5–21, and CitCII355–378) were found between the Malaysian and Swedish RA populations, despite a nearly identical percentage of patients in each population who were positive for anti–cyclic citrullinated peptide 2 antibodies. In Malaysian RA patients compared with Swedish RA patients, the frequencies of antibodies to Vim60–75 (54% versus 44%, corrected P [P corr] = 1.06 × 10−8) and CitCII355–378 (17% versus 13%, P corr = 0.02) were significantly higher, while the frequencies of antibodies to Vim2–17 (25% versus 32%, P corr = 1.91 × 10−4), Fibβ62–81b (15% versus 30%, P corr = 2.47 × 10−22), and Eno5–21 (23% versus 50%, P corr = 3.64 × 10−57) were significantly lower. Conclusion Serum ACPA fine specificities differ between RA patients in different populations, although the total proportions of individuals positive for ACPAs are similar. Differing patterns of ACPA fine specificity could be attributed to variations in genetic and/or environmental factors.

Background Recent studies have shown divergent associations between rheumatoid arthritis (RA)-associated autoantibodies and RA onset phenotype. Whereas some studies report more inflammation in seronegative patients [1, 2], others report that rheumatoid factor (RF) associates with high baseline disease activity [3]. Objectives To define how individual RA-associated autoantibodies associate with individual disease activity score (DAS28, DAS28CRP) components at the time of RA diagnosis. Methods Sixteen individual ACPA reactivities, anti-CCP2, IgA-, IgG- and IgM-RF were analyzed centrally in baseline sera from 1600 RA patients classified according to the 1987 ACR criteria. All antibody cut-offs were determined as the 98th percentile among controls. The results were related to CRP, ESR, number of swollen and tender joints, DAS28, DAS28CRP, global disease activity (visual analogue scale), and health assessment questionnaire obtained at baseline. Results Individually, anti-CCP2, IgA RF and IgM RF associated with low counts of swollen and tender joints, with strongest association to anti-CCP2, and with high ESR. The association to low joint counts was stronger to number of individual ACPA reactivities than to anti-CCP2 levels (table). In multiple regression adjusted for age, sex and inclusion year, ACPA number associated strictly with low joint counts whereas IgM RF level associated with increased inflammatory markers, especially ESR (table). Also among IgM RF negative patients, occurrence of ACPA associated with low swollen and tender joint counts. Contrary, among ACPA negative patients, IgM showed no association to elevated ESR. The effect of RF on ESR increased with the number of individual ACPA reactivities present, most prominently for IgM RF (figure). Conclusion In early RA, ACPA associate with low counts of affected joints and IgM RF associates with elevated ESR in an ACPA-dependent manner. The latter finding relates to in vitro studies showing enhanced inflammatory effect of ACPA immune complexes with addition of RF[4, 5]. Future RA studies relating autoantibodies to disease activity may benefit from evaluating the impact of individual antibodies as well as distinct DAS components separately. References [1] Barra L, et al. J Rheumatol 2014;41:2361. [2] Nordberg LB, et al. Ann Rheum Dis 2017;76:341. [3] Aletaha D, et al. Arthritis Res Ther 2015;17:229. [4] Laurent L, et al. Ann Rheum Dis 2015;74:1425. [5] Sokolove J, et al. Arthritis Rheumatol 2014;66:813. Disclosure of Interests Eleftheria Pertsinidou: None declared, Vivek Anand Manivel: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Lars Alfredsson: None declared, Linda Mathsson Employee of: employed by Thermo Fisher Scientific, Monika Hansson: None declared, Martin Cornillet: None declared, Guy Serre: None declared, Rikard Holmdahl: None declared, Karl Skriner: None declared, Per-Johan Jakobsson: None declared, Helga Westerlind: None declared, Johan Askling Grant/research support from: Karolinska Institutet (JA) has or has had research agreements with the following pharmaceutical companies, mainly in the context of the ATRIS national safety monitoring programme for rheumatology biologicals: Abbvie, BMS, MSD, Eli Lilly, Pfizer, Roche, Samsung Bioepis, and UCB., Consultant for: Karolinska Institutet has received remuneration for JA participating in ad boards arranged by Lilly, Novartis, and Pfizer., Saedis Saevarsdottir Employee of: Part-time employee at deCODE Genetics/Amgen Inc, working on genetic research unrelated to this project., Johan Ronnelid: None declared

Background The association and role of antibodies to citrullinated proteins (ACPA) on the risk of cardiovascular (CV) co-morbidity in patients with rheumatoid arthritis (RA) is incompletely understood. We have previously reported an association between high levels of CCP2 and risk of acute coronary syndrome (ACS) in RA1. Objectives To further investigate any association between specific ACPA or rheumatoid factor (RF) isotypes, and CV events (ACS, stroke, CV death, and major adverse CV event (MACE)). Methods 2533 patients with RA from the Swedish EIRA study were tested for ACPA specificities on a custom-made microarray chip. All antibodies (Ab) detected with a prevalence >= 10% were included in the analysis. “Ab load” was defined as the number of specificities expressed in an individual, and categorized into four groups. ACPA was also measured by a commercial anti-CCP2 assay and anti-CCP2 levels divided into 1500 arbitrary units/mL, where The data was linked to the National Patient Register and the Cause of Death register, and events of ACS (ICD10=I20.0, I21, or cause of death listed as “I21”), stroke (ICD10=I60-I64), CV death (cause of death listed as “I”), or MACE (any of the above) identified. We used Cox proportional hazard model, adjusted for sex, age and calendar period of RA diagnosis, to assess associations between ACPA/RF status and each CV outcome. Results Median age at diagnosis was 52 years, and median follow-up was 12 years. The incidences per 1000 person-years were 4.3 for ACS, 4.0 for stroke, 2.4 for CV deaths, and 9.4 for MACE. 66% were anti-CCP2 positive. This was associated with ACS, stroke and MACE. There was a trend with increasing CV risk with increasing anti-CCP2 levels (Table). For RF, the pattern was markedly different across isotypes; IgM RF associated with ACS, stroke and MACE whereas IgA RF associated to CV death (Table). 18 different ACPA specificities (Fil 307-324 (CPP-1), Vim 60-75, Vim 2-17, Fib β36-52, Fib alpha 580-600, Eno 5-21 (CEP-1), Fib α621-635, Fib α36-50, Fib β60-74, Ptm13, Ptm36, Pept Z1, Pept Z2, Pept 1, Pept 5, Bla26) had a frequency >10% and included in the analyses. Median Ab load was 6 (IQR:2). There was a significant p-value for trend between Ab load and CV risk for stroke and MACE. Fib β36-52 was the only Ab associated with all 4 outcomes, and, together with CPP3 and ptm13, the only ACPAs associated to ACS. No association to ACS remained when all Abs were jointly included in the model. For stroke, 13 of the 18 Abs associated with risk, but only Fib β60-74 remained (HR 1.91 (95% CI 1.07-3.42)) in the full model. For CV death and MACE, 14 Abs were associated, but no associations remained in the full model. Conclusion In patients with RA, RF and ACPA are linked to CV risk. For RF, CV risks differ with isotype. For ACPAs, very high levels, and the number of individual ACPAs, are linked to CV risk, suggesting that Ab load may be more important than individual ACPAs. Reference [1] Mantel A, et al, 2015. PMID 26138387 Disclosure of Interests Helga Westerlind: None declared, Johan Ronnelid: None declared, Monika Hansson: None declared, Lars Alfredsson: None declared, Linda Mathsson Employee of: employed by Thermo Fisher Scientific, Guy Serre: None declared, Martin Cornillet: None declared, Rikard Holmdahl: None declared, Per-Johan Jakobsson: None declared, Karl Skriner: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Saedis Saevarsdottir Employee of: Part-time employee at deCODE Genetics/Amgen Inc, working on genetic research unrelated to this project., Johan Askling Grant/research support from: Karolinska Institutet (JA) has or has had research agreements with the following pharmaceutical companies, mainly in the context of the ATRIS national safety monitoring programme for rheumatology biologicals: Abbvie, BMS, MSD, Eli Lilly, Pfizer, Roche, Samsung Bioepis, and UCB., Consultant for: Karolinska Institutet has received remuneration for JA participating in ad boards arranged by Lilly, Novartis, and Pfizer.

Background: Retrospective studies have shown that anti-citrullinated protein antibodies (ACPA) are a risk factor for the development of clinical arthritis. Objectives: We aimed to investigate in a prospective setting if ACPA and other biomarkers could predict development of ultrasound detected arthritis. Methods: Subjects with positive ACPA-test referred from primary care to the Rheumatology clinic that lacked arthritis in hands, feet and any other symptomatic joints by clinical and ultrasound examination (according to EULAR-OMERACT definition), were recruited into the Risk-RA research program during 2015-2016 and were followed up to the end of 2017. At inclusion a detailed clinical examination was performed and blood samples were analyzed for 13 specific ACPA reactivities (using a custom made peptide microarray) as well as 92 inflammation-associated protein biomarkers (using a multiplex immunoassay with Olink proximity extension technology). Presence of HLA-SE was analyzed using DR low-resolution kit. Univariate and multivariate analysis were used to investigate the association between clinical and laboratory parameters and development of ultrasound detected arthritis adjusting for the follow-up time. Results: 42% (27 out of 65) of the Risk RA subjects developed ultrasound detectable arthritis during a median follow up of 8 months. The remaining 58% (38 out of 65) were followed for a median of 25 months (range 12-44) without any signs of ultrasound detectable arthritis. Subjects developing arthritis had higher prevalence of HLA-SE (89% vs 56%) and increased occurrence of ultrasound detected tenosynovitis (44% vs 5%), as compared to those not developing arthritis. ACPA reactivities to citrullinated vimentin peptides (cit vim 2-17: 22% vs 6%; and cit vim 60-75: 70% vs 43%) and citrullinated histone peptides (cit H4 31-50: 89% vs 49%; and cit H3 21-44: 48% vs 23%) were a more common occurrence in subjects developing ultrasound detectable arthritis. Backward selection in a Cox regression model showed that ultrasound detectable arthritis could be predicted in a model including HLA-SE, tenosynovitis and ACPA reactivity to cit H4 31-50. Hazard ratio (HR) for arthritis development were 3.4 (95% CI 1.0-12, p 0.06) for HLA-SE carriers, 2.9 (95% CI 1.3-6.7, p 0.01) for tenosynovitis and 4.1 (95% CI 1.2-14, p 0.02) for Anti-citrullinated H4 31-50 positivity. Only modest differences were observed for few of the tested inflammatory markers in those developing as compared to those not developing ultrasound detectable arthritis: Interleukin- 6 (3.9 vs 3.3 AU/ML), Programmed death-ligand 1 (4.9 vs 5.2 AU/ML) and Chemokine (C-X-C motif) ligand 6 (9.2 vs 9.5 AU/ML). Conclusion: Certain ACPA fine specificities, HLA-SE and tenosynovitis predict the development of ultrasound detectable arthritis in seropositive individuals with musculoskeletal symptom who are at risk for RA. Disclosure of Interests: Aase Hensvold: None declared, Yogan Kisten: None declared, Alexandra Circiumaru: None declared, Monika Hansson: None declared, Meng Sun Grant/research support from: Yes, but not for presented project., Guozhong Fei: None declared, Nancy Vivar: None declared, Erik Af Klint: None declared, Hamed Rezaei: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Aleksandra Antovic: None declared, Anca Catrina Grant/research support from: Yes, but not for the presented study.

Background IgM RF and anti-CCP2 have been shown to associate with young age at RA diagnosis [1, 2]. By contrast, little is known about the association between other RF isotypes and age at onset, or sex. Objectives To examine the association between individual RA autoantibodies, sex and age at RA onset. Methods Anti-CCP2, IgA-, IgG- and IgM-RF were analysed centrally in baseline sera from 1600 Epidemiological Investigations in RA (EIRA) patients aged 18-70 years and classified according to the 1987 ACR criteria within one year of first symptoms. Cut-offs for RF isotypes were determined at the 98th percentile based on the EIRA RA-free controls, close to the 98.4% anti-CCP2 specificity. Results Anti-CCP2, IgA-, IgG- and IgM RF were found in 1020 (64%), 692 (43%), 529 (33%), and 916 (57%), of the patients, respectively. Mean (median) age at diagnosis among men was 54 (57) year, higher than 51 (54) years among women (p Conclusion On average, IgA RF positive (vs. negative) RA patients are almost four years older at the time of diagnosis; the opposite was found for anti-CCP2 and IgM RF. Whereas sex-differences explain the age-association for IgM RF, this is not the case for anti-CCP2 and for IgA RF. References [1] Diaz FJ, et al. Joint Bone Spine 2011;78:175. [2] Jacobsen S. Clin Rheumatol 2004;23:121. Disclosure of Interests Eleftheria Pertsinidou: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Lars Alfredsson: None declared, Linda Mathsson Employee of: employed by Thermo Fisher Scientific, Monika Hansson: None declared, Helga Westerlind: None declared, Johan Askling Grant/research support from: Karolinska Institutet (JA) has or has had research agreements with the following pharmaceutical companies, mainly in the context of the ATRIS national safety monitoring programme for rheumatology biologicals: Abbvie, BMS, MSD, Eli Lilly, Pfizer, Roche, Samsung Bioepis, and UCB., Consultant for: Karolinska Institutet has received remuneration for JA participating in ad boards arranged by Lilly, Novartis, and Pfizer., Saedis Saevarsdottir Employee of: Part-time employee at deCODE Genetics/Amgen Inc, working on genetic research unrelated to this project., Johan Ronnelid: none declared

Career situation of first and presenting author Student for a master or a PhD. Introduction The presence of anti-citrullinated protein antibodies (ACPAs) is a hallmark of rheumatoid arthritis (RA). ACPAs specifically recognize citrullinated epitopes, a result of a post-translational modification catalyzed by peptidyl arginine deiminases (PAD). Based on the unique feature of the periodontal bacteria Porphyromonas gingivalis (P.gingivalis) to express P.PAD it has been suggested that ACPA-positive RA may be precipitated in the gum mucosa. Objectives To address this hypothesis our aims were to investigate the antibody response against a citrullinated P.PAD peptide (CPP3) in patients with RA, chronic periodontitis (PD) and in controls. In addition, we generated monoclonal antibodies (mAbs) from gingival tissue B cells of RA patient aiming to investigate whether citrulline-specific B cells may reside in the gingiva. Methods Gingival tissue-derived single CD19+ B cells from an ACPA-positive RA patient with PD were sorted by flow cytometry. Immunoglobulin variable region genes were sequenced and expressed to generate recombinant mAbs. CPP3-reactivity was analysed by ELISA in serum samples from 66 PD patients, 63 periodontally healthy controls (non-PD), 200 RA patients, and 120 non-RA controls, as well as in 55 mAbs. Differences in antibody levels were examined using Mann-Whitney U test for independent groups. Results Anti-CPP3 antibody levels were low in non-PD controls, while 65% of PD patients showed elevated levels (p Conclusions This study shows that a substantial proportion of systemically healthy individuals possess ACPAs directed against P.gingivalis, and these ACPAs also bind epitopes on human proteins. Based on our data, we propose that the ACPA response may be triggered by P.gingivalis via an antibody response against CPP3, which cross-reacts with human citrullinated proteins by mechanisms of molecular mimicry. Disclosure of Interest None declared.

Career situation of first and presenting author Post-doctoral fellow Introduction Anti-citrullinated protein antibodies (ACPA) are predictive markers with pathological effects in rheumatoid arthritis (RA). Previous prospective studies have used a clinical definition of arthritis. Thus, we aimed to investigate risk factors of developing arthritis in ACPA-positive subjects with musculoskeletal complaints who did not have any of clinical and ultrasound signs of arthritis. Methods Subjects with positive ACPA-test referred from primary care to rheumatology clinic, lacking arthritis in hands and feet by clinical and ultrasound examination (according to EULAR-OMERACT synovitis definition), were recruited into the Risk-RA research program. Patient included between years 2015–2016 with clinical data up to 2017 were analysed. Blood samples from inclusion were analysed for 13 specific ACPA reactivities using a custom made ImmunoCAP ISAC microarray. Presences of HLA-SE risk gene were analysed using DR low-resolution kit. Results 41% (27 out of 66) of the Risk RA subjects developed arthritis during a median follow up of 8 months. The rest was followed 25 months in median without any signs of arthritis. Subjects developing arthritis tended to have a higher concentration of anti-CCP, more tender joints and rheumatoid factor positivity at inclusion compared to those not developing arthritis. The number of ACPA-reactivities (mean 6 vs 3), the presence of HLA-SE (89% vs 56%) and the occurrence of ultrasound detected tenosynovitis (44% vs 5%) at inclusion were significantly increased in subjects developing arthritis compared to those not developing arthritis. Univariate cox proportional hazards regression showed a hazard ratio (HR) for arthritis development of 1.1 for every increase in number of ACPA reactivities (95% CI 0.99 to 1.2, p 0.07); HR: 4.4 (95% CI 2.0 to 9.5, p 0.0002) for tenosynovitis and for HR: 4.9 (95% CI 1.5 to 16, p 0.01) for HLA-SE carriers. All subjects with tenosynovitis (n=14) prior to arthritis development were carriers of HLA-SE, except for one subject but similar to the majority this HLA-SE no-carrier also progressed to arthritis. Conclusions Subjects with ACPA-positive musculoskeletal complaints lacking any clinical and ultrasound signs of arthritis are at high risk of developing arthritis, especially carriers of HLA-SE with tenosynovitis. The role of inflammatory spreading from tendons (synovial sheath) to synovial tissue within joints need to be further investigated. Disclosure of Interest None declared

B cells are postulated to be central in seropositive rheumatoid arthritis (RA). Here, we use exploratory mass cytometry (n = 23) and next-generation sequencing (n = 19) to study B-cell repertoire shifts in RA patients. Expression of several B-cell markers were significantly different in ACPA(+) RA compared to healthy controls, including an increase in HLA-DR across subsets, CD22 in clusters of IgM(+) B cells and CD11c in IgA(+) memory. Moreover, both IgA(+) and IgG(+) double negative (IgD(-) CD27(-)) CD11c(+) B cells were increased in ACPA+ RA, and there was a trend for elevation in a CXCR5/CCR6(high) transitional B-cell cluster. In the RA BCR repertoire, there were significant differences in subclass distribution and, notably, the frequency of VH with low somatic hypermutation (SHM) was strikingly higher, especially in IgG1 (p amp;lt; 0.0001). Furthermore, both ACPA(+) and ACPA(-) RA patients had significantly higher total serum IgA and IgM compared to controls, based on serology of larger cohorts (n = 3494 IgA; n = 397 IgM). The observed elevated Ig-levels, distortion in IgM(+) B cells, increase in double negative B cells, change in B-cell markers, and elevation of unmutated IgG(+) B cells suggests defects in B-cell tolerance in RA. This may represent an underlying cause of increased polyreactivity and autoimmunity in RA. Funding Agencies|UCB PharmaUCB Pharma SA; Swedish Research CouncilSwedish Research Council; Swedish Rheumatism Association, King Gustaf Vs 80-year Foundation; Ake Wibergs stiftelse; FOREUM, Foundation for Research in Rheumatology; European Research Council (ERC) under the European UnionEuropean Research Council (ERC); EU/EFPIA Innovative Medicines Initiative (IMI) [BTCure 115142, RTCure 777357]; Karolinska InstituteKarolinska Institutet

Background:Anti-citrullinated protein antibodies (ACPA) are characteristic markers for rheumatoid arthritis (RA), developing years before disease onset. Early clinical and biological biomarkers could provide useful information on the onset of RA in predisposed individuals.Objectives:The aim of the study was to investigate whether ACPA along with inflammatory markers and musculoskeletal ultrasound changes could predict arthritis development in individuals at risk for RA.Methods:ACPA-positive individuals with musculoskeletal complaints were referred from primary care to a rheumatology clinic, recruited in the Risk-RA research program and followed-up for up to 3 years, between April 2014 and October 2019. All individuals lacked arthritis both at clinical examination by a trained rheumatologist and ultrasound assessment of hands and feet and any other symptomatic joints (according to EULAR-OMERACT definition). Blood samples were collected at inclusion and were analyzed for 15 ACPA fine specificities (by custom made peptide array), 92 inflammation-associated protein biomarkers (by multiplex immunoassay with Olink extension technology) and HLA-SE (DR low resolution kit). Statistical analysis used univariate and multivariate models with backwards selection and cox regression.Results:268 individuals with a median age of 48 (36-58) were recruited, out of which 212 (79%) were females. 75 (28%) developed arthritis within 11 months of follow-up while the median follow-up for those not developing arthritis was 21 months (14-28). Increased ACPA levels, shorter symptom duration and RF positivity were the main differences between individuals developing arthritis and those who did not. In univariate models, the presence of HLA-SE, specific ACPA reactivities, certain inflammatory markers and ultrasound-detected tenosynovitis were associated with arthritis development. In multivariate analysis the presence of anti-cit-fillagrin (HR 2.1 (95% CI 1.2-3.7, p 0.01), IL6 levels (HR 1.4 (95% CI 1.2-1.7, p 0.0001) and tenosynovitis (HR 2.9 (95% CI 1.7-5.0, p 0.0001) remained significant predictors for arthritis onset.Conclusion:Certain ACPA reactivities together with inflammatory markers and ultrasound-detected tenosynovitis predict arthritis development in predisposed individuals for developing RA.Disclosure of Interests:None declared

Background Anti-citrullinated protein antibody (ACPA) reactivities precede clinical onset of rheumatoid arthritis (RA), and it has been suggested that ACPA reactivities towards distinct target proteins may be associated with differences in RA phenotypes. We aimed to assess the prevalence of baseline ACPA reactivities in an inception cohort of patients with early RA, and to investigate their associations with disease activity, treatment response, ultrasound findings and radiographic damage. Methods Disease-modifying antirheumatic drug (DMARD)-naïve patients with early RA, classified according to the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria, were included in the ARCTIC trial and assessed in the present analysis. During follow up, patients were monitored frequently and treatment was adjusted according to a predetermined protocol, starting with methotrexate monotherapy with prednisolone bridging. Analysis of 16 different ACPA reactivities targeting citrullinated peptides from fibrinogen, alpha-1 enolase, vimentin, filaggrin and histone was performed using a multiplex chip-based assay. Samples from 0, 3, 12 and 24 months were analysed. Controls were blood donors with similar characteristics to the patients (age, gender, smoking status). Results A total of 217 patients and 94 controls were included. Median [25, 75 percentile] number of ACPA reactivities in all patients was 9 [4, 12], and were most prevalent in anti-cyclic citrullinated peptide /rheumatoid factor-positive patients 10 [7, 12]. Disease activity measures and ultrasound scores at baseline were lower in ACPA reactivity-positive compared to ACPA reactivity-negative patients. ACPA reactivity levels decreased after 3 months of DMARD treatment, most pronounced for fibrinogenβ 60–74 to 62% of baseline antibody level, with least change in filaggrin 307–324 to 81% of baseline antibody level, both p

Introduction Anti-citrullinated protein antibodies (ACPA) – a hallmark of rheumatoid arthritis – antedate joint inflammation. Based on the epidemiological association between RA and periodontal disease (PD),1 it has been suggested that break of tolerance to citrullinated proteins may occur in the gum mucosa. This hypothesis is primarily based on the unique feature of Porphyromonas gingivalis (P.g) – a keystone pathogen in PD – to express a bacterial version of the peptidyl arginine deiminase enzyme (denoted P.PAD), capable of autocitrullination.2 Objectives In the present study, we have investigated the antibody response against CPP3, a citrullinated peptide derived from P.PAD, in order to address the hypothesis that P.g may drive ACPA-production. Methods This study included 2,859 RA cases and 4864 controls from the Epidemiological Investigation of RA (EIRA) cohort; 65 PD patients and 59 periodontally healthy individuals; and 218 monoclonal antibodies, derived from RA B cells. Reactivity with citrullinated P.g and human antigens was assayed by ELISA and/or multiplex. Associations with genetic risk factors and smoking were determined by logistic regression. Monoclonal antibody mutations were analysed by IgBLAST comparison. Results Anti-CPP3 IgG was detected in 11% of RA, 10% of PD and Conclusions Based on these data, we propose that P.g infection triggers an antibody response to CPP3, which cross-reacts with citrullinated human proteins by mechanisms of molecular mimicry. Future studies should address whether anti-CPP3 IgG could serve as a biomarker to identify individuals with PD at increased risk for RA. References . Fuggle NR, Smith TO, et al. Hand to mouth: A systematic review and meta-analysis of the association between rheumatoid arthritis and periodontitis. Frontiers in Immunology2016;7:80. . Quirke AM, et al. Heightened immune response to autocitrullinated porphyromonas gingivalis peptidylarginine deiminase: A potential mechanism for breaching immunologic tolerance in rheumatoid arthritis. Annals of the Rheumatic Diseases2014;73(1):263–269. Disclosure of interest None declared

IntroductionThe second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients.MethodsWe investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array.ResultsThe prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset.ConclusionsMultiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement.

Background We have described a planar microarray for the determination of antibodies against individual citrullinated peptides (ACPA; Hansson M et al. Arthritis Res Ther 2012;14:R201). We have also developed a method for the quantification of autoantibodies in immune complexes (IC; Sohrabian et al. Ann Rheum Dis 2015;74(Suppl 1):A74). Here we have combined these techniques to determine ACPA profiles in RA IC. Objectives To investigate if measurement of specific ACPA in synovial fluids (SF) and in IC from sera and SF can provide more prognostic information than conventional measurement of total ACPA and rheumatoid factor (RF) in serum. Methods Seventy-seven RA patients with knee synovitis were treated with intra-articular triamcinolone hexacetonide, and followed until relapse. DAS28 and radiographic joint damage according to Larson-Dale were recorded. Anti-CCP2, IgM and IgA RF and circulating C1q-binding immune complexes (CIC) were determined in paired sera and SF. IC were purified from sera and SF by binding to C1q-coated beads, and thereafter eluted with a procedure developed in our laboratory. Antibodies against 19 citrullinated peptides were investigated with a custom-made microarray assay based on the ImmunoCAP ISAC system (Phadia AB, Sweden) in sera and SF as well as in IC from sera and SF. The target peptides were filaggrin 307–324 (CCP1), vimentin peptides 60–75 and 2–17, fibrinogen peptides α36–50,, α563–583, α580–600, α621–635, β36–52, β60–74, β62–81 (with citrullination in positions 72 and 74, respectively), α-enolase 5–21 (CEP-1), peptides 1, 5, Z1, Z2 and Bla26 from hnRNP, and histone 4 peptides 14–34 and 31–50. Cutoffs were established in relation to healthy controls. Backward stepwise regression was used to investigate what factors determined Larsen Dale index, DAS28, and duration of remission after steroid treatment. Independent factors were anti-CCP2, IgM RF, IgA RF, CIC, number of ACPA peptide reactivities, and number of ACPA reactivities in IC, all measured both in serum and paired SF. Results A considerable proportion of anti-CCP2 negative patients had multiple ACPA in SF, and in IC fractions. High DAS28 associated with reactivity against 7/19 peptides in serum and 9/19 in SF. High Larsen score associated with number of specific ACPA in SF IC and with CIC in SF. DAS28 levels associated with IgM RF in SF and with CIC in SF, and steroid response duration with number of specific ACPA in serum and in SF IC. Conclusions We found ACPA in SF, and especially in the IC fraction of SF, in a sizeable fraction of anti-CCP2 negative patients. Number of peptide-specific ACPA (but not anti-CCP2 levels) associated with radiological destruction and length of remission after intra-articular steroid therapy. Our data do not support a role for any unique ACPA specificity in RA pathogenesis. Instead, the number of individual ACPA specificities may be important. Disclosure of Interest None declared

Background The present study was undertaken to investigate novel anti-hnRNPs autoantibodies in rheumatic diseases. Objectives Novel mutated hnRNP A3 was cloned out of RA synovial tissue linking it directly to Warburg effect and lactate production in RA. Increased lactate production in RA synovia and tumors is klinked to alternative splicing process from PKM1 to PKM2 hnRNPs dependent. Methods After immunobloting and 2D-gel-eletrophoresis of a semipurified hnRNP fraction two protein spots were sequenced and identified to be highly similar to hnRNPA3. The hnRNP A3 variants were cloned from RA synovial tissue, which identified the isoforms found on protein level. 3700 RA sera were screened for the presence of mutated anti-hnRNP A3 autoantibodies using recombinant proteins and peptides thereof. Binding of RNA to hnRNP A3 (MA3) and mutated citrullinated A3 peptides (MCA3) the epitope recognition was investigated. Expression of hnRNP A3 in synovial tissue was analysed by Results Autoantibodies to MA3 protein were detected in 13% of RA (n=215) patients, in 9% SLE (n=154), in 27% of MCTD patients (n=44/10) and in less than 5% of 129 patients with other rheumatic disorders but not at all in healthy controls on immunoblot. When using renaturated MA3 on ELISA 22% of RA patients were detected and 87% of these patients had erosive arthritis. Same modification as in cancer cells were identified in synovial tissue and verified by MS and DNA sequencing. Using 2–3 citrullinated MCA3 peptides up to 81% of patients (n=150) with established and 67% (n=2926) of patients with an early RA with a specificity of 97% were detected. In early RA 27% and 25% in established RA of CCP2 negative and 93% of CCP2 positive patients were identified. By combining with the already established CCP2 and the new MCA3, 72% of early patients are positive. MCA3 autoantibodies predominantly occur (p Conclusions Mutated hnRNP-A3 is as a novel Toll7/9 dependent autoantigen in systemic rheumatic diseases. These mutated proteins are major components of RNA and DNA containing alternative splicing complexes leading to the Warburg effect and and autoantibodies predominantly occurre in an erosive, and severe courses of RA. Disclosure of Interest None declared

Background An increase in the number of anti-citrullinated protein antibodies (ACPA) reactivities precede RA onset, and may be involved in the pathogenesis of the disease. The presence of ACPA is associated with radiographic progression in RA, and it has been suggested that ACPAs with different reactivities may be associated with different phenotypes of RA. Objectives To assess the prevalence of baseline ACPA reactivities in an inception cohort of early RA patients, including subgroups based on anti-CCP/RF status, and to compare the findings to healthy controls. Methods 217 DMARD-naive early RA patients from the ARCTIC trial (1) were analysed. Radiographs were scored according to van der Heijde Sharp (vdHS) score. Anti-CCP status was analysed by FEIA (pos. if ≥10 IU/mL) and RF by ELISA (pos. if ≥25 IU/mL). ACPA titres (AU/ml) were considered pos. if above the 98-perc. of values in 619 non-RA subjects. Analysis of 13 ACPA reactivities targeting citrullinated peptides from fibrinogen, alpha-1 enolase, vimentin, fillagrin and histone was performed at baseline in patients and 94 controls (blood donors matched for age/gender/smoking), using a multiplex chip-based assay (2). Results Baseline characteristics are presented in the table. The figure shows the prevalence of ACPA reactivities in the subgroups, with median [IQR] number of antibody reactivities in all patients 7 [3,10], compared to 0 [0,0] in controls (p Conclusions Prevalence of ACPA reactivities differed in subgroups of DMARD-naive early RA patients according to anti-CCP and RF status. All RA subgroups, including RF- and anti-CCP- patients, had higher prevalence of ACPA reactivities compared to healthy controls. References Haavardsholm et al BMJ 2016. Hansson et al Arthr Res Ther 2012. Disclosure of Interest M. Jonsson: None declared, A. Hensvold: None declared, M. Hansson: None declared, L. Mathsson-Alm Employee of: Thermo Fisher Scientific, A.-B. Aga: None declared, J. Sexton: None declared, B.-T. Fevang Grant/research support from: Novartis, S. Lillegraven: None declared, A. Catrina: None declared, E. Haavardsholm Grant/research support from: AbbVie, MSD, Pfizer, Roche, UCB

Acpa against different citrullinated peptides identify specific phenotypes of rheumatoid arthritis

Background There is today a paucity of prospective studies to describe the natural longitudinal history of anti-ccp positive individuals developing RA or not developing RA. Further no study to investigate the detailed ACPA reactivities in such a setting is currently available. Methods Individuals at risk of developing RA were included in a cohort at Karolinska University Hospital, Stockholm. Examinations of peripheral joints was repeated at one year follow-up visit or at any time the patients experienced worsening of their symptoms. Peripheral blood samples were available at inclusion (n=70). Serum was run on a microarray based on the ImmuoCAP ISAC system testing for ACPA reactivities toward 13 different citrullinated peptides (fillagrin, fibrinogen, alpha-enolase, vimentin, histone) (1). Results Individuals referred from primary care with musculoskeletal complaints and positive anti-ccp test were systematically investigated as part of routine care at our rheumatology clinic. Individuals lacking self-reported history of suspect arthritis, clinical arthritis according to rheumatologist and signs of synovitis on ultrasound examination were included in a clinical Risk-RA program with life-style coaching and personalized information on the risk of developing RA. Seventy individuals, with a mean age of 48 years (SD 15) and 86% females, were included in the program. Twenty (29%) individuals developed arthritis during a medium follow up time of 7 months (range 1–25 months). Number of ACPA reactivities at baseline was significant higher among those developing (in mean 6 reactivities) as compared to those not developing arthritis (in mean 4 reactivities). A increased proportion of individuals were showing reactivity towards citrullinated (cit) vimentin (vim) 60–75, fibrinogen (fib) 573 and enolase (eno) (CEP-1) among those developing arthritis (80% for anti-cit-vim 45% for anti-cit-fib and 60% for anti-cit eno) as compared to those not developing arthritis (41% for anti-cit-vim, 30% for anti-cit-fib and 52% for anti-cit-eno). Increased level of anti-cit-vim and anti-cit-eno antibodies was also observed at inclusion for those individuals developing arthritis as compared to those not developing arthritis. Conclusions We describe here the pattern of ACPA reactivities in anti-CCP positive individuals with non-specific musculoskeletal symptoms at risk of developing RA and without clinical and ultrasonograph signs of synovitis and report that 30% of these patients will develop arthritis during a short follow-up. Number, frequency and titers of specific ACPA reactivities appear to be enriched already at inclusion among those patients that developed arthritis during follow-up. References Hansson et al Arthritis Res Ther 2012. Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is characterised by autoantibodies to citrullinated proteins (ACPA). Smoking and specific HLA alleles are well-established risk factors for ACPA+RA, and more recently Porphyromonas gingivalis, a major cause of periodontitis (PD), has been linked to ACPA+RA. Our ambition with this study is to clone ACPA-specific B-cells from gingival tissue of patients suffering from both PD and RA, in order to demonstrate that citrulline-specific B-cells, previously only detected in RA joints and circulation, may also reside in gingival tissue. Materials and methods Gingival tissue-derived single CD19+ B-cells from an ACPA+RA patient with PD were sorted by flow cytometry. Immunoglobulin (Ig) variable region genes were sequenced and expressed to generate recombinant monoclonal antibodies (mAbs). These mAbs will subsequently be screened for reactivity towards citrullinated antigens by ELISA and an antigen multiplex assay. Results We successfully isolated 480 CD19+ B-cells from the gingival tissue, and to this date, we have analysed 110 variable heavy chain Ig genes (IGHV). Ig gene sequences analysis demonstrated that the B-cell repertoire was predominantly polyclonal, although two clonally related B-cell populations (approximately 2%) were detected. Compared to healthy controls, RA/PD B-cells showed decrease in VH3 and JH5 genes, and increase in VH4, JH4 and JH6. By individual VH gene segments, IGHV4–31 was overrepresented compared to controls (p Alignment of VH sequences to their closest germline counterparts revealed that RA/PD B-cells exhibited extensive mutations in the IGH CDR regions and higher levels of somatic mutations in the V gene segments compared to controls (p Conclusions We have been able to successfully isolate and clone a number of gingival-tissue-derived B-cells. Based on the hypothesis that the ACPA-response may be initiated at mucosal surfaces such as gingival tissue, we now have the tools available to more directly address this etiological question.

MONOCLONAL ACPA ANTIBODIES RECOGNISING A COMMON CITRULLINE MOTIF ARE MAINLY DEPENDENT ON LIGHT CHAIN HYPERMUTATIONS FOR ANTIGEN RECOGNITION

Objective The presence of antibodies against cyclic citrullinated peptides has been demonstrated to precede the onset of symptoms of rheumatoid arthritis (RA) by several years. The aim of this stud ...

Objective To investigate the role of genetic and environmental factors in the development of anticitrullinated protein antibodies (ACPA) and ACPA-positive rheumatoid arthritis (RA) in a twin cohort. Methods A total of 12 590 twins were analysed for the presence of ACPAs (CCP2 ELISA), HLA-DRB1 shared epitope (SE) gene alleles, and exposure to smoking. Twins with established RA were identified in national public care registers. Antibody reactivities against citrullinated and native forms of α-enolase, vimentin, fibrinogen and type II collagen peptides were tested by ELISA in anti-CCP2-positive subjects and their cotwins. Structural equation models and ORs for the development of ACPA and ACPA-positive RA were computed for smokers and SE carriers. Results A total of 2.8% (350/12 590) of the twins were ACPA positive, and 1.0% (124/12 590) had ACPA-positive RA. Most of the variability in the ACPA status was accounted for by non-shared environmental or stochastic factors (78%, 95% CI 55% to 100%) rather than shared environmental and genetic factors. Analysis of specific risk factors revealed an association between smoking and SE and the presence of ACPAs. Twins with ACPA-positive RA were more frequently SE positive than twins with ACPAs without RA. Reactivities against multiple citrullinated peptides were present in most twins with ACPA-positive RA but in fewer twins with ACPAs without RA. Conclusions Environment, lifestyle and stochastic factors may be more important than genetics in determining which individuals develop ACPAs. Genetic factors (particularly SE) may have a relatively larger role in determining which ACPA-positive individuals will ultimately develop arthritis.

Background: Antibodies against citrullinated proteins (ACPA) are common in patients with rheumatoid arthritis (RA). ACPA can appear before disease onset and target many self-antigens. Citrullinated fibrin/fibrinogen represents a classical ACPA target antigen, and mass spectrometry of RA synovial fluid reveals elevated citrullinated (cit) fibrinogen (Fib) peptides compared to non-RA controls. We investigated the extent to which these less-studied peptides represent autoantibody targets and sought to visualize the corresponding cit-Fib-reactive B cells in RA patients. Methods: An in-house ELISA was established against four cit-Fib alpha-subunit peptides (cit-Fib alpha-35; cit-Fib alpha-216,218; cit-Fib alpha-263,271 and cit-Fib alpha-425,426) and serum from patients with established RA (n = 347) and disease controls with psoriatic arthritis (PsA) or ankylosing spondylitis (AS) (n = 236) were analyzed. RA patients were genotyped for HLA-DR alleles, PTPN22 R620W and screened for anti-CCP2 and cit-Fib protein antibodies. The cit-Fib peptides were also used to assemble antigen tetramers to identify cit-Fib-reactive B cells in peripheral blood by flow cytometry. Results: The frequencies of autoantibodies against different cit-Fib epitopes in RA patients compared to PsA/AS patients were: cit-Fib alpha-35 (RA 20%, vs PsA/AS 1%); cit-Fib alpha-216,218 (13% vs 0.5%); cit-Fib alpha-263,271 (21% vs 0.5%) and cit-Fib alpha-425,426 (17% vs 1%). The presence of autoantibodies against these peptides was associated with presence of anti-CCP2 and anti-cit-Fib protein antibodies. No association was found between HLA-DR shared epitope and antibodies to the different cit-Fib peptides. However, association was observed between the PTPN22 risk allele and positivity to cit-Fib alpha-35 and cit-Fib alpha-263,271. B cells carrying surface Ig reactive to these cit-Fib peptides were found in RA peripheral blood and these tend to be more common in PTPN22 risk allele carriers. Conclusions: Our data show that several cit-Fib peptides are targeted by autoantibodies in RA, but not in PsA/AS, implicating that these are not due to arthritis but more specific for RA etiology. The RA-associated anti-cit protein response is broad with many parallel immune responses. The association between cit-Fib autoantibodies and the PTPN22 R620W risk allele supports the hypothesis of altered B cell regulation, such as autoreactive B cells evading tolerance checkpoints.

Background The presence of rheumatoid factor (RF), anti-carbamylated protein antibodies (anti-CarP) and antibodies against citrullinated protein and peptides (ACPA) precedes the onset of symptoms of rheumatoid arthritis (RA) by several years. Relationships between the development of these antibodies are not obvious. Methods Three isotypes [immunoglobulin A (IgA), IgG and IgM) of RF were analysed in 321 pre-symptomatic individuals who provided 598 samples collected a median of 6.2 (interquartile range 7.2) years before the onset of symptoms, and in 492 population control subjects. All samples were donated to the Biobank of Northern Sweden. RF isotypes were analysed using the EliA system (Phadia GmbH, Freiburg, Germany) with 96 % specificity according to receiver operating characteristic curves. Ten ACPA specificities were analysed using the ImmunoCAP ISAC system, and anti-CCP2 and anti-CarP antibodies were evaluated using enzyme-linked immunosorbent assays. Results The frequencies of RF isotypes in pre-symptomatic individuals were significantly increased compared with control subjects (p

Overexpression of the human epidermal growth factor receptor type 2 (HER2) in breast carcinomas predicts response to trastuzumab therapy. Affibody molecules based on a nonimmunoglobulin scaffold have demonstrated a high potential for in vivo molecular imaging of HER2-expressing tumors. The reengineering of the molecular scaffold has led to a second generation of optimized Affibody molecules that have a surface distinctly different from the parental protein domain from staphylococcal protein A. Compared with the parental molecule, the new tracer showed a further increased melting point, stability, and overall hydrophilicity and was more amenable to chemical peptide synthesis. The goal of this study was to assess the potential effects of this extensive reengineering on HER2 targeting, using ABY-025, a DOTA-conjugated variant of the novel tracer. Methods:111In-ABY-025 was compared with previously evaluated parent HER2-binding Affibody tracers in vitro and in vivo. The in vivo behavior was further evaluated in mice bearing SKOV-3 xenografts, rats, and cynomolgus macaques (Macaca fascicularis). Results:111In-ABY-025 bound specifically to HER2 in vitro and in vivo. Direct comparison with the previous generation of HER2-binding tracers showed that ABY-025 retained excellent targeting properties. Rapid blood clearance was shown in mice, rats, and macaques. A highly specific tumor uptake of 16.7 ± 2.5 percentage injected activity per gram of tissue was seen at 4 h after injection. The tumor-to-blood ratio was 6.3 at 0.5 h and 88 at 4 h and increased up to 3 d after injection. γ-camera imaging of tumors was already possible at 0.5 h after injection. Furthermore, the repeated intravenous administration of ABY-025 did not induce antibody formation in rats. Conclusion: The biodistribution of 111In-ABY-025 was in remarkably good agreement with the parent tracers, despite profound reengineering of the nonbinding surface. The molecule displayed rapid blood clearance in all species investigated and excellent targeting capacity in tumor-bearing mice, leading to high tumor-to-organ-ratios and high-contrast imaging shortly after injection.

Introduction It has previously been shown that an increased number of antibodies against citrullinated peptides/proteins (ACPA) predate the onset of rheumatoid arthritis (RA). Over time antibody positivity expands, involving more specific responses when approaching the onset of symptoms. We investigated the impact of human leukocyte antigen-shared epitope (HLA-SE) alleles and smoking on the development of ACPA, as well as in combination with ACPA during the state of quiescent autoimmunity (before the onset of symptoms), on the development of RA. Methods Blood samples donated to the Medical Biobank of Northern Sweden from individuals prior to the onset of symptoms of RA (n = 370) and after onset (n = 203) and from population-based controls (n = 585) were used. Antibodies against 10 citrullinated peptides, fibrinogen (Fibα561-583, α580-600, ß62-81a, ß62-81b, ß36-52), vimentin (Vim2-17, 60-75), filaggrin (CCP-1/Fil307-324), α-enolase (CEP-1/Eno5-21), collagen type II (citC1359-369), and anti-cyclic citrullinated peptide (CCP)2 antibodies were analysed. Results HLA-SE-positive individuals were more frequently positive for ACPA compared with HLA-SE-negative individuals prior to the onset of symptoms of RA, particularly for antibodies against CEP-1 and Fibß62-81a (72). Smoking was associated with antibodies against Vim2-17 and citC1359-369. HLA-SE and smoking showed increasing association to the presence of the antibodies closer to disease onset. The highest odds ratio (OR) for development of RA was for the combination of HLA-SE alleles and ACPA positivity, especially for antibodies against Fibß62-81b, CCP-1/Fil307-324, and Fibβ36-52. A gene-environment additive interaction between smoking and HLA-SE alleles for the risk of disease development was found, with the highest OR for individuals positive for antibodies against Fibβ36-52, CEP-1, and Fibα580-600. Conclusions The relationships between antibodies against the different ACPA specificities, HLA-SE, and smoking showed a variable pattern in individuals prior to the onset of RA. The combination of smoking and HLA-SE alleles was significantly associated with the development of some of the antibody specificities closer to onset of symptoms, and these associations remained significant at diagnosis. An additive gene-environment interaction was found for several of the antibodies for the development of RA. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0638-x) contains supplementary material, which is available to authorized users.

Introduction: We have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA). Methods: The autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC (R) system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP) 2 using the CCPlus (R) ELISA kit. Results: Two peptides derived from the fibrinogen a chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen beta chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65 %, 15 %, 35 %, and 53 % of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5 % (Cit573), 6 % (Cit591), 8 % (Cit72), and 4 % (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84 % and 63 % respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency. Conclusions: Here we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.