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Background:anti-NXP2 antibodies is considered a serological marker of dermatomyositis (DM), with calcinosis, severe myositis and, in some series, cancer. Historically, these associations have been detected with immunoprecipitation (IP), but in the last few years commercial lineblot (LB) assay have been released.Objectives:to analyze the clinical features associated to anti-NXP2 antibodies, including the onset of concomitant cancers, both with LB and homemade IPMethods:clinical and serological data from medical charts of 213 patients with a diagnosis of inflammatory miosidites without anti-NXP2 (NXP2-), followed-up by two third-level Centers, and 61 anti-NXP2+ patients from 10 Rheumatological centers were analyzed. Anti-myositis specific (MSA) and anti-myositis associated antibodies (MAA) were detected in single centers by LB (Euroimmun Autoimmune Inflammatory Myopathies 16 antigens). Anti-NXP2 was confirmed by protein and RNA IP, as previously described (1)Results:clinical diagnosis of anti-NXP2+ positive with LB were 42 DM, 11 PM, inclusion body myositis (IBM) 4, necrotizing myositis and overlap (OM) 1 each. Anti-NXP2+ showed a lower age at onset (pIP did not confirmed anti-NXP2 antibodies in 18 sera: in 4 cases at least one MSA/MAA was identified by IP; these 18 patients did not show differences when compared with 213 anti-NXP2-.Conclusion:Protein IP confirmed anti-NXP2 antibodies in 63% of LB+ sera. Double positive cases showed more typical DM features and rarely occurred in IIM not DM. Anti-NXP2 positivity by LB should be confirmed by other methods in order to correctly diagnose and characterize IIM patients.References:[1]Arthritis Res Ther 2012,30;14:R97Acknowledgments:Forum Italiano per la Ricerca Malattie Autoimmuni (FIRMA)Disclosure of Interests:Micaela Fredi: None declared, Ilaria Cavazzana: None declared, Angela Ceribelli: None declared, Maria Grazia Lazzaroni: None declared, Simone Barsotti: None declared, Maurizio Benucci: None declared, Lorenzo Cavagna: None declared, Ludovico De Stefano: None declared, Andrea Doria Consultant of: GSK, Pfizer, Abbvie, Novartis, Ely Lilly, Speakers bureau: UCB pharma, GSK, Pfizer, Janssen, Abbvie, Novartis, Ely Lilly, BMS, Giacomo Emmi: None declared, Marco Fornaro: None declared, Federica Furini: None declared, Roberto Gerli: None declared, Maria Grazia Giudizi: None declared, Marcello Govoni: None declared, Anna Ghirardello: None declared, Luca Iaccarino Speakers bureau: GSK, Pfizer, Janssen, Novartis, Florenzo Iannone Consultant of: Speaker and consulting fees from AbbVie, Eli Lilly, Novartis, Pfizer, Roche, Sanofi, UCB, MSD, Speakers bureau: Speaker and consulting fees from AbbVie, Eli Lilly, Novartis, Pfizer, Roche, Sanofi, UCB, MSD, Maria Infantino: None declared, Alessandro Mathieu: None declared, Emiliano Marasco: None declared, Paola Migliorini: None declared, Boaz Palterer: None declared, paola parronchi: None declared, Matteo Piga: None declared, Federico Pratesi: None declared, Antonella Radice: None declared, Carlo Selmi: None declared, Valeria Riccieri: None declared, Marilin Tampoia: None declared, Giovanni Zanframundo: None declared, Angela Tincani: None declared, Franco Franceschini: None declared

OBJECTIVE—To describe the characteristics of a new set of European families with affected sib pairs (ASP) collected by the European Consortium on Rheumatoid Arthritis Families (ECRAF) to replicate the results of our first genome scan. Potential gradients for disease severity in Europe and concordance within families were studied. PATIENTS AND METHODS—From 1996 to 1998 European white families with at least two affected siblings were enrolled in the study. Demographic (sex, age at onset), clinical data (rheumatoid factor (RF), disease duration, erosive disease, extra-articular features (EF)), and HLA-DRB1 oligotyping were analysed. RESULTS—565 patients with rheumatoid arthritis (RA), belonging to 271 families including 319 affected sib pairs (ASP) were collected. Belgium, France, Italy, the Netherlands, Portugal, and Spain contributed 20, 96, 52, 24, 9, and 70 families, respectively. Sex (78% women), age at onset (mean 44 years), and RF positivity (79%) were similar among the countries. Differences were found in disease duration (11-18 years) and in the prevalence of erosive disease (70-93%), nodules (15-44%), subjective Sjögren's syndrome (5-38%), and EF (3-16%) (p

Background Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). Objective The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. Methods Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. Results RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14–34 and 31–50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. Conclusions Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.

Background Anti–citrullinated-peptid/protein antibodies (ACPA) have been detected in individuals pre-dating symptom onset of rheumatoid arthritis (RA), with an initial limited spectrum of reactivities that gradually broadens to different citrullinated substrates typical of RA (1,2). The recent discovery of NETs as source of citrullinated antigens, and namely citrullinated histones H3 and H4 (3). We have previously described deiminated sequences contained in EBV-derived proteins EBNA1 and EBNA2 as specific targets of ACPA in sera from patients with RA (4). Objectives The aim was to analyze the evolution of the antibody response predating the symptom onset using citrullinated exogenous (viral peptides) and self (H4 peptides) antigens as substrates to detect ACPA. Methods A cohort of 521 individuals sampled before symptoms of RA, and 272 population controls were identified as being donors to the Medical Biobank of Northern Sweden; 241 plasma samples from early RA-patients were also collected. ACPA were detected by ELISA on viral citrullinated peptides derived from Epstein-Barr-virus-encoded protein (EBNA1) and EBNA2 (VCP1 and VCP2) and on histone 4 derived peptides (HCP1 and HCP2). Results Anti-VCP1 antibodies were detected in 10.4% of the pre-symptomatic individuals as compared with 36.1% in early RA; anti-VCP2 in 17.1% vs. 52.3%; anti-HCP1 in 10.2% vs. 37.3%; anti-HCP2 in16.3% vs. 48.5%. Anti-VCP and -HCP concentrations were significantly increased in pre-symptomatic individuals as compared with controls (p PTPN22 T-allele. Three PADI3/PADI4 single nucleotide polymorphisms (rs3003444, rs7542629 and rs11800688) were significantly associated with anti-HCP1 antibodies after Bonferroni corrections. Conclusions Anti-VCP and -HCP antibodies predate symptom onset and predict disease development, but no hierarchy of citrullinated epitopes is clear of the ACPA response. These results suggest that no inciting citrullinated antigen so far described is common to all RA patients; rather data suggest a stochastic mechanism in ACPA production. Anti-HCP1 antibodies were associated with PADI polymorphisms. References Rantapaa-Dahlqvist S, et al. Arthritis Rheum 2003;48:2741–9. Brink M, et al. Arthritis Rheum 2013;65:899–910. Pratesi F, et al. Ann Rheum Dis 2014;73:1414–22. Pratesi F, et al Arthritis Rheum 2006;54:733–41 Disclosure of Interest None declared

Background The detection of anti-dsDNA antibodies is critical for the diagnosis and follow-up of SLE patients. The presently available assays are characterized by a non-optimal specificity (solid phase assays) or sensitivity (CLIF test). Objectives To overcome the limits of CLIFT and solid phase chromatin assays, we explored the diagnostic potential of an assay based on plasmid DNA containing a highly bent fragment of 211 bp from Crithidia Luciliae microcircles, complexed with histone peptides. Methods Electrically neutral complexes of PK201/CAT plasmid (PK) DNA and histone (H) 4 peptides were evaluated by electromobility shift assay. Complexes of H4 peptides and PK were absorbed to the solid phase to detect IgG antibodies in sera. Sera from 109 SLE patients, 100 normals and 69 disease controls (systemic sclerosis, Sjogren9s syndrome, unclassified connective tissue disease) were tested. Results H4 (14–34) containing the consensus sequence for DNA binding interacts with PK retarding its migration. H4 (14–34)-PK complexes were used to test sera by ELISA; inhibition assays show that sera react with epitopes present on DNA or on the complex, not on the peptide. Anti-H4-PK antibodies were detected in 56% SLE sera (more frequently in patients with skin or joint involvement) vs 12% disease controls; antibody titer is correlated with ECLAM score and anti-C1q antibodies, negatively with C3 levels. Anti-H4-PK antibodies compared with CLIFT and solid phase dsDNA assays display moderate concordance. Conclusions The H4-PK assay is a simple and reliable test, endowed with high specificity and sensitivity, useful for the differential diagnosis and evaluation of disease activity of SLE patients. This assay increases the variety of anti-DNA antibodies that can be measured, complementing the assays currently used for the detection of anti-DNA antibodies. Disclosure of Interest None declared

Background and objectives ACPA have been detected in individuals predating onset of rheumatoid arthritis (RA), with an initially limited spectrum of reactivities that gradually broadens to the wide recognition of different citrullinated substrates typical of established RA. Among the targets of ACPA, viral deiminated (citrullinated) proteins and antigens derived from neutrophil extracellular traps like histone H4 have been described. The aim of this study is to analyse the evolution of antibody response in pre-RA using citrullinated exogenous (viral peptides) and self (H4 peptides) antigens as substrates to detect ACPA. Patients and methods 521 individuals sampled before any symptoms of RA and 272 population controls were identified as donors to the Medical Biobank of Northern Sweden; 241 plasma of early RA-patients were also collected. ACPA were detected by ELISA on viral citrullinated peptides derived from EBNA 1 and EBNA2 (VCP1 and VCP2) and on histones 4 (H4) derived peptides (HCP1 and HCP2). Results Anti-VCP1 antibodies were detected in 10.4% of the pre-symptomatic individuals as compared with 36.1% in early RA; anti-VCP2 in 17.1% vs. 52.3%; anti-HCP1 in 10.2% vs. 37.3%; anti-HCP216.3% vs. 48.5%. Anti-VCP and anti-HCP concentrations were significantly increased in pre-symptomatic individuals as compared with controls (p < 0.001). Mean antibody concentration and frequency of positivityincreased steadily approaching symptom onset. ACPA specific for HCP or VCP appeared at the same time (median (Q1-Q3) 5.3 (2.9–8.9) years before symptom onset); positivity for anti-VCP2, HCP1 and HCP2 was associated with disease development. Combination of anti-VCP1, anti-VCP2 and anti–HCP2 and negativity for anti-HCP1 yielded a highrisk ratio (OR=18.70; 95% CI 2.53–138.47; p < 0.004). Anti-VCP2 and anti-HCP2 antibodies were associated in presymptomatic individuals with HLA-SE but not with PTPN22 T-allele. Conclusions Anti-VCP and anti-HCP antibodies start to appear long before disease onset and predict disease development, butno hierarchy of citrullinated epitopes can be drawn in the building up of the ACPA response. Taken together with previous findings, these results suggest that no inciting citrullinated antigen so far described is common to all RA patients; data are rather compatible with a stochastic mechanism in ACPA production.

Background The GAPAID (Genes And Proteins for AutoImmunity Diagnostics) consortium was created within the European Union9s Seventh Framework Programme for Research and Technological Development (FP7), with the aim of validating gene and protein biomarkers for autoimmune diseases such as systemic lupus erythematosus (SLE). This is an autoimmune disease characterized by a loss of tolerance to self-antigens, inflammation and dysregulated immune responses leading to multi-organ damage and affecting predominantly women (1). The wide spectrum of clinical manifestations shown by the disease complicates its diagnosis, treatment and research (2). Epidemiological studies suggest strong contribution of genetic factors in the pathogenesis of the disease and many SLE risk loci have been identified in several genome-wide association studies (GWAS) and other association studies in the last decades (3). Objectives To perform a replication study for several previously reported SLE susceptibility loci in an independent European sample and to assess their specificity with the different subphenotypes of the disease. Methods Previously associated 49 single nucleotide polymorphisms (SNP) were typed in a cohort of 208 SLE patients and 152 controls from Rheumatology Units of University Hospital of Pisa (Italy) and University of Pecs Medical Center (Hungary). Regression analyses were performed focused on the confirmation of disease susceptibility loci and identification of specific genes for age onset, renal, neurological and skin involvement, and presence of arthritis, secondary Sjogren syndrome and secondary antiphospholipid syndrome (APS). Results Previously described risk alleles from HLA, IRF5, BLK, ITGAM, and IRF8loci have been identified (P value Conclusions Overall, results from GAPAID project are consistent with previously established associations for HLA, IRF5, BLK, ITGAM, and IRF8 SLE susceptibility loci and report for the first time several subphenotype-specific associations. References Chung et al. PLoS Genet 2011; 7:e1001323. Eisenberg J. Autoimmun 2009; 32: 223–230. Rullo and Tsao. Ann Rheum Dis 2013; 72(Suppl 2): 56–61. Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is one of the most common autoimmune disorders affecting approximately 0.5-1.0% of the population worldwide (1), mainly females. Genetic studies of RA in European ancestry populations, including WGAS and meta-analysis-based works, have identified up to 46 RA risk loci (2-4). Most of them have been identified and validated in RA patients seropositive for ACPA autoantibodies, which are actually considered a well-established diagnostic and prognostic marker for RA. However, little is known about their contribution in seronegative RA disease. In this context, the GAPAID (Genes And Proteins for AutoImmunity Diagnostics) consortium was created within the European Union9s Seventh Framework Programme for Research and Technological Development (FP7), with the aim of validating gene and protein biomarkers for autoimmune diseases such as RA. Objectives To replicate the association of RA susceptibility loci in an independent European sample and to assess their specificity with ACPA status. Methods A selection of 64 SNP previously associated with RA have been typed in a cohort of 267 RA patients (169 ACPA-positive and 97 ACPA-negative) and 152 controls from Rheumatology Units of University Hospital of Pisa (Italy) and University of Pecs Medical Center (Hungary). Regression analyses were performed first considering overall RA patients and secondly, taking both serotype subgroups as different disease entities. Results have been adjusted for age, gender and origin of individuals. Results The well-known CD2, REL, TNFAIP3, IRF5, PTPRC, and CCR6 have been confirmed as RA disease associated loci together with recently discovered BACH2, RASGRP1, and IKZF3 loci, taking all RA patients as a unique phenotype. Results from both serological subgroups separately reflect the specificity of these susceptibility loci and show additional ACPA-positive specific associations for variants at IL6R, IL2RA, BLK, DDX6, IL6, and TLE3 genes. Conclusions Results from GAPAID project are consistent with previously established RA disease associations for CD2, PTPRC, REL, CCR6, TNFAIP3, IRF5, BLK, IL2RA, and DDX6 loci. In addition, IL6R, BACH2, RASGRP1, TLE3, and IKZF3 are replicated for the first time in an independent European population and IL6 appears to be a suggestive new RA associated locus. The stratified analysis based on ACPA status provides further support for distinct genetic aetiologies of RA subsets, which is clinically relevant since it may have therapeutic implications. References Alamanos and Drosos. Autoimmun Rev 2005;4:130–6. Stahl et al. Nat Genet. 2010;42(6):508-14. Zhernakova et al. PLoS Genet 2011;7(2):e1002004. Eyre et al. Nat Genet 2012;44(12):1336-40. Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder, characterised by a progressive joint damage, caused by a cellular infiltrate localised in the synovia. In RA, fibroblast like synoviocytes (FLS) acquire an aggressive phenotype and contribute to joint destruction also producing a variety of pro-inflammatory cytokines. In the pathogenesis of RA a pivotal role is played by fibroblast like synoviocytes (FLS), that produce cytokines that perpetuate inflammation. One of the serological markers of RA are anti citrullinated protein antibodies (ACPA), a family of autoantibodies recognising a wide variety of antigens all characterised by the deimination of arginine into citrulline. Among the different citrullinated targets, ACPA can bind viral citrullinated peptides (VCPs) and histone citrullinated peptides (HCPs). Despite their strict association with RA, so far little is known about the role of ACPA in the pathogenesis of the disease. Objective The aim of this work was to analyse the effects of anti VCPs and HCPs antibodies on in vitro cultured FLS. Methods FLS from RA and non RA patients were isolated from synovial fluid and used for experiments at 4–5th passage. Anti-VCPs and anti-HCPs antibodies were purified by affinity chromatography from RA patients sera and IgG from normal subjects were used as control antibodies. Anti-VCPs and –HCPs antibodies were incubated on subconfluent FLS; cells and supernatants were recovered at different time points. RANTES protein was quantified in the supernatants by sandwich ELISA and RANTES gene expression was evaluated by RT-PCR. To investigate intracellular signalling after ACPA stimulation, specific MAP kinase, already known to be activated during CCL5/RANTES production, were studied. Results Anti-VCPs and anti-HCPs from 5 RA patients induce RNA expression and secretion of RANTES from FLS as compared with normal IgG (p No differences were observed between RA and non RA FLS. FLS stimulation with ACPA seems to induce phosphorylation of ERK, JNK and p38. Conclusions These results suggest a novel mechanism potentially involved in damage induced by ACPA, mediated by the production of RANTES in FLS. ACPA might play a role in activation and chemotaxis of T cells in synovial tissue with a perpetuation in inflammation and immune response. Studies are in progress to clarify the intracellular pathways implicated in the synthesis of RANTES induced by ACPA in FLS and to define the target of ACPA on FLS surface.

Background The pathophysiology of rheumatoid arthritis (RA) is currently viewed as a process that starts with a pathologic activation of the immune system that eventually leads to the clinical manifestation of the disease. Systemic autoimmunity has been defined as one of the specific phases preceding the development of RA.(1) Different risk factors may be relevant for each specific phase in RA disease development. Objectives To analyse putative environmental, genetic and demographic risk factors associated with the development of systemic autoimmunity associated with RA in individuals genetically at risk. Methods This is a prospective cohort study of individuals genetically at risk of developing RA, namely first degree relatives (FDRs) of patients with known active RA. Only individuals without clinical evidence of RA were enrolled, and then followed-up yearly to detect the development of active arthritis. We included in this analysis only individuals with available anti-CCP status (anti-CCP 2.0 or anti-CCP 3.1). We pooled individuals from two separate cohorts, one from Switzerland (N=682) and one from France (N=148). We used logistic regression to analyse univariate and multivariate associations between a positive anti-CCP status and putative risk factors. Results A total of 830 FDRs of RA patients are presented, of which 41 (5%) were anti-CCP positive. FDRs had a mean age of 46 years, 79% female, 61% shared epitope positive, and 24% had a history of at least one episode of swollen joint, either at inclusion or during follow-up. In univariate analysis, a positive anti-CCP status was significantly associated with heavy tobacco smoke (>10 pack-years, Odds Ratio (OR): 2.8, p=0.02), loss of teeth (OR: 1.07, p=0.001), the presence of a swollen (OR: 2.5, p=0.04) or a tender joint (OR: 2.1, p=0.03). In multivariate adjusted analysis, only loss of teeth (OR: 1.06, p=0.005), heavy tobacco smoke (OR: 2.6, p=0.039) and the history of a swollen joints (OR: 1.16, p=0.002) were independently associated with anti-CCP positivity. Furthermore, male sex trended to be protective (OR: 0.39, p=0.089) and the effect of obesity on anti-CCP development tended to differ by sex. In males obesity was associated with an increased risk, while in women obesity was associated with a decreased risk (OR: 4.86 vs. OR: 0.18, p=0.075). No association between anti-CCP and the presence of the shared epitope, alcohol consumption or various joint symptoms was found, which may be explained by limited statistical power. Conclusions In individuals genetically at risk for RA, the development of anti-CCP antibodies was associated with tooth loss, tobacco smoke and the occurrence of a swollen joint. Taken together, these results suggest similar risk factors for the production of anti-CCP antibodies and the development of classifiable RA, suggesting that the occurrence of anti-CCP antibodies is a valid intermediate marker of RA development. References Gerlag DM et al. Ann Rheum Dis 2012. Acknowledgements All investigators and participants within the “SCREEN-RA” and the “Campagne des 1009000” studies. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5178

Background Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated proteins. Up to 50% of RA patients display synovial tertiary lymphoid structures (TLS) with functional B cell follicles supporting a germinal-centre response and local autoantibody production [1]. The nature of the main (auto)antigenic reactivity of synovial B cells is unknown. Objectives To characterize the autoreactive specificity at single cell level of synovial B cells isolated from TLS+ RA synovium. Methods Single CD19+ B cells were FACS sorted from fresh synovial cell suspension of 3 TLS+ RA joints. RNA was used to amplify Ig VH and VL genes separately and PCR products were cloned and expressed as recombinant monoclonal antibodies each displaying identical specificity of the original B cell [2]. We obtained 139 individual VH sequences (33% IgM, 40% IgG, 27% IgA) and 175 VL sequences. Recombinant monoclonal antibodies were generated from a total of 66 matching H+L chains. Monoclonal antibodies were first screened for their immunoreactivity using a RA autoantigen microarray platform [3]. Immunoreactivity was then confirmed by Luminex, ELISA and WB towards specific citrullinated antigens and by cell-based immunoassays. 30 monoclonal antibodies derived from peripheral naive or memory B cells of patients with primary Sjogren9s syndrome were used as controls. Results VH and VL mutational analysis demonstrated evidence of antigen-driven selection and intra-synovial clonal diversification. Interestingly, IgA clones displayed the highest mutational load followed by IgG and IgM. Among >300 RA autoantigens, the strongest and most frequent immunoreactivity was observed towards citrullinated histones H2A and H2B (∼30% of all RA B cell clones), followed by citrullinated vimentin. Selective reactivity towards the citrullinated but not unmodified forms of H2A, H2B and H4-derived histone peptides was confirmed by ELISA. The intensity of reactivity was directly correlated on the degree of the VH mutations and was abrogated by reverting hypermutated clones to germline sequences by reverse PCR. Citrullinated histones and vimentin are generated during neutrophil extracellular traps (NETs) formation. Indeed, WB on NET protein extracts confirmed selective reactivity towards a 14kDa band corresponding to histones H2/H4. Furthermore, immunofluorescence on neutrophils undergoing PMA-induced NETosis demonstrated specific reactivity of RA clones towards NETs but not resting neutrophils. Conclusions Here we provide evidence for the first time that highly mutated, locally differentiated B cells isolated from RA synovial germinal centre-like structures display strong immunoreactivity towards citrullinated proteins generated during NETosis, primarily citrullinated histones. Thus, synovial NETs emerge as the main antigenic force driving in situ autoreactive B cell activation, selection and differentiation within ectopic germinal centre-like structures sustaining the humoral autoimmune response within the RA joints. References Humby F et al., PLoS Med, 2009. Wardemann H et al., Science, 2003 Robinson, WH et al., Nat Med, 2002. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4422

Background Rheumatoid arthritis (RA) sera contain antibodies specific for deiminated peptide/proteins (ACPA) directed against endogenous (filaggrin, fibrin, vimentin, collagen, enolase) or exogenous antigens. Recently ACPA reacting with citrullinated peptides derived from histone H4 have been detected. It is widely accepted that HLA-DRB1 alleles encoding the shared epitope sequences are associated with the production of ACPAs. However, the genetic background underlying the production of single ACPA specificities has not been thoroughly investigated. Objectives To analyze the reactivity to two different histone H4 derived citrullinated peptides (HCP1 = citrullinated H4 14–34 and HCP2 = citrullinated H4 31–50 ) and to study their association with genetic variants within HLA-DRB1 SE alleles in a RA population. Methods One hundred and seventy-two French RA patients were characterized in terms of HLA-DRB1 genotype, anti VCP-A and anti-VCP-B antibodies. HLA-DRB1 typing was performed using the polymerase chain reaction-sequence specific primer (SSP) method, and SE alleles were classified into four SE+ groups (S1, S2, S3P, S3D) and one SE- group (X) according to the new classification of HLA-DRB1 alleles, reshaping the shared epitope (SE) hypothesis [1]. Anti-HCPs antibodies were assessed by home made ELISA on HCP1 and HCP2 coated plates. Results Setting the threshold value at the 98° percentile of the normal population, anti-HCP1 antibodies are present in 46% and anti-HCP2 antibodies in 62% of RA patients. Anti-HCP1 antibodies are not associated with HLA-SE alleles. On the contrary, anti-HCP2 antibodies are associated with HLA-DRB1 *0102 (p=0.04), *0401 (p=0.03), *0404 (p=0.01). Subgrouping the SE alleles according to the new classification, the presence of anti-HCP2 antibodies is associated with S2 allele (p Conclusions Using two histone H4 derived citrullinated peptides as probes, we showed genetic heterogeneity in the control of the immune response to single components of the ACPA family and to different portion of the same molecule. These data underline the need to type RA patients for different ACPA specificities, that may contribute to the identification of different subsets of patients. References du Montcel ST et al. Arthritis Rheum. 2005, 52(4): 1063-8 Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5450

Rheumatoid arthritis (RA) linkage has been observed on the 18q chromosome in the white population.1 The 18q locus has also shown evidence of linkage with type I insulin-dependent diabetes mellitus, systemic lupus erythematosus and Grave’s disease.2 A candidate gene on chromosome 18q, the TNFRSF11A ( RANK ) gene, which encodes the receptor activator of nuclear factor kappa B, has been shown to be involved in the osteoclast differentiation playing a pivotal role in bone resorption.3 A single RANK haplotype was reported to be associated with RA in a case–control association study.4 Furthermore, activating mutations in the RANK gene cause bone disorders such as early-onset Paget’s disease,5 expansile skeletal hyperphosphatasia6 and familial expansile osteolysis.7 The aim of this …

Background and Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated proteins. Moreover, RA patients are characterised by increased neutrophils in their synovial fluid, in particular at early stage of the disease. Recent evidence suggests a critical role of neutrophils in sustaining the inflammatory response in the RA joint. Around 40% of patients display synovial tertiary lymphoid structures (TLS) with functional B cell follicles supporting a germinal-centre response and local autoantibody production. However, the nature of the main (auto)antigenic reactivity of synovial B cells is unknown. Here we characterised the autoreactive B cell response of lesional B cells isolated from TLS + RA synovium. Materials and Methods Single CD19 + B cells were FACS sorted from synovial cell suspension of 4 TLS + RA patients. RNA was used to amplify Ig VH and VL genes and PCR products were cloned and expressed as recombinant monoclonal antibodies displaying identical specificity of the original B cells. Recombinant antibodies were then tested 1) to determine the frequency of polyreactive clones and 2) to define their immunoreactivity towards native and citrullinated antigens using a synovial antigen microarray platform. Results We obtained 139 individual VH sequences of which 33% were IgM, 40% IgG, 27% IgA and 175 VL sequences and we generated a total of 66 complete (H + L chains) recombinant monoclonal antibodies. Analysis of the VH gene somatic mutation rate showed evidence of antigen selection and intra-synovial clonal diversification. No skewed distribution of the VH and VL gene usage was observed. Around 30% of synovial monoclonal antibodies were reactive towards citrullinated histones in the antigen microarray, in particular citH2A and citH2B. This reactivity was confirmed by citH2A and citH2B ELISA. Moreover, when the synovial monoclonal antibodies were tested on neutrophil extracellular traps (NETs), which contained citrullinated histones, reactivity towards NETs proteins but not neutrophil nuclear antigens was observed. Conclusions Here we provided novel evidence that highly mutated, locally differentiated B cells within RA synovial germinal centre-like structures display a strong immunoreactive bias towards citrullinated histones which likely derived from neutrophil NETs that continuously form in the RA synovial fluid. This suggests that citrullinated histones are the main antigens driving in situ B cell activation and differentiation sustaining the humoral autoimmune response within the RA joints.

Background Psoriatic arthritis (PsA) is an inflammatory joint disease associated with psoriasis, classified within the familyof spondyloarthropathies. The events underlying the pathogenesis of PsA have not yet been fully elucidated. It is known that angiogenesis is an early event in the joint inflammation during PsA, resulting in cartilage degradation and bone destruction. Previous studies have demonstrated an increase of circulating pro-angiogenic mediators, including vascular endothelial growth factor (VEGF) in PsA. The role of endogenous inhibitors of angiogenesis in PsA is unknown. Objectives The aim of our study is to investigate the potential involvement of Endostatin (ES) and Thrombospondin-1 (TSP-1), extracellular matrix (ECM) fragments with anti-angiogenic activity, in patients with PsA. Methods Fifty four adult patients with PsA were studied. The diagnosis of PsA was established according to the CASPAR criteria. Endostatin (ES) and Thrombospondin-1 (TSP-1) were measured, by enzyme immunoassays, in the sera of these patients and of 52 patients with Rheumatoid Arthritis (RA), diagnosed according to the 2010 ACR-EULAR classification criteria, and of 39 healthy subjects. Results The serum level of ES resulted significantly higher in the PsA group than in the control subjects (P Conclusions The observed increase of ECM fragments with anti-angiogenic properties suggests a reduction of pro-angiogenic activity in PsA. It is possible that this reduction is due to underlying treatments. Disclosure of Interest None Declared

Background The spondyloarthritis (SpA), including ankylosing spondylitis (AS) psoriatic arthritis, reactive arthritis, arthritis associated with inflammatory bowel disease and “undifferentiated” SpA (uSpA), are characterised by inflammatory back pain (IBP), sacroiliitis, peripheral arthritis, and enthesitis together with an association with HLA B27. Undifferentiated SpA is a diagnosis of exclusion, and can include patients with IBP who may have early ankylosing spondylitis but do not yet fulfil the classification criteria for AS. Angiogenesis, the formation of new capillaries from pre-existing vessels, isan early event in inflammatory joint diseases. Objectives The aim of our study is to investigate the regulation of pro-angiogenic mediators and their endogenous inhibitors in uSpA and AS. Methods Forty one adult patients with uSpA and 9 patients with definitive diagnosis of AS (according to the 1984 modified New York criteria) were studied. Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF-2) and their inhibitors Endostatin (ES) and Thrombospondin-1 (TSP-1) were measured in the sera of these patients and of 39 healthy subjects by enzyme immunoassays. Results The serum levels of VEGF but not FGF-2 resulted significantly higher in the uSpA group than in the control subjects (P=0.0052). ES and TSP-1 were significantly higher in both uSpA (P Conclusions Our results suggest that a disregulation of circulating angiogenic and angiostatic mediators takes place both in uSpA than in AS. Further investigations on angiogenic and angiostatic mediators in a larger number of patients with different subtypes of SpA would be useful to understand better their role in the pathophysiology of this group of disorders. Disclosure of Interest None Declared

Background Histone deimination is a crucial event in cell biology, regulating gene function and contributing to antimicrobial response, through the formation of neutrophil extracellular traps (NETs.) Deiminated proteins are target of ACPA in rheumatoid arthritis. Objectives Aim of the present study is to to investigate the presence in RA sera of a new subset of ACPA, antibodies reactive with deiminated histones. Methods Neutrophils from peripheral blood were stimulated with calcium ionophore; NETosis was induced by phorbol myristate acetate and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were digested with trypsin and subjected to MALDI-TOF analysis, before and after derivatization to detect citrullinated peptides. Results RA sera reacted with an antigen of 11 KDa expressed in nuclei of activated neutrophils, identified as H4. MALDI TOF analysis indicated that this antigen, present also in NETs, is citrullinated. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14-34 and 31-50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titer was correlated with anti-CCP2. Conclusions Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA. Disclosure of Interest None Declared

Background Ectopic lymphoid structures (ELS) are preferential sites for Epstein-Barr virus (EBV) latency and reactivation in the brain of multiple sclerosis (1) and are functional sites of autoreactive B cell activation in the synovial tissue of rheumatoid arthritis (RA) patients (2) leading to the in situ production of anti-citrullinated protein/peptide antibodies (ACPA). However, it is unknown whether ELS are responsible for the generation of antibodies against native and/or post-translationally modified EBV proteins. Objectives In this work we aimed: 1) To investigate the presence of anti-VCA, anti-EBNA1, anti-EA and ACPA antibodies in the serum vs synovial fluid (SF) of RA patients; 2) To provide in vivo evidence of the synovial production of antibodies against unmodified and citrullinated EBV-proteins (3) in the HuRA/SCID chimeric model; 3) To investigate whether EBV-infected synovial plasma cells produce ACPA. Methods Anti-EBNA1, anti-VCA, anti-EA and ACPA IgG were measured in paired serum and SF from RA and OA patients. The local production of unmodified and citrullinated (VCP1 and VCP2) anti-EBV antibodies (3) was investigated by screening the serum of 30 SCID mice transplanted with ELS+ vs ELS- RA synovium. Finally, sequential double immunofluorescence for biotinylated citrullinated fibrinogen (BCf), BFRF1 (EBV lytic antigen) and CD138 was used to detect EBV reactivation in ACPA-producing synovial plasma cells. Results Serum and SF levels of anti-EBNA1, anti-VCA and ACPA IgG were significantly correlated. Sera of SCID mice transplanted with ELS+ (but not ELS-) RA synovia displayed high levels of human IgG ACPA, anti-EBNA1 and anti-VCA but not anti-EA antibodies. Interestingly, a proportion of SCID mice sera displaying high reactivity against anti-CCP and anti-EBNA1 antibodies were also found positive for anti-VCP1 and VCP2. Finally, in ELS+ RA synovia a subset of locally differentiated ACPA+ plasma cells surrounding ectopic germinal centres displayed EBV lytic infection as detected by reactivity for BFRF1. Conclusions Here we showed that antibodies against unmodified and citrullinated EBV antigens are locally produced in the RA synovium in the presence of ELS and are closely associated with in situ ACPA production. Evidence of ACPA+ plasma cells showing lytic EBV infection strongly supports an important role for EBV in the activation and differentiation of autoreactive B cells within the RA synovium. References Serafini B, Rosicarelli B, Franciotta D, Magliozzi R, Reynolds R, Cinque P, Andreoni L, Trivedi P, Salvetti M, Faggioni A, Aloisi F. Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain. J Exp Med. 2007 Nov 26;204(12):2899-912. Humby F, Bombardieri M, Manzo A, Kelly S, Blades MC, Kirkham B, Spencer J, Pitzalis C. Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium. PLoS Med. 2009 Jan 13;6(1):e1 Pratesi F, Tommasi C, Anzilotti C, Chimenti D, Migliorini P. Deiminated Epstein-Barr virus nuclear antigen 1 is a target of anti-citrullinated protein antibodies in rheumatoid arthritis. Arthritis Rheum. 2006 Mar;54(3):733-41. Disclosure of Interest None Declared

Objectives Rheumatoid arthritis (RA) is associated with an increased Epstein-Barr virus (EBV) blood DNA load, a robust immune response to EBV and cross-reactive circulating antibodies for viral and self-antigens. However, the role of EBV in RA pathogenesis remains elusive. Here we investigated the relationship between synovial EBV infection, ectopic lymphoid structures (ELS) and immunity to citrullinated self and EBV proteins. Methods Latent and lytic EBV infection was investigated in 43 RA synovial tissues characterised for presence/absence of ELS and 11 OA samples by RT-PCR, in situ hybridisation and immunohistochemistry/immunofluorescence. Synovial production of anti-citrullinated proteins (ACPA) and anti-citrullinated EBV peptides (VCP1/VCP2) antibodies was investigated in situ or in vivo in the SCID/RA chimeric model. Results EBV dysregulation was observed exclusively in ELS+ RA, but not OA, synovia as revealed by presence of EBV latent [LMP2A, EBV-encoded small RNA (EBER)] transcripts and EBER+ cells and immunoreactivity for EBV latent (LMP1, LMP2A) and lytic (BFRF1) antigens in ELS-associated B cells and plasma cells, respectively. Importantly, ~20% of synovial plasma cells producing ACPA were infected with EBV. Furthermore, ELS-containing RA synovia transplanted into SCID mice supported production of ACPA and anti-VCP1/VCP2 antibodies cross-recognised by ACPA. Analysis of CD4+ and CD8+ T-cell localisation and granzyme B expression suggests that EBV persistence in ELS-containing synovia is favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Conclusions We demonstrated active EBV infection within ELS in the RA synovium that appears to contribute to local growth and differentiation of ACPA-reactive B cells.