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International audience; Rheumatoid arthritis (RA) is associated with HLA-DRB1 alleles expressing the "shared epitope". RA is usually preceded by the emergence of anti-citrullinated protein autoantibodies (ACPAs). ACPAs recognize citrulline residues on numerous proteins. Conversion of arginine into citrulline is performed by enzymes called peptidyl arginine deiminases (PADs). We have previously demonstrated that C3H mice immunized with PADs can produce ACPAs by a hapten-carrier mechanism. Here, we address the influence of HLA-DR alleles in this model in mice expressing RA-associated HLA-DRB1*04:01 (KO/KI*04:01) or HLA-DRB1*04:04 (KO/KI*04:04) or non RA-associated HLA-DRB1*04:02 (KO/KI*04:02) after murine PAD2 (mPAD2) immunization. Immunization with mPAD2 triggers production of ACPAs in wild type (WT) and HLA-DR4 C57BL/6 mice. Both I-Ab and HLA-DR are involved in the activation of mPAD2 specific T lymphocytes. Among HLA-DR4 mice, mice expressing RA-associated HLA-DRB1*04:01 are the best responders to mPAD2 and the best anti-citrullinated peptide antibody producers. This article is protected by copyright. All rights reserved.

International audience

International audience; Psoriatic arthritis (PsA) is a type of inflammatory arthritis associated with psoriasis. HLA association studies performed in northern Europe, comparing patients with control populations, have shown that the highest risk for PsA is carried by HLA-C*06, HLA-B*57 and HLA-B*27 alleles. This retrospective association study compared HLA-A, -B, -C, and -DR alleles of 500 patients from southern France, who fulfilled the CASPAR criteria for Psoriatic Arthritis (PsA), with 2346 controls from healthy blood donors, using the chi-square test. We classified PsA patients into three different subgroups according to disease: purely axial, purely peripheral and combined axial and peripheral. The 'axial' subgroup was associated with HLA-B*27 (OR = 16.3, p = 2.7 × 10-28) and its haplotypes: HLA- B*27-C*01 (OR = 12.4, p = 1.7 × 10-12) and HLA-B*27-C*02 (OR = 8.7, p = 10 × 10-9). The 'axial and peripheral' and the 'peripheral' subgroups were associated with HLA-C*06 (respectively OR = 1.5, p = 3.6 × 10-10 and OR = 2.4, p = 3.6 × 10-12) and its haplotypes HLA-C*06-B*13 (respectively OR = 2.4, p = 1.2 × 10-6 and OR = 2.8, p = 6.4 × 10-11). This association study on a southern French PsA cohort identifies HLA-C*06 as a marker for peripheral PsA and HLA-B*27 as a marker for purely axial PsA.

Purpose Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding a five amino acid basic motive, the shared epitope SE). Each HLA-DRB1 genotype defines a genotype specific risk of developing RA. RA is preceded by the emergence of anti citrullinated protein antibodies (ACPAs). Citrullin is a neutral version of arginin, a basic amino acid, formed after post translational modification by Peptidyl Arginyl Deiminases (PADs). HLA-DRB1 genes associated with RA are also associated with ACPAs. Two models might explain this association: - RA associated HLA-DR molecules might bind citrullinated peptides better than non RA associated HLA-DR molecules. - RA associated HLA-DR molecules might bind PAD4 peptide(s) better than non RA associated HLA-DR molecules. Here we tested both models for prediction of HLA-DRB1 genotypic risks of developing RA. Methods We calculated the likelihoods for the 2 HLA-DR molecules encoded by 12 common HLA-DRB1 genotypes to bind at least one randomly chosen peptide from PAD4 or fibrinogen(native or citrullinatd) and compared them with the 12 respective HLA-DRB1genotypic risks of developing RA. Results HLA-DRB1 Genotypic risks of developing RA correlate with likelihoods of binding PAD4 peptides, not citrullinated Fibrinogen peptides. Thus, the molecular basis for the association of HLA-DR and ACPA positive RA is most likely the capability for RA associated HLA-DR molecules to bind peptides(s) from PAD4.

Objective.To assess the prevalence of axial articular manifestations (AAMs) in patients with primary Sjögren syndrome (pSS), to investigate whether these symptoms reveal an associated spondyloarthritis (SpA), and to assess their therapeutic management.Methods.Among 148 consecutive patients with pSS fulfilling European League Against Rheumatism (EULAR)/American College of Rheumatology 2019 classification criteria followed between 2010 and 2018, we selected those who presented with AAMs. The association with SpA was retained when patients fulfilled Assessment of SpA international Society criteria.Results.A total of 29 patients (20%, 28 women) with a median age of 43 years (range 15–65 yrs), were identified. The main extraglandular features were peripheral arthralgia and arthritis in 93% and 90% of patients, respectively. Positive anti-Ro/SSA (anti-SSA) antibody was reported in 62%. AAMs were inaugural in 7%, delayed from the diagnostic of pSS in 7%, and occurred concomitantly in 86% of patients. AAMs were not associated to multisystemic involvement of pSS. Radiographic sacroiliitis was mentioned in 65%, and HLA-B27 was positive in 13%. The diagnosis of SpA was retained in 23/29 patients (79%), among which 74% and 26% fulfilled psoriatic arthritis and ankylosing spondylitis criteria, respectively. There was no phenotypic difference according to the anti-SSA antibody status. With a median follow-up of 60 months (range: 5–96), 61% of patients with associated SpA required biotherapies, mainly of anti–tumor necrosis factor-α or anti–interleukin 17A molecules with a good clinical outcome in 64% and no effect on pSS.Conclusion.AAMs are not uncommon in patients with pSS and may reveal an associated SpA. Treatment of AAMs, especially when clearly associated with SpA, may necessitate biologics, following SpA-management therapeutic guidelines.

International audience; No abstract available

Resume Avec une prevalence mondiale de 0,5 %, la polyarthrite rhumatoide (PR) est une des maladies auto-immunes les plus frequentes. L’origine de la PR n’est pas connue, cependant des facteurs genetiques et environnementaux pourraient contribuer a son developpement. Le virus d’Epstein-Barr (EBV) constitue l’un de ces facteurs. Nous abordons dans cette etude plusieurs aspects du lien etroit entre EBV et la PR. Chez les patients atteints de PR, le controle de l’infection par EBV est altere. Ces patients presentent en effet des titres eleves d’anticorps diriges contre les antigenes d’EBV. Leurs lymphocytes T du sang peripherique sont moins efficaces pour controler la proliferation des lignees de lymphocytes B infectees par EBV. Les patients atteints de PR ont une charge virale en EBV dans leurs PBMCs augmentee d’un facteur 10 par rapport aux temoins. Le syndrome lymphoproliferatif post-transplantation (SLP-PT) correspond a une proliferation polyclonale de lymphocytes B infectes par EBV susceptible d’evoluer en un lymphome malin non Hodgkinien a lymphocytes B EBV+. Les patients atteints de PR presentent egalement un risque plus eleve de developper des syndromes lymphoproliferatifs (SLP) qui sont parfois associes a EBV. Il etait donc necessaire de surveiller la charge en EBV chez les patients atteints de PR lors de l’arrivee des nouveaux traitements immunosuppresseurs (biotherapies). EBV, un virus tres repandu, fortement reconnu par les anticorps mais jamais elimine, est un candidat ideal comme cible de la reponse immune dans les maladies immunitaires chroniques complexes. Les reponses sous forme d’anticorps anti-EBV doivent etre considerees comme partie integrante des reponses chroniques par autoanticorps liees au developpement de la PR, de meme que les anticorps anti-proteine citrullinee.

Introduction Les patients atteints de polyarthrite rhumatoide (PR) ont des anticorps anti proteines citrullinees (ACPA) qui reconnaissent des epitopes multiples. Cinq peptides du fibrinogene citrulline (Fbcit) suffisent a representer tous ces epitopes. L’objectif de ce travail est d’etudier si les caracteristiques cliniques et le terrain HLA-DR des PR ACPA positives sont associes a une reactivite particuliere vis a vis de ces 5 epitopes. Materiels et methodes 184 patients atteints de PR repondant aux criteres ACR/EULAR 2010, ACPA positifs (anti-CCP2 et/ou AhFibA positifs) ont ete inclus dans cette etude prospective entre janvier 2018 et decembre 2018. Les caracteristiques cliniques des patients et leur genotypage HLA-DRB1 ont ete recueillis. La reactivite de leurs serums pour les peptides du Fbcit β60–74cit60,72,74, α36–50cit38,42, α621–635cit621,627,630, α501–515cit510,512 et α171–185cit178,181, AhFibA, facteurs rhumatoides, et anti-CCP2 a ete testee par ELISA. Tous les patients ont donne leur consentement eclaire pour l’etude. La collection d’echantillon etait approuvee par le comite d’ethique sous le numero DC-2008-327. Resultats Correlation entre les taux d’anticorps (ac) antiCCP2 et AhFiba, et specificites Fbcit : – Les taux d’ac antiCCP2 et AhFiba sont correles (Pearson, p : 5,6 × 10-29, R :0,7). Les taux d’ac antiCCP2 sont correles a la specificite Fbcit β60–74cit60,72,74, (Pearson, p : 5,4 × 10-14, R :0,5), Fbcit α621–635cit621,627,630, (Pearson, p : 0,0001 R :0,2), Fibcit α501–515cit510,512 (Pearson, p : 3,8 × 10-5, R :0,3) et Fbcit α171–185cit178,181, (Pearson, p : 2,4 × 10-23, R :0,6) mais ne sont pas correles a la specificite Fbcit α36–50cit38,42 (Pearson, p : 0,9, R :0,007). Association de reactivite contre les epitopes du Fbcit avec HLA-DR : – La presence a double dose de l’epitope partage de HLA-DR est associee a une reactivite anti α501–515 (p = 0,002) et a la reconnaissance de plus de trois epitopes du Fbcit (p = 0,034). La presence de l’epitope QKRAA de HLA-DR est associee a une reactivite anti α501–515 (p = 0,0003) et anti α621-635 (p = 0,036). Association de reactivite contre les epitopes du Fbcit avec les facteurs rhumatoides : – Les facteurs rhumatoides sont associes a une reactivite anti α501–515 (p = 0,026) et a un antecedent de tabagisme (p = 0,022). La reconnaissance de plus de 3 epitopes citrullines est associee aux facteurs rhumatoides (p = 0,069 ; ns). Association de reactivite contre les epitopes du Fbcit avec les donnees cliniques : – Les nodules rhumatoides sont associes a une reactivite moyenne ou forte anti α501–515 (p = 0,044). La reconnaissance de plus de 3 epitopes citrullines est associee a une meilleure reponse aux anti TNF alpha (p = 0,025), et a une polyarthrite rhumatoide peu erosive (p = 0,051). Nous confirmons l’association deja decrite de QKRAA de HLA-DR avec les nodules rhumatoides (p = 0,081 ; ns). Conclusion Le statut HLA-DR des patients atteints de PR ACPA positive influence les specificites epitopiques des ACPA. Parmi les differentes specificites des ACPA, la presence d’une specificite anti peptide α501–515 du fibrinogene citrulline est associee a la presence de nodules rhumatoides et a HLA-DRB1*04 :01 (QKRAA).

International audience

The X chromosome, hemizygous in males, contains numerous genes important to immunological and hormonal function. Alterations in X-linked gene dosage are suspected to contribute to female predominance in autoimmunity. A powerful example of X-linked dosage involvement comes from the BXSB murine lupus model, where the duplication of the X-linked Toll-Like Receptor 7 (Tlr7) gene aggravates autoimmunity in male mice. Such alterations are possible in men with autoimmune diseases. Here we showed that a quarter to a third of men with rheumatoid arthritis (RA) had significantly increased copy numbers (CN) of TLR7 gene and its paralog TLR8. Patients with high CN had an upregulated pro-inflammatory JNK/p38 signaling pathway. By fluorescence in situ hybridization, we further demonstrated that the increase in X-linked genes CN was due to the presence of an extra X chromosome in some cells. Men with RA had a significant cellular mosaicism of female (46,XX) and/or Klinefelter (47,XXY) cells among male (46,XY) cells, reaching up to 1.4% in peripheral blood. Our results present a new potential trigger for RA in men and opens a new field of investigation particularly relevant for gender-biased autoimmune diseases.

Objective The presence of autoantibodies to citrullinated proteins (ACPAs) often precedes the development of rheumatoid arthritis (RA). Citrullines are arginine residues that have been modified by peptidylarginine deiminases (PADs). PAD4 is the target of autoantibodies in RA. ACPAs could arise because PAD4 is recognized by T cells, which facilitate the production of autoantibodies to proteins bound by PAD4. We previously found evidence for this hapten–carrier model in mice. This study was undertaken to investigate whether there is evidence for this model in humans. Methods We analyzed antibody response to PAD4 and T cell proliferation in response to PAD4 in 41 RA patients and 36 controls. We tested binding of 65 PAD4 peptides to 5 HLA–DR alleles (DRB1*04:01, *04:02, *04:04, *01:01, and *07:01) and selected 11 PAD4 peptides for proliferation studies using samples from 22 RA patients and 27 controls. Peripheral blood lymphocytes from an additional 10 RA patients and 7 healthy controls were analyzed by flow cytometry for CD3, CD4, CD154, and tumor necrosis factor expression after PAD4 stimulation. Results Only patients with RA had both antibodies and T cell responses to PAD4. T cell response to peptide 8, a PAD4 peptide, was associated with RA (P = 0.02), anti‐PAD4 antibodies (P = 0.057), and the shared epitope (P = 0.05). Conclusion ACPA immunity is associated with antibodies to PAD4 and T cell responses to PAD4 and PAD4 peptides. These findings are consistent with a hapten–carrier model in which PAD4 is the carrier and citrullinated proteins are the haptens.

Background:Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding the shared epitope (SE), a 5 amino acid motive. RA is usually preceded by the emergence of anti-citrullinated protein antibodies (ACPAs) detected by anti-CCP2 tests. Citrullin is a neutral amino acid resulting from post translational modification of arginin by Peptidyl Arginyl Deiminases (PADs). ACPAs recognize epitopes from citrullinated human fibrinogen (Fib-cit) and can be specifically detected by the AhFibA assay. Five peptides derived from Fib-cit together represent almost all of the epitopes recognized by patients with ACPA-positive RA: β60–74cit, α36–50cit, α621–635cit, α501–515cit and α171–185cit. As RA is a pleiomorphic disease, whose evolution is difficult to predict, the use of antibody fine specificity as a marker of clinical phenotypes has become a major challenge.Objectives:Our objective was to study whether clinical characteristics and HLA-DRB1 genetic background were associated with a specific reactivity against these epitopes.Methods:184 ACPA positive RA patients fulfilling the 2010 ACR/EULAR criteria were studied. Patients characteristics, including HLA-DRB1 genotype, were collected from their medical files. Anti-CCP2, AhFibA, Rheumatoid Factors (RF), and antibodies against the five major Fib-cit peptides were analyzed using ELISA assays.Results:Anti-CCP2 and AhFibA titres were strongly correlated (rs: 0.7037; p = 5.69x10-29, Pearson’s). Anti-α505-515cit antibodies were associated with HLA-DRB1*04:01 (OR = 5.52 [2.00 – 13.64]; p = 0.0003). High level anti-α505-515cit antibodies were significantly associated with rheumatoid nodules (OR = 2.71 [1.00 – 7.16], p= 0.044). Anti α501–515cit antibodies were associated with RF (OR=2.31 [1.10 – 4.78], p= 0.026).Conclusion:Immune complexes containing anti-α501–515cit antibodies and rheumatoid factors might be involved in the development of rheumatoid nodules on the HLA-DRB1*04:01 background. These findings highlight the role played by the HLA-DRB1*04:01 molecule and its rapid intracellular route into the lysosomes, enabling original antigen processing. Finally, purifying these epitope specific antibodies might be a new therapeutic opportunity for rheumatoid nodules.Disclosure of Interests:None declared

OBJECTIVE: Rheumatoid arthritis (RA)-associated autoantibodies include those directed at the kinase site of BRAF (v-Raf murine sarcoma viral oncogene homolog B1), a serine-threonine kinase involved in the MAPK signaling pathway. To understand anti-BRAF immunization, we sought to identify BRAF mutations in the peripheral blood lymphocytes (PBLs) of patients with RA. METHODS: We first cloned the major BRAF region known for mutations in the pCR2.1 vector, using genomic DNA from the PBLs of 8 RA patients. For each patient, 100 clones were sequenced. In 5 of 8 patients, we detected a new BRAF mutation in 1 clone. The frequency of this new mutation was evaluated by droplet digital polymerase chain reaction in PBLs from RA patients and controls. To test whether p.Val600Ala influences the kinase activity of BRAF, we developed an in vitro assay based on phosphorylation of MEK-1, a major BRAF substrate. RESULTS: A BRAF mutation, p.Val600Ala, was identified in 1 of 8,000 PBLs and 1 of 6,000 T lymphocytes from RA patients and in 1 of 12,500 PBLs and 1 of 12,500 T lymphocytes from controls. The BRAF p.Val600Ala mutation was not correlated with the presence of anti-BRAF autoantibodies. The p.Val600Ala mutation activated phosphorylation of MEK-1 in vitro. CONCLUSION: Most RA patients have a p.Val600Ala mutation in the BRAF gene. This mutation activates the kinase activity of BRAF. The p.Val600Ala mutation could activate the MAPK pathway, leading to the activation of T lymphocytes.

Objective Ankylosing spondylitis (AS) is a chronic inflammatory disease affecting the spine and pelvis of young adults. On the HLA-B27 genetic background, the occurrence of AS is influenced by the intestinal microbiota. The goal of our study was to test whether breast feeding, which influences microbiota, can prevent the development of AS. Methods First, 203 patients with HLA-B27-positive AS fulfilling the modified New York criteria were recruited in the Department of Rheumatology, Ste Marguerite hospital in Marseilles. A total of 293 healthy siblings were also recruited to make up a control group within the same families. Second, 280 healthy controls, and 100 patients with rheumatoid arthritis and their siblings were recruited. The data collected were age, gender, number of brothers and sisters, age at disease onset, type and duration of feeding (breast or bottle). Results Patients with AS had been breast fed less often than healthy controls. In families where children were breast fed, the patients with AS were less often breast fed than their healthy siblings (57% vs 72%), giving an OR for AS onset of 0.53 (95% CI (0.36 to 0.77), p value=0.0009). Breast feeding reduced familial prevalence of AS. The frequency of breast feeding was similar in the AS siblings and in the 280 unrelated controls. However, patients with AS were less often breast fed compared with the 280 unrelated controls (OR 0.6, 95% CI (0.42 to 0.89), p Conclusions Our study suggests a breastfeeding-induced protective effect on the occurrence of AS. To our knowledge, this is the first study of breastfeeding history in patients with AS.

Introduction Currently, there is no biomarker available to diagnose psoriatic arthritis (PsoA). Genome wide association studies showed that the majority of PsoA loci are shared with PsoC.1 However, PsoA has a strong familial predisposition, more so than PsoC as was shown in an Icelandic population.2 In the GWAS performed in UK and Germany, the strongest genetic locus is located within the major histocompatibility complex (MHC).3,4 No studies have been performed in southern Europe Objectives The primary objective of our study was to focus on the HLA class I and II alleles found in a cohort of 600 French patients with PsoA clinically well characterised, compared to a control cohort. The secondary objective was to compare two clinical subsets of PsoA, one axial and one peripheral, to test which genotype determines these phenotypes. Methods 600 patients from the Rheumatology department, St. Marguerite’s Hospital, Marseilles, who fulfilled the CASPAR criteria for PsoA, underwent clinical, radiographic and laboratory investigations. HLA Class I and Class II alleles were genotyped. A cohort of 2346 healthy blood donors (HBD) was also tested. Results Comparison between the PsoA population and controls showed one set of alleles significantly associated with PsoA; HLA-B*27, B*21(B*50), C*06 and with a weak significance HLA-A*01, A*25, B*13. Within the PsoA population, two genetically different subsets determine two different clinical subtypes: Peripheral disease was significantly associated with -C*06 in disequilibrium linkage with -B*13 and -DR*07, independently associated with HLA-A*01, -B*21 and -B*17. Axial disease was significantly associated with HLA-B*27 in disequilibrium linkage with C*01 and -C*02. Conclusions This is the first and the largest study ever realised in southern Europe. It lead to conclude that PsoA is genetically heterogeneous. We found that PsoA is divided in two genetically and phenotypically relevant subgroups, one axial group with HLA-B*27 predominance very close to Ankylosis spondylitis and one peripheral group with HLA-C*06 predominance. Other HLA alleles within the MHC are also implied. The clinical utility of HLA typing in PsoA should be further addressed in larger studies. References . Stuart PE, Nair RP, Tsoi LC, et al. Genome-wide association analysis of psoriatic arthritis and cutaneous psoriasis reveals differences in their genetic architecture. Am J Hum Genet2015;97:816–36. doi:10.1016/j.ajhg.2015.10.019 . Chandran V, Schentag CT, Brockbank JE, et al. Familial aggregation of psoriatic arthritis. Ann Rheum Dis2009;68:664–7. doi:10.1136/ard.2008.089367 . Rahman P, Elder JT. Genetics of psoriasis and psoriatic arthritis: A report from the GRAPPA 2010 annual meeting. J Rheumatol2012;39:431–3. doi:10.3899/jrheum.111242 . Bowes J, Budu-Aggrey A, Huffmeier U, et al. Dense genotyping of immune-related susceptibility loci reveals new insights into the genetics of psoriatic arthritis. Nat Commun2015;6:6046. doi:10.1038/ncomms7046 Disclosure of interest None declared

Introduction Autoantibodies to citrullinated proteins (ACPAs) are highly associated with rheumatoid arthritis (RA). ACPAs are produced in the absence of T cell responses to citrullinated proteins. Objectives Peptidyl arginine deiminase 4 (PAD4), which binds numerous different proteins and citrullinates them, is the target of autoantibodies in early RA. This suggests a model to explain the production of ACPAs in the absence of T cells to citrullinated proteins. ACPAs could arise because, at first, PADs are recognised by T cells, which, in turn help the production of autoantibodies to proteins being citrullinated by PADs, through a hapten/carrier model. Here, we test this model in mice. Methods HLA-DRB1*04:01 transgenic mice were immunised subcutaneously with PADs or phosphate buffered saline (PBS) in Freund’s complete adjuvant (CFA). Three booster injections of PAD or PBS in Freund’ incomplete adjuvant (IFA) were given subcutaneously 15, 35 and 55 days later. Mice were: tested for anti-PAD antibodies by ELISA. tested for T cell responses to PADs, native or citrullinated fibrinogen 65 days after PAD immunisation. tested for anti-citrullinated fibrinogen antibodies by ELISA using fibrinogen peptides under citrullinated and native form. Results HLA-DRB1*04:01 transgenic mice immunised with PADs developed antibodies and T cells to PADs and IgG antibodies to citrullinated peptides from fibrinogen, in the absence of T cell response to native or citrullinated fibrinogen. Conclusions T cell immunisation to PAD proteins triggers ACPAs through a hapten carrier mechanism in which the carrier is PAD which performs citrullination and the hapten any protein being citrullinated by PAD. Disclosure of interest None declared

Introduction The production of IgG ACPA can be helped by T lymphocytes specific for PADs, the enzymes that transform arginin into citrullin. Thus, the molecular basis for the HLA-DRB1 association with RA might be the capability for the two alleles encoded by a given HLA-DRB1 genotype to bind PAD4 derived peptides.1 We recently published a table showing that the relative risk to develop ACPA positive RA for the 106 most common genotypes.2 French HLA-DRB1 genotypes varies from 28 to 0.2. For a given HLA-DRB1 genotype, the risk to develop RA should be correlated with the probability for the two HLA-DRB1 molecules encoded by this genotype to bind peptides from PAD peptides. Objectives To test whether the risk of developing ACPA positive RA for each HLA-DRB1 genotypes correlates with the probability for the 2 HLA-DR alleles encoded by each genotype to bind at least one PAD4 peptide. Methods 65 Synthetic peptides (20 mers) of human PAD4 and 167 peptides encompassing the A and B chains of human fibrinogen were synthesised in solid phase. Whenever there was an arginine residue, both the arginine and the citrullin variant were synthesised. In the end, we had 25 fibrinogen peptides containing neither arginin nor citrulline, 71 citrullinated and 71 arginine peptides from fibrinogen. HLA-DRB1 peptide binding studies were performed by adding one microgram of purified HLA-DRB1 to ELISA wells coated with 10 micrograms PAD peptide. Bound HLA-DR was revealed by biotinylated anti HLA-DR antibody followed by peroxidase conjugated avidin. Statistical analyses Correlation between HLA-DRB1 genotypic risk for RA and Likelihood to bind PAD4 for a given genotype was evaluated by Spearman’s Results HLA-DRB1 genotypic risks to develop RA correlate with likelyhood to bind PAD4 peptides (p=0.06, Pearson’s), not citrullinated Fibrinogen peptides (p>0.6 and p>0.9). Conclusions HLA-DRB1 genotypes are associated with a risk to develop RA and a likelihood to bind at least one of 65 overlapping PAD4 peptides. The strong correlation between these two parameters suggest that PAD4 peptide binding to HLA-DRB1 may be the basis of the HLA-DRB1 RA association. Such correlation is not observed when testing the binding of citrullinated or native peptides from Fibrinogen to HLA-DRB1 molecules. References . Fanny Arnoux, Charlotte Mariot*, Elisa Peen*, Nathalie Lambert, Nathalie Balandraud, Jean Roudier, Isabelle Auger. Peptidyl arginine deiminase immunisation induces anti-citrullinated protein antibodies in mice with particular MHC types. PNAS 2017. . Nathalie Balandraud, Christophe Picard, Denis Reviron, Cyril Landais, Eric Toussirot, Nathalie Lambert, Emmanuel Telle, Caroline Charpin, Daniel Wendling, Etienne Pardoux, Isabelle Auger, Jean Roudier. HLA DR and the risk of developping RA. Plos One2013. Disclosure of interest None declared

Background Several risk loci for Rheumatoid Arthritis (RA) have been identified by Genome Wide Association Studies (GWAS), but they do not include Allograft Inflammatory Factor 1 (AIF1). Nevertheless, a few studies have shown that AIF1 rs2269475 (C/T) is associated with RA1,2. Objectives We propose 1) To examine associations in French Caucasian patients with RA, of the seven most described AIF1 SNPs; 2) To study their linkage disequilibrium with HLA-DRB1 alleles; 3) To evaluate whether AIF1 single nucleotide polymorphisms (SNPs) could predict first line treatment responses in RA. Methods We amplified the AIF1 gene region containing the 7 SNPs and sequenced PCR products on a total of 469 individuals, including 95 Anti-Citrullinated Protein Antibody (ACPA) positive RA patients, 146, patients with scleroderma, 132 healthy controls and 96 additional healthy controls selected from a large database of volunteer bone marrow donors (VBMD) for carrying at least one RA-associated allele. Patients and controls were HLA-DRB1 genotyped. Patients with RA were divided into 2 groups, a first group called “non responders” was defined as patients who did not respond to first-line methotrexate (MTX) combined with Etanercept and a second group called “good responders” was defined as patients who did respond to methotrexate combined with Etanercept. Results Two SNPs were associated with RA: rs4711274 (G/A) and rs2269475 (C/T). The frequency of minor allele carriers was respectively 37% (A) and 36% (T) in patients with RA versus 18% among controls (p=0.0014 and p=0.001). Furthermore, patients with RA-associated HLA-DRB1 alleles carried more often minor alleles for both SNPs (p=0.0005). Preliminary clinical data show that 56% of non-responders (N=16) carried the minor alleles of both rs4711274 and rs2269475 compared to only 21% of good responders (N=24, p=0.02). Conclusions AIF is an inflammation-responsive protein encoded within the HLA class III region on chromosome 6 (6p21.3). As already described in British and Polish Caucasians, we found a significant AIF1 Rs2269475 association with RA. We also found an association with Rs4711274 in linkage disequilibrium with the former. The increased frequency of minor AIF1 alleles in RA was not associated with a particular HLA-DRB1 allele, but to any HLA-DRB1 allele carrying the shared epitope. Finally, patients who failed to respond to Etanercept and MTX carried more often minor alleles of the 2 described AIF1 SNPs. Further analysis on a larger groups of patients is required to confirm whether AIF1 SNPs can predict response to therapy with Etanercept and Methotrexate. References Harney SM, Vilarino-Guell C, Adamopoulos IE, Sims AM, Lawrence RW, et al. (2008) Fine mapping of the MHC Class III region demonstrates association of AIF1 and rheumatoid arthritis. Rheumatology 47: 1761–1767. Pawlik A, Kurzawski M, Szczepanik T, Dziedziejko V, Safranow K, et al. (2008) Association of allograft inflammatory factor-1 gene polymorphism with rheumatoid arthritis. Tissue antigens 72: 171–175. Disclosure of Interest None declared

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, with a 0.5% worldwide prevalence. The cause of RA remains unknown, however both genetic and environmental factors may contribute to its development. Among these is the Epstein-Barr virus (EBV). Here, we discuss several aspects of the close relationship between EBV and RA. Patients with RA have impaired control of EBV infection. Indeed, they have high titres of antibodies against EBV antigens. Their peripheral blood T lymphocytes are less efficient at controlling the outgrowth of EBV-infected B cells. RA patients have more EBV-infected B cells than normal controls, leading to a 10-fold systemic EBV overload. Post-transplant lymphoproliferative disorder (PTLPD) is a polyclonal EBV-positive B lymphocyte proliferation, which can evolve into an EBV-positive B cell lymphoma. RA patients also have an increased risk of developing EBV-associated lymphoproliferative disorder (LPD). Hence the need to monitor EBV load when treating RA patients with immunosuppressors. EBV, a widespread virus, highly recognized by antibodies but never eliminated, is an ideal candidate to trigger chronic immune complex disease. Anti-EBV antibody responses should be considered as one of the chronic autoantibody responses linked to the development of RA, in the same way as anti-citrullinated protein antibodies.

International audience; BACKGROUND:Epstein-Barr Virus (EBV) is a widely disseminated lymphotropic herpes virus implicated in benign and malignant disorders. In transplant patients, immunosuppressive drugs (cyclosporine) diminish control of EBV replication, potentially leading to lymphoproliferative disorders (LPD). Rheumatoid arthritis (RA) patients have impaired control of EBV infection and have EBV load ten times higher than controls. As post transplant patients, patients with RA have increased risk of developing lymphomas. Immunosuppressive drugs used to treat RA (conventional disease modifying drugs cDMARDs or biologics bDMARDs) could enhance the risk of developing LPD in RA patients. We have previously shown that long term treatment with Methotrexate and/or TNF alpha antagonists does not increase EBV load in RA. Our objective was to monitor the Epstein-Barr Virus load in RA patients treated with Abatacept (CTLA4 Ig), a T cell coactivation inhibitor, and Tocilizumab, an anti IL6 receptor antibody.METHODS:EBV load in the peripheral blood mononuclear cells (PBMCs) of 55 patients under Abatacept (in 34% associated with Methotrexate) and 35 patients under Tocilizumab (in 37% associated with Methotrexate) was monitored for durations ranging from 6 months to 3 years by real time PCR. The influences of treatment duration and disease activity score 28 (DAS28) index on EBV load were analyzed.RESULTS:Abatacept did not significantly modify EBV load over time. Tocilizumab significantly diminished EBV load over time. No patient (of 90) developed EBV associated lymphoma.CONCLUSION:Long term treatment with Abatacept or Tocilizumab does not increase EBV load in the PBMNCs of patients with RA.

International audience; In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10(-4)) and 60% versus 28% (P = 3.10(-8)). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10(-10)) and 82% (P = 5.10(-13)). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P< 0.0001) and correlated with disease activity (p< 0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3'-5' exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis.

We report the case of a 40-year old man diagnosed with a scleroderma-like disease. Clinical similarities with graft versus host disease prompted initial testing for chimerism employing fluorescence in situ hybridization (FISH). Female cells were observed within peripheral blood mononuclear cells from the patient. Because maternal cells have been detected in healthy immunologically competent adults and patients with autoimmune conditions, we hypothesized that these cells were of maternal origin. Contrary to our expectations, HLA-specific quantitative PCR (QPCR) ruled out maternal microchimerism. However, HLA-specific QPCR testing was positive for the paternal HLA haplotype that the patient did not inherit. We reasoned that the most likely origin of chimerism with non-inherited paternal HLA alleles was from an unrecognized "vanished" twin. The patient had never received a blood transfusion. This report suggests that cells from a vanished twin are a possible source of chimerism. The frequency of chimerism fr...

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Objectives. Male microchimerism (Mc) persisting from pregnancy has been found at greater frequencies and/or higher quantities in women with scleroderma (SSc) compared with controls, suggesting a possible role in disease development. Moreover, women with an HLA-compatible child have a higher risk to develop SSc. We tested the hypothesis, on our French SSc cohort, that women with lcSSc and dcSSc, two distinct clinical subsets, have a different profile in terms of Mc and HLA compatibility in families. Methods. We studied 98 women (52 lcSSc and 46 dcSSc) for male Mc, by real-time PCR, in their whole blood and/or peripheral blood mononuclear cells (PBMC). Similarly, 91 matched healthy women were analysed. Complete HLA-DRB1 typing was obtained for 58 SSc and 68 control families (proband/children). Results. Women with lcSSc (N ¼ 50) had male Mc more often in their whole blood than women with dcSSc (N ¼ 40, 20 vs 5%, P ¼ 0.038), but not significantly more than controls. By contrast, women with dcSSc (N ¼ 36) hold Mc more often in PBMC (25 vs 9%), but not significantly and have greater quantities than controls (N ¼ 82, P ¼ 0.048). This contrast is also visible in feto-maternal HLA-DRB1 compatibility, which was increased only among women with lcSSc (N ¼ 33) compared with controls (N ¼ 68, P ¼ 0.003). Conclusion. For the first time, we showed that women with lcSSc and dcSSc hold male Mc in different blood compartments. Furthermore, a distinct pattern between the two SSc subtypes is observed for feto-maternal HLA-DRB1 compatibility. These results suggest a different mechanism behind each type of disease.

Objective: To test whether HLA-DR alleles influence the production of particular autoantibodies in rheumatoid arthritis (RA) patients, we screened synovial proteins with sera of RA patients homozygous for different HLA-DR alleles by using 2D blots. We found that sera of RA patients homozygous for HLA-DRB1*0404 recognised a 100-kDa synovial protein identified as calpastatin. We studied B and T cell epitopes on calpastatin and their association with HLA-DRB1*0404. Methods: The frequency of positive sera in patients expressing different RA-associated HLA-DR allele combinations was calculated by inhouse ELISA using purified synovial calpastatin or calpastatin peptides encompassing the entire calpastatin protein as immunosorbent. Interaction between calpastatin peptides and HLA-DR alleles was tested by a direct binding assay. T cell responses to calpastatin were measured in RA patients and controls. Results: We found that RA-associated HLA-DR alleles are associated with presence of autoantibodies to synovial calpastatin in RA patients’ sera. HLA-DRB1*0404 is strongly associated with antisynovial calpastatin in RA sera. One linear B cell epitope is preferentially associated with HLA-DRB1*0404. Multiple peptides from calpastatin bind every tested HLA-DR allele associated or not with RA. Peptides from domain 1 and 4 of calpastatin are the best HLA-DR allele binders. The T cell response to calpastatin is frequent in RA patients and independent of the HLA-DR background. Conclusions: HLA-DRB1*0404 is strongly associated with anticalpastatin antibodies in rheumatoid arthritis.

Resume Il existe de multiples relations entre le virus d'Epstein-Barr (EBV) et la polyarthrite rhumatoide (PR) : les malades atteints de PR ont des titres d'anticorps diriges contre les antigenes d'EBV plus eleves par rapport a des sujets sains ; la PR est caracterisee par un defaut de controle cellulaire de l'infection par EBV ; des similitudes antigeniques entre EBV et des composants du soi ont ete observees (sequences glycine–alanine d'EBNA-1 et proteines de la synoviale ; glycoproteine gp110 et epitope partage) ; des reponses cellulaires dirigees contre des proteines du cycle replicatif et contre gp110 ont ete observees au niveau du liquide articulaire de PR ; EBV (ses antigenes ou son genome) a ete mis en evidence au niveau de la synoviale de PR par hybridation in situ ou PCR, avec un rendement variable ; la charge virale en EBV des cellules mononucleees du sang circulant est elevee dans la PR comparativement aux temoins; EBNA-1 est susceptible de subir une citrullination et des anticorps antipeptides citrullines peuvent etre induits par EBV ; des lymphomes compliquent la PR et certains sont associes a EBV. Ainsi, les liens EBV–PR sont nombreux et complexes, mais la relation de causalite directe n'a pas ete formellement etablie. Le virus d'Epstein-Barr pourrait agir indirectement dans le schema physiopathologique de la PR, par l'intermediaire du mauvais controle de l'infection et d'une exposition chronique a ses antigenes, sources de reaction inflammatoire chronique. L'effet des nouvelles therapeutiques de la PR (biotherapies) sur la cohabitation EBV-hote necessite d'etre analyse.

International audience; OBJECTIVE: To determine whether the -308 A/G tumor necrosis factor alpha (TNFalpha) gene polymorphism can predict the outcome of etanercept therapy in 86 patients with rheumatoid arthritis (RA), as already observed in patients treated with infliximab. METHODS: Eighty-six RA patients treated with etanercept were genotyped for -308 A/G TNFalpha gene polymorphism by polymerase chain reaction and melting curve analysis, using specific gene primers and probes. Patients were subdivided into group A (G/A genotype) and group G (G/G genotype). We compared clinical responses to etanercept between groups A and G after 6 months, using the Disease Activity Score in 28 joints (DAS28). After 12-month treatment, 48 of 86 patients were evaluated again. RESULTS: Of 86 patients, 18 (21%) belonged in group A and 68 (79%) belonged in group G. After 6-month treatment, 55.6% of patients in group A and 82.4% of patients in group G had DAS28 improvement >1.2 (P = 0.027 by chi-square). The mean +/- SD DAS28 improvement was 1.69 +/- 1.31 in group A and 2.23 +/- 1.19 in group G (P = 0.098 by t-test). After 1-year treatment 48 patients were tested again: 10 (21%) belonged in group A and 38 (79%) belonged in group G. Forty percent of patients in group A and 87% in group G had DAS28 improvement >1.2 (P = 0.005 by chi-square). The mean +/- SD DAS28 improvement was 1.334 +/- 1.37 in group A and 2.29 +/- 1.47 in group G (Mann-Whitney U test = 115, P = 0.0057). CONCLUSION: RA patients with a -308 G/G TNFalpha genotype respond to etanercept better than patients with a -308 A/G genotype.