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Journal of Autoimmunity 37 (2011) 263e272

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Journal of Autoimmunity

journal homepage: www.elsevier .com/locate/ jaut imm

The antigen specificity of the rheumatoid arthritis-associated ACPA directedto citrullinated fibrin is very closely restricted

Cristina Iobagiu a,b, Anna Magyar c,1, Leonor Nogueira a,b,1, Martin Cornillet a, Mireille Sebbag a,Jacques Arnaud a, Ferenc Hudecz d, Guy Serre a,b,*

a Laboratory of “Epidermis Differentiation and Rheumatoid Autoimmunity”, UMR 5165 CNRS-Toulouse III University, Purpan Hospital, Place du Dr Baylac,TSA 40031, 31059 Toulouse cedex 9, Franceb Laboratory of Cell Biology and Cytology, Purpan Hospital, Place du Dr Baylac, TSA 40031, 31059 Toulouse cedex 9, FrancecResearch Group of Peptide Chemistry, Hungarian Academy of Sciences, ELTE Budapest, Pázmány Péter sétány 1/A, 1117 Budapest, HungarydResearch Group of Peptide Chemistry, Department of Organic Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1A, H-1117 Budapest, Hungary

a r t i c l e i n f o

Article history:Received 15 June 2011Received in revised form21 July 2011Accepted 28 July 2011

Keywords:Anti-citrullinated protein autoantibodies(ACPA)CitrullinationB-Cell epitopesFibrinRheumatoid arthritis

Abbreviations: RA, rheumatoid arthritis; ACPA, anttibodies; Cit, citrulline; AhFibA, anti-human citrullinate* Corresponding author. Laboratory of “Epidermis

toid Autoimmunity”, UMR 5165 CNRS-Toulouse IIIPlace du Dr Baylac, TSA 40031, 31059 Toulouse cedex401; fax: þ33 561 499 360.

E-mail addresses: cristina.iobagiu@ch-roanne.fr ((A. Magyar), nogueira.l@chu-toulouse.fr (L. Nogueira)fr (M. Cornillet), mireille.sebbag@udear.cnrs.fr (M. S(J. Arnaud), fhudecz@elte.hu (F. Hudecz), serre.sec@ch

1 These authors contributed equally to the work.

0896-8411/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.jaut.2011.07.003

a b s t r a c t

The major targets of the disease-specific autoantibodies to citrullinated proteins (ACPA) in synovium ofrheumatoid arthritis (RA) patients are borne by the citrullinated a- and b-chains of fibrin. We demon-strated that ACPA target a limited set of citrullinated fibrin peptides and particularly four multi-citrullinated peptides which present the major epitopes.

In this study, we established the clear immunodominance of the peptides a36e50Cit38,42 and b60e74Cit60,72,74 which were recognised by 51/81 (63%) and 61/81 (75%) of ACPA-positive patients, respectively,more than 90% recognising one, the other or both peptides. We also identified the citrullyl residuesaCit42, bCit72 and bCit74 as essential for antigenicity, and at a lesser degree aCit38. Then, we assayed onoverlapping 7-mer peptides encompassing the sequences of the two peptides, 3 series of sera recog-nising either a36e50Cit38,42 or b60e74Cit60,72,74 or both peptides. In each series, the reactivity profiles ofthe sera, largely superimposable, allowed identification of the two 4/5-mer overlapping epitopes(a: VECit42HQ and a0: Cit38VVE), and the single 5-mer epitope (b: GYCit72ACit74), all located to a flexibleglobular domain of fibrin on a topological 3D model.

In conclusion, we demonstrated that only 3 immunodominant epitopes are targeted by ACPA on cit-rullinated fibrin stressing their actual oligoclonality. However, the reactivity to the 3 epitopes distin-guishes three subgroups of patients. The closely restricted antigen specificity suggests that theautoimmune reaction to citrullinated fibrin is antigen-driven. The accessibility of the epitopes reinforcesthe hypothesis of a pathogenic role for ACPA via immune complexe formation in the synovial tissue.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Rheumatoid arthritis (RA), an autoimmune disease affecting0.5e1% of the worldwide population, still requires elucidation of its

i-citrullinated protein autoan-d fibrinogen autoantibodies.Differentiation and Rheuma-University, Purpan Hospital,9, France. Tel.: þ33 561 158

C. Iobagiu), magyar@elte.hu, martin.cornillet@udear.cnrs.ebbag), arnaud@udear.cnrs.fru-toulouse.fr (G. Serre).

All rights reserved.

aetiology. In the complex autoimmune dysregulation admitted tobe responsible for the chronic inflammation, various genetic factorshave been incriminated such as HLA haplotypes or polymorphismsof the citrullinating enzyme peptidylarginine deiminase (PAD), andalso environmental factors such as smoking [1,2]. However, theirprecise role in the pathophysiology of RA has still to be clarified.One definitively established association to RA is the presence ofautoantibodies to citrullinated proteins (ACPA). Indeed, ACPA arepresent in the sera of almost 80% of patients with a very highdiagnostic specificity of 98% [3,4]. In addition, they can be detectedearly, even before the onset of symptoms [5], and are associatedwith a more aggressive disease course [6]. Characterisation of theantigens targeted by ACPA led to the finding that the ACPA epitopesare sequential peptidic epitopes which contain citrullyl (Cit) resi-dues, generated by posttranslational deimination (citrullination) ofarginyl residues [7,8]. Soon after, citrullinated forms of the a- and

C. Iobagiu et al. / Journal of Autoimmunity 37 (2011) 263e272264

b-chains of fibrin, abundant in the inflamed synovial tissue of thejoints, were identified as major in vivo targets of ACPA [9].Accordingly, use of in vitro citrullinated human fibrinogen asimmunosorbent proved to be very efficient for ACPA detection.Indeed, this assay (AhFibA-ELISA) presented a very high diagnosticvalue, closely similar to that of the CCP2-ELISAwhich uses syntheticcyclic citrullinated peptides and is recognised as the most per-formant assay for ACPA [3,10].

The presence in diseased joints of both ACPA and their target[11] suggests that these antibodies play a role in the pathophysi-ology of RA. We hypothesised that, in vivo, the interaction of cit-rullinated fibrin deposits with locally produced ACPA results in theformation of immune complexes able to chronically maintaininflammation. Indeed, these immune complexes probably activatepro-inflammatory effectormechanisms, the resulting inflammationleading to formation of fibrin deposits secondarily citrullinated bya locally expressed PAD and therefore becoming new targets forACPA [12]. Accordingly, we demonstrated that, in vitro, immunecomplexes containing ACPA and citrullinated fibrinogen triggerTNFa secretion bymacrophagesmainly via engagement of their Fcgreceptor IIa [13]. Moreover, we also showed that among the 5 PADisotypes, only PAD type 2 and type 4 are expressed in the rheu-matoid synovial tissue and probably both involved in the citrulli-nation of fibrin [14].

The profiling of RA autoantibodies performed with autoantigenmicroarrays confirmed that fibrinogen-derived citrullinatedpeptides are targeted in RA [15]. Moreover, we mapped thesequential epitopes recognised by ACPA on the a- and b-chains ofcitrullinated fibrin [16]. Among seventy-one 15-mer citrullinatedpeptides, only 18 were specifically immunodetected by at least oneof 20 ACPA-positive RA sera. Among them, four peptides, termeda36e50Cit38,42, a501e515Cit510,512, a621e635Cit621,627,630 andb60e74Cit60,72,74, were shown to bear the major ACPA epitopes,since each of themwas recognised by at least 9 out of the 20 ACPA-positive RA sera [16] and were selected for further analysis.Moreover, the groups of RA sera reactive with the two mostimmunodominant a36e50Cit38,42 and b60e74Cit60,72,74 peptides(individually recognised by 14 and 12 out of the 20 sera, respec-tively) only weakly overlapped. All the 20 sera exhibited reactivityto at least one of the 2 peptides.

The first aim of the present study was to confirm the immu-nodominance of a36e50Cit38,42 and b60e74Cit60,72,74 by evaluatingthe proportion of sera containing autoantibodies to the peptides ina larger cohort of RA patients. The second one was to refine epitopemapping and search for the minimal sequential epitopes targetedby ACPA on a36e50Cit38,42 and b60e74Cit60,72,74. Therefore, wetested the reactivity of series of RA sera to overlapping heptapep-tides derived from their respective sequences, and analysed theprofiles of reactivity. Our results show that a36e50Cit38,42 and/orb60e74Cit60,72,74 are recognised by more than 90% of the ACPA-positive sera and confirm them as bearing the immunodominantepitopes of citrullinated fibrin. Moreover we identified thoseepitopes as three 4/5-mer citrullinated peptides whereas two otherputative citrullinated epitopes were unreactive, demonstrating thatthe antigen specificity of ACPA is very closely restricted. Never-theless, the recognition of the three immunodominant epitopesallowed three subgroups with different ACPA antigen specificitiesto be distinguished among RA patients.

2. Material and methods

2.1. Serum samples

Sera were obtained from patients attending the departments ofrheumatology of Purpan and Rangueil hospitals (Toulouse, France).

They were sent to the Laboratory of Cell Biology and Cytology ofPurpan hospital for serological investigation of ACPA. Afterwards,the serum leftovers were stored at �80 �C until anonymouslyassayed in research protocols in accordance to national ethical laws.Clinical diagnoses were established by rheumatologists accordingto the relevant international classification criteria.

For ELISA testing of 15-mer fibrin-derived peptides, 100 serafrom patients with RA and 200 sera from patients with non-RArheumatic diseases (control samples) were selected. The controlgroup was comprised of 38 inflammatory diseases (6 ankylosingspondylitis, 7 psoriatic arthritis, 6 systemic lupus erythematosus, 3systemic sclerosis and 13 other inflammatory diseases) and 162noninflammatory diseases (55 osteoarthritis, 28 Paget’s disease, 25malignant bone diseases, 16 reflex sympathetic dystrophy, 13compressive neuropathy and 25 other noninflammatory diseases).These two groups (RA and control) included 83 (83%) and 98 (49%)women respectively. The mean age of patients was 59 years for RA(SD: 13) and 63 (SD: 11) for controls. This series was prepared to berepresentative for the presence of ACPA assayed with variousreference tests, i.e. immunofluorescence on rat esophagus cry-osections for antikeratin antibodies, immunoblotting on humanfilaggrin-enriched epidermis extracts for antifilaggrin antibodies,AhFibA-ELISA and anti-CCP2-ELISA (Immunoscan RA; Euro-diagnostica, Arnhem, The Netherlands). For all the tests, the diag-nostic indexes were similar to those usually observed in cohorts ofwell-characterised rheumatic diseases [4,17]. In addition, 76/100 RAand 22/200 control sera were rheumatoid factor-positive whenassayed by nephelometry (IMMAGE Immunochemistry System,Beckman Culter, Fullerton, CA) using the threshold of 20 IU/ml.

The variously citrullinated variants of the 15-mer a36e50Cit38,42and b60e74Cit60,72,74 fibrin-derived peptides were tested by ELISAwith a previously described pool of RA sera formed bymixing equalvolumes of 10 ACPA-positive RA sera, chosen among 115 RA serareactive to both the Aa- and Bb-chains of citrullinated fibrinogen byimmunoblotting [16]. In addition,with each peptide, a pool of ACPA-negative sera, prepared as described in [16], was also tested.

For the testing of pin-bound peptides, 3 groups of RA sera wereselected according to their reactivity to the a36e50Cit38,42 and/orb60e74Cit60,72,74 fibrin-derived 15-mer peptides as assayed byELISA on microtitration plates (see subsection 2.3.), the sera beingconsidered positive when their titre reached thresholds corre-sponding to a diagnostic specificity of 95%, as established using theabove-mentioned representative series of sera. Ten sera werechosen that were reactive to a36e50Cit38,42 only and were referredto as the “a-sera”, 10 sera were reactive to b60e74Cit60,72,74 only(“b-sera”), and 10 RA sera were reactive with both peptides(“ab-sera”). In addition a pool formed by mixing equal volumes of10 AhFibA-negative sera also unreactive with a36e50Cit38,42 andb60e74Cit60,72,74, was used as the negative control (“ab-negativepool”).

2.2. Synthetic peptides

Four citrullinated fibrin-derived 15-mer peptides and their non-citrullinated counterparts harbouring arginyl instead of citrullylresidues were purchased from NeoMPS (Strasbourg, France). Thefour a36e50Cit38,42, a501e515Cit510,512, a621e635Cit621,627,630 andb60e74Cit60,72,74 peptides were named according to the fibrinogenchain from which their sequence was derived (a or b), withnumbers indicating the position of their N- and C-terminal aminoacid (aa) residues in the related fibrinogen chain and indexnumbers giving the position of citrullyl residues, as previouslydescribed [16]. Using the standard one-letter code for common aaresidues, and “Cit” for citrullyl residues, their sequences areGPCitVVECitHQSACKDS, SGIGTLDGFCitHCitHPD, CitGHAKSCitPVCit

C. Iobagiu et al. / Journal of Autoimmunity 37 (2011) 263e272 265

GIHTS and CitPAPPPISGGGYCitACit, respectively. The C-terminalcitrulline residue was synthesized with a terminal carboxamideinstead of a free carboxylic group.

On the other hand, we synthesized 2 sets of heptapeptidescovalently attached to pin-supported crowns (Chiron Mimotopes,Clayton, Australia) and referred to as “pin-bound peptides”, thatsequentially overlapped with each other by 5 aa residues andencompassed the sequences of either the aa residues 34e48 of thefibrinogen Aa-chain or the aa residues 58e77 of the fibrinogen Bb-chain. The sets also incorporated the 15-mer a36e50Cit38,42 andb60e74Cit60,72,74 peptides. All possible variants of peptides, con-taining either arginyl or one or several citrullyl residues, weresynthesized on b-alanine-functionalized polyethylene pins withFmoc/tBu chemistry according to Geysen’s method [18] with somemodifications. We used t-butyl (tBu) for Ser, Tyr, Asp and Glu,acetamidomethyl (Acm) for Cys, trityl (Trt) for Gln and His,2,2,4,6,7-pentamethyldihydrobenzo-5-sulfonyl (Pbf) for Arg, andt-butoxylcarbonyl (Boc) for Lys as side-chain protecting groups. The9-fluorenylmethoxycarbonyl (Fmoc) a-amino protecting groupwasremoved with 20% piperidine/dimethylformamide (v/v). Thecoupling was performed with diisopropylcarbodiimide (DIPCI)/1-hydroxybenzotriazole (HOBt) methodology and monitored withbromophenol blue added to the reaction mixture [19]. After thefinal coupling cycle, the Fmoc protecting groups were removed andpeptides were acetylated with a mixture of acetic anhydride:N,N-diisopropylethylamine (DIEA) or left with free N-terminus. Theside-chain protecting groups were removed with trifluoroaceticacid containing 2.5% ethanditiol and 2.5% thioanisole (v/v/v) exceptAcm on Cys. The unprotected peptides remained covalentlyattached to the pins. All peptides were synthesized in duplicates.Peptide coating was estimated at w66 nmol/pin. Control peptidesfor purity and efficiency of synthesis [20] and negative controls(crowns without peptide) were included on each set.

2.3. Testing of peptide reactivity

ELISA testing of each fibrin-derived 15-mer peptides was per-formed on microtitration plates as previously described [16]. Inaddition, testing of the association of a36e50Cit38,42 andb60e74Cit60,72,74 (a36e50Cit38,42 þ b60e74Cit60,72,74) was per-formed with the same protocol, using a mixture of both peptides at5 mg/mL each for coating the plates. Pooled sera, as well as indi-vidual sera, were tested diluted to 1:50.

For the testing of pin-bound peptides, the pins fitting into 96-well microtitration plates were held on blocks, one block per setof a36e50Cit38,42- or b60e74Cit60,72,74-derived peptides. Afterblocking for 1 h under agitation in PBS pH 7.4 containing 2% BSA(SigmaeAldrich, Lyon, France), hereafter referred to as PBS-BSA,followed by three washes in PBS 0.1% Tween-20, the pins wereincubated overnight at 4 �C under agitation with the serum dilutedin PBS-BSA containing 2 M NaCl. Individual serum dilutions wereadjusted so as to equalize their avidities to the 15-mer peptiderelated to the peptide set on which they were tested (titrez OD of1.00 in the ELISA on the 15-mer peptide). After washing, complexedIgG were incubated with peroxidase-conjugated rabbit anti-humanIgG antibodies (Dako, Denmark) diluted to 1/2000 in PBS-BSA, for1 h at room temperature. After a newwashing step, the presence ofperoxidase activity was revealed by a 4-min incubation with OPD(SigmaeAldrich) in citrate buffer (pH 5). The reaction was stoppedand ODs were measured at 492 nmwith an automated plate reader(Multiskan RC; Labsystems, Life Sciences International SA, CergyPontoise, France). Removal of complexed antibodies and storage ofpins were done according to the manufacturer’s recommendations.

In all assays, titres were considered as the difference betweenthe OD obtained with the citrullinated peptide and that obtained

with its non-citrullinated counterpart (DOD). Interassay variationswere corrected by linear regression analysis using a referencepeptide and a known reactive pool of sera giving a 4-point standardcurve and tested on each plate. Sera were tested twice with twoindependent dilutions and the results were averaged. When theresults were discordant, the experiments were repeated two (ormore) times and the results from at least 3 concordant experimentswere averaged.

2.4. Topological 3D model

A topological 3D model of the central E domain of human fibrinincluding portions corresponding to the uncitrullinated a36e50and b60e74 peptides was produced. First, 3D structures of thea36e50 and b60e74 peptides were produced by the BioinformaticTool Server @TOME V2 (@utomatic Threading OptimisationModeling & Evaluation) at the CBS Informatics Team [http://abcis.cbs.cnrs.fr/ATV2/meta.html] [21]. They were refined by energyminimization (18000 iterations) and validated by Ramachandranplot analyses using MSI Insight II modules Biopolymer, CHARMMand Viewer on an O2 SGI workstation and finally analysed on the“Swiss-Pdb Viewer 4.0.1” [http://www.expasy.org/spdbv/] [22].Then, the only E domain of human fibrin with atomic coordinatesavailable in the Protein Data Bank [http://www.wwpdb.org] (PDBaccession code 2A45) was selected. This model does not include thesequences of the a36e44 and b45e83 [23] and was completed. Aa44e50 were removed from the a-chain and aa 45e59 and 75e83were added to the beta chain before the selected 3D model ofa36e50 and b60e74 were incorporated, in their respective chains.After further refinement by energy minimization (6000 iterations),the model was finally validated using MSI Insight II modules.

2.5. Statistical analysis

Data analyses were performed using Medcalc software (Maria-kerke, Belgium). Median differences were tested with the Man-neWhitney U test and diagnostic sensitivities computed on ourcohort of patients were comparedwith the chi2 test. p values� 0.05were considered significant.

3. Results

3.1. ELISA testing of 4 citrullinated fibrin-derived peptides,a36e50Cit38,42, a501e515Cit510,512, a621e635Cit621,627,630and b60e74Cit60,72,74, with a large cohort of patients

The reactivity of each peptide was tested by ELISA with 100 RAsera and 200 control sera from patients with perfectly characterisednon-RA rheumatic diseases. In this series of patients, the diagnosticsensitivity of the AhFibA-ELISA was 81% for a 95% diagnosticspecificity and 78% for a 98.5% diagnostic specificity (Table 1). Thisdiagnostic performance is similar to that we previously reported[4,10].

The ELISA titre of the reactivity to a citrullinated peptide wastaken as the difference between the OD (optical density) obtainedwith the citrullinated peptide and that obtained with itsnon-citrullinated counterpart (DDO; Supplementary Figure). In theRA sera, the titres ranged from 0 to 3.82 (median 0.110) fora36e50Cit38,42, from0 to 3.50 (median 0.077) for a501e515Cit510,512,from 0 to 3.44 (median 0.059) for a621e635Cit621,627,630 and from0 to 4.65 (median 0.264) for b60e74Cit60,72,74. In the control sera, thetitres ranged from0 to 0.766 (median0.055) for a36e50Cit38,42, from0 to 2.366 (median 0) fora501e515Cit510,512, from0 to 0.485 (median0) for a621e635Cit621,627,630 and from 0 to 0.281 (median 0.035) forb60e74Cit60,72,74. For the 4 peptides, the titres were found to be

Table 1Diagnostic sensitivities (95% confidence intervals) of autoantibodies to 4 fibrin-derived citrullinated peptides, compared to those of autoantibodies to human cit-rullinated fibrinogen (AhFibA), computed at 2 diagnostic specificity thresholds (95and 98.5%).

Specificity

95% 98.5%

AhFibA 81(73.3e88.7) 78(69.9e86.1)a36e50Cit38,42 55(45.2e64.8) 46(36.2e55.8)a501e515Cit510,512 13(6.4e19.6) 6(1.3e10.7)a621e635Cit621,627,630 37(27.5e46.5) 31(21.9e40.1)b60e74Cit60,72,74 63(53.5e72.5) 55(45.2e64.8)a36e50Cit38,42 þ b60e74Cit60,72,74a 72(63.2e80.8) 66(56.7e75.3)

a The association of a36e50Cit38,42 and b60e74Cit60,72,74was tested on a mixtureof the same amounts of a36e50Cit38,42 and b60e74Cit60,72,74.

C. Iobagiu et al. / Journal of Autoimmunity 37 (2011) 263e272266

significantly higher in RA than in control sera (p � 10�3). Two posi-tivity thresholds, allowing a specificity of 95% (10 positive controlsera) or 98.5% (3 positive control sera) to be reached, were calculatedwith the control sera tested on each peptide. As shown in Table 1, ata diagnostic specificity of 95%, the diagnostic sensitivities obtainedwith b60e74Cit60,72,74 and a36e50Cit38,42 (63% and 55%, respec-tively) were both significantly higher than those obtained witha621e635Cit621,627,630 and a501e515Cit510,512 (37% and 13%,respectively; p < 0.05). Their higher sensitivity was confirmed ata 98.5% specificity (p < 0.05).

At 95% specificity, 80% of the RA sera were found to be reactiveto either a36e50Cit38,42 or b60e74Cit60,72,74, and when we evalu-ated the diagnostic performance of a mixture of a36e50Cit38,42 andb60e74Cit60,72,74 used together as the immunosorbent, 72% of thesera were found to be positive. This sensitivity is not different fromthat of the AhFibA-ELISA (p ¼ 0.18; non significant; Table 1).

Analysing the individual profile of reactivity of each AhFibA-positive RA serum to the 4 peptides (Table 2), we found thatsera reactive with a621e635Cit621,627,630 constituted a subgroup ofthose reactive with b60e74Cit60,72,74. Indeed, 33/37 (89%) of serarecognising a621e635Cit621,627,630 were also reactive withb60e74Cit60,72,74 whereas 28/61 (46%) sera reactive withb60e74Cit60,72,74 did not recognise a621e635Cit621,627,630. Similarly,12/13 (92%) sera reactive with a501e515Cit510,512 were also reactivewith a36e50Cit38,42 whereas 39/51 (76%) sera recognisinga36e50Cit38,42 did not recognise a501e515Cit510,512, demonstrating

Table 2Distribution of the 81 AhFibA-positive RA sera, according to the presence (þ) or theabsence (blank) of autoantibodies to a36e50Cit38,42, b60e74Cit60,72,74,a621e635Cit621,627,630 or a501e515Cit510,512 detected by ELISA (the thresholds usedare those giving a 95% diagnostic specificity).

a36e50Cit38,42

b60e74Cit60,72,74

a621e635Cit621,627,630

a501e515Cit510,512

Numberof sera

Total

a-seraa þ 8 13þ þ 2þ þ 3

ab-serab þ þ 11 38þ þ þ 18þ þ þ þ 3þ þ þ 6

b-serac þ 11 23þ þ 12

ab-negativeserad

þ 2 7þ 1

4

a a-sera contain antibodies to a36e50Cit38,42 and not to b60e74Cit60,72,74.b ab-sera contain antibodies to both a36e50Cit38,42 and b60e74Cit60,72,74.c b-sera contain antibodies to b60e74Cit60,72,74 and not to a36e50Cit38,42.d ab-negative sera are unreactive to both a36e50Cit 38,42 and b60e74Cit60,72,74.

that sera reactive with a501e515Cit510,512 constituted a subgroup ofthose reactive with a36e50Cit38,42. Finally, among the AhFibA-positive RA sera, 74 (91.3%) were reactive with eithera36e50Cit38,42 or b60e74Cit60,72,74 or with both peptides, and only 7recognised neither of them. Among the reactive sera, three maingroups couldbedescribed:13 (16%) sera containing autoantibodies toa36e50Cit38,42 only (group referred to as “a-sera”), 23 (28.4%) serawith autoantibodies to b60e74Cit60,72,74 only (“b-sera” group) and 38(46.9%) sera with autoantibodies to both peptides (“ab-sera”).

Altogether, those data show that more than 90% of the AhFibA-positive RA sera exhibit reactivity to a36e50Cit38,42 or/andb60e74Cit60,72,74. Since this is true only for those 2 citrullinatedfibrin-derived peptides among 71 possible, this confirms thatthey bear the immunodominant epitopes recognised by ACPA onthe a- and b-chains of citrullinated fibrin.

We next underwent to further define these epitope(s).

3.2. Unequal contribution to ACPA reactivity of the citrullylresidues of a36e50Cit38,42 and b60e74Cit60,72,74

In order to evaluate how each citrullyl residue of the multi-citrullinated a36e50Cit38,42 and b60e74Cit60,72,74 peptidescontributed to their immunoreactivity, all their possible citrulli-nated variants were probed by ELISA. With a pool of ACPA-negativesera, the specific immunoreactivity to each of the 9 variously cit-rullinated peptides (DOD) was negative, the mean DOD neverreaching values of 0.1. With a pool of ACPA-positive RA sera, asshown in Fig. 1, the titre obtained with the monocitrullinateda36e50Cit42 peptide (citrullyl residue in position 42; Cit42) reachedthe same level as that obtained with the fully citrullinateda36e50Cit38,42 peptide, suggesting a major contribution of Cit42 inthe immunoreactivity of the latter peptide. Nonetheless, that thea36e50Cit38 peptide exhibited 30% of the reactivity of the fullycitrullinated peptide suggested some contribution of Cit38. Con-cerning b60e74Cit60,72,74, absence of reactivity of the b60e74Cit60peptide indicated no contribution of Cit60. On the other hand, thereactivities of b60e74Cit60,74, b60e74Cit72,74 and b60e74Cit74reached levels similar to that of the fully citrullinatedb60e74Cit60,72,74 and the reactivity of b60e74Cit72 reached only 5%

Fig. 1. Immunoreactivity of the monocitrullinated a36e50Cit38, a36e50Cit42,b60e74Cit60, b60e74Cit72 and b60e74Cit74, and multicitrullinated a36e50Cit38,42,b60e74Cit60,74, b60e74Cit72,74 and b60e74Cit60,72,74 peptides analysed by ELISA witha pool of ACPA-positive RA sera. Results are expressed as the ratio of the DOD obtainedwith each peptide to that obtained with the fully citrullinated related peptide (%DOD).

C. Iobagiu et al. / Journal of Autoimmunity 37 (2011) 263e272 267

of this value, suggesting a major contribution of Cit74 and a veryweak contribution of Cit72.

These data indicate that Cit42 and Cit74 predominantlycontribute to the ACPA reactivity, suggesting they belong to theepitopes targeted by ACPA on a36e50Cit38,42 and b60e74Cit60,72,74.

3.3. Identification of the epitope(s) targeted by ACPAon a36e50Cit38,42 and b60e74Cit60,72,74

Our next goal was to precisely define the epitope(s) targeted byACPA on a36e50Cit38,42 and b60e74Cit60,72,74, and to characterisethe diversity of ACPA specificities toward these epitopes withinindividual RA sera. To this aim, we scanned the reactivity of RA serato two sets of pin-bound heptapeptides that overlapped with eachother by 5 aa residues and included at least one citrullyl residue,allowing to sequentially cover the sequences of a36e50Cit38,42 andb60e74Cit60,72,74, respectively. These sets included pin-boundforms of the original 15-mer peptides and all the possible citrulli-nation variants of 15- or 7-mer peptides (see Supplementary Tablefor a full description of each set). Ten sera were chosenwithin each

Fig. 2. Reactivity profiles of individual RA sera containing either antibodies to a36e50Cit3tested with pin-bound peptides derived from a36e50Cit38,42. A. Homogenous profiles obtaiVECit42HQ sequence). B. Sera presented in panel A were tested against each bicitrullinatedprofiles of reactivity obtained with the remaining 2 a-sera and 9 ab-sera leading neverthpresented in panel C were tested against each bicitrullinated peptide comparatively to its twpeptide sequences for aa residues, and Cit indicates a citrullyl residue. All sera were tested

of the three a-sera, b-sera and ab-sera groups (see subsection 3.1.),and each serumwas individually tested with the 2 sets of peptides.

The reactivity obtainedwith each serum on the peptides derivedfrom a36e50Cit38,42 are depicted in Fig. 2. Eight out of 10 a-seraand 1 out of 10 ab-sera recognised only two of the 7-mer peptides,Cit38VVECit42HQ and VECit42HQSA, whereas the other peptideswere poorly or not reactive (Fig. 2A). The common parts of theirsequences allowed us to define the minimal sequential epitopetargeted mainly by the a-sera, the “a-epitope”. Its sequence,VECit42HQ, harbours Cit42 in agreement with the results presentedin Fig. 1. Consistent with these results, the analysis of the reactivityof each pin-bound monocitrullinated peptide (Fig. 2B) compared tothat of its bicitrullinated counterpart, showed that (1) the level ofreactivity of the monocitrullinated 15-mer peptide including Cit42was similar to that of the bicitrullinated counterpart (includingCit38,42) whereas the monocitrullinated form including Cit38 wasnot reactive; (2) the bicitrullinated peptide GPCit38VVECit42 wasnot reactive, suggesting that a terminally situated Cit42 lacks therequired aa environment able to confer immunoreactivity; (3)whereas Cit38VVERHQ lacking Cit42, was not reactive, Cit42conferred the same level of reactivity to RVVECit42HQ than to

8,42 only (a-sera) or antibodies to both a36e50Cit38,42 and b60e74Cit60,72,74 (ab-sera)ned with 8 a-sera and 1 ab-serum, allowing a minimal a-epitope to be defined (boxedpeptide comparatively to its two monocitrullinated counterparts. C. Less homogenouseless to the definition of an additional a0-epitope (boxed Cit38VVE sequence). D. Serao monocitrullinated counterparts. Below the panels, standard one-letter code is used inat least twice in two independent dilutions and the obtained results were averaged.

C. Iobagiu et al. / Journal of Autoimmunity 37 (2011) 263e272268

Cit38VVECit42HQ. Taken together, these results confirmed that thepresence of Cit42, but not that of Cit38 is important for reactivity toa36e50Cit38,42.

The remaining sera, 9 out of 10 ab-sera and 2 out of 10 a-serarecognised the a36e50Cit38,42-derived 7-mer peptides with lesshomogenous profiles (Fig. 2C). The heptapeptides VRGPCit38VV,GPCit38VVECit42, Cit38VVECit42HQ and VECit42HQSA were all rec-ognised with various intensities, indicating that, in addition to theabove-mentioned a-epitope, these remaining sera also targeted an“a0-epitope”, encompassing Cit38 and included in the GPCit38VVEsequence. In addition, as shown is Fig. 2D depicting the reactivity ofthese sera to the various citrullination variants of multicitrullinatedpeptides, (1) Cit38 is conferring to GPCit38VVER the same level ofreactivity than to GPCit38VVECit42 suggesting that it is responsibleof the immunoreactivity of the a0-epitope; (2) 7 out of 10 serareacting with Cit38VVECit42HQ are also reactive not only withRVVECit42HQ (a-epitope) but also with Cit38VVERHQ (overlappingwith the sequence containing the a0-epitope); (3) the levels ofreactivity obtained with GPCit38VVER and Cit38VVERHQ are similar,allowing to narrow the sequence including the a0-epitope toCit38VVE.

In total, two minimal epitopes were defined on a36-50Cit38,42:the a-epitope, VECit42HQ, targeted by a majority of a- and ab-seraand the a0-epitope, Cit38VVE, mainly targeted by the ab-sera. Asexpected, none of the b-sera was reactive with any of thea36e50Cit38,42-derived peptides (data not shown).

The reactivity profiles obtained with the 7-mer peptides derivedfrom b60e74Cit60,72,74 are shown in Fig. 3. All b-sera and 8 out of 10ab-sera recognised not only GGYCit72ACit74P, including Cit72 andCit74, but also the overlapping YCit72ACit74PAK peptide, althoughreactivity to the latter was less intense (Fig. 3A). The sequence theyshare allowed the minimal “b-epitope” to be localised in theGYCit72ACit74P sequence. Comparisons of the reactivities of thevarious citrullination variants of the peptides (Fig. 3B) showed that:

Fig. 3. Reactivity profiles of individual RA sera containing either antibodies to b60e74Cit60tested with pin-bound peptides derived from b60e74Cit60,72,74. A. Homogenous profiles odefined (boxed GYCit72ACit74 sequence). B. Each multicitrullinated peptide was tested companel A.

(1) the reactivity to b60e74Cit74 is higher than that to b60e74Cit60and than that to b60e74Cit72, in agreement with the results of Fig. 1demonstrating the greater contribution of Cit74 to the peptidereactivity; (2) however, the reactivity of the bicitrullinated GGYCi-t72ACit74P peptide was most often higher than that of each of its 2monocitrullinated counterparts. The same could be observed withYCit72ACit74PAK, although the levels of reactivity were weaker.Thus, the quasi-exclusive contribution of Cit74 to the reactivity ofthe 15-mer b60e74Cit60,72,74 peptide deduced from Figs. 1 and 3was not confirmed with the pin-bound 7-mer peptides. On thecontrary, the results depicted in Fig. 3B showed that Cit72 and Cit74are both necessary to form a complete b-epitope; (3) the level ofreactivity of GGYCit72ACit74P is similar to that obtained with the 15-mer peptide lacking the last proline residue. All taken together,these results allowed defining the sequential b-epitope as corre-sponding to GYCit72ACit74.

The two remaining ab-sera presented different patterns ofrecognition of the b60e74-derived peptides with reactivity to theb-epitope and an additional variable reactivity (data not shown).Lastly, as expected, 8 out of 10 a-sera did not show any reactivity onb60e74-derived peptides and 2 unexpectedly but weakly reactedwith the b-epitope (data not shown).

Altogether, our results led to define three 4 to 5 aa-longsequential citrullinated epitopes on the two fibrin-derivedsequences, a36e50Cit38,42 and b60e74Cit60,72,74, previously iden-tified as bearing the major ACPA epitopes (Fig. 4). On the a-chain,the VECit42HQ a-epitope is specifically recognised by a largemajority of the a-sera and the ab-sera, whereas an additionala0-epitope, Cit38VVE, is specifically recognised only by a majority ofthe ab-sera. On the b-chain, the GYCit72ACit74 b-epitope is recog-nised by a large majority of the b- and ab-sera.

These results demonstrated the very limited diversity of theACPA specificity to citrullinated fibrin since it is restricted to threeimmunodominant epitopes located around Cit38 and Cit42 on the

,72,74 only (b-sera) or antibodies to both a36e50Cit38,42 and b60e74Cit60,72,74 (ab-sera)f reactivity obtained with 9 b-sera and 7 ab-sera allowing a minimal b-epitope to beparatively to its various mono- and bicitrullinated variants with the sera presented in

Fibrin -chainGYCit72ACit74

-sera-sera

’ -sera

VECit42HQCit38

Fibrin -chain

αβαβ

α

ααα

ββ

β

Fig. 4. Schematic representation of recognition of the immunodominant fibrinepitopes by ACPA. On the a-chain of fibrin, the a-sera specifically recognise theminimal a-epitope while the ab-sera recognise the a0-epitope in addition to the a-epitope. On the b-chain of fibrin, the b- and ab-sera recognise the minimal b-epitope.

C. Iobagiu et al. / Journal of Autoimmunity 37 (2011) 263e272 269

a-chain, and around Cit72 and Cit74 on the b-chain of the protein.They stressed the tight oligoclonality of ACPA. Moreover, thehomogeneity of the individual profiles of ACPA reactivity (Figs. 2and 3) showed that inter-individual variations in ACPA specificityto citrullinated fibrin exist but are limited to 3 main epitopes.

3.4. Structural model of the three immunodominant epitopeswithin the central E domain of fibrin (Fig. 5)

A schematic representation of the 3D structure of the central Edomain of the human fibrin molecule [(a-chain 36e92, b-chain45e135, g-chain 32e71) � 2] shows that the 3 immunodominantepitopes are located in the periphery of the central globulardomain, outside the coiledecoiled region of the molecule (Fig. 5A).Moreover, their arginyl residues point to the outside part of thefibrin molecule (Fig. 5B), and therefore are clearly accessible to PADbut also, once citrullinated, to ACPA.

4. Discussion

One way to shed light on the specificity of antigeneantibodyinteraction is the molecular mapping of the targeted antigen. Thegoal of the present study was to determine the fine specificity of

Fig. 5. Location of the three a, a0 and b immunodominant epitopes targeted by ACPA on thedomain of fibrin derived from the crystal structure of native fibrin was refined with structuragrey. The structures presented are those of the arginyl-containing sequences onwhich the lob-epitope in blue. (B) Side view of the same representation observed along the axis of the

ACPA to citrullinated fibrin and to analyse the diversity of ACPAsubsets [16]. In a previous epitope mapping performed witha restricted number of RA sera, we identified two fibrin-derivedpeptides, a36e50Cit38,42 and b60e74Cit60,72,74, harbouring immu-nodominant epitopes. In the present study, we first confirmed thisresult with a series of 100 RA sera and 200 controls. Consistent withour previous results [16], we demonstrated that the proportionsof RA sera containing autoantibodies to a36e50Cit38,42 or tob60e74Cit60,72,74 (55% or 63%, respectively) were significantlyhigher than those containing autoantibodies to the 2 other bestautoantigenic fibrin-derived peptides a501e515Cit510,512 ora621e635Cit621,627,630 (13% or 37% respectively). In addition, weconfirmed that the groups of sera recognising a501e515Cit510,512 ora621e635Cit621,627,630 constitute subgroups of those recognisinga36e50Cit38,42 or b60e74Cit60,72, respectively. Although nosequence identities exist between the peptides reacting with thesame sera, these results suggest that the epitopes borne bya501e515Cit510,512 and a621e635Cit621,627,630 most probablyrepresent molecular mimics of those borne by a36e50Cit38,42 andb60e74Cit60,72,74, respectively.

In agreement with these results, among the 81 AhFibA-positivesera from our series of 100 RA sera, it could be calculated that 74(91.4%) contained autoantibodies to a36e50Cit38,42 and/or tob60e74Cit60,72,74. Therefore almost all the sera containing AhFibAcould be classified as "a-sera", reactive only with a36e50Cit38,42,“besera”, reactive only with b60e74Cit60,72,74 or "ab-sera", reactivewith both peptides. This demonstrates that the reactivity ofa36e50Cit38,42 and b60e74Cit60,72,74 almost totally overlaps withthat of the complete molecule of citrullinated fibrinogen. Moreover,previous data showing that a36e50Cit38,42 and b60e74Cit60,72,74used together in absorption experiments were able to abolish thereactivity to citrullinated fibrinogen of a pool of 38 ACPA-positive RAsera, also strongly sustain that they bear the immunoreactivity toACPApresentedby thewholefibrinmolecule [13]. Thus, they supportthe hypothesis that the autoantibodies reacting with a36e50Cit38,42and b60e74Cit60,72,74 crossreact with a501e515Cit510,512 anda621e635Cit621,627,630, respectively. Only absorption experiments oflarge series of sera will confirm whether this crossreactivity isconstant or whether, in some rare patients, some ACPA targetinga501e515Cit510,512 or a621e635Cit621,627,630 coexist with ACPA tar-geting the immunodominant epitopes as they rarely exist as the onlyACPA specificity (3 out of 81 patients in that study).

Our results with a621e635Cit621,627,630 are consistent with thoseof Perez et al. [24], who showed that a617e631Cit621,627,630 bearingthe same citrullyl residues was only detected by 24% of RA sera ata 94% diagnostic specificity. Interestingly, the same group developedan original ELISA using a chimeric fibrin/filaggrin peptide containing

central domain of human fibrin. (A) Ribbon representation (front view) of the central El models of the non-citrullinated N terminal portions of the a, b and g chains, shown incations of the a- and a0-epitopes are coloured in red and purple, respectively, that of thea-helixes shown in green.

C. Iobagiu et al. / Journal of Autoimmunity 37 (2011) 263e272270

a617e631Cit627,630 and cfc-1, the cyclic citrullinatedfilaggrinpeptideof the commercial CCP1 assay, which proved to be more sensitivethan the commercial CCP2 assay [25,26]. In the study of Perez, noother a-chain-derived peptidewas found to be reactive among thosepredicted to be antigenic by computational methods [24], however,no peptides overlapping with a36e50Cit38,42 or a501e515Cit510,512were predicted and therefore evaluated. Concerning the b-chain, thereactivity of Cit72 and Cit74 contained within the b60e74Cit60,72,74peptide was not identified by these authors, instead, a major anti-genic region around aa 40e60 was predicted in which the mostreactive b43e62Cit47,60 peptide detected only 30% of 111 RA sera(95% specificity). It is likely that the aa environment of the citrullylresidue in the tested peptides explains the discrepancy with ourresults [24,26].

Analysing the epitope core of the two major peptide sequencesby two different approaches (ELISA of pooled AhFibA-positive seraon coated 15-mer peptides and individual sera on 7-mer pin-boundpeptides), we demonstrated, as previously shown on filaggrin-derived peptides [8,16], that citrullyl residues were not equallyreactive on citrullinated fibrin.We then demonstrated that on theseepitope-bearing sequences, ACPA targeted three minimal immu-nodominant epitopes: the a-epitope and the partially overlappinga0-epitope on a36e50Cit38,42, and one single b-epitope onb60e74Cit60,72,74. In addition, we showed that the diversity of ACPAcontained in individual sera, analysed by their pattern of recogni-tion of the 7-mer peptides encompassing the sequence ofa36e50Cit38,42 and b60e74Cit60,72,74 was very restricted. Indeed,a large majority of the a-sera recognised the minimal a-epitopeonly, and the b-sera recognised the b-epitope only. The ab-sera hada less restricted profile, recognizing the a0-epitope in addition tothe a-epitope and the b-epitope.

To date, itwas admitted that theACPA response presented a largeinter-individual heterogeneity in its fine specificity [8,27]. Indeed,a large variability from one RA serum to another in the patterns ofrecognition of variously citrullinated filaggrin-derived peptides [8]or various recombinant citrullinated polypeptides had beenobserved [7]. Surprisingly, the present data contrarily demonstratea very limited diversity of the ACPA reactivity to citrullinated fibrinwhich is their most abundant target in vivo. This sustains that ACPApresent a tight oligoclonality. We hypothesise that very few ACPAspecificities exist in vivo almost exclusively directed to the 3immunodominant epitopes, and that other reactive peptides rep-resenting molecular mimics of the major epitopes are targeted bythe same ACPA subsets. We think that such crossreactive epitopesare particularly numerous on citrullinated filaggrin, explaining whyfilaggrin and filaggrin-derived peptides are good substrates forACPA detection [17,28]. ELISA screening after direct coating ofpeptides permitted identification of continuous and essentiallysequential epitopes fromvarious ACPA protein targets. However, nosequence identity exists among the reactive peptides derived fromthe various targets. The citrullyl residues are essential in theconstitution the ACPA epitopes but have to be located withina specific aa environment in a short linear peptide. We suggest thatimmunoreactivity of the epitopes might accept certain aa substi-tutions by aa sharing chemical properties. Our previous report thatsome fibrin-derived citrullinated synthetic peptides were reactiveto ACPAwhile theywere not generated in vitro following fibrinogenincubationwith a recombinant human PAD argues for the existenceof structural homologues crossreactive with the truly antigenicepitopes. Only a huge work of comparative absorption experimentsusing the reactive peptides of the citrullinated proteins identified asACPA targets will give the definitive response regarding the actualcrossreactivity of ACPA and their degree of polyclonality.

Although various antibody specificities are generally describedafter a viral infection, numerous studies show that they are most

often directed to a restricted number of immunodominantepitopes. For example, it was demonstrated that the antibodyresponse to the envelope glycoproteins gp120 and gp41 of thehuman immunodeficiency virus (HIV) was restricted to limitedregions of these proteins since only nine peptides (19- to 36-mer)were recognised by a pool of anti-HIV-positive sera [29]. Refiningthe mapping, Palker et al. identified, a gp120-derived peptide(aa 504e518) recognised by 45% of anti-HIV-positive sera [30] andGnann et al. identified a gp41-derived peptide (aa 598e609) rec-ognised by 100% of infected individuals on which the minimalepitope was found to correspond to aa 603e609 [31]. Similarly, onVP1 and VP2 structural proteins and the non-structural NS proteinof the human B19 parvovirus, only 6 B-cell antigenic regions wereidentified within which only two immunodominant domains, aa271e286 of NS and aa 555- 571 of VP2, were shown to be targetedby 71e78% and 62% of seropositive sera, respectively [32]. Otherexamples of restricted diversity come from the epitope mapping ofthe 8 ORF 65-encoded protein of the human herpesvirus, in whicha single epitope was mapped to aa 162e169 [33] and, concerningbacteria, from the mapping of the IgA1 protease of streptococcuspneumoniae in which a single 37 aa-long domain was shown to bebroadly and specifically recognised [34]. Several epitope mappingstudies have also been conducted on autoantigens (for review see[35]) demonstrating the presence of restricted numbers of immu-nodominant epitopes. For example, on two 15-mer peptidesderived from the sequence of the centromere-associated proteinCENP-A targeted by a large majority of sera containing anti-centromere autoantibodies, it was shown that all sera recognisetwo immunologically related motifs harbouring a consensussequence. Other examples are given by autoantibodies to Ro60found to recognise a unique immunodominant 12-mer epitopein almost all the positive sera [36] and by autoantibodies tothe heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2),mainly directed to 4 peptides of which one is immunodominant(aa 155e175) [37]. All together, data in the literature show that theimmunodominance of a restricted number of epitopes is a generalfeature of the B immune response. Therefore, although our resultsdo not directly point towards a particular origin of ACPA, they arecompatible with the hypothesis of a still elusive infectious origin.The observed restricted diversity of ACPA which would not beexpected in the case of a non-specific polyclonal activationdemonstrates their oligoclonality. Such an oligoclonality probablyresulting from somatic mutations and affinity maturation suggeststhat the autoimmune reaction to citrullinated fibrin is antigen-driven. However, recognition of citrullinated fibrin could only bea secondary crossreactive phenomenonwhile the initiating antigenmight be of infectious origin. Unfortunately, searching for homol-ogies between the sequences of the minimal epitopes and those ofexogenous proteins led to the spotting of very numerous infectiousagents but identified no obvious candidate.

It was shown that antibodies produced against peptides fromhighly mobile regions of a protein react more strongly with thewhole protein than antibodies directed to peptides from well-ordered regions, demonstrating that mobility is a major factor inthe recognition of proteins [38]. In agreement, the topographical 3Dmodel we developed confirms that the 3 immunodominantepitopes are located in a flexible globular domain of fibrin,constituted of non-ordered regions and distant from the orderedcoiledecoiled region of the molecule. In addition, the epitopes areexposed to the outside of the molecule, and therefore easilyaccessible on the monomeric form of fibrin. The accessibility,probably preserved on polymerized fibrin, is compatible within vivo fixation of ACPA on the fibrin deposits of the synovial tissueand supports the hypothesis of a pathogenic role of ACPA viaformation of immune complexes.

C. Iobagiu et al. / Journal of Autoimmunity 37 (2011) 263e272 271

5. Conclusion

In the present studywe established precisemajor features of theACPA response. Firstly, we confirmed the immunodominance ofa36e50Cit38,42 and b60e74Cit60,72,74 on citrullinated fibrin.Secondly, we defined the three major epitopes that they bear:Cit38VVE and VECit42HQ on the a-chain, and GYCit72ACit74 on theb-chain, stressing the restricted antigenic specificity of ACPA andtherefore their very tight oligoclonality. However, we showed thatthe reactivity to the 3 epitopes distinguishes three subgroups ofpatients with different antigen specificity. It will be therefore ofmajor interest to evaluate on larger cohorts of patients whetherthese 3 subgroups of patients present any clinical difference andwhether they have different disease outcome. Finally, the closelyrestricted antigenic specificity of ACPA sustains that the autoim-mune reaction to citrullinated proteins is antigen-driven. Thirdly,we showed that the ACPA epitopes are easily accessible on thefibrin monomers and probably able to form immune complexeswith ACPA in the synovial tissue of the patients, reinforcing theautoantibodies as pathogenic actors in RA.

Conflict of interestNone.

Acknowledgements

The study was supported by research grants from the “Uni-versité Paul Sabatier-Toulouse III”, the “Centre National de laRecherche Scientifique” (CNRS) and grants allocated by jointHungarian-French intergovernmental scientific and technicalcooperation programmes: “Partenariat Hubert Curien”, “Agencenationale de la recherche (ANR) programme blanc France-Hongrie2009” (ANR-09-BLAN-0398-01 and TET_09_FR_ANR_BIO).

We thank Pr. B. Fournié and Pr. A. Cantagrel (Centre de rhu-matologie, Hôpital Purpan, Toulouse), for providing patient data.We also thank Frédéric Pont, Marie-Françoise Isaïa andMarie-PauleHenry for kindly providing technical support.

Appendix. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jaut.2011.07.003.

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