Characterization of Autoreactive T Cells to the Autoantigens Heterogeneous Nuclear Ribonucleoprotein A2 (RA33) and Filaggrin in Patients with This information is current as of September 26, 2021. Ruth Fritsch, Daniela Eselböck, Karl Skriner, Beatrice Jahn-Schmid, Clemens Scheinecker, Barbara Bohle, Makiyeh Tohidast-Akrad, Silvia Hayer, Josef Neumüller, Serafin Pinol-Roma, Josef S. Smolen and Günter Steiner

J Immunol 2002; 169:1068-1076; ; Downloaded from doi: 10.4049/jimmunol.169.2.1068 http://www.jimmunol.org/content/169/2/1068

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Characterization of Autoreactive T Cells to the Autoantigens Heterogeneous Nuclear Ribonucleoprotein A2 (RA33) and Filaggrin in Patients with Rheumatoid Arthritis1

Ruth Fritsch,* Daniela Eselbo¨ck,* Karl Skriner,*† Beatrice Jahn-Schmid,*‡ Clemens Scheinecker,2* Barbara Bohle,‡ Makiyeh Tohidast-Akrad,¶ Silvia Hayer,* Josef Neumu¨ller,§ Serafin Pinol-Roma,ʈ Josef S. Smolen,*¶ and Gu¨nter Steiner3*†

The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% Downloaded from of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33- induced cytokine secretion revealed high IFN-␥ and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 http://www.jimmunol.org/ phenotype and secreted higher amounts of IFN-␥ than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA. The Journal of Immunology, 2002, 169: 1068–1076.

heumatoid arthritis (RA)4 is a chronic inflammatory joint Already in early disease stages the inflamed rheumatoid syno- disease of unknown etiology characterized by hyperpla- vial membrane is characterized by massive infiltration of T cells

sia of the synovial membrane, degradation of cartilage, and other immune cells including B cells, macrophages, and mast by guest on September 26, 2021 R ϩ and bone erosion. Overexpression of proinflammatory cytokines cells (3Ð5). The majority of synovial T cells are CD4 memory such as IL-1 and TNF-␣ is considered to drive the destructive cells that typically express activation markers such as HLA-DR, processes, but the causes for this disregulated cytokine production CD69, or CD40L but, remarkably, may be deficient in CD28 ex- are unknown. Although it is generally assumed that RA belongs to pression (6). Cytokine analyses performed in situ revealed a pre- the group of systemic autoimmune diseases, the pathogenetic role dominance of IFN-␥ over IL-4-producing T cells, although the of cellular and humoral autoimmune reactions is still incompletely numbers of cytokine-secreting T cells were found to be relatively understood (1, 2). low (7Ð9). Furthermore, the T cell repertoire of RA patients shows features of clonal expansion (6) and most T cell clones (TCC) obtained from synovial tissue or fluid could be functionally attrib- *Division of Rheumatology, Department of Internal Medicine III, and Institutes of uted to the Th1 subset (10Ð12), which is generally considered to †Medical Biochemistry, ‡Pathophysiology, and ¤Histology, University of Vienna, Vi- constitute a driving force of pathologic autoimmune reactions (13Ð enna, Austria; ¶Ludwig Boltzmann Institute for Rheumatology and Balneology, Vi- enna, Austria; and ʈDepartment of Cell Biology and Anatomy, Mount Sinai School of 15). However, in vitro, synovial T cells generally show some fea- Medicine, New York, NY 10029 tures of anergy such as reduced responsiveness to mitogens or Received for publication July 19, 2001. Accepted for publication May 10, 2002. recall Ags, which has been attributed to the effects of chronic The costs of publication of this article were defrayed in part by the payment of page TNF-␣ exposure and oxidative stress in the joint (16, 17). charges. This article must therefore be hereby marked advertisement in accordance The abundance of T cells and the association of RA suscepti- with 18 U.S.C. Section 1734 solely to indicate this fact. bility with particular MHC class II alleles suggests an important if 1 This work was supported by the Interdisciplinary Cooperation Project of the Aus- trian Federal Ministry for Education, Science, and Culture and in part by Austrian not pivotal role of T cells in the pathogenesis of RA, which has National Bank Grant 7021. been widely discussed in recent years (2, 18Ð22). Moreover, the 2 Current address: Laboratory of Immunology, National Institute of Allergy and In- presence of high-titer rheumatoid factor (RF) and other autoanti- fectious Diseases, National Institutes of Health, Bethesda, MD 20892. bodies (Aab) in the sera of RA patients is considered a further 3 Address correspondence and reprint requests to Dr. Gu¬nter Steiner, Division of indication for involvement of autoimmune processes, although di- Rheumatology, Department of Internal Medicine III, University Hospital of Vienna, Wa¬hringer Gu¬rtel 18-20, A-1090 Vienna, Austria. E-mail address: Guenter.Steiner@ rect evidence for a role of Aab in the pathophysiology of RA is akh-wien.ac.at scarce (23). Based on these observations, an autoimmune model of 4 Abbreviations used in this paper: RA, rheumatoid arthritis; RF, rheumatoid factor; RA pathogenesis has been suggested in which autoreactive T cells Aab, ; hnRNP, heterogeneous nuclear ribonucleoprotein; Fil, filaggrin; are initially activated by Ags presented via disease-associated AFA, anti-Fil Aab; OA, osteoarthritis; PSA, psoriatic arthritis; cFil, citrullinated Fil; TT, tetanus toxoid; SFMC, synovial fluid mononuclear cell; SI, stimulation index; HLA molecules (2). These T cells subsequently activate autoreac- SN, supernatant; TCC, T cell clone; CRP, C-reactive protein. tive B cells, macrophages, and synoviocytes, which may further

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 1069 stimulate T cell responses. Thus, a vicious circle is induced and tigation patients were treated with nonsteroidal anti-inflammatory drugs maintained that leads to chronic inflammation and finally results in (n ϭ 32), disease-modifying antirheumatic drugs (n ϭ 34), and/or low- ϭ irreversible destruction of the joint. dose glucocorticoids (n 18). While 37 patients had moderately active disease with fewer than five swollen joints and C-reactive protein (CRP) The search for Ags triggering pathogenic T cell responses has Յ2 mg/dl, 13 patients had active RA (i.e., more than six swollen joints and led to the identification of a number of joint-specific candidate CRP Ͼ2 mg/dl). Synovial fluids were obtained during routine joint tapping proteins including collagen, cartilage link protein, and gp39 (23, from seven of the patients using heparinized tubes. The control population 24). However, even though arthritis may be induced in susceptible consisted of 18 patients with OA (all female, mean age 59.2 Ϯ 12 years), 11 patients with PSA (four male, seven female, mean age 45 Ϯ 17 years), mouse and rat strains by immunization with these Ags, their patho- and 21 healthy individuals (10 female, 11 male, mean age 39.3 Ϯ 13.4 genetic role has remained elusive. In addition to joint-specific Ags, years), including two otherwise healthy persons with osteoporosis. Four of several other proteins have been identified as targets of Aab of the PSA patients had active disease with more than two swollen joints patients with RA. Interestingly, even though these Ags are more or and/or elevated serum CRP levels. HLA-DR genotyping as assessed by less ubiquitously expressed, the Aab directed to them seem to be more PCR revealed that 66% of the RA patients and 30% of the tested controls carried HLA-DR4 and/or DR1. specific for RA than anti-collagen Aab or RF (23). These include the following: Aab to the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, also known as the RA33 Ag (A2/RA33) (25); anti- Antigens keratin Aab, which recognize the cytokeratin-aggregating protein Recombinant full-length hnRNP-A2/RA33 was used throughout this study. filaggrin (Fil) (26); anti-Sa Aab, whose target moiety is still unknown The cDNA encoding the Ag (45) was cloned by ligation-independent clon- (27); and Aab to the H chain binding stress protein (28). ing into the pET-30 LIC vector (Novagen, Madison, WI) and expressed as Aab to A2/RA33 can be detected in about one-third of RA pa- His-tagged fusion protein. Purification from bacterial lysates was achieved Downloaded from by nickel-nitrilotriacetic acid affinity chromatography (Qiagen, Hilden, tients and may also occur in patients with systemic erythem- Germany) followed by polymyxin B Sepharose adsorption (Bio-Rad, Her- atosus or mixed connective tissue disease, usually in association cules, CA) and anion exchange chromatography on DEAE Sepharose with anti-spliceosomal Aab (such as anti-Sm or anti-U1RNP) (Pharmacia Biotech, Uppsala, Sweden). Endotoxin content was determined which are not found in RA (29, 30). A2/RA33 is an abundant by the lympholytic amebocyte lysate assay (BioWhittaker, Verviers, Bel- Ͼ mRNA binding protein that, like other hnRNP proteins, shows a gium). By this procedure a 99% pure, endotoxin-free preparation was obtained. The optimum concentration for proliferation assays was found to modular structure consisting of two conserved RNA binding do- be 0.35 ␮g/ml. http://www.jimmunol.org/ mains and a glycine-rich auxiliary domain assumed to be involved Recombinant His-tagged Fil and cFil were a kind gift from Dr. A. Union in interactions with other proteins (31, 32). It has a predominant (Innogenetics, Ghent, Belgium) and were used at a concentration of 2.5 nuclear localization and exerts multiple functions, including reg- ␮g/ml. In addition, two synthetic cFil-derived peptides as well their un- modified isoforms were provided and used at 2.5 ␮g/ml. These peptides ulation of alternative splicing and transport of mRNA (33, 34). were selected on the basis of recognition by AFA (46) and had the fol- A2/RA33 appears to be ubiquitously expressed, although the level lowing sequences (X ϭ citrulline, i.e., deiminated arginine): HSAS of expression may greatly vary between different tissues (35). QDGQDTIXGHPGSS (filpep 1) and DSGHXGYSGSQASQDNEGH Anti-keratin or anti-Fil Aab (AFA), respectively, can be de- (filpep 2). tected by indirect immunofluorescence or immunoblotting in 40Ð Tetanus toxoid (TT) as control Ag was obtained from Pasteur Merieux

Connaught (Willowdale, Ontario, Canada) and used at a concentration of by guest on September 26, 2021 50% of RA patients (36). They are rather specific for RA and 0.5 U/ml as previously described (47). recognize the citrullinated form of Fil (cFil), which is generated by posttranslational deimination of certain arginine residues (37). Fil is exclusively expressed in terminally differentiated epithelial Ab detection cells, where it is involved in aggregation of cytokeratin filaments RF was determined by nephelometry. Anti-A2/RA33 Aab were detected by and presumably plays a role during apoptosis (38). Remarkably, ELISA (IMTEC, Berlin, Germany) and by immunoblotting as described when citrullinated peptides were used instead of the whole protein, (29). AFA were detected by an ELISA (Eurodiagnostics, Arnhem, The Ͼ Netherlands) using cFil-derived peptides (39). RF, anti-A2/RA33 Aab, and 60% of the patients were found to be reactive, even with peptides AFA were present in 57, 22, and 50% of the RA patients, respectively. not derived from Fil (39, 40). This has led to speculations on the existence of other citrullinated targets, and recently strong evi- dence has been provided that such proteins are indeed present in T cell stimulation assays synovial tissue of RA patients (41). PBMC or synovial fluid mononuclear cells (SFMC) were isolated from Anti-A2/RA33 and AFA are already present in early stages of fresh heparinized blood or synovial fluid samples of RA patients and con- the disease (40, 42Ð44) and are predominantly of the IgG isotype, trols by centrifugation on Ficoll-Hypaque (Pharmacia Biotech). After which is indicative of T cell-driven processes. To gain more in- washing and counting, cells were either immediately used or frozen in RPMI medium containing 10% DMSO and 20% FCS. Cells were cultured sight into such processes and their potential pathogenetic role, we for 5 days at 37¡C in triplicate in 96-well plates (Costar, Cambridge, MA) investigated the spontaneous T cell responses to A2/RA33 and Fil in a total of 200 ␮l (105 cells/well) in the presence of the Ags. Culture in patients with RA and in control subjects including patients with medium consisted of Ultra Culture serum-free medium (BioWhittaker, osteoarthritis (OA) and psoriatic arthritis (PSA) and healthy per- Walkersville, MD) containing 2 mM glutamine and 0.02 mM 2-ME sup- plemented with 100 U/ml penicillin/streptomycin (Life Technologies, Pais- sons. The data obtained suggest that A2/RA33 may constitute an ley, U.K.). PHA (Life Technologies) and IL-2 (Roche Molecular Bio- important T cell autoantigen in patients with RA, whereas Fil does chemicals, Mannheim, Germany) were used as polyclonal stimuli. During not seem to be a major T cell autoantigen and therefore presum- the last 16 h of culture, 0.5 ␮Ci/well [3H]TdR (Amersham Biosciences ably does not drive the humoral autoimmune response to Europe, Freiburg, Germany) was added and the incorporated radioactivity citrullinated Ags. was measured by scintillation counting. Results were expressed as stimu- lation index (SI) defined as the ratio of mean cpm obtained in cultures with Ag to mean cpm obtained in cultures incubated in the absence of Ag. SI Ն2 Materials and Methods and ⌬cpm Ͼ1000 (mean cpm obtained in cultures with Ag minus mean Patients and controls cpm obtained in cultures incubated in the absence of Ag) was regarded as a positive response. Peripheral blood from 50 patients with RA (37 female, 13 male, mean age Cytokines were measured by ELISA (BioSource, Fleurus, Belgium) in 55 Ϯ 17.7 years) classified according to the established criteria of the supernatants (SN) after a 24-h incubation with A2/RA33 or 0.5 U/ml TT as American College of Rheumatology was drawn into heparinized test tubes. control Ag. Detection limits were 5 pg/ml for IL-2, 4 pg/ml for IL-4, and Informed consent was obtained from all patients. At the time of the inves- 9 pg/ml for IFN-␥. 1070 AUTOREACTIVE T CELLS IN RA

Blocking experiments Inhibition of Ag-induced T cell activation was investigated by incubating PBMC with A2/RA33 in the presence or absence of 5 ␮g of mAb specific for HLA-DR (clone G46-6) or HLA-DR/DP/DQ (clone TU¬ 39), respec- tively (BD PharMingen, San Diego, CA) known to block MLR. An iso- type-matched mAb to ␤-galactosidase was used as control Ab (Zymed Laboratories, San Francisco, CA). Proliferation was measured by [3H]TdR uptake and IFN-␥ production was measured by ELISA. T cell lines and clones Ag-specific T cell lines were obtained using a previously established pro- tocol (48, 49). In brief, 2 ϫ 106 PBMC were stimulated with A2/RA33 for 5 days in 24-well flat-bottom culture plates. On day 5 of culture 20 U/ml IL-2 was added and the culture was continued for an additional 7 days. To generate TCC, T cell lines were restimulated with A2/RA33 and after 2 more days viable T cell blasts were separated by Ficoll-Hypaque and seeded in limiting dilution (0.5 cells/well) in 96-well plates. T cell blasts FIGURE 1. Proliferative responses of PBMC from RA patients and were cultured in the presence of 1 ϫ 105 irradiated (5000 rad) allogeneic controls to A2/RA33. Proliferation was measured by [3H]TdR incorpora- PBMC as feeder cells, 0.5 ␮g/ml PHA, and 20 U/ml IL-2 in medium tion in PBMC from 50 RA patients and 50 controls (18 patients with OA, containing 1% heat-inactivated human AB sera. Growing microcultures 11 patients with PSA, and 21 healthy subjects) in the presence or absence were then expanded at weekly intervals with fresh feeder cells in the pres- Ն ⌬ Ͼ of A2/RA33. A SI 2 and a cpm 1000 cpm were considered a positive Downloaded from ence of IL-2. The specificity of TCC was assessed by proliferation assays Ϯ ϫ 4 ␮ response; the cut-off value is indicated by a solid line. Mean SI was 4 3.5 incubating 2 10 T cell blasts with A2/RA33 (0.35 g/ml) in the pres- Ϯ Ͻ ence of 105 autologous irradiated PBMC. After a 48-h incubation and puls- for RA patients and 1.5 1.1 for controls (p 0.00002); no significant Ϯ ing with [3H]TdR for additional 16 h, cells were harvested and the incor- differences were seen among the three control groups (mean SI was 1.6 porated radioactivity was measured by scintillation counting. Production of 1.2 in OA patients, 1.2 Ϯ 1.5 in PSA patients, and 1.5 Ϯ 0.8 in healthy IL-4 and IFN-␥ was measured by ELISA in SN collected after 24 h of subjects). incubation. 4 5 For phenotyping, cloned T cells (5 ϫ 10 Ð10 ) were washed twice with http://www.jimmunol.org/ ice-cold FACS buffer (PBS, 5% FCS, 0.01% NaN3) by centrifugation for 5 min at 1000 ϫ g. The washed cells were incubated for 30 min at 4¡C with SI did not differ among the three control groups (see Fig. 1). More- a FITC- or PE-conjugated mAb (BD PharMingen). Anti-TCR␣␤ and anti- over, SI Ն4 were seen in 18 RA patients (maximum SI ϭ 15) but ␥␦ CD4 mAb were FITC conjugated, and anti-TCR and anti-CD8 mAb in only two controls (maximum SI ϭ 5.8). were PE conjugated. Afterward, cells were washed again in FACS buffer and analyzed with a FACScan flow cytometer (BD Biosciences, Franklin In RA patients the T cell response did not correlate with the Lakes, NJ) supported by PC Lysis software (BD Biosciences). presence of anti-A2/RA33 Aab, which were detected in 22% of the RA patients but in neither healthy controls nor patients with OA or Immunohistochemistry PSA, confirming previous observations (29, 44). Thus, T cell re-

Immunohistochemical analysis of synovial tissue was performed as de- activity to A2/RA33 was seen in 8 of the 11 (73%) anti-A2/RA33 by guest on September 26, 2021 scribed previously (9). To assess expression of A2/RA33, cryostat sections Aab-positive patients and also in 18 of the 39 (46%) anti- from synovial tissue of five patients with RA and three patients with OA were incubated for 60 min with the anti-A2/RA33 mAb 10D1 (50). After A2/RA33-negative patients. Although 73% of the reactive RA pa- rinsing and blocking of endogenous peroxidase, sections were incubated tients vs 57% of the nonreactive ones carried HLA-DR4 and/or for 30 min with a biotinylated horse anti-mouse IgG Ab followed by in- DR1, this association did not reach the level of statistical cubation with the Vectastain@ABC reagent (Vector Laboratories, Burlin- significance. game, CA) for another 30 min using diaminobenzidine (Sigma-Aldrich, St. Louis, MO) as substrate leading to brown staining of A2/RA33-expressing Proliferative responses of synovial T cells to A2/RA33 cells. Finally, slides were counterstained with hematoxylin (Merck, Darm- stadt, Germany). To investigate expression of A2/RA33 in macrophages, To address whether cellular reactivity against A2/RA33 was also tissues were double stained with 10D1 and an anti-CD68 mAb (DAKO, present in the synovial compartment, proliferation was measured Glostrup, Denmark) using an alkaline phosphatase-based detection system in SFMC obtained from seven RA patients; proliferation of the (DAKO) leading to blue staining of the CD68-positive cells. Additional double stainings were performed with an anti-CD3 mAb (BD PharMingen) corresponding PBMC was determined in parallel. In six patients, and the fibroblast-specific mAb ASO2 (Dianova, Hamburg, Germany). Iso- both SFMC and PBMC proliferated significantly, while in one type-matched mAb (DAKO) served as negative controls. patient only SFMC were responsive (Fig. 2). Of note, with one Statistical analysis Unless stated otherwise, SI or cytokine concentrations, respectively, are indicated as mean Ϯ SD. Student’s t test was used to determine differences between groups. Where appropriate, Bonferroni corrections were done. A p value Ͻ0.05 was regarded as significant. Results Proliferative responses of PBMC to A2/RA33 Cellular reactivity against A2/RA33 was investigated by measur- ing proliferation of PBMC obtained from 50 RA patients and 50 controls, including 18 patients with OA, 11 patients with PSA, and 21 healthy subjects. As shown in Fig. 1, pronounced responses were observed in 58% of the RA patients and in 20% of the con- FIGURE 2. Proliferative responses of SFMC from RA patients to A2/ trols (six healthy subjects, three OA patients, and one PSA pa- RA33. SFMC and PBMC obtained from seven RA patients were cultured tient). In RA patients mean SI was 4 Ϯ 3.5 (median SI ϭ 2.6), in parallel in the presence or absence of A2/RA33 and proliferation was whereas in controls mean SI amounted to 1.5 Ϯ 1.1 (median SI ϭ determined by measuring [3H]TdR incorporation. Except for patient 1, 1.1). This difference was highly significant ( p Ͻ 0.00002), while SFMC always showed a stronger response than the corresponding PBMC. The Journal of Immunology 1071

FIGURE 3. Cytokine production of PBMC from RA patients and controls in response to A2/RA33. PBMC from 15 RA patients and eight controls were incubated in the presence or absence of A2/RA33, and secretion of IL-2, IFN-␥, and IL-4 was measured in culture SN by ELISA. Back- ground levels were generally below the de- tection limit. Mean values are indicated by solid lines. A significant difference be- tween RA patients and controls was seen only for IL-2.

exception, the SI of SFMC was always higher than the SI of the was obtained with the second anti-HLA class II mAb (data not corresponding PBMC, although the difference was not significant. shown). In contrast, an isotype-matched control Ab had no signif- icant effect (Fig. 4, C and D). Cytokine secretion by PBMC in response to A2/RA33

To further characterize A2/RA33-induced T cell responses, the T cell reactivity to Fil Downloaded from cytokines IL-2, IL-4, and IFN-␥ were measured by ELISA in cul- The proliferative responses to both Fil and cFil were investigated ture SN of PBMC from 15 responsive RA patients and eight con- in PBMC of 19 RA patients and 20 healthy controls. As shown in trol subjects stimulated with A2/RA33 or the control Ag TT, re- Fig. 5, proliferative responses to either form of Fil were observed spectively (Fig. 3). These investigations revealed significantly in only four RA patients and two controls: three patients responded higher IL-2 production by PBMC of RA patients (10.3 Ϯ 9.1 vs to both forms and the fourth patient recognized only Fil. Interest-

4.1 Ϯ 3.3 pg/ml, p Ͻ 0.03), with IL-2 detectable in 10 SN of RA ingly, cFil always elicited a lower response than Fil, indicating that http://www.jimmunol.org/ patients and in three SN of controls (Fig. 3A). A similar result was arginine deimination may affect T cell recognition. Of the two obtained with IL-4, but here the difference between the two groups controls, one responded to Fil and the other one responded to cFil. did not reach the level of statistical significance (Fig. 3B). In con- Mean SI of PBMC of RA patients was 1.7 Ϯ 1.4 for Fil and 1.1 Ϯ trast to IL-2 and IL-4, high IFN-␥ levels were measured in virtu- 0.7 for cFil, respectively, which did not significantly differ from ally all SN of both RA patients and controls (Fig. 3C). Levels were the SI values obtained in controls (1.1 Ϯ 0.6 for Fil and 1.3 Ϯ 0.6 similar in both groups ( p ϭ NS) and were an order of magnitude for cFil, respectively). In contrast, the proliferative responses to higher than IL-4 levels. However, when A2/RA33-induced IFN-␥ A2/RA33 of these 19 RA patients were significantly higher, with production was compared with TT-induced production, a remark- a mean SI of 4.1 Ϯ 3.5 ( p Ͻ 0.02 vs Fil and p Ͻ 0.002 vs cFil, able difference became apparent: thus, in SN of RA patients IFN-␥ respectively). by guest on September 26, 2021 levels were markedly higher upon A2/RA33 stimulation than upon To learn whether citrullinated peptides could elicit a better T stimulation with TT (484 Ϯ 379 vs 117 Ϯ 97 pg/ml, p Ͻ 0.003), cell response, two synthetic cFil-derived peptides harboring major whereas PBMC from controls responded to both Ags in a compa- B cell epitopes (46) as well as their corresponding unmodified rable manner (875 Ϯ 672 vs 1376 Ϯ 1918 pg/ml, p ϭ NS). There- isoforms were used in proliferation assays involving PBMC from fore, TT-induced IFN-␥ production was significantly higher in seven RA patients, three of whom had responded to the whole controls than in RA patients ( p Ͻ 0.02). Concerning IL-4, A2/ RA33 induced a stronger response than TT only in RA patients, although compared with IFN-␥ the difference was less pronounced ( p Ͻ 0.05). However, with respect to IL-2, no difference between the two stimulants was seen in RA patients, whereas in controls TT induced a stronger IL-2 response than A2/RA33 ( p Ͻ 0.01). It is noteworthy that this response was comparable to the IL-2 response induced in RA patients by either A2/RA33 or TT, respectively. Taken together, these data suggested that A2/RA33-responsive T cells belong predominantly to the Th1 subset in both RA patients and controls. However, in RA patients these cells secreted IL-2 and appeared to be expanded as indicated by increased IFN-␥ pro- duction upon A2/RA33 stimulation relative to TT-induced stimulation.

MHC restriction of the A2/RA33-induced T cell response To investigate whether A2/RA33-induced T cell activation was dependent on Ag presentation by MHC class II molecules, two ␥ mAbs directed to HLA-DR or to HLA-DR/DP/DQ, respectively, FIGURE 4. Suppression of A2/RA33-induced IFN- production and proliferation by an anti-HLA-DR mAb. PBMC from eight RA patients and were used in inhibition experiments performed with PBMC of three controls were incubated with Ag in the presence of a mAb against eight responsive patients and three responsive controls (Fig. 4). As HLA-DR (A and B) or an isotype-matched control mAb (C and D). IFN-␥ shown in Fig. 4A, addition of the anti-HLA-DR Ab led to a marked secretion (A and C) was measured by ELISA, and proliferation (B and D) reduction of IFN-␥ secretion in all patients and controls (43Ð90% was determined by measuring [3H]TdR incorporation (cpm). The anti- decrease, p Ͻ 0.02). Proliferation was inhibited to an even higher HLA-DR mAb caused significant reduction of IFN-␥ secretion and prolif- degree (63Ð94% decrease, p Ͻ 0.0003) (Fig. 4B). A similar result eration, whereas the control mAb had no significant effect. 1072 AUTOREACTIVE T CELLS IN RA

Table II. Phenotype, proliferation, and cytokine production of A2/ RA33-reactive TCC derived from RA patients and healthy controls

IFN-␥ IL-4 Clone Phenotype SI (pg/ml) (pg/ml)

RA patientsa HWRA.10 CD4ϩCD8Ϫ 3.7 Ͼ2000 HWRA.33 CD4ϩCD8Ϫ 3.4 Ͼ2000 HWRA.43 CD4ϩCD8Ϫ 4.0 Ͼ2000 HWRA.91 CD4ϩCD8Ϫ 3.8 Ͼ2000 HWRA.14 CD4ϩCD8Ϫ 29.0 1014 5 HWRA.151 CD4ϩCD8ϩ 2.8 318 KBRA.113 CD4ϩCD8Ϫ 3.0 1033 15 KBRA.15 CD4ϩCD8Ϫ 3.4 136 KBRA.110 CD4ϩCD8Ϫ 9.4 59 FIGURE 5. Proliferative responses of PBMC from RA patients and KBRA.107 CD4ϩCD8Ϫ 3.1 48 3 controls to Fil. Proliferation was measured by [ H]TdR incorporation in LLRA.114 ND 47.0 Ͼ2000 PBMC from 19 RA patients and 20 controls in the presence or absence of LLRA.75 ND 2.3 33 either Fil or cFil. A SI Ն2 and a ⌬cpm Ͼ1000 cpm was considered a SSRA.130 CD4ϩCD8Ϫ 3.3 Ͼ2000 ϩ Ϫ positive response. The cut-off value is indicated by a solid line. Mean SI of SSRA.41 CD4 CD8 3.1 Ͼ2000 Ϯ Ϯ WWRA.175 CD4ϩCD8Ϫ 2.2 Ͼ2000 PBMC of RA patients was 1.7 1.4 for Fil and 1.1 0.7 for cFil; mean ϩ Ϫ Downloaded from Ϯ Ϯ KRRA.2 CD4 CD8 2.8 SI of PBMC of controls was 1.1 0.6 for Fil and 1.3 0.6 for cFil. These b differences were not significant. Controls RMHC.200 CD4ϩCD8Ϫ 2.6 Ͼ2000 RMHC.80 CD4ϩCD8Ϫ 2.3 154 RMHC.126 CD4ϩCD8ϩ 3.6 Ͼ2000 12 molecule, and 13 controls (Table I). Among the three responsive RMHC.140 CD4ϩCD8ϩ 2.7 Ͼ2000 RMHC.116 CD4ϩCD8ϩ 2.3 Ͼ2000 patients, only patient A responded selectively to the citrullinated Ϫ ϩ

RMHC.87 CD4 CD8 4.7 Ͼ2000 http://www.jimmunol.org/ form of peptide 1 (and to both forms of peptide 2), whereas pa- BSHC.102 CD4ϩCD8Ϫ 37.0 39 tients B and C responded only to the unmodified peptides. One of BSHC.83 CD4ϩCD8Ϫ 5.5 36 the four nonresponders (patient D) was reactive with both forms of BSHC.99 CD4ϩCD8Ϫ 2.1 12 Ϫ ϩ peptide 2 and one of the 13 controls responded to unmodified BSHC.16 CD4 CD8 4.5 53 WiWiHC.11 CD4ϩCD8Ϫ 19.0 360 peptide 1. Thus, these data confirmed that deimination can affect T ϩ Ϫ WiWiHC.41 CD4 CD8 2.2 68 cell recognition. a Sixteen TCC were derived from six RA patients. Mean SI was 7.9 Ϯ 12.3; ϩ Ϫ A2/RA33-reactive TCC median SI was 3.4. All but one clone showed a CD4 CD8 phenotype (clone HWRA.151 was CD4ϩCD8ϩ). The majority of clones showed a pronounced Th1 phenotype (IFN-␥ Ն100 pg/ml) and secreted significantly more IFN-␥ than T cell lines specific for A2/RA33 were established from the pe- by guest on September 26, 2021 CD4ϩCD8Ϫ control clones ( p Ͻ 0.04). ripheral blood of six RA patients and three healthy controls. Using b Twelve TCC were derived from three healthy controls. Mean SI was 7.4 Ϯ 10.4; the limiting dilution technique, between one and six Ag-specific median SI was 3.2. Seven TCC showed a CD4ϩCD8Ϫ phenotype, two clones were Ϫ ϩ TCC per individual could be generated (Table II). Sixteen TCC CD4 CD8 (RMHC.87 and BSHC.16), and three clones were double positive (RMHC.126, 140, and 116). Only three CD4ϩCD8Ϫ clones (RMHC.200, RMHC.80, were derived from RA patients, the majority of which (i.e., 13 of and WiWiHC.11) showed a pronounced Th1 phenotype (IFN-␥ Ն100 pg/ml). the 14 TCC analyzed) were CD4ϩCD8Ϫ and thus belonged to the Th cell subset. The TCC showed high variability in their prolifer- ative responses, with SI ranging from 2.2 to 47 and a mean SI of ϩ ϩ were CD4 CD8 (all from donor RM). Although all TCC pro- 7.9 Ϯ 12.3. Analysis of the cytokine secretion pattern, in contrast, duced IFN-␥ (and no IL-4), only five of them secreted high revealed high IFN-␥ production (i.e., Ն100 pg/ml) by the majority amounts (Ͼ1 ng/ml), which, interestingly, were all derived from of the clones with levels in the SN exceeding 1 ng/ml in 10 of donor RM. However, only one of these high producers them, while IL-4 was generally not detectable. Cytokine produc- ϩ Ϫ (RMHC.200) showed a Th1 phenotype (CD4 CD8 ), while the tion did not correlate with proliferation because even poorly pro- Ϫ ϩ remaining four clones were either CD4 CD8 or double positive. liferating TCC secreted large amounts of IFN-␥. ϩ Ϫ Among the six remaining CD4 CD8 clones only two secreted Proliferation of the 12 TCC derived from control individuals IFN-␥ Ͼ100 pg/ml (RMHC.80 and WiWiHC.11). Thus, Th1 was comparable to patient-derived clones with SI ranging from 2.1 clones from controls produced significantly less IFN-␥ than Th1 to 37 with a mean SI of 7.4 Ϯ 10.4. In contrast, phenotyping clones from RA patients ( p Ͻ 0.04). revealed some differences because only seven TCC were ϩ Ϫ Ϫ ϩ CD4 CD8 , while two clones were CD4 CD8 and three clones Expression of A2/RA33 in synovial tissue Expression at the protein level was studied in synovial tissue from five RA and three OA patients using a mAb against A2/RA33 (Fig. Table I. Proliferative responses of PBMC from RA patients to Fil- derived peptidesa 6). These investigations revealed the autoantigen to be abundantly expressed in RA synovial tissue, particularly in the lining layer, Patient cFil Fil cfilpep 1 filpep 1 cfilpep 2 filpep 2 where macrophage-like type A synoviocytes are the predominating cell population, and in the sublining area (Fig. 6A). Double stain- A36 4.1 1.7 5.7 5.8 ing experiments confirmed abundant expression by CD68-positive B22.3 1.8 2.2 1.8 2.2 macrophages (Fig. 6, B and C) and CD68-negative fibroblast-like C 0.8 4 1.4 3.5 1.2 2.1 D 0.9 1.9 1.2 1.4 3.2 2.8 synoviocytes, as well as by endothelial cells, whereas A2/RA33 expression was scarcely detectable in T and B cells (data not a Two synthetic peptides (for sequences refer to Materials and Methods) contain- ing dominant B cell epitopes were employed in citrullinated (cfilpep) and unmodified shown). Remarkably, A2/RA33, which in cultured cells shows a (filpep) forms. SI Ն 2 are underlined. predominant nuclear localization (51), was detected not only in the The Journal of Immunology 1073

FIGURE 6. Immunohistochemical analy- sis of A2/RA33 expression in synovial tissue. Cryosections from a patient with RA (AÐC and F) and a patient with OA (D and E) were stained with a mAb against A2/RA33 (AÐE) or an isotype- matched control mAb (F). Cells were counterstained with either hematoxylin (A, D, and F) or an anti-CD68 mAb (B, C, and E) to identify synovial macro- phages. Cells expressing A2/RA33 are stained brown; CD68-bearing cells are Downloaded from blue. Pronounced expression of A2/ RA33 is visible in the lining layer and in the sublining areas of RA tissue (AÐC), whereas in OA tissue many fewer cells are stained (D and E). Note both nuclear and cytoplasmic localization of A2/

RA33 in RA synovial cells. Magnifica- http://www.jimmunol.org/ tion: 100-fold (B, E, and F), 200-fold (A and D), and 400-fold (E). by guest on September 26, 2021

nucleus but also in the cytoplasm of RA synovial cells (Fig. 6, A bition experiments with mAb against MHC class II molecules. and C). In contrast, in OA synovial tissue only few cells stained Studies are currently in progress to investigate interaction of A2/ positive and the degree of cellular expression appeared to be gen- RA33 peptides with RA-associated HLA-DR molecules. erally weaker (Fig. 6, D and E). Cytokine analysis revealed abundant IFN-␥ secretion in re- sponse to A2/RA33 by the majority of patient and control PBMC, Discussion whereas production of IL-4 was considerably lower or undetect- The two Ags investigated in this study, A2/RA33 and cFil, repre- able. The data obtained with A2/RA33-specific TCC bolster these sent well-characterized target structures of the humoral autoim- observations because almost all TCC derived from RA patients mune response of RA patients. Because the factors driving the Aab showed a strong Th1 phenotype that was more pronounced than response to these two proteins are unknown, we were interested to that of control-derived Th1 clones. Taken together, these data in- search for the presence of autoreactive T cells in blood and syno- dicate a strong preponderance of Th1 cells among A2/RA33-reac- vial fluid of RA patients, assuming that autoreactive T cells might tive T cells, which is in agreement with previous observations on have more pathogenetic relevance than Aab, whose contribution to Th1:Th2 ratios in RA patients (9Ð12, 53). Remarkably, IFN-␥ pro- the pathophysiology of RA is still unclear. The data obtained in the duction of PBMC of RA patients was much higher in response to course of this study seem to support such an assumption, because A2/RA33 than to TT, whereas PBMC from control subjects re- T cell responses to A2/RA33 were observed in peripheral blood of sponded to both Ags in a comparable manner. Therefore, in RA ϳ60% of the RA patients and in all synovial fluids investigated patients A2/RA33-reactive T cells appeared to be more prevalent and thus occurred more frequently than anti-A2/RA33 Aab. Fur- in relation to TT-responsive T cells, suggesting Ag-driven expan- thermore, cellular responses to A2/RA33 were seen in only 20% of sion. This conclusion is supported by the observation that A2/ the controls and were significantly weaker than in RA patients, RA33-induced IL-2 production appeared to be largely restricted to even in patients suffering from PSA, an inflammatory and destruc- PBMC of RA patients. tive arthropathy showing some similarities with RA, particularly The presence of autoreactive T cells to A2/RA33 in some with respect to TNF-␣ production (52). healthy persons was not unexpected and is consistent with obser- Although no significant correlation was found between the A2/ vations repeatedly made by other investigators in both humans and RA33 response and the MHC susceptibility alleles HLA-DR4 and animal models (54Ð60). Under normal physiological conditions DR1, it should be noted that Ͼ70% of the reactive patients carried autoreactive T cells are under tight control and may even play the shared epitope. Importantly, this autoimmune response ap- beneficial roles (61, 62). The lack of significant IL-2 secretion as peared to be largely HLA-DR restricted as demonstrated by inhi- well as the low proliferative capacity of control PBMC in response 1074 AUTOREACTIVE T CELLS IN RA to A2/RA33 may indicate an anergic state preventing these cells ity of the T cells that actually drive the Aab production to citrul- from becoming expanded. Furthermore, TCC derived from con- linated targets. trols were phenotypically more heterogeneous, with some of them At the present time it is unclear whether any of the autoantigens being CD4ϩCD8ϩ, indicating the existence of immature subpopu- recognized by RA patients or any of the Ags eliciting chronic lations of A2/RA33-reactive cells in the blood of healthy persons. arthritis in animal models (such as cartilage Ags or the mycobac- Thus, it will be particularly interesting to see whether there exist terial 65-kDa heat shock protein) are of relevance in the patho- differences in epitope recognition between RA patients and healthy genesis of RA (23, 24, 74). This is particularly true for type II subjects as has been observed in organ-specific autoimmune dis- collagen, even though collagen-induced arthritis is the most widely ease (63). used model of RA and even though autoimmunity to collagen II The pronounced overexpression and cytoplasmic localization of occurs early in the course of RA (75, 76). An interesting novel A2/RA33 observed in synovial membranes of RA patients was candidate Ag is the glycolytic enzyme glucose-6 phosphate striking and was not seen in tissue of OA patients, who also did not isomerase, which has been identified as the disease-inducing au- show a significant autoimmune response to A2/RA33. Remark- toantigen in the KRN ϫ NOD model of RA (77, 78). This finding ably, abundant cytoplasmic expression was particularly seen in demonstrates once more the potential pathogenetic importance of synovial macrophages and may form a prerequisite for presenta- autoimmune reactions to ubiquitously expressed targets that are tion of this autoantigen to autoreactive T cells. It is well known not joint specific. Similar to the observations made for A2/RA33 or that the stressful conditions in inflamed tissues lead to activation, citrullinated fibrin, glucose-6 phosphate isomerase was recently overexpression, aberrant localization, or deposition of numerous described to be present in a high concentration in synovial tissue of Downloaded from molecules, some of which may function as autoantigens (64). At RA patients who also had anti-glucose-6 phosphate isomerase Aab present the molecular mechanisms leading to aberrant localization in their serum (79). This is suggestive of pathogenetic involvement of A2/RA33 are unknown, but, interestingly, a similar observation also in human RA; however, this remains to be proven. has recently been made in pathological tissue of lung cancer pa- Taken together, the frequent presence of A2/RA33-reactive tients (65). Thus, under conditions of chronic cellular stress trans- Th1-like cells in the blood of RA patients, their presence in the location of A2/RA33 to the cytoplasm seems to occur in which this synovial compartment in conjunction with the observed aberrant protein may exert a hitherto unknown function. A2/RA33 is a shut- expression of A2/RA33 in the inflamed synovium, and the occur- http://www.jimmunol.org/ rence of Aab to A2/RA33 early in the course of RA (42Ð44) sug- tling protein involved in transport of certain mRNAs and possibly gest that this protein may be an important autoantigen in RA. also in regulation of their translation as suggested for myelin basic Moreover, A2/RA33 Aab are among the earliest detectable Aab in protein or glucose transporter 1 (66, 67). Like other shuttling pro- MRL/lpr lupus mice (80), which, in addition to anti-DNA and teins, A2/RA33 is known to undergo posttranslational modifica- other systemic lupus erythematosus-specific Aab, also produce RF tions such as phosphorylation or methylation (68, 69). Therefore, and may develop erosive arthritis (81), and have recently been it is conceivable that the cytoplasmic form differs from the nuclear found to occur also in TNF-␣ transgenic mice (Ref. 82 and S. one and might in fact be more immunogenic, taking into consid- Hayer, B. Jahn-Schmid, D. Plows, K. Skriner, H. Erlandsson-Har- eration that posttranslational modifications may lead to the forma- by guest on September 26, 2021 ris, S. Haralambous, F. Monneaux, S. Trembleau, G. Schett, W. tion of neoepitopes and render a protein autoantigenic (70). Thus, van Venrooij, et al., manuscript in preparation), which represent a it may well be possible that the Aab are primarily directed to post- well-established model of RA (83). Although the significance of translationally modified (i.e., the cytoplasmic) forms of A2/RA33 these observations is not yet clear, they may be considered as and escape detection in our assays where Ag is used that is either additional indications for pathogenetic relevance of this autoim- bacterially expressed or derived from cultured cells in which ex- mune response. Further studies in patients and experimental ani- pression is largely restricted to the nucleus (51). To address this mals will show whether A2/RA33 is indeed part of the inflamma- issue, A2/RA33 must be isolated from human synovial tissue and tory and deleterious cascade of events characteristic of RA. subjected to a detailed biochemical and immunological analysis. Although Aab to the second Ag investigated, Fil, can be more Acknowledgments frequently found in RA patients than anti-A2/RA33 Aab (26, 36, We thank Dr. Ann Union for generously providing us with Fil and Fil 39), cellular reactivity to Fil was seen in only few RA patients. peptides, Elisabeth Ho¨fler for expert determination of anti-A2/RA33 and Moreover, these responses were substantially weaker than those AFA, and Walter Knapp and the other members of the Interdisciplinary against A2/RA33, which could be detected in all patients reactive Cooperation Project for their interest in our work and stimulating to Fil. Thus, these data suggest that Fil may not be the T cell Ag discussions. driving the humoral autoimmune response to citrullinated Ags. Nevertheless, B cells secreting AFA have been detected in syno- References vial tissue and fluid of RA patients (71, 72), and citrullinated pro- 1. Feldmann, M., F. Brennan, and R. N. Maini. 1996. Rheumatoid arthritis. 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B. A. de Jong, W. J. van Venrooij, F. C. Breedveld, and C. L. Verweij. 2001. tumor necrosis factor and receptors in models of multiorgan failure, rheumatoid http://www.jimmunol.org/ Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in arthritis, multiple sclerosis and inflammatory bowel disease. Immunol. Rev. rheumatoid arthritis. Arthritis Rheum. 44:41. 169:175. by guest on September 26, 2021