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ARTHRITIS & RHEUMATISM Vol. 63, No. 10, October 2011, pp 3153–3162 DOI 10.1002/art.30503 © 2011, American College of Rheumatology

Impaired Suppression of Synovial Fluid CD4⫹CD25⫺ T Cells From Patients With Juvenile Idiopathic Arthritis by CD4⫹CD25⫹ Treg Cells Susanne Haufe,1 Markus Haug,2 Carsten Schepp,3 Jasmin Kuemmerle-Deschner,1 Sandra Hansmann,1 Nikolaus Rieber,1 Nikolay Tzaribachev,4 Toni Hospach,5 Jan Maier,5 Guenther E. Dannecker,5 and Ursula Holzer1 Objective. Natural CD4ⴙCD25ⴙFoxP3ⴙ Treg cells play a crucial role in maintaining immune homeostasis and controlling autoimmunity. In patients with juvenile idiopathic arthritis (JIA), inflammation occurs despite the increased total numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB). This study was undertaken to investigate the phenotype of CD4ⴙ T cells in PB and SF from JIA patients, the function of synovial Treg cells, and the sensitivity of PB and SF CD4ⴙCD25ⴚ effector T cells to the immunoregulatory properties of Treg cells, and to study the suppression of cytokine secretion from SF effector T cells by Treg cells. Methods. The phenotypes of effector T cells and Treg cells of PB and SF from JIA patients and healthy donors were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in 3H-thymidine proliferation assays. Cytokine levels were analyzed using Bio-Plex Pro assay.

Results. Compared to PB, SF showed significantly elevated numbers of activated and differentiated CD4ⴙCD45ROⴙ T cells. Sensitivity of SF effector T cells to the suppressive effects of Treg cells from both PB and SF was impaired, correlating inversely with the expression of CD69 and HLA–DR. However, SF effector T cell cytokine secretion was partly suppressed by SF Treg cells. Conclusion. Our findings indicate that regulation is impaired in the SF of patients with JIA, as shown by the resistance of effector T cells to immunoregulation by functional Treg cells. This resistance of the SF effector T cells might be due to their activated phenotype. Juvenile idiopathic arthritis (JIA), the most common rheumatic disease in childhood, is defined as local inflammation in one or more joints persisting for more than 6 weeks and beginning before the age of 16 years (1). This clinically heterogeneous group of chronic pediatric arthritides with unknown etiology is characterized by T cell–dependent inflammation of the synovium, resulting in progressive cartilage destruction, bone damage, and finally, joint destruction (2). The reason for this autoimmune reaction in JIA is unclear. Naturally occurring CD4⫹CD25⫹FoxP3⫹ Treg cells, which develop in the thymus, are some of the key players in immunoregulation (3). This T cell subpopulation constitutes 5–15% of peripheral CD4⫹ T cells in humans (4), but only 2–3% of the CD4⫹CD25⫹ T cells are truly regulatory, expressing the antigen CD25 at high levels and the intracellular transcription factor FoxP3 (5,6). To date, several studies have demonstrated accumulating numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB) of both patients

Supported by the DFG (grant HO 2340/2-2), the RosemarieGermscheid-Stiftung, the Juergen Manchot Stiftung (scholarship to Ms Haufe), and Wyeth/Pfizer Pharma (unrestricted research grant). 1 Susanne Haufe, MSc, Jasmin Kuemmerle-Deschner, MD, Sandra Hansmann, MD, Nikolaus Rieber, MD, Ursula Holzer, MD: University Children’s Hospital Tuebingen, Tuebingen, Germany; 2 Markus Haug, PhD: NTNU, Trondheim, Norway; 3Carsten Schepp, MD, PhD: University Hospital Regensburg, Regensburg, Germany; 4 Nikolay Tzaribachev, MD: Center for Rheumatic Diseases, Bad Bramstedt, Germany; 5Toni Hospach, MD, Jan Maier, MD, Guenther E. Dannecker, MD: Olgahospital, Clinical Center Stuttgart, Stuttgart, Germany. Dr. Tzaribachev has received consulting fees, speaking fees, and/or honoraria from Pfizer, Chugai, and MSD (less than $10,000 each). Dr. Dannecker has received consulting fees from Wyeth and Pfizer (less than $10,000 each). Address correspondence to Ursula Holzer, MD, University Children’s Hospital Tuebingen, Hoppe-Seyler-Strasse 1, 72076 Tuebingen, Germany. E-mail: [email protected]. Submitted for publication August 19, 2010; accepted in revised form June 9, 2011. 3153

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with rheumatoid arthritis (RA) and patients with JIA (4,7,8). Despite the presence of these immunoregulatory cells, inflammation occurs. One possible explanation for this seeming paradox might be a defect in the ability of Treg cells to suppress autologous CD4⫹CD25⫺ effector T cells, as seen in patients with RA (9) and type 1 diabetes mellitus (DM) (10). Another potential explanation could be linked to the finding that effector T cells display decreased susceptibility to the regulatory effect of functional autologous CD4⫹CD25⫹ Treg cells in patients with RA (4), type 1 DM (11), and systemic lupus erythematosus (12) and a murine model of inflammatory bowel disease (13). Taken together, these findings of Treg cell dysfunction in autoimmune diseases are a matter of controversy. In this study, we investigated the function of synovial Treg cells and the sensitivity of effector T cells in the PB and SF of patients with JIA to the immunoregulatory properties of Treg cells. We found that effector T cells isolated from SF were significantly less sensitive to the suppressive function of Treg cells from both PB and SF. Furthermore, the expression of CD69 and HLA–DR within the CD4⫹CD25⫺ population of SF negatively correlated with the degree of inhibition by Treg cells, indicating the activated phenotype of SF effector T cells as a cause of the observed resistance to immunoregulation. MATERIALS AND METHODS Donors. Mononuclear cells were isolated from paired samples of PB and SF from patients with oligoarticular JIA (n ⫽ 9) and patients with polyarticular JIA (n ⫽ 7) fulfilling the International League of Associations for Rheumatology criteria for JIA (14) and from healthy individuals (n ⫽ 13) for phenotype analysis. For proliferation assays, PB samples (n ⫽ 10) and SF samples (n ⫽ 21) were obtained from patients with JIA at the time of clinically indicated arthrocentesis. Patients

were included from the University Children’s Hospital Tuebingen, Olgahospital Stuttgart, and the Center for Rheumatic Diseases, Bad Bramstedt. The characteristics of the patients are shown in Table 1. Mononuclear cells obtained from the PB of 10 healthy donors who had no history of autoimmune disease were used as controls. Approval for this study was obtained from the independent ethics committee of the University of Tuebingen. Informed consent to participate in the study was obtained from all donors. Cell isolation. Mononuclear cells were prepared from heparinized PB and SF by Ficoll-Hypaque (Biochrom) densitygradient centrifugation. CD4⫹ T cells were isolated from peripheral blood mononuclear cells (PBMCs) by negative selection using a CD4⫹ T cell isolation kit II (Miltenyi Biotec). Synovial mononuclear cells were positively isolated via a CD4 Multisort kit according to the recommendations of the manufacturer (Miltenyi Biotec). For proliferation assays, CD4⫹ T cells were separated into CD25⫹ and CD25⫺ populations using anti-CD25 microbeads (Miltenyi Biotec). The positive fraction of CD4⫹ cells sorted by a no-touch magnetic technique were used as antigen-presenting cells (APCs) in proliferation assays. The purity of isolated cells, as assessed by flow cytometry using anti-CD3, anti-CD4, anti-CD25, and antiFoxP3 antibodies, was ⬎85%. Phenotype analysis. Cells were stained with fluorochrome-conjugated monoclonal antibodies and their appropriate isotype controls. The stained cells were incubated for 10 minutes in the dark at 4°C and then washed with phosphate buffered saline containing 2% bovine serum albumin (Biochrom). For intracellular staining of FoxP3, cells were fixed and permeabilized according to the recommendations of the manufacturer after the cells were stained for surface expression. An isotype-matched control monoclonal antibody (mAb) was used to determine nonspecific staining. Expression of cell surface or intracellular proteins was quantified using a flow cytometer (FACSCalibur; BD Biosciences). Data were analyzed using CellQuest Pro software (BD Biosciences). Antibodies and reagents. The following mAb for flow cytometric analysis were used: fluorescein isothiocyanate (FITC)/peridinin chlorophyll A protein (PerCP)–conjugated anti-CD3 (UCHT-1, SK7), phycoerythrin (PE)–conjugated anti-CD25 (M-A251), PE-conjugated anti-CD69 (FN50), PEconjugated anti–HLA–DR (L243) (all from BD Biosciences),

Table 1. Clinical characteristics of the patients with JIA who contributed samples for the suppression assays*

No. female/no. male Age at time of sample, mean ⫾ SEM years ANA positive Treatment at time of sample MTX NSAIDs TNF inhibitor Prednisolone None

Persistent oligoarticular JIA (n ⫽ 14)

Extended oligoarticular JIA (n ⫽ 2)

Polyarticular JIA (n ⫽ 5)

14/0 10.6 ⫾ 1.37 10 (71.43)

2/0 8.0 ⫾ 0.75 2 (100)

4/1 8.6 ⫾ 2.85 3 (60)

5 (35.71) 10 (71.42) 1 (7.14) 1 (7.14) 1 (7.14)

1 (50) 2 (100) 0 (0) 0 (0) 0 (0)

3 (60) 3 (60) 2 (40) 0 (0) 1 (20)

* Except where indicated otherwise, values are the number (%) of patients. JIA ⫽ juvenile idiopathic arthritis; ANA ⫽ antinuclear antibody; MTX ⫽ methotrexate; NSAIDs ⫽ nonsteroidal antiinflammatory drugs; TNF ⫽ tumor necrosis factor.

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Figure 1. Increased numbers of CD4⫹CD45RO⫹ memory T cells with activated phenotype and expression of CD4⫹CD45RO⫹CD25bright T cells in synovial fluid (SF) compared to peripheral blood (PB). Mononuclear cells were isolated from the PB and SF of patients with oligoarticular (oligo) juvenile idiopathic arthritis (JIA) and patients with polyarticular (poly) JIA and from the PB of healthy donors. Cells were stained for CD4, CD45RO, CD69, HLA–DR, and CD25. The percentage of CD4⫹ T cells from SF and PB mononuclear cells coexpressing A, CD45RO, B, CD45RO and CD69, C, CD45RO and HLA–DR, or D, CD45RO and CD25 was determined by flow cytometric analysis. Symbols indicate individual subjects (n ⫽ 19 patients with oligoarticular JIA, 9 patients with polyarticular JIA, and 19 healthy donors in A; 9 patients with oligoarticular JIA, 7 patients with polyarticular JIA, and 13 healthy donors in B–D). Horizontal lines indicate the mean. ⴱⴱⴱ ⫽ P ⬍ 0.001 by Student’s t-test.

PerCP/allophycocyanin–conjugated anti-CD4 (RPA-T4), FITC-conjugated anti-CD25 (BC96), PE-conjugated antiCD45RO (UCHL-1), FITC-conjugated anti-CD69 (FN50), FITC-conjugated anti–HLA–DR (L243), and Alexa Fluor 647–conjugated anti-FoxP3 (259D) with its IgG1 control (MOPC-21) (all from BioLegend) and anti-CD27 (clone O323) (from eBioscience). Relevant isotype-matched control antibodies were used. T cells were activated with soluble murine anti-human CD3 antibody (IgG2a) OKT3 (JanssenCilag). Cells were cultured in RPMI 1640 media (Biochrom) containing 10% pooled human serum (University Children’s Hospital Tuebingen), 100 units/ml (100 ␮g/ml) penicillin/ streptomycin, 2 mM L-glutamine, and 10 mM HEPES buffer (all from Biochrom). 3 H-thymidine proliferation assays. Varying numbers of CD4⫹CD25⫹ Treg cells were cocultured with autologous or allogeneic CD4⫹CD25⫺ effector T cells (5 ⫻ 105) and APCs (5 ⫻ 105) (irradiated with 3,000 rads) along with 0.5 ␮g/ml soluble anti-CD3 mAb in 96-well round-bottomed plates (Greiner Bio-One). As controls, 5 ⫻ 105 irradiated APCs were cultured either alone or with the same number of autologous effector T cells and Treg cells, respectively. After 72 hours, 1 ␮Ci/well of 3H-thymidine (GE Healthcare) was added for an

additional 17 hours. Cell proliferation was determined by scintillation counting on day 4 with 1450 MicroBeta Wallac TriLux (PerkinElmer). The percent suppression was calculated as 1 ⫺ (counts per minute incorporated in cocultures/cpm of effector T cells cultured alone) ⫻ 100. Cytokine assay. Cytokine levels in the supernatants of PB and SF CD4⫹CD25⫺ effector T cells cultured alone or with autologous CD4⫹CD25⫹ Treg cells at a Treg cell: effector T cell ratio of 0.25:1, and of PB and SF CD4⫹CD25⫹ T cells cultured alone, were measured after 48 hours of incubation by Bio-Plex Pro human cytokine 27-plex assay (Bio-Rad). Data analysis was performed using Bio-Plex Manager software. Statistical analysis. Data analysis was performed using Prism 4.0 software (GraphPad). Student’s paired t-test was used to compare the percentage of activation markers, the mean percent inhibition between PB and SF from JIA patients and healthy controls, and cytokine secretion in PB and SF effector T cells. Pearson’s correlation was used to statistically analyze the interrelation of inhibition of SF effector T cell proliferation by Treg cells and the expression of CD69, HLA– DR, and CD45RO. P values less than 0.05 were considered significant.

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RESULTS Increased numbers of CD4ⴙCD45ROⴙ memory T cells with activated phenotype and expression of CD4ⴙCD45ROⴙCD25bright T cells in SF compared to PB. Since the CD4⫹ population comprises a mixture of cells in different conditions, we analyzed whether CD4⫹ T cells of the SF and PB of patients with JIA and the PB of healthy controls differed in their relative expression of CD45RO, a member of the leukocyte common antigen family that is expressed largely on previously activated or memory T cells (15). In the SF of both patients with oligoarticular JIA and patients with polyarticular JIA, the number of CD4⫹CD45RO⫹ T cells was highly increased compared with the number in PBMCs from JIA patients and healthy donors (Figure 1A). To examine whether the accumulated CD4⫹CD45RO⫹ T cells in SF demonstrate an increased activation and regulatory phenotype, CD4⫹CD45RO⫹ T cells from SF and PB were analyzed for the expression of CD69, HLA– DR, and CD25. CD4⫹CD45RO⫹ T cells from the PB of JIA patients with oligoarticular or polyarticular disease had levels of CD69 (Figure 1B) and HLA–DR (Figure 1C) similar to those in controls, whereas CD4⫹CD45RO⫹ SF cells showed a significant increase in markers of activation compared to the PB of patients with JIA and the PB of healthy controls. CD4⫹ Treg cells preferentially reside within the CD4⫹CD25 bright population (5). We defined CD4⫹CD25⫹ Treg cells as the 3% of cells with the highest level of CD25bright among the CD4⫹CD45RO⫹ T cells in the PB of JIA patients and set the gate in evaluating SF cells accordingly. In SF samples obtained from patients with oligoarticular JIA and patients with polyarticular JIA, a mean ⫾ SEM of 14.41 ⫾ 1.70% and 14.00 ⫾ 1.28%, respectively, of CD4⫹CD45RO⫹ T cells coexpressed CD25bright on their surface (Figure 1D). This percentage was significantly higher than that in the PB of patients with either oligoarticular or polyarticular JIA and healthy controls. In summary, CD4⫹CD45RO⫹ T cells in the PB of JIA patients displayed a similar phenotype to those in control PB. In SF samples from patients with oligoarticular JIA and patients with polyarticular JIA, CD4⫹CD45RO⫹ T cells showed an increased expression of activation markers as well as of CD25bright cells. However, no significant difference was seen between the 2 subsets of JIA. Similar findings regarding the expression of CD45RO, CD69, and HLA–DR were obtained by analyzing the CD4⫹CD25⫺ T cell subset in PB and SF (data not shown).

Phenotypic characterization of isolated PB and SF CD4ⴙCD25ⴚ and CD4ⴙCD25ⴙ T cells. For suppression assays, PB and SF mononuclear cells were sorted into CD4⫹CD25⫺ and CD4⫹CD25⫹ T cells as described above. Cells were stained for CD4, CD25, CD27, and FoxP3 to analyze their phenotype and purity by flow cytometry (Figure 2). Representative dot plots of PB and SF CD4⫹CD25⫺ and CD4⫹CD25⫹ T cells stained for CD25 and FoxP3 are displayed in Figure 2A to illustrate the gating strategy used to obtain the results shown in Figure 2B. The purity of isolated CD4⫹CD25⫹ T cells from the PB of healthy individuals (n ⫽ 8) and JIA patients (n ⫽ 5) and from SF (n ⫽ 16) coexpressing FoxP3 was ⬎85%, whereas ⬍2% of CD4⫹CD25⫺ T cells coexpressed FoxP3 (Figure 2B). No significant difference in the expression of FoxP3 was observed between CD4⫹CD25⫺ T cell populations in PB samples and CD4⫹CD25⫺ T cell populations in SF samples or between CD4⫹CD25⫹ T cell populations in PB samples and CD4⫹CD25⫹ T cell populations in SF samples (Figure 2B). Analyzing the expression of the surface molecule CD27 may discriminate between FoxP3-expressing CD4⫹CD25⫹ Treg cells and activated CD4⫹CD25⫹ T cells in the SF, since among synovial CD4⫹CD25⫹ T cells, Treg cells are CD27 positive, whereas CD27negative cells are nonsuppressive (16). Figure 2C shows coexpression of FoxP3 and CD27 in PB and SF CD4⫹CD25⫺ and CD4⫹CD25⫹ T cells as dot plots. The percent of isolated CD4⫹CD25⫹ T cells from the PB of healthy donors (n ⫽ 6) and from SF (n ⫽ 6) that expressed CD27 was at least 86% and at least 89%, respectively, and the percent of CD4⫹CD25⫺ cells from PB and SF samples that expressed CD27 was at least 86% and at least 85%, respectively (Figure 2D). However, only the isolated Treg cell population coexpressed CD27 and FoxP3. No significant difference in CD27 expression was observed between CD4⫹CD25⫹ T cells isolated from the SF and PB of JIA patients and from the PB of healthy donors (Figure 2D). Suppression of effector T cells in PB samples from JIA patients and healthy donors by autologous Treg cells and impairment of the suppression of effector T cells in SF samples. To determine the suppressive potential of synovial and peripheral Treg cells from JIA patients and healthy donors, CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell populations were isolated and subjected to a proliferation assay. CD4⫹CD25⫹ Treg cells were cocultured with autologous CD4⫹CD25⫺ effector T cells (at ratios of 1:1, 0.5:1, 0.25:1, and 0.125:1 Treg cells:effector T cells), irradiated APCs, and soluble

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Figure 2. FoxP3 and CD27 expression in isolated CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell populations from the PB of healthy donors and JIA patients and from the SF of JIA patients. T cells were stained for CD4, CD25, CD27, and FoxP3. A, Flow cytometric dot plots showing CD25 and FoxP3 expression in CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells from the PB of healthy donors and JIA patients (grouped together) and from the SF of JIA patients. B, Percentages of CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell populations from the PB of healthy donors (n ⫽ 8) and JIA patients (n ⫽ 5) (grouped together) and from the SF of JIA patients (n ⫽ 16) coexpressing FoxP3. Symbols indicate individual subjects. Horizontal lines indicate the mean. C, Flow cytometric dot plots showing CD27 and FoxP3 expression in CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells from the PB of healthy donors and JIA patients (grouped together) and from the SF of JIA patients. D, Percentages of CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell populations from the PB of healthy donors (n ⫽ 6) and the SF of JIA patients (n ⫽ 6) coexpressing CD27. Symbols indicate individual subjects. Horizontal lines indicate the mean. NS ⫽ not significant (see Figure 1 for other definitions).

anti-CD3 mAb for 89 hours. Inhibition of proliferation was analyzed as a percentage of the proliferative response to soluble anti-CD3 mAb of effector T cells cultured in the presence of different numbers of Treg cells compared to effector T cells cultured alone. PB samples from 10 JIA patients and 10 healthy donors and SF samples from 21 JIA patients were tested. The characteristics of the patients are shown in Table 1. CD4⫹CD25⫹ Treg cells derived from the PB of JIA patients, the PB of healthy controls, or the SF of JIA patients were anergic upon stimulation with soluble anti-CD3 mAb (Figure 3A). There was no significant difference in the proliferation rate between effector T cells derived from SF, the PB of JIA patients, and the PB of healthy donors when cultured alone and when stimulated with soluble anti-CD3 mAb. The PB CD4⫹CD25⫹ T cell populations from both JIA patients and healthy individuals suppressed the proliferation of autologous effector T cells comparably, in a dosedependent manner (Figure 3B). However, the suppression of SF effector T cells was significantly impaired compared to autologous PB effector T cells (Figure 3C).

This significant difference was observed at all Treg cell:effector T cell ratios tested. No significant difference in the inhibition of SF effector T cells was detectable with regard to disease subtype (oligoarticular or polyarticular JIA), treatment, or antinuclear antibody (ANA) status (see Table 1). Next, we addressed the issue of whether the lower suppressive capacity seen in the coculture assays of SF cells was due to an impaired suppressor function of Treg cells or a resistance of effector T cells to Treg cell– mediated suppression. To test these possibilities, SF Treg cells from JIA patients were cocultured with PB effector T cells from healthy donors at a Treg cell: effector T cell ratio of 0.25:1 and vice versa (effector T cells from SF samples from JIA patients and Treg cells from PB samples from healthy donors) (Figure 3D). Although Treg cells from healthy individuals effectively inhibited the proliferation of autologous effector T cells, Treg cells from healthy donors showed impaired suppressive ability in cocultures with synovial effector T cells. Interestingly, SF Treg cells from JIA patients, which exhibited reduced suppressive activity in cultures

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Figure 3. Impaired suppression of SF effector T cells (Teffs) by autologous and allogeneic Treg cells. A, Proliferation of Treg cells and effector T cells from the PB of JIA patients (n ⫽ 10), the SF of JIA patients (n ⫽ 21), and the PB of healthy donors (n ⫽ 10). Bars show the mean ⫾ SEM. B and C, Inhibition of the proliferation of effector T cells from the PB of JIA patients (n ⫽ 10) and the PB of healthy donors (n ⫽ 10) (B) or the SF of JIA patients (n ⫽ 21) (C) by autologous Treg cells. For suppression assays, PB and SF effector T cells from JIA patients and healthy donors were cultured with antigen-presenting cells (APCs) alone or with APCs and autologous Treg cells at Treg cell:effector T cell ratios of 1:1–0.125:1. The percent inhibition was determined using the formula 1 ⫺ (cpm incorporated in cocultures/cpm of effector T cells cultured alone) ⫻ 100. Values are the mean ⫾ SEM. Data are presented as a nonlinear regression curve. Broken lines show 95% confidence intervals. D, Inhibition of the proliferation of PB effector T cells from healthy donors (n ⫽ 12) cocultured with autologous PB Treg cells or allogeneic SF Treg cells at a Treg cell:effector T cell ratio of 0.25:1 and SF effector T cells from JIA patients (n ⫽ 12) cocultured with autologous Treg cells or allogeneic PB Treg cells. Symbols indicate individual samples. Horizontal lines indicate the mean. ⴱⴱ ⫽ P ⬍ 0.01 by Student’s t-test. NS ⫽ not significant (see Figure 1 for other definitions.)

with autologous effector T cells, were competent to inhibit PB effector T cells from healthy donors comparable to PB Treg cells. These findings indicate that the impaired effector T cell regulation is due to the low sensitivity of SF effector T cells to the activity of functional Treg cells. Inverse correlation between the suppression of SF effector T cell proliferation by Treg cells and the activated phenotype of SF effector T cells. A correlation study was performed to investigate whether the percentage of SF CD4⫹CD25⫺ effector T cell inhibi-

tion by autologous Treg cells correlates with the activated phenotype of SF effector T cells as determined by expression of CD69, HLA–DR, and the memory marker CD45RO (Figure 4). The percentages of CD4⫹CD25⫺CD69⫹ and CD4⫹CD25⫺HLA–DR⫹ T cells were significantly inversely correlated with the suppression of CD4⫹CD25⫺ effector T cell proliferation at a Treg cell:effector T cell ratio of 0.25:1 (Figures 4A and B). However, no significant correlation was found between the percentage of CD4⫹CD25⫺CD45RO⫹ T cells and effector T cell inhibition (Figure 4C).

Figure 4. Negative correlation between suppression by synovial fluid (SF) Treg cells and the activated phenotype of SF effector T cells. Correlations between the percent inhibition of SF effector T cell proliferation by autologous Treg cells at a Treg cell:effector T cell ratio of 0.25:1 and the percentage of A, CD4⫹CD25⫺CD69⫹ T cells (n ⫽ 17; P ⫽ 0.0042), B, CD4⫹CD25⫺HLA–DR⫹ T cells (n ⫽ 17; P ⫽ 0.0068), and C, CD4⫹CD25⫺CD45RO⫹ T cells (n ⫽ 17; P ⫽ 0.2938) are shown. Statistical analysis was performed using Pearson’s correlation.

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Figure 5. Cytokine secretion in SF effector T cells (Teffs) from patients with JIA and in PB effector T cells from healthy donors, and suppression of cytokine secretion by SF Treg cells. A, Cytokine levels in PB and SF CD4⫹CD25⫺ effector T cells. Cells were cultured in the presence of irradiated antigen-presenting cells and 0.5 ␮g/ml soluble anti-CD3 monoclonal antibody. Supernatants were collected 48 hours after stimulation, and cytokines were measured by Bio-Plex Pro human cytokine 27-plex assay. B, Cytokine levels in SF effector T cells cultured alone and SF effector T cells cocultured with autologous CD4⫹CD25⫹ Treg cells at a Treg cell:effector T cell ratio of 0.25:1. Supernatants were collected 48 hours after stimulation and cytokines were measured by Bio-Plex Pro human cytokine 27-plex assay. Bars show the mean ⫾ SEM of 3 independent experiments. ⴱ ⫽ P ⬍ 0.05 by Student’s t-test. NS ⫽ not significant; IL-2 ⫽ interleukin-2; TNF␣ ⫽ tumor necrosis factor ␣; IFN␥ ⫽ interferon-␥; ND ⫽ not detected (see Figure 1 for other definitions).

Selective suppression of enhanced proinflammatory cytokine production by SF effector T cells after coculture with autologous Treg cells. To investigate cytokine secretion of SF effector T cells in comparison to PB effector T cells, supernatants of SF and PB CD4⫹CD25⫺ effector T cells cultured in the presence of irradiated APCs and 0.5 ␮g/ml soluble anti-CD3 mAb (n ⫽ 3 PB samples and 3 SF samples) were collected 48 hours after stimulation. Significantly enhanced cytokine secretion was detected in the supernatants of SF effector T cells compared to PB effector T cells for the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor ␣ (TNF␣), CCL3, and interferon-␥ (IFN␥) and the antiinflammatory cytokines IL-5, IL-10, and IL-13 (Figure 5A). No differences were observed in IL-2 secretion. The addition of synovial Treg cells to SF effector T cells at a ratio of 0.25:1 significantly inhibited production of the cytokines IL-2, TNF␣ and IFN␥ (Figure 5B), but failed to suppress IL-6 and IL-10 production. A slight but not significant reduction in IL-5, IL-13, and CCL3 secretion was detected after coculture of SF effector T cells with SF Treg cells. Minimal IL-2 and no IFN␥ cytokine production was detected in PB and SF Treg cells cultured alone (data not shown). DISCUSSION In this study, we characterized T cells in SF and PB samples from JIA patients, demonstrating increased

numbers of CD4⫹CD45RO⫹ memory T cells with activated phenotype as well as elevated numbers of CD4⫹CD45RO⫹CD25bright T cells in the SF compared to the PB of JIA patients and healthy donors. Furthermore, we showed a hyporesponsiveness of SF effector T cells to the regulatory properties of highly enriched and functional synovial and peripheral Treg cells, which was inversely correlated with the activated phenotype of SF effector T cells. However, SF effector T cell cytokine production was selectively suppressed by SF Treg cells. Several studies have demonstrated increased numbers of activated CD4⫹ T cells and CD4⫹CD25⫹ Treg cells in the SF of patients with RA and JIA (17–21). In an analysis of the CD4 subpopulation, we found increased numbers of activated and differentiated CD4⫹ T cells in the SF compared to the PB of JIA patients and healthy donors, consistent with the results of previous studies (19,20,22). Since the majority of SF CD4⫹ T cells express CD45RO, the increased detection of activation markers within the CD4 population of SF may reflect the surplus of CD45RO⫹ memory T cells. Hence, we analyzed the CD4⫹CD45RO⫹ T cell subpopulation of PB and SF for the expression of CD69, HLA–DR, and CD25. We found a significant increase in all tested activation markers in CD4⫹CD45RO⫹ T cells in the SF compared to those in the PB of JIA patients and healthy donors, indicating an activated memory phenotype of SF effector T cells.

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Additionally, we found an increased expression of CD25bright among the CD4⫹CD45RO⫹ T cells in the SF with no significant difference between samples from patients with oligoarticular JIA and those from patients with polyarticular disease. Expression of CD25 is not limited to Treg cells; it is also found transiently on activated T cells. Intracellularly expressed FoxP3, a critical transcription factor for the development and function of Treg cells, is the most specific marker for Treg cells. However, activated effector T cells can also express FoxP3 (23), which can result in misinterpreting recently activated effector T cells as Treg cells (24). CD27, a TNF family member, has been identified by Ruprecht et al as an additional marker to discriminate activated effector T cells from Treg cells within the synovial CD4⫹CD25⫹ population (16). In the present study, the isolation of CD4⫹CD25⫹ T cells from the PB and SF of patients with JIA resulted in a cell population that highly coexpressed FoxP3 and CD27, indicating highly enriched Treg cells, which were used for further experiments. Numerous studies have investigated the function of Treg cells in developing autoimmunity with different results. Reduced suppressive capacities of peripheral CD4⫹CD25⫹ Treg cells have been described in patients with type 1 DM, psoriasis, myasthenia gravis, and multiple sclerosis (10,25–27). Earlier studies investigating the suppressive capacity of Treg cells from the PB and SF of patients with RA revealed no defect in suppression (7,18,28). A defect in the ability of Treg cells to inhibit the secretion of IFN␥ and TNF in effector T cells isolated from the PB of patients with RA was demonstrated in a different study (9). The described functional deficiency of Treg cells may be due to insufficient expression of cell surface molecules (e.g., CTLA-4 [29], CD39, and CD95), defects in the production of soluble factors, such as transforming growth factor ␤ and IL-10, involved in suppression, or the local constitution of the milieu, including cytokines (30–32) and APCs (24). In patients with JIA, SF CD4⫹CD25⫹ Treg cells were found to efficiently suppress SF CD4⫹CD25⫺ effector T cell proliferation, with increased suppressive capacity of Treg cells in the SF due to maturation of the Treg cells with high expression of CD25, CTLA-4, glucocorticoid-induced TNF receptor, and HLA–DR. However, SF CD4⫹CD25bright T cells showed a higher suppressive efficiency compared to CD4⫹CD25intermediate T cells (8). In this study, we investigated the SF of 21 individuals with JIA and demonstrated impaired T cell regulation by CD4⫹CD25⫹FoxP3⫹ Treg cells in the SF of individuals with JIA. There was no difference in the

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behavior of effector T cells and Treg cells between ANA-positive and ANA-negative patients or between patients who received different treatments. The suppressive properties of SF Treg cells seem to be comparable to those of PB Treg cells, whereas the defect in regulation is a result of effector T cells, which are resistant to Treg suppression. This hyporesponsiveness of SF effector T cells to Treg suppression has also been reported for RA (4). The ability of Treg cells to suppress effector T cells was shown to be dependent on the activation status of the CD4⫹ responder T cells, as in vitro preactivated CD4⫹ T cells become resistant to regulation by CD4⫹CD25⫹ T cells depending on the strength and duration of the stimulus (33). This finding was consistent with results showing that SF effector T cells of patients with RA had a highly activated phenotype associated with reduced susceptibility to Treg suppression (4). In the present study, we determined the degree of inhibition by Treg cells and the activation status of SF effector T cells by assessing the expression of the activation markers CD69 and HLA–DR and found a negative correlation between inhibition and activation of the effector T cells. There was no correlation between the percentage of SF effector T cells expressing CD45RO and the suppression of SF effector T cell proliferation, which supports the notion that the highly activated status rather than the memory condition of effector T cells is a main factor in the observed hyporesponsiveness of SF effector T cells in JIA. We confirmed a significant increase in the production of the proinflammatory cytokines/chemokines IL-6, TNF␣, CCL3, and IFN␥ by SF effector T cells compared to PB effector T cells, as has also been demonstrated by other studies of RA and JIA (4,32). SF Treg cells from patients with JIA significantly suppressed the production of the proinflammatory cytokines IL-2, TNF␣, and IFN␥ but failed to inhibit IL-6 and IL-10 released by autologous effector T cells. Efficient inhibition of TNF␣ and IFN␥ production in the presence of autologous PB and SF Treg cells from RA patients was demonstrated in 2 studies (4,21). A functional defect of isolated PB CD4⫹CD25⫹ T cells from patients with active RA in their ability to suppress proinflammatory IFN␥ and TNF cytokine production, which was abrogated after anti-TNF therapy, was observed by Ehrenstein et al (9). SF Treg cells failed to inhibit the proinflammatory cytokine IL-6 and the chemokine CCL3. A resistance to the inhibitory effects of Treg cells in the presence of IL-6 has been described (34). Therefore, the resulting imbalance between proinflammatory and anti-

IMPAIRED SUPPRESSION OF SYNOVIAL T CELLS IN JIA

inflammatory cytokines may allow SF effector T cells to bypass the suppression of proliferation by autologous Treg cells. In conclusion, we demonstrated an impaired susceptibility of activated synovial effector T cells to the suppressive effects of autologous, functional, highly purified CD4⫹CD25⫹FoxP3⫹ Treg cells in JIA patients, with a selective suppression of proinflammatory cytokines. Inhibition of effector T cells was negatively correlated with the expression of CD69 and HLA–DR on SF effector T cells, indicating that the highly activated status of SF effector T cells is a main factor in the observed hyporesponsiveness. Our finding that SF effector T cells are hyporesponsive to functional SF Treg cells may contribute to a better understanding of the immunopathogenic processes involved in JIA.

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5.

6.

7.

8.

9.

ACKNOWLEDGMENTS We are grateful to the patients and control subjects who participated in the study. We would like to thank Anya Schneider (Benaroya Research Institute at Virginia Mason, Seattle, WA) for helping with statistical analysis, Christiane Zimmer and Monika Moll for performing arthrocentesis at the University Children’s Hospital Tuebingen, Andreas Wirth for technical assistance with the Bio-Plex Pro assay, and Dominik Hartl for critical review of this manuscript.

10.

11.

12.

AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Holzer had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Haufe, Haug, Schepp, Dannecker, Holzer. Acquisition of data. Haufe, Schepp, Kuemmerle-Deschner, Hansmann, Rieber, Tzaribachev, Hospach, Maier, Dannecker, Holzer. Analysis and interpretation of data. Haufe, Schepp, Rieber, Dannecker, Holzer.

13. 14. 15.

16.

ROLE OF THE STUDY SPONSOR Wyeth/Pfizer Pharma had no role in the study design or in the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication. Publication of this article was not contingent upon approval by Wyeth/Pfizer Pharma.

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