Effector CD8+ T cells in systemic sclerosis ... - Wiley Online Library

ARTHRITIS & RHEUMATISM Vol. 60, No. 4, April 2009, pp 1119–1128 DOI 10.1002/art.24432 © 2009, American College of Rheumatology

Effector CD8⫹ T Cells in Systemic Sclerosis Patients Produce Abnormally High Levels of Interleukin-13 Associated With Increased Skin Fibrosis Patrizia Fuschiotti, Thomas A. Medsger, Jr., and Penelope A. Morel Conclusion. Dysregulated IL-13 production by effector CD8ⴙ T cells is important in the pathogenesis of SSc and is critical in the predisposition to more severe forms of cutaneous disease. Our study is the first to identify a specific T cell phenotype that correlates with disease severity in SSc and can be used as a marker of immune dysfunction in SSc and as a novel therapeutic target.

Objective. T lymphocytes play an important role in systemic sclerosis (SSc), a connective tissue disease characterized by inflammation, fibrosis, and vascular damage. While their precise role and antigen specificity are unclear, T cell–derived cytokines likely contribute to the induction of fibrosis. The aim of this study was to establish the role of cytokine dysregulation by T cells in the pathogenesis of SSc. Methods. To identify relationships between a specific cytokine, T cell subset, and the disease course, we studied a large cohort of patients with diffuse cutaneous SSc (dcSSc) or limited cutaneous SSc (lcSSc). Using Luminex analysis and intracellular cytokine staining, we analyzed the intrinsic ability of CD4ⴙ and CD8ⴙ T cell subsets to produce cytokines following in vitro activation. Results. High levels of the profibrotic type 2 cytokine interleukin-13 (IL-13) were produced following activation of peripheral blood effector CD8ⴙ T cells from SSc patients as compared with normal controls or with patients with rheumatoid arthritis. In contrast, CD4ⴙ T cells showed a lower and more variable level of IL-13 production. This abnormality correlated with the extent of fibrosis and was more pronounced in dcSSc patients than in lcSSc patients.

Systemic sclerosis (SSc; scleroderma) is an autoimmune connective tissue disease characterized by vascular damage, inflammation, and progressive fibrosis of the skin and internal organs (1). Patients with SSc are commonly classified into 2 subsets based on the pattern of skin involvement (2). Patients with diffuse cutaneous SSc (dcSSc) have rapidly progressive fibrosis of the skin, lungs, and other internal organs. In contrast, in limited cutaneous SSc (lcSSc) the most prominent features are vascular manifestations, with generally mild skin and internal organ fibrosis. Several lines of evidence suggest that T cells are important in the pathogenesis of SSc, including observations that ␣/␤ and ␥/␦ T lymphocytes exhibit increased expression of activation markers (3,4), show signs of antigen-driven expansion (5), and dominate the inflammatory infiltrates in the skin and involved tissues during the early stages of the disease (6,7). T cells have been found to be necessary for the production of anti– topoisomerase I antibodies in SSc (8). Autoantibodies against a variety of nuclear proteins are a nearly uniform feature of patients with SSc (9). Abnormal levels of T cell–derived cytokines, including transforming growth factor ␤ (TGF␤) (10), tumor necrosis factor ␣ (TNF␣), interleukin-6 (IL-6), IL-10 (11), IL-17 (12), IL-4, and IL-13 (13–15), have been found in the serum of SSc patients. Among other functions, these cytokines are thought to promote the overproduction of collagen by fibroblasts, resulting in excessive fibrosis (16).

Supported by the University of Pittsburgh School of Medicine, the Scleroderma Research Fund (University of Pittsburgh Division of Rheumatology and Clinical Immunology), the Taub Fund (Chicago, IL), the Zale Foundation (Dallas, TX), and the Shoemaker Fund (Arthritis Foundation, Western Pennsylvania Chapter). Dr. Fuschiotti is recipient of the 2008 Marta Marx Eradication of Scleroderma award from the Scleroderma Foundation. Patrizia Fuschiotti, PhD, Thomas A. Medsger, Jr., MD, Penelope A. Morel, MD: University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Address correspondence and reprint requests to Patrizia Fuschiotti, PhD, Department of Immunology, University of Pittsburgh School of Medicine, 200 Lothrop Street, BST W1052, Pittsburgh, PA 15261. E-mail: [email protected]. Submitted for publication August 6, 2008; accepted in revised form January 10, 2009. 1119

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An emerging hypothesis concerning the pathogenesis of fibrotic disorders implicates an altered balance between type 1 and type 2 cytokines (17). Type 2 cytokines are important regulators of extracellular matrix remodeling, leading to enhanced collagen deposition and tissue fibrosis, whereas type 1 cytokines inhibit this process. Type 2 polarized T cells secrete large amounts of IL-4, IL-5, and IL-13 and low levels of type 1 cytokines, such as interferon-␥ (IFN␥) (18). Findings in animal studies provide support for the role of a polarized immune response in the pathogenesis of fibrosis (19,20). Although reports concerning the balance of type 1 and type 2 cytokines in SSc patients are conflicting, most studies suggest that the immunopathologic response is dominated by type 2 cytokines (21–30). Therefore, the skewing of the immune response toward the type 2 pattern of cytokine secretion may contribute to the pathogenesis of tissue fibrosis in SSc. Understanding the mechanisms that generate this pattern of immune response in SSc is of significant interest, since at the present time, it is not known which specific cell types are the major source(s) of pathogenic cytokines. Reduced numbers of certain T cell subsets, such as natural killer (NK) and ␥/␦ T cells, in the peripheral blood of SSc patients have been reported (31). The results of that study also showed a correlation between the reduced number of NKT cells and increased inflammation, suggesting that these cells play a regulatory role in SSc. The absolute and relative numbers of CD4⫹ T cells in SSc patients are normal, whereas those of CD8⫹ T cells are decreased (6). Since CD4⫹ T cells represent the predominant T cell infiltrate in the skin of SSc patients during the inflammatory phase of the disease and since there is a strong HLA class II major histocompatibility complex association with the autoantibody profile, clinical subsets (32,33), and T cell responses (34), most studies to date have been focused on CD4⫹ T cells. Little is known about the role of CD8⫹ T cells in the pathogenesis of SSc, apart from the finding of an increased number of CD8⫹ T cells in bronchoalveolar lavage fluid from SSc patients with lung fibrosis as compared with that from healthy controls (23). The aim of this study was to determine whether SSc patients exhibited abnormalities in the production of type 2 cytokines by CD4⫹ and CD8⫹ T cells, as compared with normal controls and with patients with other inflammatory conditions. We observed that CD8⫹ T cells from SSc patients produced high levels of IL-13 following in vitro activation. Furthermore, we found that it was the effector CD8⫹ T cell population that was responsible for the dysregulated production of IL-13 and

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that this was associated with a more severe disease phenotype. Thus, we have identified a specific T cell subset that is likely to be an important factor in the pathogenesis of SSc and that may be the target of future therapeutic interventions. PATIENTS AND METHODS Study participants. A total of 53 SSc patients (12 men and 41 women) with a mean age of 48.6 years (range 19.8–72.8 years) were enrolled in the study. These individuals were new and consecutive returning patients seen in our weekly Scleroderma Clinic. All patients fulfilled either the criteria for scleroderma proposed by the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) (35) (n ⫽ 50 of 53 patients) or the criteria for early SSc proposed by LeRoy and Medsger (36) (n ⫽ 3 of 53 patients). Patients were classified as having dcSSc or lcSSc according to the system proposed by LeRoy et al (37) and were then divided into those with early or late disease as previously described (2). To better understand the natural history of this disease, dcSSc patients were arbitrarily subdivided according to early (⬍3 years) and late (ⱖ6 years) disease and lcSSc patients according to early (⬍5 years) and late (ⱖ10 years) disease, both of which were measured from the time of the first symptom attributable to SSc (2). Published international consensus recommendations for each organ system were used to determine disease severity (38). The time of disease onset was defined as the date of the first symptom attributable to SSc. Diffuse cutaneous SSc was defined as skin thickening proximal to the elbows or knees occurring at any time during the illness. Pulmonary fibrosis was defined according to findings on chest radiography or high-resolution computed tomography scan of the lungs. Organ system involvement was defined as described elsewhere (39). Serologic studies were performed by the Research Laboratory of the Division of Rheumatology and Clinical Immunology using established techniques (9). All demographic, clinical, and laboratory data were abstracted from the medical files, recorded on standardized data collection forms, and entered into the Pittsburgh Scleroderma Databank time-oriented computer system (MEDLOG) for analysis. Medications taken by the patients at the time the blood was drawn were recorded. Six of the 53 patients were treated with low-dose corticosteroids (3–10 mg/day of prednisone). In addition to steroid treatment, 3 patients were also taking low-dose D-penicillamine (250 mg/day). Six patients were taking D-penicillamine alone (250–500 mg/day; in 1 patient, 1,500 mg/day). None of the patients received other immunosuppressive therapy. Twenty-three patients with rheumatoid arthritis (RA) who met the ACR classification criteria (40) were included in the study, and their concomitant medications were recorded. Most were treated with a combination of immunosuppressive drugs (prednisone and methotrexate) and other diseasemodifying antirheumatic drugs (DMARDs) including leflunomide, hydroxychloroquine, and anti-TNF␣ agents, such as etanercept and infliximab. A total of 33 age- and sex-matched healthy controls were also studied. These were either blood

ASSOCIATION OF Teff CELLS WITH IL-13 AND SKIN FIBROSIS IN SSc

Table 1.

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Clinical and serologic characteristics of the patients with SSc, according to SSc subtype*

Age at diagnosis of SSc, years Mean ⫾ SD Range Sex, no. female/male Duration of SSc at first visit to our clinic (from SSc diagnosis), years Mean ⫾ SD Range Organ system involvement at any time during the disease course Vascular, no. (%) Skin, mean ⫾ SD total skin score Joint/tendon, no. (%) GI tract, no. positive/no. tested (%) Lung, radiologic fibrosis, no. positive/no. tested (%) Kidney Serologic characteristics, no. (%) Anti–topoisomerase I antibody Anticentromere antibody Anti–RNA polymerase I/III antibody Anti–Ku antibody Anti–U1 RNP antibody ANA of undefined specificity

All patients (n ⫽ 53)

Patients with dcSSc (n ⫽ 31)

Patients with lcSSc (n ⫽ 22)

48.6 ⫾ 12.2 19.8–72.8 41/12

48.4 ⫾ 13.9 19.8–72.8 23/8

48.9 ⫾ 9.4 25.2–62.1 18/4

2.5 ⫾ 4.9 0–24.2

1.6 ⫾ 2.7 0–10.9

3.7 ⫾ 6.8 0–24.2

53 (100) 18.2 ⫾ 11.7 46 (87) 29/37 (78) 17/50 (34) 2 (4)

31 (100) 25.8 ⫾ 9.2 28 (90) 14/19 (74) 11/29 (38) 2 (6)

22 (100) 7.4 ⫾ 3.5† 18 (81) 15/18 (83) 6/21 (29) 0 (0)

22 (42) 20 (38) 3 (6) 1 (2) 1 (2) 6 (11)

17 (55) 5 (16) 3 (10) 1 (3) 0 (0) 5 (16)

5 (23)‡ 15 (68)§ 0 (0) 0 (0) 1 (5) 1 (5)

* SSc ⫽ systemic sclerosis; dcSSc ⫽ diffuse cutaneous SSc; lcSSc ⫽ limited cutaneous SSc; GI ⫽ gastrointestinal; ANA ⫽ antinuclear antibody. † P ⬍ 0.001 versus patients with dcSSc, by chi-square test or analysis of variance. ‡ P ⬍ 0.05 versus patients with dcSSc, by chi-square test or analysis of variance. § P ⬍ 0.001 versus patients with dcSSc, by chi-square test or analysis of variance.

donors or employees of the Central Blood Bank of Pittsburgh, and were recruited from the Pittsburgh area. All participants signed a written consent document. Clinical information and biologic specimens were deidentified and coded. Research protocols involving human subjects were approved by the Institutional Review Board of the University of Pittsburgh. Collection of specimens. Blood was collected by venipuncture. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on a Ficoll-Hypaque gradient and frozen in liquid nitrogen prior to the assays or were used fresh. We compared frozen and fresh PBMCs to verify that they had similar functional abilities to differentiate and to produce cytokines. For all relevant cytokines, frozen and fresh PBMCs were found to be equivalent. CD8⫹ and CD4⫹ T cells were purified from the PBMC samples by negative selection using an EasySep enrichment cocktail according to the manufacturer’s instructions (StemCell Technologies, Vancouver, British Columbia, Canada). CD8⫹ T cells were subsequently separated into CD45RA⫹ (naive and effector) and CD45RA– (central and effector memory) subpopulations by positive selection with a magnetically coupled monoclonal antibody (mAb) against CD45RA⫹ according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Effector CD8⫹ T cells were purified from PBMCs in 2 steps, first, by negative selection using CD27 microbeads (Miltenyi Biotec), and then, using an EasySep enrichment cocktail for human CD8⫹ T cells (Stem-

Cell Technologies). Finally, human naive T cells were purified with a Naive CD8⫹ T Cell Isolation kit (Miltenyi Biotec). All of these subset purifications were conducted according to the manufacturers’ instructions. The purity of each T cell subpopulation was ⬎90%, as determined by flow cytometry. Flow cytometry. For phenotype analysis of human CD8⫹ T cells from SSc patients and age-matched normal controls, freshly isolated PBMCs were stained with a combination of the following anti-human antibodies: Pacific Blue– conjugated CD8, Alexa Fluor 700–conjugated CD3, phycoerythrin–Cy5–conjugated CD45RA, and Alexa Fluor 750–conjugated CD27 (all from eBioscience, San Diego, CA). After incubation at 4°C for 30 minutes, the cells were washed twice with phosphate buffered saline (PBS) containing 10% fetal calf serum and resuspended in PBS/2% paraformaldehyde. Flow cytometry was performed on a 3-laser, 9-detector LSR II system (Becton Dickinson, San Jose, CA), and data were analyzed with FlowJo software (Tree Star, Ashland, OR). Cell culture and measurement of cytokine production. In order to examine cytokine production by T cells from SSc patients, we performed in vitro activation of purified CD8⫹ and CD4⫹ T cells. Briefly, 1 ⫻ 106 purified CD8⫹ or CD4⫹ T cells were activated in vitro in the presence of beads that had been coated with anti-CD3 and anti-CD28 mAb (Dynabeads CD3/CD28 T cell Expander; Dynal Biotech–Invitrogen, Carlsbad, CA) and recombinant human IL-2 (20 units/ml; PeproTech, Rocky Hill, NJ). After 5 days of culture, the cells were restimulated for 24 hours with anti–T cell receptor and soluble

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anti-CD28. The supernatants and cells were collected, and cytokine levels were determined by Luminex technique and intracellular cytokine staining, respectively. In the supernatants, we evaluated type 1 and 2 cytokines (IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, granulocyte–macrophage colonystimulating factor, IFN␥, TNF␣) using Luminex technology (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. At the single-cell level, we measured IL-13 and IFN␥ production by intracellular cytokine staining. A commercially available kit was used according to the manufacturer’s protocol (eBioscience). Briefly, anti-CD3/CD28–activated CD8⫹ and CD4⫹ T cells were harvested and stimulated with a combination of 25 ng/ml of phorbol 12-myristate 13-acetate and 1 ␮g/ml of ionomycin (Sigma-Aldrich, St. Louis, MO) in the presence of 10 ␮g/ml of brefeldin A (Epicentre Biotechnologies, Madison, WI) for 5 hours at 37°C in a humidified atmosphere containing 5% CO2. Activated cultures were stained for the expression of CD4 and CD8 using a mixture of Pacific Blue–conjugated anti-CD8 or Pacific Blue–conjugated anti-CD4 and allophycocyanin-conjugated anti-CD3 mAb for 30 minutes at 4°C, fixed, and permeabilized. Cells were then incubated for 20 minutes at room temperature with the following cytokine-specific mAb (all from eBioscience) diluted in permeabilization buffer: fluorescein isothiocyanate– conjugated anti-human IL-13 (clone PVM13-1) and phycoerythrin-conjugated anti-human IFN␥ (clone 4S.B3). Labeled cells were analyzed by flow cytometry as described above. Statistical analysis. All values are expressed as the mean ⫾ SD. When indicated, we performed an analysis of significance using Student’s 2-tailed t-test for comparison of 2 groups, or by analysis of variance with post hoc Dunnett’s or Tukey-Kramer’s multiple comparison analysis for multiple groups, and nonparametric analysis of variance for nonnormally distributed data (Kruskal-Wallis). Differences in proportions were examined using the chi-square test. P values less than 0.05 were considered significant, those less than 0.01 were considered very significant, and those less than 0.001 were considered highly significant. Data were analyzed with the use of InStat software (GraphPad Software, San Diego, CA).

RESULTS IL-13 dysregulation by peripheral blood CD8ⴙ T cells from SSc patients. Activated CD4⫹ or CD8⫹ T cells in the skin (22) and lung (23) of SSc patients were previously observed to produce more IL-4 than IFN␥ and were therefore considered profibrotic. Based on this finding, we hypothesized that profibrotic cytokine production by peripheral blood CD4⫹ and/or CD8⫹ T cells is dysregulated, predisposing patients to more severe forms of SSc. We studied a well-defined population of SSc patients (n ⫽ 53) evaluated at our Scleroderma Clinic (Table 1). Controls consisted of healthy normal donors (n ⫽ 33) and patients with RA (n ⫽ 23). In order to evaluate cytokine production by peri-

Figure 1. Cytokine dysregulation in CD8⫹ and CD4⫹ T cells from patients with systemic sclerosis (SSc). Levels of cytokine production were measured by Luminex techniques, as described in Patients and Methods. A, Ratio of interferon-␥ (IFN␥) to interleukin-13 (IL-13) levels in CD8⫹ T cells from SSc patients, normal donors (ND), and rheumatoid arthritis (RA) patients. The decreased ratio in SSc patients was highly significant (ⴱⴱⴱ ⫽ P ⬍ 0.001) compared with normal donors and with RA patients, by Kruskal-Wallis test (nonparametric analysis of variance [ANOVA]). B, Ratio of IFN␥ levels to IL-13 levels in CD4⫹ T cells from SSc patients and normal controls. The difference was significant (ⴱ ⫽ P ⬍ 0.05, by Student’s unpaired 2-tailed t-test). C and D, Production of IL-13 (C) and IFN␥ (D) by CD8⫹ T cells from SSc patients, normal donors, and RA patients. IL-13 levels were significantly increased in SSc patients compared with normal donors (ⴱⴱ ⫽ P ⬍ 0.01) and with RA patients (ⴱ ⫽ P ⬍ 0.05), by ANOVA with Dunnett’s post hoc multiple comparison test. Each data point represents a single study subject. Horizontal lines show the mean.

pheral blood T cells from SSc patients, we examined the ability of purified CD4⫹ and CD8⫹ T cells to produce cytokines following in vitro anti-CD3/anti-CD28 activation for 5 days. Our results showed significantly higher levels of IL-13 production in CD4⫹ T cells and particularly CD8⫹ T cells from SSc patients than from normal donors. When production was expressed as the ratio of IFN␥ to IL-13, we noted a highly significant (P ⬍ 0.001), 7-fold reduction in the ratio in the CD8⫹ T cells from SSc patients (Figure 1A). CD4⫹ T cells also showed a reduced IFN␥:IL-13 ratio in SSc patients compared with normal donors (Figure 1B) that was statistically significant (P ⫽ 0.011) but was more variable than that in the CD8⫹ T cells. The reduced ratio in CD8⫹ T cells appears to be due to augmented production of IL-13, which we noted to be significantly increased (P ⫽ 0.005) in SSc patients compared with normal controls (Figure


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Figure 2. Cytokine expression at the single-cell level. A, Expression of interleukin-13 (IL-13) and interferon-␥ (IFN␥) in CD8⫹ and CD4⫹ T cells from a representative patient with systemic sclerosis (SSc) and a representative normal donor (ND). Intracellular cytokine production was determined by flow cytometry with 4-color analysis. Cells were first stimulated for 5 days with anti-CD3, anti-CD28, and IL-2, and then for 5 hours with phorbol 12-myristate 13-acetate, ionomycin, and brefeldin A. Plots represent live cells gated for expression of CD8 or CD4 and CD3. Percentages of positive cells are indicated in the respective compartments, which were set according to the staining of isotype control monoclonal antibodies (data not shown). B and C, Frequencies of CD8⫹ (B) and CD4⫹ (C) T cells producing IL-13 and IFN␥ in SSc patients and normal donors. The frequencies were increased in SSc patients compared with normal donors (ⴱⴱⴱ ⫽ P ⬍ 0.001, and ⴱⴱ ⫽ P ⬍ 0.01, by Student’s unpaired 2-tailed t-test). Each data point represents a single study subject. Horizontal lines show the mean.

1C), while IFN␥ production was not significantly different between the 2 groups (P ⫽ 0.211) (Figure 1D). No differences were detected in the production of other cytokines, such as IL-4 or IL-5, in the Luminex panel by CD4⫹ or CD8⫹ T cells from SSc patients compared with normal donors (data not shown), suggesting that this defect is specific to IL-13. To determine whether the cytokine abnormality seen in CD8⫹ T cells was specific to SSc or was associated with other autoimmune/inflammatory diseases, we examined samples from patients with RA, which is characterized by type 1 cytokine production (39). As shown in Figure 1A, no decrease in the IFN␥: IL-13 ratio was observed in CD8⫹ T cells from RA patients. This ratio was significantly higher than that in SSc patients (P ⬍ 0.001) and was no different from that in normal controls (P ⫽ 0.269). The absolute amounts of IL-13 produced by CD8⫹ T cells from RA patients were similar to those produced by normal donors (P ⫽ 0.176) and were significantly lower than those produced by SSc patients (P ⫽ 0.03) (Figure 1C). Furthermore, the levels of IFN␥ produced by CD8⫹ T cells from RA patients were comparable to those produced by SSc patients (P ⫽ 0.110) and normal donors (P ⫽ 0.863) (Figure 1D). In an effort to exclude any effect on cytokine production by immunosuppressive medications or DMARDs, we compared the medications taken at the time blood samples were obtained (see Patients and Methods) with the levels of cytokines produced by SSc

and RA patients. On average, RA patients took a larger number of medications (combined immunosuppressants and DMARDs) than did SSc patients. Only 12 of 53 SSc patients were treated, and only 3 of them took a combination of prednisone and methotrexate. We found no correlation between treatment and no treatment and IL-13 levels, suggesting that the therapies had no effect on IL-13 production, at least in the SSc patients. We next performed single-cell intracellular cytokine staining to determine whether increased cytokine production was associated with changes in the frequencies of cytokine producing T cells. A significant increase (P ⬍ 0.01) in the number of IL-13⫹ cells was found in both CD8⫹ and CD4⫹ compartments of the SSc patients as compared with the normal donors (Figure 2) or with the RA patients (data not shown), confirming the Luminex data. Although the number of IFN␥⫹ cells varied more widely among the individuals tested, the frequency among the CD8⫹ and CD4⫹ T cells was not significantly different between the SSc and normal donor groups (Figures 2B and C). Effector CD8ⴙ T cells are responsible for abnormal IL-13 production in the peripheral blood of SSc patients. To further characterize the phenotype of the IL-13–producing CD8⫹ T cells, we performed multicolor flow cytometry of peripheral blood CD8⫹ T cells from several SSc patients and age-matched healthy controls. Previously, 2 distinct populations of antigenexperienced CD8⫹ T cells, called T effector (Teff) cells

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Table 2. Phenotypic characterization of peripheral blood CD8⫹ T cells in SSc patients and age-matched normal donors, by multicolor flow cytometry* CD8⫹ T cell subset CD45RA⫹CD27⫹ CD45RA⫺CD27⫹ CD45RA⫹CD27⫺ CD45RA⫺CD27⫺

(naive) (memory) (effector) (effector/memory)

Normal donors SSc patients (n ⫽ 13) (n ⫽ 30) 41.4 ⫾ 15.7 36.9 ⫾ 14.7 10.3 ⫾ 11.5 8.4 ⫾ 5.3

39.8 ⫾ 22.3 21.0 ⫾ 15.8† 28.9 ⫾ 21.8† 8.6 ⫾ 9.6

* CD8⫹ cells were ⬎98% CD3⫹. Values are the mean ⫾ SD percentage of positive cells. SSc ⫽ systemic sclerosis. † P ⬍ 0.01 versus normal donors, by Student’s t-test.

and T central memory cells, were described, and coexpression of cell surface molecules, such as CD27 and CD45RA, allows the identification of naive, memory, and effector CD8⫹ T cell subtypes (41). The proportions of naive, memory, effector, and effector/memory subsets in the peripheral blood CD8⫹

T cells from SSc patients and normal donors are reported in Table 2. Our data showed an increase in the effector cell subpopulation (mean ⫾ SD 28.9 ⫾ 21.8 versus 10.3 ⫾ 11.5; P ⬍ 0.01) and a lower percentage of the memory T cell subset (mean ⫾ SD 21.0 ⫾ 15.8 versus 36.9 ⫾ 14.7; P ⬍ 0.01) in SSc patients compared with healthy age-matched controls, indicating that an active CD8⫹ T cell immune response is ongoing in the peripheral blood of SSc patients. To address the possibility that previously activated or memory CD8⫹ T cells in the periphery were responsible for the increase in IL-13 levels, we examined specific CD8⫹ T cell subsets. We purified CD8⫹CD45RA⫹ T cells from total CD8⫹ T cells isolated from SSc patients and determined the number of IL-13⫹ cells in the CD45RA⫹ (naive and effector) and CD45RA– (memory) CD8⫹ T cell subsets (41) (Figures 3A and B). In all samples tested, we found that a higher number of IL-13⫹ cells was present in the

Figure 3. Cytokine expression in CD8⫹ T cell subsets. A, Interleukin-13 (IL-13) production by CD8⫹, CD8⫹CD45RA⫹, and CD8⫹CD45RA– T cells from patients with systemic sclerosis (SSc). Most IL-13–producing cells were CD45RA⫹. Differences were significant for CD8⫹ versus CD8⫹CD45RA– cells (ⴱⴱ ⫽ P ⬍ 0.01) as well as for CD8⫹CD45RA⫹ versus CD8⫹CD45RA– cells (ⴱⴱⴱ ⫽ P ⬍ 0.001) by analysis of variance (ANOVA) with Tukey-Kramer’s post hoc multiple comparison test. B, Representative histogram from the experiments shown in A. C, IL-13⫹ cells in the naive and Teff cell subsets from a representative patient with SSc. CD8⫹ naive and Teff cells were first purified from peripheral blood mononuclear cells and then activated in vitro as described in Figure 2A. Intracellular cytokine staining was used to determine the proportions of IL-13⫹ and interferon-␥ (IFN␥)–positive cells. D, Comparison of the number of IL-13⫹ cells (left) and the number of IFN␥⫹ cells (right) in naive and Teff cells subsets from SSc patients and age-matched normal donors (ND). Differences in the numbers of IL-13⫹ cells were significant (ⴱⴱⴱ ⫽ P ⬍ 0.001) for Teff cells versus naive cells from SSc patients and for SSc Teff cells versus Teff cells and naive cells from normal donors by ANOVA, with Tukey-Kramer’s multiple comparison test. Each data point in A and D represents a single study subject. Horizontal lines show the mean.


CD8⫹CD45RA⫹ subset than in the CD8⫹CD45RA– memory subpopulation (mean ⫾ SD 6.64 ⫾ 2.23 versus 1.12 ⫾ 0.4; P ⬍ 0.001), excluding the possibility that preactivated/memory CD8⫹ T cells with abnormal IL-13 production were present in the peripheral blood CD8⫹ T cells in SSc patients. Since CD8⫹CD45RA⫹ T cells include both naive and effector cells, we identified the CD8⫹ T cell subset responsible for increased IL-13 production by further purifying peripheral blood CD8⫹ T cells into naive (CD8⫹CD45RA⫹CD27⫹) and effector (CD8⫹CD45RA⫹/–CD27–) cells and analyzing their ability to produce cytokines after in vitro activation. All of the excess IL-13 was produced by effector, and not naive, CD8⫹ T cells (Figure 3C). This was confirmed in a larger analysis of several SSc patients and age-matched normal donors (Figure 3D), clearly showing that the effector subset is the source of IL-13⫹ cells (mean ⫾ SD 7.42 ⫾ 3.3 Teff cells versus 1.8 ⫾ 1.6 naive cells in SSc patients, and 1.7 ⫾ 0.4 versus 1.6 ⫾ 0.6 in normal donors). Interestingly, variable proportions of IFN␥⫹ cells were present in naive and Teff cells from SSc patients, and normal donors (Figure 3D), but the differences were not statistically significant. Further characterization of the Teff cells IL-13⫹ subset in SSc patients demonstrated that these cells express high levels of perforin and granzyme B, but do not express the chemokine receptor CCR7, suggesting that these are cytotoxic effector cells (41) (data not shown). Our results suggest that the increased propensity for IL-13 production by CD8⫹ T cells is an immune dysfunction associated with SSc and not the result of an increased presence of preactivated/memory CD8⫹ T cells and that CD8⫹ T cells are likely involved in the pathogenesis of the disease. Association of CD8ⴙ T cell IL-13 dysregulation and clinical features. We next attempted to determine whether this abnormality of CD8⫹ T cell cytokine production in SSc patients was associated with any particular clinical features. Interestingly, the lower IFN␥:IL-13 ratios in CD8⫹ T cells were associated predominantly with dcSSc as compared with lcSSc (Figure 4A). Patients with dcSSc had significantly higher levels of skin fibrosis (P ⬍ 0.001), and the proportion with lung fibrosis was higher than in patients with lcSSc (Table 1). Since IL-13 is involved in tissue remodeling in some pulmonary diseases, including asthma (42), we compared the IFN␥:IL-13 values in SSc patients with and without pulmonary fibrosis. We found only a minimal decrease in these ratios in patients with pulmonary fibrosis compared with those without (mean ⫾ SD 2.4 ⫾

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Figure 4. Association of cytokine production with clinical features of systemic sclerosis (SSc). A, Ratio of interferon-␥ (IFN␥) to interleukin-13 (IL-13) in patients with limited cutaneous SSc (lcSSc) and those with diffuse cutaneous SSc (dcSSc). Ratios were calculated from the levels of IFN␥ and IL-13 measured by Luminex techniques in the supernatant of unfractionated CD8⫹ T cells from the SSc patients. Differences were significant (ⴱⴱ ⫽ P ⬍ 0.01) by Student’s unpaired 2-tailed t-test. B, Ratio of IFN␥ to IL-13 in dcSSc patients with early disease (symptoms ⬍3 years) and those with late disease (symptoms ⱖ6 years). C, Ratio of IFN␥ to IL-13 in SSc patients according to antinuclear antibody expression: anticentromere antibodies (ACA), anti–topoisomerase I antibodies (ATA), or other antinuclear antibodies (ANA). Each data point represents a single study subject. Horizontal lines show the mean.

1 versus 3.1 ⫾ 1.9), suggesting that IL-13 is associated more with the extent of skin fibrosis than with lung fibrosis, although this result remains to be confirmed in larger numbers of patients. In comparing the IFN␥:IL-13 ratios with disease duration (Figure 4B), we found no significant difference between dcSSc patients who had symptoms for ⬍3 years (early) or ⱖ6 years (late) (2). For lcSSc patients, data from the early stage of disease are rare, since these patients typically do not come to medical attention during this period of disease (2). However, we found no difference in the levels of cytokine produced by patients with early (duration ⬍5 years) versus late (duration ⱖ10 years) lcSSc (data not shown). IL-13 dysregulation is therefore not dependent on disease duration but is an intrinsic characteristic of SSc. IL-13 levels are also independent of autoantibody expression (Figure 4C) but are associated with the severity of skin fibrosis (Figure 4A). Despite the fact that patients with serum anti– topoisomerase I antibodies more often have dcSSc, a few in our cohort presented with lcSSc. Similarly, although anticentromere antibody–positive patients almost all have lcSSc, several in our study population had dcSSc and lung involvement (Table 1). Thus, correlation between IL-13 dysregulation in CD8⫹ T cells and the clinical manifestations in our patient population indicates that IL-13 dysregulation is

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an intrinsic characteristic of SSc patients and appears to correlate with the degree and extent of cutaneous fibrosis. DISCUSSION Although activation of the immune system has long been recognized, the mechanisms responsible for initiation of autoimmunity and the role of immune effector pathways in the pathogenesis of SSc remain incompletely understood. Our results provide new insights into potential pathogenetic pathways in SSc by showing that peripheral blood effector CD8⫹ T cells from SSc patients demonstrate a shift toward a type 2 phenotype, characterized by up-regulation of the important profibrotic cytokine IL-13. Although IL-13 was previously associated with SSc through the observation of higher serum levels (13,15), we were able for the first time to associate dysregulated IL-13 production with a specific cell type in the blood of SSc patients and, furthermore, to indicate a direct role of this effector CD8⫹ T cell type in the pathogenesis of SSc. This may allow the development of novel therapeutic strategies aimed at targeting these cells and the cytokine they produce. It has been proposed that the pathogenesis of SSc is mediated by an imbalance in Th1 and Th2 cytokines, such that CD4⫹ T cells contribute to disease by increased production of type 2 cytokines (1). This concept has been supported by the observations that CD4⫹ T cells infiltrate early lesions (22), that there are strong HLA–DR/DQ associations with certain forms of the disease (32), and that distinct and specific autoantibodies are present. In addition, several reports have suggested that circulating T cells produce higher levels of type 2 cytokines, as determined by short-term activation of whole blood (21,24–26) and the detection of IL-4 and IL-13 in the serum (13–15). Few studies have examined the intrinsic ability of purified CD4⫹ and CD8⫹ T cells from SSc patients to produce cytokines following in vitro activation. Our study is unique in demonstrating a novel relationship between CD8⫹ T cells and IL-13 production in SSc. IL-13 is an immunoregulatory cytokine that is predominantly secreted by activated Th2 cells. It is known to play prominent roles in mediating tissue fibrosis and participating in the pathogenesis of fibrosis in many diseases (17). The profibrotic activities of IL-13 involve both direct fibroblast activation and indirect mechanisms due to stimulation of TGF␤ (17). Although elevated levels of IL-13 in the serum of patients with SSc

have been reported, the source of this important cytokine had not been determined. IL-13 shares some functions with IL-4, and they share a common receptor. Unlike IL-4, IL-13 does not have a direct effect on T cell differentiation, but rather, it appears to be necessary during the effector phase of inflammation and fibrosis. However, IL-13 may contribute to or perpetuate the type 2 response via several pathways, such as by inducing the expression of Th2attracting chemokines, namely, CCL17 (thymus and activation–regulated chemokine) and CCL22 (macrophage-derived chemokine) (43). Furthermore, it appears to promote its own production via regulation of several mediators, including adenosine and histamine, which in turn, stimulate cells such as eosinophils, mast cells, basophils, and smooth muscle cells to produce more IL-13. Thus, through stimulation of these pathways, IL-13 may be an important contributor to the chronic nature of fibrotic disorders. The importance of IL-13 in SSc is supported by recent studies that implicate polymorphisms in the IL-13 (44) and IL-13 receptor ␣2 (45) genes in dcSSc, providing further evidence that the control of this important profibrotic cytokine may be abnormal in SSc. In the studies presented here, IL-13 levels were elevated in both CD8⫹ and CD4⫹ T cells from SSc patients, but the abnormality was significantly more pronounced and consistent in CD8⫹ T cells. Our study is the first to associate dysregulated IL-13 production with CD8⫹ T cells in the blood of SSc patients. Furthermore, our data show that CD8⫹ Teff cells are the source of IL-13 and that this increased IL-13 production is more prevalent in dcSSc than in lcSSc patients. Previous studies have demonstrated the presence of CD8⫹ T cells producing IL-4 in bronchoalveolar lavage fluid from patients with SSc, and this finding was correlated with the development of lung fibrosis (23). In addition, CD8⫹ Teff cells were shown to be a source of IL-13 in a mouse model of airway hyperresponsiveness and airway inflammation (46), supporting the idea that CD8⫹ T cells can play important roles in type 2–driven immune responses. Analysis of peripheral blood CD8⫹ T cells indicates that not only is the number of IL-13⫹ Teff cells increased, but the total number of circulating Teff cells in the peripheral blood of SSc patients is also elevated compared with that in normal donors. In addition, these cells express cytolytic effector molecules, such as perforin and granzyme B, and they are also CCR7–, indicating an increased ability to migrate to sites of inflammation. Thus, these cells are likely to be present in SSc


lesions of the skin and lung, and this is an area of investigation that we are actively pursuing. The biologic significance of CD8⫹ Teff cells in autoimmune disorders such as SSc has not been well studied, but recent studies, including our own, suggest that these cells may play an important role. Teff cells may exert cytolytic activities and produce high levels of cytokines with immunomodulatory potential and profibrotic activities. Indeed, inhibition of endothelial cell growth in SSc by vascular injury appears to be mediated by granzyme B and perforin (47). Interestingly, selfprotein fragments (47) generated by granzyme B are recognized by autoantibodies in a subset of SSc patients (48). Therefore, the activation of cytotoxic cell– mediated pathways may be involved in early vascular damage in SSc and potentially in initiating and propagating the specific autoimmune response in this disease. While several studies have identified CD4⫹ T cells specific for autoantigens such as topoisomerase I (49), to date, there have been few reports describing autoantigen-specific CD8⫹ T cells in SSc (50). The question has been difficult to investigate because of the inaccessibility of target tissues for analysis and because of the low number of antigen-specific precursors in the peripheral blood. The presence of distinct and restricted T cell receptor signatures indicates oligoclonal expansion of T cell populations in response to antigens (5). However, their precise role and antigen specificity remain to be established. Nonetheless, the identification of an abnormal population of circulating IL-13–producing CD8⫹ T cells associated with SSc may provide a new source of potentially autoreactive T cells. Taken together, our results suggest that CD8⫹ Teff cells in the peripheral blood of SSc patients might represent the effector arm of the immune response in SSc because of IL-13 overproduction, direct cellular cytotoxicity, and their tissue-infiltrating potential. These novel results suggest that dysregulated IL-13 production by CD8⫹ T cells plays an important role in human SSc and could be a critical predisposing factor for the disease. Elucidation of the phenotype of T lymphocytes in the peripheral blood of patients with SSc and identification of an intrinsic/genetic defect in the control of a specific cytokine such as IL-13 provide new insights into the pathogenesis of the disease. We expect to use these observations to explore the establishment of one or more biomarkers of immune dysfunction in SSc and pursue avenues for the development of novel therapeutic agents that will benefit patients with this serious disease.

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ACKNOWLEDGMENTS We are very grateful to Prof. Olivera Finn for her support and insightful comments. We thank Huijie Sun and Dewayne H. Falkner (Department of Immunology) for helping with the Luminex and flow cytometry analyses, and Mary Lucas (Division of Rheumatology and Clinical Immunology) for providing the clinical details of the patients. AUTHOR CONTRIBUTIONS Dr. Fuschiotti 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 design. Fuschiotti, Medsger, Morel. Acquisition of data. Fuschiotti, Medsger. Analysis and interpretation of data. Fuschiotti, Medsger, Morel. Manuscript preparation. Fuschiotti, Medsger, Morel. Statistical analysis. Fuschiotti, Medsger.

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