Expanded T cell receptor V restricted T cells from patients with sporadic inclusion body myositis are proinflammatory and cytotoxic CD28null T cells.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 62, No. 11, November 2010, pp 3457–3466 DOI 10.1002/art.27665 © 2010, American College of Rheumatology Expanded T Cell Receptor V␤–Restricted T Cells From Patients With Sporadic Inclusion Body Myositis Are Proinflammatory and Cytotoxic CD28null T Cells Jayesh M. Pandya,1 Andreas E. R. Fasth,1 Mei Zong,1 Snjolaug Arnardottir,2 Lara Dani,1 Eva Lindroos,1 Vivianne Malmström,1 and Ingrid E. Lundberg1 Objective. Sporadic inclusion body myositis (IBM) is characterized by T cell infiltrates in muscle tissue, but their functional role is unclear. Systemic signs of inflammation are lacking, and the absence of beneficial effects following immunosuppression has challenged the notion of a role for the immune system. This study was undertaken to investigate the phenotype and functionality of T cells, specifically a subset of proinflammatory, cytotoxic, and apoptosis-resistant T cells defined as CD28null T cells, in the pathogenesis of sporadic IBM. Methods. A cohort of 27 patients with sporadic IBM was analyzed for the frequency of circulating and muscle-infiltrating CD28null T cells. The T cell receptor (TCR) V␤ usage was determined using flow cytometry and immunohistochemistry. Anti-CD3–stimulated peripheral blood mononuclear cells were analyzed for intracellular interferon-␥ and cytotoxic potential by flow cytometry. Results. We found striking accumulations of both CD8ⴙCD28null and CD4ⴙCD28null T cells, which represented the TCR V␤–expanded T cells in sporadic IBM. Such CD28null T cells were abundant both in the inflamed muscle tissue and in the circulation. Although the specific TCR V␤ expansions varied between patients, both CD8ⴙCD28null and CD4ⴙCD28null T cells consistently displayed a highly proinflammatory and cytotoxic potential. Conclusion. Our results suggest that CD28null T cell expansions represent the previously described expanded T cell subsets in sporadic IBM, and their proinflammatory capacity and presence in both muscle tissue and the circulation may imply a role of immune activation in sporadic IBM. In addition, CD4ⴙCD28null T cells may exert cytotoxic effects directly on muscle fibers due to a cytotoxic potential similar to that in CD8ⴙ T cells. Sporadic inclusion body myositis (IBM) is a chronic progressive myopathy that leads to severe disability, and presently, there is no successful long-term treatment. Sporadic IBM is characterized clinically by skeletal muscle weakness and muscle atrophy, and histologically by T cell infiltrates, vacuolar degeneration, and accumulation of amyloid-related proteins in muscle fibers (1,2). Although the cause of sporadic IBM is unclear, 2 main mechanisms have been suggested: muscle degeneration and/or autoinflammation. A role for an autoimmune component in this puzzling disease is supported by accumulating evidence, such as the strong association with specific HLA alleles (e.g., DRB1*0301, DRB3*0101, and DQB1*0201) (3); association with immune-mediated conditions and the presence of autoantibodies in some patients (4); and the presence of clonally expanded T cells (5–7), plasma cells (8), and antigen-presenting cells in muscle tissue (9). In particular, cytotoxic CD8⫹ T cells invading class I major histocompatibility complex (MHC)– expressing muscle fiber have been implicated in the pathogenesis of sporadic IBM (10,11). These autoinvasive CD8⫹ T cells have been demonstrated to be clonally expanded in the muscle tissue of patients with Supported by grants from the Swedish Research Council, the Swedish Rheumatism Association, King Gustaf V’s 80-Year Foundation, Karolinska Institutet, the European Union Sixth Framework Programme (project AutoCure; LSH-018661), and by the Stockholm County Council and Karolinska Institutet through a regional agreement on medical training and clinical research (ALF). 1 Jayesh M. Pandya, MSc, Andreas E. R. Fasth, PhD, Mei Zong, MD, Lara Dani, MD, Eva Lindroos, MLT, Vivianne Malmström, PhD, Ingrid E. Lundberg, MD, PhD: Karolinska University Hospital, Solna, and Karolinska Institutet, Stockholm, Sweden; 2Snjolaug Arnardottir, MD, PhD: Karolinska Institutet, Stockholm, Sweden. Address correspondence and reprint requests to Jayesh Pandya, MSc, Rheumatology Unit, CMM L8:04, Karolinska Universit y Hospital, SE-171 76 Stockholm, Sweden. E-mail: Jayesh.Pandya@ki.se. Submitted for publication March 18, 2010; accepted in revised form July 13, 2010. 3457 3458 PANDYA ET AL sporadic IBM, with a restricted variation in the third complementarity-determining region of the T cell receptor (TCR), which persists over several years (7,12). In addition to muscle, clonal expansions of CD8⫹ T cells have been found in the peripheral blood of patients with sporadic IBM (13), and peripheral blood and muscle tissue have been shown to share T cell expansions (14). However, an enigma is the observation that sporadic IBM is mostly resistant to conventional immunosuppressive treatment, including high doses of glucocorticoids in combination with other disease-modifying agents, which challenges the notion that T cells and immune components play a role in this disease (15,16). In this context, the proinflammatory CD28null T cells are of particular interest, since they are long-lived and resistant to apoptosis (17–19). These cells are terminally differentiated T cells lacking CD28, are often clonally expanded, and have acquired new effector functions and up-regulated a set of activating receptors mostly associated with natural killer (NK) cells (20,21). CD8⫹CD28null T cells emerge from repeated antigen stimulation and are increased in the peripheral blood of individuals with chronic viral infections, such as EpsteinBarr virus, human cytomegalovirus (CMV), and human immunodeficiency virus (22,23). CD4⫹CD28null T cells are less frequently found in the general population, but are overrepresented in the peripheral blood of patients with inflammatory and autoimmune diseases, such as multiple sclerosis, cardiovascular diseases, rheumatoid arthritis, and Crohn’s disease (24–26). Recently, we demonstrated the presence of CD28null T cells of both the CD4⫹ and CD8⫹ subtypes in the muscle tissue of patients with dermatomyositis (DM) and polymyositis (PM) and showed that these cells correlated with disease variables (27). Despite a number of reports indicating a role of T cells in the pathogenesis of sporadic IBM, the specific phenotype and functionality of T cells in this condition is poorly understood. In this study, we provide evidence that the TCR V␤–restricted T cells in patients with sporadic IBM are proinflammatory and cytotoxic CD28null T cells, belonging to both the CD8⫹ and the CD4⫹ T cell compartments, and that these cells dominate the T cell infiltrates within muscle tissue. PATIENTS AND METHODS Patients and healthy subjects. Twenty-seven consecutive patients (16 men and 11 women) who had sporadic IBM according to published criteria (28) and regularly visited the rheumatology clinic (n ⫽ 20 patients) or the neurology clinic (n ⫽ 7 patients) at Karolinska University Hospital were enrolled in the study. At the time of blood sampling, their median age was 76 years (range 46–85 years). The median age of the men was 76 years (range 46–84 years), and the median age of the women was 70 years (range 63–85 years). The median disease duration was 2.5 years (range 0–12 years) for the entire group, 2.5 years (range 0.1–12 years) for the men, and 2.5 years (range ⫺0.04–8.9 years) for the women. At the time of blood sampling, 13 patients were untreated, 9 were receiving prednisolone (either alone [n ⫽ 1] or in combination with methotrexate [n ⫽ 5], azathioprine [n ⫽ 2], or intravenous immunoglobulin [n ⫽ 1]), 2 were receiving methotrexate, and 3 were receiving anakinra. Immunoregulatory treatment in the 3 months before blood sampling was considered to be ongoing treatment. Patient records were scrutinized for any infection in the 6 months preceding blood sampling. Information was available for 25 patients. Muscle biopsy specimens obtained at the same time as blood sampling were available for 13 patients, and were included in this study. Peripheral blood was analyzed from 29 healthy subjects with a median age of 54 years (range 46–82 years). The study was approved by the local Ethics Committee of the Karolinska University Hospital, and both patients and healthy subjects gave informed consent. Flow cytometric analysis. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll separation (Ficoll-Paque Plus; GE Healthcare Biosciences) and screened for CD8⫹CD28null and CD4⫹CD28null phenotypes by flow cytometry. Antibodies used were CD3 Pacific Blue (clone UCHT1), phycoerythrin–Cy7 (PE-Cy7)–conjugated CD4 (SK3), allophycocyanin (APC)–conjugated CD28 (CD28.2; BD PharMingen), and peridinin chlorophyll A protein (PerCP)–conjugated CD8 (SK1; Becton Dickinson). Cells were acquired by flow cytometry on a CyAn ADP Analyzer (Beckman Coulter) and analyzed with Flow Jo software (Tree Star). The percentages of CD8⫹CD28 null and CD4⫹CD28null T cells were calculated as the frequencies of CD28-negative cells in the gated CD3⫹CD8⫹ or CD3⫹CD4⫹ populations, respectively. TCR V␤ analysis. The TCR V␤ usage was determined by the IOTest1 ␤ Mark kit (Beckman Coulter) for 8 selected patients with a high frequency (exceeding the median) of CD8⫹CD28null T cells and/or CD4⫹CD28null T cells (Table 1). The TCR V␤ stainings were combined with CD3 PB, PerCP-conjugated CD8, PE-Cy7–conjugated CD4, APC– conjugated CD28, APC-Cy7–conjugated CD14 (MphiP9), and APC-Cy7–conjugated CD19 (SJ25C1; BD PharMingen) fluorochrome-conjugated antibodies. CD14 and CD19 antibodies were used to exclude monocytes and B cells in order to increase the purity of the CD3⫹ population. The cells were analyzed by 7-color flow cytometry, using a CyAn ADP Analyzer and analyzed with Flow Jo software. A frequency of ⬎2 SD from the frequency found in healthy subjects was considered to be a significant TCR V␤ restriction. Functional assays. PBMCs from 4 patients analyzed for TCR V␤ were cultured for 6 hours of 72 hours at a density of 200,000 cells/well in 96-well plates (Sarstedt) coated with anti-CD3 antibodies (OKT3) at a 2.5 g/ml concentration in RPMI with 5% heat-inactivated pooled male human AB serum from the Karolinska University Hospital Blood Center. APCconjugated antibody to CD107a (Becton Dickinson) was added at time point 0 for 6-hour stimulation cultures and during the last 5–6 hours for 72-hour stimulation cultures. BD GolgiStop (containing monensin) and brefeldin A (Sigma-Aldrich) were added for the last 4 hours of all of the stimulations at a final CD28null T CELLS IN SPORADIC INCLUSION BODY MYOSITIS 3459 Table 1. TCR V␤ expansions and demographic characteristics of the patients with sporadic inclusion body myositis who were screened for TCR V␤ repertoire in peripheral blood* CD8⫹ CD28null cells, % CD4⫹ CD28null cells, % TCR V␤ expansions in CD8⫹ T cells† TCR V␤ expansions in CD4⫹ T cells† TCR V␤ analysis in muscle biopsy specimen Patient/ age/sex Disease duration, months 1/80/M 2/66/M 16 73 93 31 72 17.5 V␤14, V␤22 V␤7.2 V␤13.6, V␤5.1 V␤5.1 Yes Yes 3/81/M 35 85 70 V␤2, V␤22 V␤7.1, V␤5.1, V␤16 NA 4/62/F 107 40 13.8 V␤1, V␤22 V␤21.3 Yes 5/83/F 6/84/F‡ ⫺0.5 3 40 88 15 37 V␤20, V␤21.3 V␤21.3, V␤13.2 No Yes 7/76/M§ 8 70.5 V␤17 V␤22, V␤5.1, V␤21.3, V␤7.1 None Yes 8/78/F¶ 7 75 V␤20 Yes 1.6 18 V␤2 V␤21.3, V␤13.1, V␤1, V␤13.2, V␤13.6 Treatment at the time of sampling Infections in the 6 months prior to sampling No treatment Prednisolone 2.5 mg/day Prednisolone 10 mg/day, MTX 20 mg/week Prednisolone 1.25 mg/day; MTX 15 mg/ week, IVIG No treatment Prednisolone 7.5 mg/day Upper airways None Prednisolone 2.5 mg/day; AZA 100 mg/day; anakinra 100 mg/day Prednisolone 20 mg/day Herpes zoster None None Pneumonia Urinary tract Urinary tract * NA ⫽ not available; MTX ⫽ methotrexate; IVIG ⫽ intravenous immunoglobulin; AZA ⫽ azathioprine. † Expansions are shown in sequence, from the most restricted T cell receptor (TCR) V␤ to the least restricted TCR V␤ in peripheral blood. ‡ Also diagnosed as having systemic lupus erythematosus. § Also diagnosed as having Bechterew’s disease. ¶ Also diagnosed as having Sjögren’s syndrome. concentration of 6 g/ml and 10 g/ml, respectively. Cells were fixed and permeabilized using a BD Cytofix/Cytoperm Fixation/Permeabilization kit and stained for intracellular interferon-␥ (IFN␥) using PE-Cy7–conjugated IFN␥ antibody (B27; Becton Dickinson). The stainings were combined with fluorochrome-conjugated antibodies to CD3, CD8, CD4, CD28, CD14, CD19 (as described above), TCR V␤, and PE-conjugated CD244 (2B4) (clone C1.7; Immunotech). In addition, aqua fluorescent reactive dye from Live/Dead Fixable Dead Cell Stain kit (Invitrogen) was added to all of the stainings in order to exclude dead cells from the analysis. Cells were acquired by a CyAn ADP Analyzer and analyzed by Flow Jo. Muscle biopsy specimens and immunostaining. Biopsy specimens were obtained from the vastus lateralis or anterior tibialis muscle by a “semi-open” technique under local anesthesia (29). The biopsy specimens were immediately frozen in isopentane chilled by liquid nitrogen and stored at ⫺80°C. The presence of CD28null T cells in muscle tissue should be investigated by expression of a positive marker to allow direct quantification and reduce the risk of inclusion of recently activated T cells temporarily down-regulating CD28. We have previously demonstrated the use of CD244 as a surrogate marker for CD28null T cells in the muscle tissue of DM and PM patients (27). A similar immunohistochemistry protocol was used in the present study, combining CD3 and TCR V␤ with CD244 (27). An extra blocking step was introduced to block endogenous biotin or biotin-binding activity in tissue sections using an Avidin/Biotin Blocking kit (Vector). Primary antibodies included goat anti-human CD244 (R&D Systems), mouse monoclonal anti-CD3 (clone SK7; Becton Dickinson), and mouse and rat monoclonal antibodies against human TCR V␤ (all from Immunotech/Beckman Coulter). Respective isotype control antibodies were irrelevant mouse IgG1 (Dako), mouse IgG2a (Sigma), mouse IgG2b (BD Biosciences), and rat IgG1 (R&D Systems). Biotinylated secondary antibodies included horse anti-mouse IgG (Vector), goat anti-rat IgG (Vector), or donkey anti-sheep/goat IgG (The Binding Site). After the secondary antibody step, sections were washed, incubated with peroxidase-conjugated ExtrAvidin (1:2500; Sigma-Aldrich), developed using a Peroxidase Substrate kit (Vector) containing diaminobenzidine, and counterstained with hematoxylin. Stained tissue sections were examined with a Leica DM RXA2 microscope (Leica Microsystems) equipped with a Leica DC digital color video camera 300F (Leica Microsystems) connected to a PC computer. The number of cells expressing CD244 and CD3 per unit area was assessed quantitatively using a computer-assisted Quantimet 600 image analyzer (Leica). Sections containing ⬎50 CD3⫹ T cells/mm2 were categorized as having a large T cell infiltrate. Sections containing ⬍50 CD3⫹ T cells/mm2 positive for CD3 were categorized as having small T cell infiltrates. Statistical analysis. The Mann-Whitney 2-tailed test was used to compare the frequencies of CD4⫹CD28null and CD8⫹CD28null T cells in peripheral blood between healthy subjects and patients with sporadic IBM, and to compare the frequencies of CD28null T cells between peripheral blood and 3460 PANDYA ET AL muscle tissue. The correlations between muscle-infiltrating CD3⫹ and CD244⫹ cells per area were investigated by Spearman’s 2-tailed correlation test. The computer software program GraphPad Prism, version 5.02 was used for all graphing and curve fitting. RESULTS ber of CD3 and CD244 positively stained cells per area (Spearman’s r ⫽ 0.89, P ⫽ 0.012; n ⫽ 7) (Figure 1D). The ratio between the number of CD244⫹ cells/mm2 and CD3⫹ cells/mm2 was considered to be the frequency of CD28null T cells in muscle tissue (median 72%, range 45.5–96%). Comparing the frequencies of circulating and muscle-infiltrating CD28null T cells in High frequencies of CD28null T cells in the peripheral blood of patients with sporadic IBM. Patients with sporadic IBM displayed significantly higher median frequencies of circulating CD8⫹CD28null T cells than did healthy subjects (67% in patients with IBM versus 32% in healthy subjects; P ⬍ 0.01) (Figure 1A). The frequency of circulating CD4⫹CD28null T cells was also significantly higher in patients with sporadic IBM (median 11.5% in patients versus 2% in healthy subjects; P ⫽ 0.0001) (Figure 1B). There was a significant correlation between the size of the circulating CD4⫹CD28null and CD8⫹CD28null T cell populations (Spearman’s r ⫽ 0.64, P ⫽ 0.001). The sizes of both CD28null T cell populations were stable over a followup period of up to 2 years in repeated blood samples from 6 patients (data not shown). Clonal T cell expansions could be due to recent infections; however, we did not observe any significant difference between the frequencies of CD8⫹CD28null T cells in the group of patients with a preceding infection (median 72.2%; n ⫽ 10) and the group of patients with no infections (median 56.2%; n ⫽ 15) (P ⫽ 0.211). Similarly, no significant difference was observed between the frequencies of CD4⫹CD28null T cells in the group of patients with infections (median 8.1%) and the group of patients with no infections (median 12.0) (P ⫽ 0.867). Higher frequency of CD28null T cells in inflamed muscle compared with peripheral blood. CD244, in combination with CD3, is a surrogate marker for CD28null T cells in muscle tissue. We used these markers in immunohistochemistry to analyze the muscle tissue of patients with sporadic IBM for the presence and frequency of CD28null T cells. Seven of 13 muscle biopsy specimens obtained from patients contained large CD3⫹ T cell infiltrates with ⬎50 CD3⫹ cells/mm2, and these were used for detailed phenotyping of the CD3⫹ T cells. In consecutive sections of muscle biopsy specimens, we found that most of the areas that were positive for CD3 were also positive for the CD244 marker. A representative immunohistochemistry staining depicting the same area as positive for CD3 and CD244 in 2 consecutive sections of a muscle biopsy specimen from a patient with sporadic IBM is shown in Figure 1C. We found a significant linear correlation between the num- Figure 1. Increased frequency of CD28null T cells in the peripheral blood and muscle tissue of patients with sporadic inclusion body myositis (IBM). A and B, Significantly higher frequencies of CD28null T cells in the CD8⫹ (A) and CD4⫹ (B) T cell populations in peripheral blood mononuclear cells (PBMCs) from patients with sporadic IBM than in PBMCs from healthy controls (HC). Each data point represents a single subject; horizontal lines show the median. C, Staining of a muscle biopsy sample from a representative patient with IBM. Muscle biopsy specimens from patients with sporadic IBM (n ⫽ 13) were subjected to immunohistochemical analysis for the presence of CD3 and CD244. Most cells that were positive for CD3 were also positive for CD244 in 7-m–thick consecutive sections from specimens with mononuclear infiltrates (original magnification ⫻ 320). D, Significant correlation between the number of CD3⫹ and the number of CD244⫹ cells per unit area within the muscle tissue of patients with sporadic IBM, as determined by Spearman’s correlation test. E, Significantly higher frequency of CD28null T cells in muscle tissue (ratio of CD244⫹ cells per area to CD3⫹ cells per area on consecutive muscle sections) than of CD28null T cells in peripheral blood. P values were determined by the Mann-Whitney test. CD28null T CELLS IN SPORADIC INCLUSION BODY MYOSITIS Figure 2. Higher proinflammatory and cytotoxic functions in CD28null T cells than in CD28⫹ subsets, in both the CD4⫹ and CD8⫹ compartments. For functional analysis, peripheral blood mononuclear cells from patients with sporadic inclusion body myositis (IBM; n ⫽ 4) were stimulated with plate-bound anti-CD3 for 6 hours, and the frequencies of intracellular interferon-␥ (IFN␥)– and extracellular CD107a–expressing T cells were determined by multicolor flow cytometry. A, Frequency of IFN␥-containing cells in the CD28null subset (upper panel), and surface expression of CD107a⫹ on CD28null T cells (lower panel), compared with CD28⫹ counterparts within the CD8⫹ T cell compartment. While the frequency of IFN␥-containing cells in the CD28null subset was significantly higher, the surface expression of CD107a⫹ was not always found to be increased compared with the CD8⫹CD28⫹ T cell subset. B, Higher frequencies of both intracellular IFN␥-containing and CD107a⫹ cells in CD4⫹CD28null T cell subsets compared with their CD28⫹ counterparts within the CD4⫹ T cell compartment for all patients. C and D, Flow cytometry plots of cells from a representative patient for CD8⫹ (C) and CD4⫹ (D) T cell compartments. The percentages displayed on the plots are the percentages of CD28null or CD28⫹ T cells that were IFN␥⫹ or CD107a⫹ cells, after the frequency of positive cells in unstimulated samples was deducted. patients with sporadic IBM, we found a significantly higher ratio of CD28null T cells in muscle tissue (median 72%) than in peripheral blood (median 35%; P ⬍ 0.05) (n ⫽ 7) (Figure 1E). Taken together, these data demonstrate that the 3461 higher frequencies of CD28null T cell subsets in the peripheral blood of patients with sporadic IBM are higher than those in healthy individuals and that the majority of muscle-infiltrating T cells in these patients are of the CD28null phenotype. Higher IFN␥ secretion and degranulation potential in circulating CD8ⴙCD28null and CD4ⴙCD28null T cell subsets compared with the CD28ⴙ T cell subset. Next, we investigated whether the CD28null T cells were proinflammatory in function. PBMCs from 4 patients with sporadic IBM with a high frequency (exceeding the median) of CD8⫹CD28 null T cells and/or CD4⫹CD28null T cells were stimulated with anti-CD3 and stained for intracellular IFN␥ and CD107a surface expression. The latter is an indirect measure of cytotoxicity and degranulation, i.e., release of perforin and granzyme B (30,31). Following 6 hours of stimulation, the CD8⫹CD28null T cell populations displayed higher frequencies of intracellular IFN␥-containing cells (median 5.6%) compared with their CD28⫹ counterparts (median 1.4%; P ⬍ 0.05) (Figure 2A, upper panel). The frequency of CD107a⫹CD8⫹CD28null T cells (median 2.6%) was similar to that of the CD8⫹CD28⫹ T cell subset (median 1.4%) (Figure 2A, lower panel). Within the CD4⫹ compartment, CD4⫹ CD28null T cell populations displayed much higher frequencies of both intracellular IFN␥-containing cells (median 8.8%; P ⬍ 0.05) and CD107a⫹ cells (median 1.9%; P ⬍ 0.05) compared with their CD28⫹ counterparts (median 0.7% for IFN␥ and 0.2% for CD107a⫹) (Figure 2B). Flow cytometry plots from the 6-hour anti-CD3 stimulation experiments with specimens obtained from a representative patient are shown in Figures 2C and D. The data from the 72-hour stimulation experiments were similar with respect to the relative differences between CD28null and CD28⫹ subsets, although there were magnitude differences compared with the results from the 6-hour cultures (data not shown). Overall, these results demonstrate that both the CD8⫹CD28null and CD4⫹CD28null T cell subsets are true effector cells with a capacity to act both by cytokine release and by direct cytotoxicity. Pronounced TCR V␤ restriction of CD28null T cells in both peripheral blood and muscle infiltrates. Previous studies have shown clonal expansions in the CD8⫹ T cell populations of patients with sporadic IBM (12,14), a feature shared with CD28null T cells (19). To investigate whether the CD28null T cell populations in our patient cohort were restricted in their TCR V␤ usage, we analyzed the TCR V␤ repertoire in 8 patients with high frequencies of CD8⫹CD28null T cells and/or CD4⫹CD28null T cells in the blood (Table 1). Not only CD8⫹, but also CD4⫹ T cell 3462 PANDYA ET AL Figure 3. Skewed T cell receptor (TCR) V␤ repertoire in patients with sporadic inclusion body myositis (IBM), due to TCR V␤ restriction in the CD28null population. Patients with sporadic IBM (n ⫽ 8) with high frequencies of CD28null T cells were screened for TCR V␤ restriction. All patients with sporadic IBM were found to display a skewed TCR V␤ repertoire within CD8⫹ and CD4⫹ T cells compared with mean frequencies in healthy controls (HC). A and C, TCR V␤ that were found to be significantly increased in CD8⫹ T cells (A) and CD4⫹ T cells (C) in any of the patients screened are displayed. Shaded bars show expanded TCR V␤; open bars show nonexpanded TCR V␤. B and D, Comparison of the CD28null versus CD28⫹ T cells within CD8⫹ T cell compartments (B) and CD4⫹ T cell compartments (D). The expansions were mainly observed within the CD28null T cell populations, leading to an overall expansion in the CD8⫹ or CD4⫹ T cell population. Results from 2 representative patients (patient 7 and patient 2) are shown. E and F, Immunohistochemistry staining of specimens from 2 representative patients, displaying the presence of the same TCR V␤–containing cells in muscle tissue as were dominant in circulating CD8⫹ (E) or CD4⫹ (F) T cell populations. Immunohistochemistry staining for CD3, isotype control for TCR V␤ antibodies and nondominant TCR V␤ on consecutive muscle biopsy sections is shown along with dominant TCR V␤ staining (original magnification ⫻ 250). populations from screened patients with sporadic IBM were found to have a skewed TCR repertoire. Figure 3 summarizes the TCR V␤ restriction patterns in CD8⫹ and CD4⫹ T cells in peripheral blood. In total, 10 and 8 expansions were found in the CD8⫹ and CD4⫹ T cells, respectively, for all patients. Different patients were found to be restricted in different TCR V␤ (Table 1). Also, CD8⫹ and CD4⫹ T cell populations displayed different TCR V␤ restrictions within the same patient. Moreover, the clonal expansions were found to be consistent over a period of up to 2 years (n ⫽ 3) (data not shown). Importantly, when we compared the CD28null versus CD28⫹ T cells within the CD8⫹ and CD4⫹ T cell compartments, the expansions were confined within CD28null T cell populations (Figure 3). In fact, we found that the TCR V␤ restrictions observed in the total CD8⫹ and CD4⫹ T cell populations were mainly due to clonally expanded CD28null T cells. This was consistent for all patients investigated. To investigate whether the clonally expanded, dominant TCR V␤ in peripheral blood were also present in inflamed muscle tissue, immunohistochemistry analyses of muscle biopsy specimens were performed in 6 patients with known TCR V␤ profiles in peripheral blood and where muscle biopsy samples were available. The dominant TCR V␤ from peripheral blood CD8⫹ and CD4⫹ T cells were also dominant in muscle tissue. Immunohistochemistry stainings of samples from representative patients are displayed in Figures 3E and F for CD8⫹ and CD4⫹ T cells, respectively. As a control experiment, nondominant TCR V␤ in peripheral blood were analyzed in muscle tissue, and none of these were found in significant numbers in the muscle biopsy specimens (Figures 3E and F). Taken together, these results demonstrate that CD28null T cells display a limited TCR V␤ repertoire in blood and that the same T cell populations are also CD28null T CELLS IN SPORADIC INCLUSION BODY MYOSITIS found in the inflamed muscles of patients with sporadic IBM. These expanded TCR V␤ subsets are indicative of persistent antigen-specific activation. Proinflammatory and cytotoxic functions of CD28null T cells expressing dominant TCR V␤. Persistent antigen-specific activation and clonal expansion could also indicate functional senescence of T cells. Therefore, we next investigated whether the TCR V␤– dominant CD28null T cells were senescent cells, exhausted in their effector functions due to repeated division, or if they were still capable of exerting proinflammatory and cytotoxic functions. To this end, functional analyses were performed in anti-CD3–stimulated Figure 4. Dominant T cell receptor (TCR) V␤–expressing CD28null T cells are not functionally senescent. In multicolor flow cytometry–based functional analysis experiments, peripheral blood mononuclear cells from 3 patients with sporadic inclusion body myositis (IBM) were stained for circulating dominant TCR V␤. A and B, Similar interferon-␥ (IFN␥) secretion and degranulation capacity in CD28null T cells with dominant TCR V␤ and CD28null cells with nondominant TCR V␤ in both CD8⫹CD28null (A) and CD4⫹CD28null (B) T cell compartments. C and D, Flow cytometry plots of CD8⫹CD28null (C) and CD4⫹CD28null (D) T cells from a representative patient. The percentages displayed on the plots are the percentages of dominant V␤⫹ or dominant V␤⫺ CD28null T cells that were IFN-␥⫹ or CD107a⫹ cells, after the frequency of positive cells in unstimulated samples was deducted. 3463 PBMCs from 3 patients with sporadic IBM where the dominant TCR V␤ were stained in parallel. Interestingly, CD28null T cells with dominant TCR V␤ were found to display equal capacity with regard to IFN␥ secretion and degranulation as nondominant CD28null T cells in both the CD8⫹ and CD4⫹ T cell compartments (Figures 4A and B). Flow cytometry plots of cells from a representative patient are displayed in Figures 4C and D. The results indicate that CD28null T cells expressing dominant TCR V␤ are proinflammatory and cytotoxic effector cells and are not driven to functional senescence. DISCUSSION It is still debated whether T cells play a role in the pathogenesis of sporadic IBM or whether sporadic IBM is merely a degenerative muscle disease. In this study, we demonstrated that proinflammatory CD28null T cells are the dominant T cell subset in peripheral blood and in the inflamed muscles of patients with sporadic IBM. The CD28null expansions were not confined to CD8⫹ only, but were also prominent in the CD4⫹ T cell populations. These cells displayed a strong proinflammatory and cytotoxic phenotype, more so than the corresponding CD28⫹ subsets. Additionally, both the CD8⫹CD28null and the CD4⫹CD28null T cell populations were highly TCR V␤–restricted in the peripheral blood, indicative of persistent stimulation by a limited number of antigens. Strikingly, these cells were not functionally senescent but retained a highly proinflammatory and cytotoxic function, and the same TCR V␤–expressing subsets were also abundant in inflamed muscle tissue. Previous reports have described the clonal expansions of CD8⫹ T cells in both the muscle and blood of patients with sporadic IBM (13,14) and the persistent nature of expanded CD8⫹ T clones in the muscle tissue of patients with sporadic IBM (7,12). In the present study, we demonstrated that the observed clonal T cell expansions in patients with sporadic IBM are due to expansions in the CD28null T cell populations. Importantly, this feature is not limited to CD8⫹ T cells, but is also found in the CD4⫹ compartment. Expanded T cells with the same TCR V␤ expression were found in the peripheral blood and muscle. Furthermore, in muscle tissue, many of these cells were surrounding muscle fibers and were localized close to the muscle fiber membrane, which could indicate a role of TCR V␤– dominant CD8⫹CD28null and CD4⫹CD28null T cells in muscle fiber destruction in sporadic IBM. In addition, our findings support the notion of an exchange between 3464 blood and muscle and/or specific recruitment of CD28null T cells to muscle tissue. An accumulation of CD4⫹CD28null T cells in tissue has previously been demonstrated in the vessel walls in inflammatory cardiovascular disorders and shown to correlate with clinical disease variables (32,33). Recent results from our group demonstrated that CD28null of both the CD4⫹ and CD8⫹ T cells dominate the T cell infiltrates within the muscle tissue of patients with DM and PM (27). Similarly, we showed in the present study that in patients with sporadic IBM, the T cell infiltrates in inflamed muscle tissue are dominated by CD28null populations. Using a surrogate marker, such as CD244, requires caution in interpretation since other cells, such as NK cells, may also express CD244. However, the colocalization and strong correlation between CD3 and CD244 expression in the muscle tissue strongly indicate that CD244⫹ cells in this compartment are CD3⫹ T cells. This was also shown in DM and PM, where CD244 was mainly expressed by cells coexpressing CD3 (27). Of note, CD244 expression on T cells is not a general phenomenon at the site of inflammation, since only occasional CD3⫹ T cells in the synovial membrane of RA patients express CD244 (34). The factors responsible for the observed CD8⫹CD28null and CD4⫹CD28null T cell expansions may be common to both subsets or may differ. Possible common factors include chronic viral infections. Indeed, the results of a number of recent studies indicate a link between retroviral infection and the pathogenesis of sporadic IBM (35,36). However, we could not explain the CD28null T cell expansions by the presence of previous infections in our patient cohort. The cooccurrence of both CD8⫹CD28null and CD4⫹CD28null T cells could also indicate a common cause, such as accelerated telomere erosion in an IFN␣-rich environment (37,38). Interestingly, plasmacytoid dendritic cells are known to produce high levels of IFN␣ in response to human CMV, and IFN␣ has been shown to accelerate phenotypic differentiation and telomere erosion in CD4⫹ T cells (39). Strikingly, recent studies have demonstrated a link between the long-term use of IFN␣, particularly for the treatment of chronic hepatitis C virus infection, and the development or exacerbation of numerous autoimmune phenomena and a spectrum of myopathies, including IBM (40,41). The factors described above are possible mechanisms of CD28null T cell expansions in patients with sporadic IBM. There are several possible mechanisms by which CD8⫹CD28null and CD4⫹CD28null T cells could harm the muscle fibers in sporadic IBM. In patients with sporadic IBM, the endomysial inflammatory cells have PANDYA ET AL been demonstrated to be predominated by activated CD8⫹ T cells (10); therefore, the autoimmune pathogenesis in sporadic IBM is believed to be mediated mainly by class I MHC–restricted CD8⫹ T cells invading non-necrotic muscle fibers, a typical histopathologic feature of sporadic IBM (10,11). Although CD4⫹ T cells have also been detected in endomysial infiltrate in previous studies, they were considered less important in the pathogenesis of sporadic IBM, since CD4⫹ T cells are not generally cytotoxic. However, our data demonstrate that a vast number of the CD4⫹ T cells in the muscle tissue of patients with sporadic IBM are of the CD4⫹CD28null phenotype and have cytotoxic potential similar to that of CD8⫹ T cells. Therefore, the CD4⫹CD28null T cells may also exert cytotoxic effects directed to muscle fibers. We also demonstrated that CD28null T cells are potent producers of IFN␥, which alone or in combination with other cytokines can induce a chain of inflammatory events: up-regulation of class I or class II MHC on muscle fibers and Fas-mediated apoptosis of muscle cells, leading to myopathic conditions (42,43). Expression of MHC molecules on muscle fibers could allow T cell–muscle cell interaction. However, based on the results of previous studies, this has been regarded as unlikely because the costimulatory molecules CD80/86, which are normally required for functional interaction, are not expressed by inflamed muscle fibers (44,45). Nonetheless, CD28null T cells, which are independent of costimulation by CD80/86, could be activated following interaction with MHC-expressing muscle fibers and cause muscle fiber damage. The function of CD28null T cells could be potentiated by unconventional costimulatory molecules, such as CD244. We have recently demonstrated that de novo expressed NK receptors on CD28null T cells in an additive pattern compensate for suboptimal TCR stimulation (21). In addition, decreased susceptibility to immunoregulation by Treg cells can potentially further strengthen the pathogenic role of CD28null T cells (46). The present study has some limitations. The absence of large T cell infiltrates despite myopathic features in some muscle biopsy specimens from patients with sporadic IBM strengthens the general belief that the role of T cells may be more important in certain stages of the disease. Nevertheless, we cannot rule out the possibility that we have missed infiltrates by chance due to the patchy nature of infiltrates. The age ranges of patients and healthy controls were similar, but the median age of the patients was higher than for the controls. However, we did not observe a significant correlation between CD28null T cell frequencies and age CD28null T CELLS IN SPORADIC INCLUSION BODY MYOSITIS in healthy subjects or in patients; therefore, it is not likely that the difference in the frequencies of CD28null T cells between patients and controls could be explained by the age difference at the beginning of the study. A pathogenic role of CD28null T cells may not be a general feature of sporadic IBM in all patients, since we observed variable frequencies of CD28null T cells. Instead, we suggest that CD28null T cell expansions in patients with sporadic IBM could serve as a biomarker for the presence of proinflammatory T cells and might support an indication to choose therapies targeting CD28null T cells for a subgroup of patients with sporadic IBM. A recent proof-of-principle study concluded that in patients with sporadic IBM one series of alemtuzumab infusions (a humanized monoclonal antibody to CD52 that causes depletion of or a severe reduction in peripheral blood lymphocytes and certain leukocyte subsets) could slow down disease progression for up to 6 months, improve the strength of some patients, and reduce endomysial inflammation and stressor molecules in muscle tissue (47). These results strengthen the notion of a role of inflammation and T cells in patients with sporadic IBM. At the same time, we do not rule out the possibility that other suggested mechanisms contribute to disease pathogenesis, especially in patients with sporadic IBM with low CD28null T cell frequencies (48). In conclusion, we identified CD28null T cells to be the TCR V␤–expanded T cells in muscle and peripheral blood in patients with sporadic IBM. CD28null T cells constituted a majority of the T cells in muscle cell infiltrates. Since these cells are proinflammatory and have NK cell properties, our findings support the notion that immune mechanisms play a role in the pathogenesis of sporadic IBM and indicate that selective targeting of CD28null T cells may provide effective therapy for patients with sporadic IBM. ACKNOWLEDGMENTS We are grateful to Ms Adina Nisell for contributing to the collection of patient demographic data, Mrs. Annika van Vollenhoven for outstanding technical help with the flow cytometry, and Mrs. Eva Jemseby and Mrs. Gull-britt Almgren for organizing blood sampling and the biobank. 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. Pandya 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. Fasth, Malmström, Lundberg. Acquisition of data. Pandya, Zong, Arnardottir, Dani, Lindroos, Lundberg. 3465 Analysis and interpretation of data. Pandya, Fasth, Malmström, Lundberg. REFERENCES 1. Oldfors A, Fyhr IM. Inclusion body myositis: genetic factors, aberrant protein expression, and autoimmunity. 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