Tc17 express neither granzyme B nor perforin, and have no cytolytic activity against antigen (Ag)-loaded targets [11]. Tc17 have been shown to be involved in various conditions. Tc17 can protect mice against lethal influenza challenge, while Tc17 protection is accompanied by greater neutrophil influx into the lung than in Tc1-injected mice [11]. Tc17 have also been shown to play an important role in controlling the growth of B16 malignant melanoma in mice via the recruitment of neutrophils [12]. Moreover, Tc17 promote acute graft-versus-host disease (aGVHD) in bone marrow transplantation, and the occurrence of aGVHD is reduced through decreased IL-17 secretion by T cells [13]. Tc17 are also implicated in the pathogenesis of some human autoimmune diseases, such as psoriasis [14] and systemic lupus erythematosus (SLE) [15]. However, the characteristics of Tc17 and the correlation between Tc17 and Th17 in ITP patients have not been systematically investigated. To further investigate the possible role of Tc17 and Th17 cells in the pathogenesis of ITP, we measured the number of Tc17 and correlated the number to Th17 cells and evaluated the clinical relevance. Methods Ethics Statement Our research was approved by the Medical Ethical Committee of Qilu Hospital of Shandong University. An informed consent document was obtained from each participant. The informed consent stated that the unused portion or excess of the peripheral blood drawn from patients for complete blood cell counts was as the source of Tc17 in ITP research, or the peripheral blood drawn from healthy subjects for this research was voluntary. Patients and Controls Forty-seven ITP patients (25 females and 22 males; age range, 16–80 years; median age, 41 years), including 20 newly-diagnosed and 27 ITP patients who had complete response (CR) after glucocorticoid therapy, intravenous gamma globulin, and/or splenectomy were enrolled in this study. Enrollment took place between May 2010 and June 2011 in the Department of Hematology of Qilu Hospital (Jinan, China). The platelet counts ranged between 1 and 706109/L, with a median count of 24.576109/L. The megakaryocyte counts in the bone marrow smear (1.5 cm63.0 cm) ranged between 26 and 560, with a median count of 173. Patients with diabetes, hypertension, cardiovascular diseases, pregnancy, active or chronic infections, or connective tissue diseases, such as SLE, were excluded. All of the patients met the diagnostic criteria of ITP, as previously described [16]. Thirty-four healthy controls were included (18 females and 16 males; age range, 20–58 years; median, 32 years). The platelet counts of healthy controls ranged from 132 to 3086109/L, with a median count of 2296109/L. Flow Cytometric Analysis Intracellular cytokines were studied by flow cytometry to identify the cytokine-producing cells. Briefly, heparinized periph- eral whole blood (400 ml) with an equal volume of Roswell Park Memorial Institute (RPMI)-1640 medium was incubated for 4 h at 37uC in 5% CO2 in the presence of 25 ng/mL of phorbol myristate acetate (PMA), 1 mg/mL of ionomycin, and 1.7 mg/mL of monensin (all from Alexis Biochemicals, San Diego, CA, USA). PMA and ionomycin are pharmacologic T cell-activating agents that mimic signals generated by the T cell receptor (TCR) complex and have the advantage of stimulating T cells of any Ag specificity. Monensin was used to block intracellular transport Tc17 in ITP mechanisms, thereby leading to an accumulation of cytokines in the cells. After incubation, the cells were stained with PE-Cy5-conjugated anti-CD3 and fluorescein isothiocyanate-conjugated anti-CD8 monoclonal antibodies at room temperature in the dark for 15 min to delimit CD4+ T cells because CD4 was down- modulated when cells were activated by PMA. After surface staining, the cells were stained with PE-conjugated anti-IL17 monoclonal antibodies after fixation and permeabilization accord- ing to the manufacturer’s instructions. Isotype controls were given to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FAC Scan cytometer equipped with CellQuest software (BD Bioscience PharMingen, San Jose, CA, USA). Addition of dexamethasone (DEX) to peripheral blood mononuclear cells (PBMCs) from patients with ITP PBMCs of ITP patients were isolated from heparinized blood by gradient centrifugation (4006g for 20 min) on Ficoll-Paque (Pharmacia Diagnostics, Uppsala, Sweden), and washed twice. Then, PBMCs were adjusted to 16106/ml in RPMI-1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum at a density of 26106 cells/well in a 6-well culture plate and incubated in humidified air in 5% CO2 at 37uC in the presence of 0, 0.25, 0.5, or 1 mmol/L of DEX. After 72 h, the PBMCs were collected to enumerate Tc17 and Th17 by flow cytometric analysis. Anti-platelet autoantibody determination All plasma samples were stored at 220uC prior to use. The specific anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies were analyzed by modified monoclonal antibody-specific immo- bilization of platelet antigens (MAIPA), which was carried out as previously described in detail by Hou et al. [17]. Statistical Analysis The results are expressed as the median (range). Comparisons between the two groups were assessed by the Wilcoxon rank-sum test, and Spearman’s test was used for correlation analysis. All tests were performed by SPSS (version 16.0; SPSS, Inc., Chicago, IL, USA). P value,0.05 was considered statistically significant. Results Elevated Tc17 and Th17 cells in ITP patients We analyzed the frequencies of Tc17 and Th17 cells based on cytokine patterns after in vitro activation by PMA/ionomycin in short-term cultures. The expression of a typical dot plot of IL-17 gated on CD3+ T cells in ITP patients and healthy controls is shown in Fig. 1. IL-17 was expressed on CD3+CD82 and CD3+CD8+ T cells. Compared with healthy controls (median, 0.30%; range, 0.04– 1.80%), the percentage of Tc17 was significantly increased in the newly-diagnosed patients (median, 0.79%; range, 0.25–3.58%; P,0.0001), while there was no significant change in the patients with CR (median, 0.39%; range, 0.10–1.54%). Tc17 were decreased significantly in patients with CR compared to newly- diagnosed patients (P = 0.0017; Fig. 2a). We also demonstrated a statistically significant difference in Th17 between newly-diagnosed patients (median, 2.24%; range, 0.72–8.18%) and healthy controls (median, 1.19%; range, 0.04–4.53%; P = 0.0016; Fig. 2b), which was similar to the results we previously reported [9]. We noted a difference in the Th17:Tc17 ratio among newly- diagnosed patients with CR and the healthy control group. As shown in Fig. 2c, the Th17:Tc17 ratio in newly-diagnosed patients (median, 2.5850; range, 1.4221–3.0714) was 0.76-fold lower than PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26522