Ange, 6.01?4.02 ) in this study (Fig. 1 E, F, G and Fig. 2B).IL-22 and IL-17 Enzyme-Linked Immunosorbent Assay (ELISA)PB and BM were collected into heparin-anticoagulant vacutainer tubes. Plasma was obtained by centrifugation and stored at 280uC for determination of cytokines. IL-22 and IL-17 levels were determined with a quantitative sandwich enzyme immunoassay technique in accordance with the manufacturer’s recommendations (lower detection limit 9 pg/ml; eBioscience).mRNA Expression Levels of RORC, IL-6, TNF-a and IL-23 in E-MDS, L-MDS Cohort and ControlsRORC, the key transcription factor directing 22948146 Th17 lineage commitment, was determined by real-time PCR. Our result demonstrated that RORC transcript was notably higher in PBMCs of E-MDS patients, consistent with the flow 520-26-3 biological activity cytometryStatistical AnalysisResults were expressed as mean 6 SD or median (range). Comparisons between two groups were assessed by the non-pairedTh22 and Th17 Cells in Different Stages of MDSFigure 1. Circulating percentages of Th17, Th1 and Th22 cells in representative healthy controls, E-MDS and L-MDS patients. Heparinized peripheral whole blood from 37 MDS(E-MDS, n = 17; L-MDS, n = 20)patients and 20 healthy PB donors were stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h, and then stained with labeled antibodies for FACS analysis. (A) Lymphocytes were gated by flow cytometry. (B, C, D) Representative FACS dot plots of circulating Th17 (CD4+IL-17+) cells from healthy controls, E-MDS and L-MDS patients. 1662274 (E, F, G) Representative FACS dot plots of circulating Th1 (CD4+IFNc+) cells from healthy controls, E-MDS and L-MDS patients. (H, I, J) Representative FACS dot plots of circulating Th22 (CD4+IL-22+IL-172IFNc2 ) cells from healthy controls, E-MDS and L-MDS patients. Numbers in plots indicate relative percentages per quadrant. doi:10.1371/journal.pone.0051339.gdata. The relative amount of RORC mRNA in E-MDS patients was increased 4.7- and 3.3-fold compared 
with healthy (-)-Indolactam V site controls (HC) and L-MDS patients (P = 0.0007; P = 0.002, respectively) (Fig. 4A). In view of increased frequencies of Th22 cells in L-MDS and Th17 cells in E-MDS, we also determined the mRNA expressionlevels of regulatory factors IL-6, TNF-a and IL-23 in MDS patients and controls. The relative amount of mRNA of IL-6 in L-MDS patients was 2.4-(P,0.05) and 5.3-fold (P,0.001) of that in EMDS patients and healthy controls, respectively (Fig. 4B). TNF-a mRNA level was also present at higher levels in L-MDS comparedTh22 and Th17 Cells in Different Stages of MDSwith E-MDS and controls (3.5-fold, P,0.05 and 10.6-fold, P,0.005, respectively) (Fig. 4C). IL-23 is a heterodimeric cytokine consisting of two subunits: p40, which is also a component of IL-12, and p19, a unique subunit of IL-23. So we examined IL-23p19 mRNA expression as representative of IL-23. Quantitative real-time PCR analysis showed that there was no significant difference in IL-23p19 mRNA expression levels among E-MDS, L-MDS and healthy controls (P.0.05) (Fig. 4D).Clinical Relevance of Peripheral Th22, Th17 and Circulating IL-22 in MDS PatientsPeripheral Th17 percentage significantly increased in BM blasts ,5 patients compared with healthy donors (1.7660.80 vs. 0.9860.30 , P = 0.003), while no significant difference was seen between BM blasts 5 patients and healthy donors (1.3860.80 vs. 0.9860.30 , P.0.05) or between blasts 5 and ,5 patients (1.3860.80 vs. 1.7660.80 , P.0.05) (Fig. 2D). As for the correlations bet.Ange, 6.01?4.02 ) in this study (Fig. 1 E, F, G and Fig. 2B).IL-22 and IL-17 Enzyme-Linked Immunosorbent Assay (ELISA)PB and BM were collected into heparin-anticoagulant vacutainer tubes. Plasma was obtained by centrifugation and stored at 280uC for determination of cytokines. IL-22 and IL-17 levels were determined with a quantitative sandwich enzyme immunoassay technique in accordance with the manufacturer’s recommendations (lower detection limit 9 pg/ml; eBioscience).mRNA Expression Levels of RORC, IL-6, TNF-a and IL-23 in E-MDS, L-MDS Cohort and ControlsRORC, the key transcription factor directing 22948146 Th17 lineage commitment, was determined by real-time PCR. Our result demonstrated that RORC transcript was notably higher in PBMCs of E-MDS patients, consistent with the flow cytometryStatistical AnalysisResults were expressed as mean 6 SD or median (range). Comparisons between two groups were assessed by the non-pairedTh22 and Th17 Cells in Different Stages of MDSFigure 1. Circulating percentages of Th17, Th1 and Th22 cells in representative healthy controls, E-MDS and L-MDS patients. Heparinized peripheral whole blood from 37 MDS(E-MDS, n = 17; L-MDS, n = 20)patients and 20 healthy PB donors were stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h, and then stained with labeled antibodies for FACS analysis. (A) Lymphocytes were gated by flow cytometry. (B, C, D) Representative FACS dot plots of circulating Th17 (CD4+IL-17+) cells from healthy controls, E-MDS and L-MDS patients. 1662274 (E, F, G) Representative FACS dot plots of circulating Th1 (CD4+IFNc+) cells from healthy controls, E-MDS and L-MDS patients. (H, I, J) Representative FACS dot plots of circulating Th22 (CD4+IL-22+IL-172IFNc2 ) cells from healthy controls, E-MDS and L-MDS patients. Numbers in plots indicate relative percentages per quadrant. doi:10.1371/journal.pone.0051339.gdata. The relative amount of RORC mRNA in E-MDS patients was increased 4.7- and 3.3-fold compared with healthy controls (HC) and L-MDS patients (P = 0.0007; P = 0.002, respectively) (Fig. 4A). In view of increased frequencies of Th22 cells in L-MDS and Th17 cells in E-MDS, we also determined the mRNA expressionlevels of regulatory factors IL-6, TNF-a and IL-23 in MDS patients and controls. The relative amount of mRNA of IL-6 in L-MDS patients was 2.4-(P,0.05) and 5.3-fold (P,0.001) of that in EMDS patients and healthy controls, respectively (Fig. 4B). TNF-a mRNA level was also present at higher levels in L-MDS comparedTh22 and Th17 Cells in Different Stages of MDSwith E-MDS and controls (3.5-fold, P,0.05 and 10.6-fold, P,0.005, respectively) (Fig. 4C). IL-23 is a heterodimeric cytokine consisting of two subunits: p40, which is also a component of IL-12, and p19, a unique subunit of IL-23. So we examined IL-23p19 mRNA expression as representative of IL-23. Quantitative real-time PCR analysis showed that there was no significant difference in IL-23p19 mRNA expression levels among E-MDS, L-MDS and healthy controls (P.0.05) (Fig. 4D).Clinical Relevance of Peripheral Th22, Th17 and Circulating IL-22 in MDS PatientsPeripheral Th17 percentage significantly increased in BM blasts ,5 patients compared with healthy donors (1.7660.80 vs. 0.9860.30 , P = 0.003), while no significant difference was seen between BM blasts 5 
patients and healthy donors (1.3860.80 vs. 0.9860.30 , P.0.05) or between blasts 5 and ,5 patients (1.3860.80 vs. 1.7660.80 , P.0.05) (Fig. 2D). As for the correlations bet.