Representative plots showed that healthy individual and bladder carcinoma patients had similar Th17 numbers in the PBMCs (Fig. 1a). As shown in Fig. 1b, the mean frequency of peripheral blood Th17 cells in bladder carcinoma patients was comparable with that in healthy individuals (1·2 ± 0·7% versus 1·3 ± 0·6%). The population of Th17 cells in the TILs isolated from resected tumour specimens of patients with bladder carcinoma (n = 20) was also evaluated. Strikingly, as representative data showed, the percentage of Th17 cells in the TILs was higher than that in the PBMCs in the same patient (Fig. 1a and c). The mean percentage of Th17 cells in the CD4+ population was significantly

higher in TILs (8·2 ± 4·6%) compared with that in the PBMCs from bladder carcinoma patients (1·2 ± 0·7%, P < 0·01, Fig. 1b) or healthy individuals (1·3 ± 0·6%, P < 0·01, Fig. 1b). In some patients up Pexidartinib ic50 to 11% of the CD4+ TILs secreted IL-17 upon brief stimulation, suggesting that IL-17+ T cells may be differentiated predominantly in the tumour microenvironment. To characterize more effectively the CD4+ T cell population producing IL-17 ex

vivo, we also analysed their phenotype and cytokine profile in the tumour microenvironment. Our data showed that the CCR6 surface expression on Th17 cells in TILs was similar to that in PBMCs from patients or healthy controls (Fig. 2a), whereas CCR4 expression on Th17 cells in TILs was significantly higher than Protein tyrosine phosphatase that in PBMCs from patients

or healthy controls (Fig. 2a). Our results showed that most of the tumour-infiltrating IL-17+ T cells expressed high levels Palbociclib ic50 of homing molecules, which might be involved in regulating lymphocyte migration. We further analysed the cytokine profile of human Th17 cells in TILs and PBMCs. Representative plots showed that Th17 cells in PBMCs from a healthy individual and a bladder carcinoma patient had similar lower levels of polyfunctional effector cytokines, including TNF-α and IFN-γ (Fig. 2b). In contrast, Th17 cells in TILs expressed high levels of TNF-α and IFN-γ. Almost half of the tissue Th17 cells were able to produce TNF-α or IFN-γ (Fig. 2c), which implied the possible existence of a developmental and/or functional relationship between Th17 and Th1 cells in bladder tumours. Treg were identified as CD4+CD25high T cells by selecting those CD4+ cells whose CD25 expression exceeded the level of CD25 positivity seen on the CD4 negative population [24] (Fig. 3a). The phenotypic characteristics of CD4+CD25–, CD4+CD25int and CD4+CD25high subsets from cancer patients and healthy donors were then analysed further by flow cytometry. The highest percentage of CD45RO+CTLA-4+ was detected in the CD4+CD25high subsets and the percentage, respectively, was 92% ± 2·5% (range: 89–94%) and 94% ± 3·6% (range: 85–99%), but CD127 and CD69 were not expressed in the CD4+CD25high subsets (Fig. 3b).