The percentage of Treg cells in the tumour tissue was 15·4%, with a standard deviation (s.d.) of 9·9% (range: 7·2–23·6%). There were multiple immune cell populations in the tumour microenvironment. The relationships were evaluated further between Th17 cells and other immune cell subsets, such as IFN-γ+ CD4+ T cells and Treg Pifithrin-�� supplier cells in the same tumours. Flow cytometry analysis revealed that the proportion of Th17 cells was correlated positively with that of IFN-γ+ CD4+ T cells, but correlated inversely with Treg cells in the same tumour microenvironment (Fig. 6a). Several studies suggested

that instillations of IL-2 into the urinary bladder might be effective for treatment of superficial bladder cancer, and recent data also indicated that IL-2 might play a role in regulating the TH17/Treg balance in the tumour microenvironment, so we investigated the potential effects of IL-2 on Th17 and Treg cell differentiation in vitro. A Treg subset from tumour

samples was sorted ex vivo by flow cytometry cell sorting and the purity of the separated cells subset was confirmed to be >97%. Next, we analysed IL-17 production of sorted Treg after stimulation with the autologous irradiated CD3– fraction in the presence of IL-2 for 10 days. As shown in Fig. 6b, Th17 cells were clearly Selleck TSA HDAC detectable in populations from the purified Treg cell fractions. However, no proliferation or IL-17 production was observed after culture of tumour Treg stimulated by the

autologous irradiated CD3– fraction in the absence of IL-2. We also failed to detect any significant proliferation or IL-17 production when the purified tumour Treg cells were cultured with IL-2 alone. To characterize further the tumour Treg after in vitro expansion, we assessed IL-17 production and FoxP3 expression simultaneously by these cells stimulated by the autologous irradiated CD3– fraction in the presence of IL-2. As shown in Fig. 6c, the sorted Treg gradually expressed IL-17 and lost FoxP3 expression. The proportion of Treg co-expressing FoxP3 and IL-17 was increased gradually in the early days, but decreased as culture time went on. Co-culture with responder CD4+CD25– cells and Treg was used to evaluate the function change of tumour Treg after conversion. As shown in Fig. 6d, compared with the tumour Treg before stimulation, the tumour Sirolimus mouse Treg after conversion exhibited hampered inhibition of responder CD4+CD25– cell proliferation, which may be associated with down-regulated FoxP3 expression. Little IFN-γ production was found in the Treg cultures (Fig. 6e). Studies have shown that tumour is potentially immunogenic and that the host immune response influences survival [27]. It has been shown that tumour-infiltrating effector T cells correlates with improved prognoses of several types of cancer, whereas tumour-infiltrating Treg cells are associated negatively with patient outcome [28,29].