Cells were fixed and permeabilized with Perm/Fix solution (eBioscience, San Diego, CA, USA), and intracellularly stained with anti-IL-17, anti-FoxP3, anti-tumour necrosis factor (TNF)-α and anti-interferon (IFN)-γ (all from BD Biosciences, San Jose, find more CA, USA, except anti-IL-17; eBioscience). Flow cytometric analysis was performed on a fluorescence activated cell sorter (FACS)Calibur cytometer. Data processing was performed with CellQuest software (Becton Dickinson, San Jose, CA, USA). CD4+CD25- and CD4+CD25+ T cells were isolated from peripheral blood mononuclear cells

and tumour-infiltrating lymphocytes by sorting with the FACSCalibur system after staining with anti-CD4 and anti-CD25 monoclonal antibodies (mAbs). The purity of the isolated CD4+CD25- and CD4+CD25+ T cells was greater than 97%. FoxP3 mRNA expression was quantified by real-time PCR using ABI PRISM 7700 Sequence Detector Ulixertinib mw (Applied Biosystems, Foster City, CA, USA). The human housekeeping gene β-actin primers and probe set was used as a reference for sample normalization. Total RNA isolated from CD4+CD25high T cell was reverse-transcribed into cDNA using random hexamer primers. The primer set for FoxP3 was 5′-TTCGAAGAGCCAGAGGACTT-3′ and 5′-GCTGCTCCAGAGACTGTACC-3′. The probe for FoxP3 was 5′-FAM-CTCAAGCACTGCCAGGCGGACCATC-TAMRA-3′. The primer set for β-actin was 5′-ATCTGCTGGAAGGTGGACAGCGA-3′

and 5′-CCCAGCACAATGAAGATCAAGATCAT-3′. The probe for β-actin almost was 5′-FAM-TGAGCGCA AGTACTCCGTGTGGATCGGCG-TAMRA-3′. The primers and probes used in the real-time PCR were ordered from Sangon (Shanghai, China) and designed not to amplify genomic DNA. Standard curves were generated from serial dilutions of purified plasmid DNA encoding the respective genes with a linear regression R greater than 0·99 and used to quantify mRNA copy numbers for each sample. The amplification protocol used was described as follows: 1 µl of synthesized cDNA product was subsequently added into PCR mix containing

25 µl of TaqMan 2 × PCR master mix (Applied Biosystems), 30 pmol human FoxP3 primer with 10 pmol probe, 2·5 µl β-actin primer/probe set, and distilled water was added to make a total reaction volume of 50 µl. The PCR was programmed as an initial incubation for 10 min at 95°C followed by 40 thermal cycles of 15 s at 95°C and 1 min at 60°C. The normalized values in each sample were calculated as the relative quantity of FoxP3 mRNA expression divided by the relative quantity of β-actin mRNA expression. All reactions were confirmed by at least one additional independent run. The suppressor capacity of Treg was studied in a co-culture suppression assay. A 96-well U-bottomed plate was treated by coating with 10 µg/ml anti-CD3 (UCHT1) and 10 µg/ml anti-CD28 (clone 28·2) monoclonal antibodies in sodium hydrogen carbonate buffer (pH = 9·2) for 2 h. The buffer was washed off with PBS and the plates blocked using T cell media.