The percentages of suppression were determined based on the proliferation index for RAD001 cell line effector cells cultured alone (100% proliferation, 0% suppression) compared with the proliferation

index of effector cells co-cultured with Treg cells. Statistical analysis was performed using SPSS version 19 and the normality of the data was assessed by the Shapiro–Wilk test. Differences between independent data sets, with normal distribution, were analysed using the Student’s unpaired t-test with the assumption of equal variance assessed by Levene’s test and for data sets without normal distribution the Mann–Whitney U-test was used. Differences between related data sets were analysed using the Student’s paired t-test and the Wilcoxon Signed Rank test for data sets normally or not normally distributed, respectively. Values were considered significant when P < 0·05. The frequency of 5-Fluoracil CD4+ CD25inter CD127low/− (termed CD25inter) and CD4+ CD25high CD127low/− (termed CD25high) Treg cells in the peripheral circulation of newly presenting HNSCC patients as a whole cohort (8·59 ± 0·41% and 6·67 ± 0·45%) was similar to that of healthy controls (8·77 ± 0·85% and 5·81 ± 0·66%). However, the expression of Foxp3 by both

CD25inter and CD25high Treg cells was significantly greater in HNSCC patients (n = 9; 30·08 ± 3·47% and 81·67 ± 2·21%, respectively) compared with healthy controls (n = 6; 15·83 ± 2·26% and 70·63 ± 3·17%), P ≤ 0·01. Additionally, the expression of Foxp3 in the CD25high Treg cell population was significantly greater compared with the CD25inter Treg cells in both HNSCC patients and healthy controls, P ≤ 0·01. Dividing the HNSCC patient cohort by tumour subsite demonstrated that patients with cancer of the larynx and oropharynx had similar percentages of circulating Treg cells irrespective of whether the level of expression of CD25 was intermediate or high (Fig. 2a). However, on analysis of tumour stage, patients with advanced stage tumours had a significantly elevated level of CD25high cells

compared with early stage patients, a trend mirrored, although not significantly, in both the the laryngeal and oropharyngeal subgroups (Fig. 2b). It was also observed that patients with tumours that had metastasized to the lymph nodes had significantly elevated levels of CD25high Treg cells compared with patients without nodal involvement, a trend shared by CD25inter Treg cells but not reaching significance (Fig. 2c). The functional activity of CD25inter and CD25high Treg cells from HNSCC patients (n = 28) and healthy controls (n = 9) was assessed by their ability to suppress the proliferation of two distinct autologous effector T-cell populations (CD4+ CD25− CD127−/+ and CD4+ CD25+ CD127+).