Figure 3
Induction of Th17 cells in HNSCC microenvironment. (A) Expression of the Th-17-promoting cytokines IL-1β, -6 and -23 in tumour cells and TILs ex vivo analysed by flow cytometry. The bars show the mean of the positive cells±s.d. (B) Induction of Th17 cells in HNSCC microenvironment. T cells were incubated with tumoursupernatants from single cell suspension of tumour tissue for 7 days (n=30) with P<0.05. The cells were stained with CD3-FITC, CD4-PerCP, IL-17-APC and analysed by flow cytometry. The data is expressed as the frequency of Th17 cells in the CD4+ T cell population. The box plots show the median (middle line), 25th and 75th percentiles (box), the extreme values (whiskers, which indicate the 90th and 10th percentile) and the outliers (circles). (C) Blocking of Th17-inducing cytokines with blocking antibodies diminishes the Th17 induction through HNSCC tumourmilieu. T cells were incubated with tumoursupernatants from single cell suspension of tumour tissue for 7 days in the presence of blocking antibodies (n=15). First column cytokines are not blocked, columns 2–4 block one of the cytokines IL-1β, -6, -23, columns 5–7 block two of the Th17 cell-inducing cytokines, column 8 block all three cytokines. The error bars show the s.d. (D) PGE(2) contributes to Th17 cell induction in HNSCC microenvironment. T cells were incubated with tumoursupernatants from single cell suspension of tumour tissue for 7 days in the presence of a PGE(2) blocking antibody (n=15). Blocking of PGE(2) (column 3) leads to a decrease in Th17 induction in HNSCC milieu. Blocking of IL-1β, -6, -23 and PGE(2) leads to a significant downregulation of Th17 cells compared with the number of Th17 cells induced through tumour supernatants alone. The error bars show the s.d.
