Fig. 4
The diminished responses of PD-L1241-265-specific CD4+ T-cell lines against tumor cell lines transfected with PD-L1-specific siRNA. a Expression levels of PD-L1 on tumor cell lines were examined by flow cytometry. Black: isotype control, Red: PD-L1 on parental cell lines, Blue: PD-L1 on tumor cell lines transfected with PD-L1-specific siRNA (upper panels) or mock siRNA (lower panels). Each tumor cells was treated with IFN-γ (500 U/ml) for 48 h. b Evaluation of PD-L1 in tumor cell lines was performed by Western blot. c PD-L1 241-265-specific CD4+ T-cell lines were co-cultured with HLA-DR-matched tumor cell lines after transfection of PD-L1-targeting siRNA or mock. Supernatants were collected and analyzed by ELISA for IFN-γ release. The results shown are representative of two separate experiments. d The reactivity of PBMCs of five HNSCC patients and two healthy donors (HD) against PD-L1241-265 peptides was assessed. Isolated PBMCs were cultured in the presence of PD-L1241-265 or PADRE peptides (10 μg/mL) for 10 days. Supernatants were collected and analyzed by ELISA for IFN-γ release. Bars and error bars indicate the mean and SD of triplicate determinations, respectively. Each data is representative of two separate experiments