Fig. 3
Recognition of PD-L1241-265-specific CD4+ T-cell lines against tumor cell lines and DCs expressing PD-L1. Expressions of PD-L1 and HLA-DR on tumor cell lines were examined by flow cytometry after treatment with IFN-γ for 48 h. Representative flow histograms for a PD-L1 and b HLA-DR expressions were shown in upper panels and lower panels, respectively. Black: isotype control (MOPC-21 for PD-L1 and MOPC-173 for HLA-DR), Red: untreatment, Blue: IFN-γ treatment (500 U/ml). c PD-L1241-265-specific CD4+ T-cell lines were cocultured with HLA-DR-matched and unmatched tumor cell lines expressing PD-L1 with/without anti-HLA-DR mAbs as indicated. Supernatants were collected after 48 h and analyzed for IFN-γ by ELISA. d The reactivity of PD-L1241-265-specific CD4+ T-cells (G1 and G2) to autologous DCs and tumor cell lines. DCs and tumor cells (HSC4 or SAS) were treated with IFN-γ (500 U/ml, 48 h), and were cocultured with each PD-L1241-265-specific CD4+ T-cells for 24–48 h. Anti-HLA-DR antibody was used for blocking HLA-DR-specific reaction. 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