Fig. 5.

TGF-β and IFN-γ repress trastuzumab-elicited ADCC by upregulating PD-L1/PD-1. a FCM assay for determining the expression of PD-L1 on breast cancer cells incubated with IFN-γ for the indicated times. b NK cells were cocultured with breast cancer cells supplemented with control or PD-L1-blocking antibody, and the cytotoxicity of NK cells was measured by FCM. c FCM assay for determining the expression of PD-1 on NK cells incubated with TGF-β for the indicated times. d NK cells cocultured with SK-BR-3 cells were treated with TGF-β (50 ng/ml) for 6 h. Trastuzumab together with a control or PD-1-blocking antibody was then added, and the cytotoxicity of the NK cells was measured by FCM. e NK cells were cocultured with WT or GKO #3 SK-BR-3 cells in the presence of trastuzumab, and were treated with or without TGF-β (50 ng/ml) for 6 h, and a cytotoxicity assay was performed via FCM. f NK cells cocultured with wild-type SK-BR-3 cells in the presence of trastuzumab were treated with or without TGF-β (50 ng/ml, 6 h) and/or incubated with the indicated antibodies, and then a cytotoxicity assay was performed via FCM. All experiments were performed three times. Statistical significance was obtained by Student’s t test. *P < 0.05 and **P < 0.01. n.s. Nonsignificant