Fig. 4.

TGF-β and IFN-γ upregulate HLA-G on breast cancer cells and KIR2DL4 on NK cells, respectively. a NK cells were cultured alone or with SK-BR-3 cells (E:T = 1:1) supplemented with the indicated antibodies. The supernatant was harvested and measured for cytokine levels using a cytokine antibody array. b NK cells and SK-BR-3 cells were cocultured or cultured alone for 48 h with or without trastuzumab, followed by measurement of IFN-γ and TGF-β production via ELISA. c FCM assay for determining the trastuzumab-elicited cytotoxicity of NK cells to cocultured HER2-positive breast cancer cells when treated with or without TGF-β (50 ng/ml) for 6 h. d Cells were incubated with TGF-β for the indicated times and were subjected to FCM assay for determining HLA-G level. e, f FCM assay for the expression of KIR2DL4 and ILT2 on NK cells incubated with IFN-γ and different inhibitors for the indicated times. g NK cells were incubated with IFN-γ (50 ng/ml) for the indicated times and were subjected to Western blot analysis. h ChIP assay for determining the enrichment of STAT1 on the KIR2DL4 promoter using NK cells prepared from different donors. All experiments were performed three times. Statistical significance was obtained by Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s. Nonsignificant