Fig. 2

Anti-HER2 antibody-dependent NK cell activation triggers the CCL5/IFN-CXCL9/10 axis in in vitro ADCC assays. A, B Purified primary NK cells were cocultured with SKBR3, BT474 and HCC1954 breast cancer cell lines (E:T ratio 1:1) in the presence of trastuzumab (210 ng/ml) for 24 h. In some experiments blocking/neutralizing antibodies for IFNAR (5 µg/ml), IFN-ɣ (5 µg/ml) and TNF-α (50 µg/ml) were included in the culture. CCL5, IFN-ɣ, CXCL9 and CXCL10 production was analysed in cell-free culture supernatants by ELISA. A Average amount of CCL5, IFN-ɣ, CXCL9 and CXCL10 in each indicated condition, in two independent experiments. B Production of CXCL9 and CXCL10 upon IFN-ɣ, TNF-α and IFNAR individual or combined blockade in in vitro ADCC assays, as analysed by ELISA. Data from three independent experiments normalized to the production of each cytokine/chemokine in the absence of blocking antibodies. Asterisks label statistically significant differences by Mann Withney U test. C HCC1954 and SKBR3 cells were treated with recombinant IFN-ɣ (10 ng/ml), TNF-α (10 ng/ml), IFN-β (1000 U/ml) and their combinations for 24 h. Cell culture supernatants were analysed for the presence of CCL5, CXCL9 and CXCL10 by ELISA. HeatMap showing the absolute amount each chemokine in each condition in pg/ml for CCL5 and ng/ml for CXCL9 and CXCL10