Fig. 3: IFN-γ response is required for efficient killing by redirected lymphocytes.

a Left, cocultures of PBMCs with BT474 or BT-R cells were treated with different concentrations of HER2-TCB in presence of an IgG control or an IFN-γ blocking antibody for 72 h. Then, viable cells were quantified by flow cytometry using EpCAM as a marker. Right, BT474 cells were grown in 3D and treated with HER2-TCB in presence of an IgG control (−) or an IFN-γ blocking antibody (+) for 72 h. Viable BT474 cells were quantified by flow cytometry using EpCAM as a marker. Results were normalized to untreated cells. b Parental BT474 cells were cocultured with different ratios of CAR T cells for 48 h in the presence of an IgG control or an IFN-γ blocking antibody. Then, cell numbers were calculated and expressed as in (a). c Parental BT474 or resistant BT-R cells were treated with different concentrations of IFN-γ for 5 days. Cell numbers were estimated with the crystal violet staining assay. d Parental BT474 or resistant BT-R cells were treated with 1 μg/ml of IFN-γ and the percentages of apoptotic cells were determined by means of Annexin V⁺ cells measured by flow cytometry. e Cells were stained with anti-IFNGR1 or isotype antibody and analyzed by flow cytometry. IFR1 stands for IFNGR1. f Sensitivity of the indicated cells to IFN-γ was analyzed as in (c). g The indicated cells were treated with different concentrations of HER2-TCB and analyzed as in (a). h The indicated cell lines were cocultured with CAR Ts and analyzed as in (b). i Totally, 6.5 × 10⁷ BT474 cells or the same cells knock-out for IFNGR1 were injected orthotopically into NSG mice. Mice were treated as described in Fig. 1c. Tumor volumes are represented as averages ± SD (empty, n = 8; IFR1 KO, n = 4). a Left, **p = 0.001, *p = 0.05; right, *p = 0.01. b ***p < 0.001, **p = 0.002. c ***p < 0.001. d **p = 0.007. f Left, **p = 0.002, ***p < 0.001, **p = 0.003, **p = 0.0013; right, ***p < 0.001, **p = 0.002 (shIFNGR1 #31); **p = 0.009, **p = 0.006, ***p < 0.001, **p = 0.003 (shIFNGR1 #92); *p = 0.02, **p = 0.004, ***p < 0.001 (shIFNGR1 #97). g Left, ***p < 0.001; right, ***p < 0.001, **p = 0.004, *p = 0.02 (shIFNGR1 #31); *p = 0.046, *p = 0.047, *p = 0.03 (shIFNGR1 #92); *p = 0.05, *p = 0.02, *p = 0.02 (shIFNGR1 #97). h Left, ***p < 0.001; right *p = 0.02, ***p < 0.001 (shIFNGR1 #31); ***p < 0.001, *p = 0.02 (shIFNGR1 #92); ***p < 0.001, **p = 0.005, **p = 0.005 (shIFNGR1 #97). Two-tailed t test. i **p < 0.01, ***p < 0.001, two-way ANOVA and Bonferroni correction. Data are presented as mean ± SD of three (a–c, f, g right, h), four (d), or six (g left) independent experiments. Source data are provided as a Source Data file.