Fig. 6. Model of resistance to IFN-γ.

a Parental BT474 cells or resistant BT-R or BT-RG cells were treated with different concentrations of IFN-γ for 5 days. Cell numbers were estimated with the crystal violet staining assay. b Cocultures of PBMCs with the indicated cells were treated with different concentrations of HER2-TCB for 72 h. Then, viable target cells were quantified by flow cytometry using EpCAM as a marker. c The same cells as in (a) were treated with different ratios of CAR T:Tumor cell. Cell numbers were obtained by means of EpCAM counts by flow cytometry. d Totally, 10⁷ BT474 or resistant BT-RG cells were injected orthotopically into NSG mice. When tumors reached ~300 mm³ (dark background), 10⁷ PBMCs were injected i.p. Then animals were treated i.v. with 0.125 mg/kg HER2-TCB as indicated (red arrows). Tumor volumes are represented as averages ± SD (BT474, n = 8; BT-RG, n = 6). e The levels of JAK2 (mRNA and protein) as determined by quantitative real-time PCR (left) or Western blot (right). Results were normalized to BT474 cells. f–h The indicated cells were analyzed as in (a–c), respectively. i BT-RG cells were treated with indicated compounds for 48 h. Then, the levels of the mRNA encoding JAK2 were determined by RT-PCR and normalized to the levels in cells treated with vehicle. j Levels of H3K27Me3 and H3K27Ac histone marks in the promoter of JAK2 in BT474 and BT-RG cells as measured by ChIP followed by quantitative real-time PCR. Results were normalized to the levels of an IgG control antibody in each sample, and then normalized to the levels in BT474 cells. k Schematic drawing summarizing our findings. a **p = 0.002, ***p < 0.001. b **p = 0.009, **p = 0.006, *p = 0.013, **p = 0.002. c **p = 0.008, ***p < 0.001. e ***p < 0.001. f *p = 0.02, **p = 0.004, **p = 0.002, ***p < 0.001, **p = 0.002. g **p = 0.005, ***p < 0.001. h **p = 0.0015, ***p < 0.001. i, j ***p < 0.001. Two-tailed t test. d *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA and Bonferroni correction. Data are presented as mean ± SD of three (a–c, e–i), two (j left) or four (j right) independent experiments. Source data are provided as a Source Data file.