Figure 7HER-2-E0771 mammary tumours cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl;Stat5fl/+ or Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth. Lymphocytes were isolated from the tumours on day 3 post adoptive transfer and mCherry+CD45+ CD8+ CAR-T cell numbers were determined by flow cytometry. HER-2-E0771 mammary tumours cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl;Stat5fl/+ or Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth. HER-2-E0771 mammary tumours cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl;Stat5fl/+ or Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth. Lymphocytes were isolated from the tumours on day 16 post adoptive transfer and mCherry+CD45+ CD8+ CAR-T cell numbers were determined by flow cytometry. In (c) TILs were stained for intracellular IFNγ and TNF after PMA/Ionomycin treatment. HER-2-E0771 cells generated to inducibly overexpress PTPN2 in response to doxycycline (E0771-HER-2-PTPN2hi) were pre-incubated (24 h) with vehicle or doxycycline (DOX) subsequently stimulated with IFNγ for the indicated times. STAT-1 Y701 phosphorylation (p-STAT-1) and PTPN2 levels were assessed by immunoblotting. Cxcl9 and Cxcl10 mRNA levels in vehicle versus DOX-treated and IFNγ-stimulated HER-2-E0771 cells were assessed by quantitative real time PCR. E0771-HER-2-PTPN2hi mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6x106 FACS-purified central memory Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells. E0771-HER-2-PTPN2hi mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6x106 FACS-purified central memory Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells. Mice were then injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer In (g) CD45+CD8+mCherry+ TILs were quantified by flow cytometry at day 4 post adoptive transfer. E0771-HER-2-PTPN2hi mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6x106 FACS-purified central memory Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells. Mice were then injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer and (h) tumour growth was monitored.