Extended Data Fig. 4. Role of IFN-γ production for the activity of CAR4 and CAR8 T cells.
(a-f) In vitro quantification of CAR4 T cell cytotoxic activity. (a) Pro-B-DEVD tumors were co-cultured with WT CAR4 T cells at 1:1 effector-to-target ratio or left untreated for 24 hours. When indicated, TRAIL and FasL were neutralized using a blocking antibody. Tumor cell death was assessed using fixable viability dye (Zombie NIR). Each dot represents one technical replicate. Data are representative of n = 2 independent experiments. (b, c) Pro-B-DEVD tumors were co-cultured with WT, IFN-γ−/− or Prf1−/− CAR4 T cells at 1:1 effector-to-target ratio or left untreated for 24 hours. (b) Tumor cell apoptosis was assessed using the FRET-based caspase 3 reporter. (c) Tumor cell death was assessed using fixable viability dye (Zombie NIR) and normalized to untreated tumor cells. (b–c) Each dot represents one technical replicate from n = 2 independent experiments for Prf1−/− CAR4 T cells and n = 3 independent experiments for WT and IFN-γ−/− CAR4 T cells. One-way ANOVA and Holm-Sidak’s multiple comparisons were used for statistical analysis. (d) Representative histograms of IFN-γ-R1 expression by WT (white) and CRISPR/Cas9 edited pro-B-cell tumors (black). (e) Functional validation of CRISPR/Cas9-mediated deletion of IFN-γ-R1 on pro-B-cell tumors. Pro-B-cell tumors were incubated with the indicated IFN-γ concentrations in vitro for 24 hours. H2-Kb surface expression analyzed by flow cytometry and expressed as a fold change relative to untreated cells. Each dot represents the mean of 3 technical replicates (n = 1 experiment). gMFI, geometric mean fluorescence intensity. (f) IFN-γ-R1−/− pro-B-cell tumors were co-cultured with WT or IFN-γ−/− CAR4 T cells at 1:1 effector-to-target ratio. Tumor cell death was assessed using fixable viability dye (Zombie NIR) and normalized to untreated tumor cells. Each dot represents one technical replicate (n = 1 experiment). (g) In vivo experimental setup. Pro-B-cell tumors were established by intravenous injection of 0.5 × 106 of pro-B cells expressing the FRET-based caspase 3 reporter in IFN-γ−/− mice after sublethal irradiation. Six days later, mice were injected intravenously with WT or IFN-γ−/− CAR4 T cells. Three days after CAR T cell transfer, bone marrow cells were processed using flow cytometry. (h, i) CAR4 T cell-derived IFN-γ increases H2-Kb and PD-L1 levels in tumor and tumor-infiltrating immune cells. Surface expression of H2-Kb (left) and PD-L1 (right) was assessed on CD11b+ myeloid cells (H) and tumor cells (I). gMFI, geometric mean fluorescence intensity. Each dot represents one mouse (n = 4 mice per group from n = 1 experiment). One-way ANOVA, Tukey’s and Holm-Sidak’s multiple comparisons were used for statistical analysis. (j) Percentage of tumor cells recovered from the bone marrow. Each dot represents one mouse (n = 4 mice per group from n = 1 experiment). One-way ANOVA and Holm-Sidak’s multiple comparisons were used for statistical analysis. (k) In vitro quantification of CAR8 T cell cytotoxic activity. Pro-B-DEVD tumors were co-cultured with WT, IFN-γ−/− or Prf1−/− CAR8 T cells or untransduced control CD8+ T cells (CTRL8) at 1:1 effector-to-target ratio for 24 hours. Tumor cell death was assessed using fixable viability dye (Zombie NIR). Each dot represents one technical replicate (n = 1 experiment). (l) Pro-B-cell tumors were established by intravenous injection of 0.5 × 106 pro-B-DEVD cells in C57BL/6 mice after sublethal irradiation. Six days later, mice were injected intravenously with WT or IFN-γ−/− CAR8 T cells. (m) Percentage of tumor cells recovered from the bone marrow 3 days after the transfer of CAR8 T cells. Each dot represents one mouse (n = 11 untreated and n = 10 WT CAR8 T cell-treated mice from n = 3 independent experiments, n = 7 IFN-γ−/− CAR8 T cell-treated mice from n = 2 independent experiments). One-way ANOVA and Tukey’s multiple comparisons were used for statistical analysis. Data are expressed as mean ± SEM. ***P<0.001; **P<0.01; *P<0.05; ns, not significant.