Extended Data Fig. 9. Murine and human CAR4 T cell-derived IFN-γ promotes the bystander killing of CD19− target cells.
(a) Representative histogram of CD19 expression by antigen-positive (black) and CRISPR-edited antigen-negative (white) pro-B cell tumors. (b) Experimental setup. Antigen-negative (CD19−) pro-B tumors were co-cultured with antigen-positive (CD19+) pro-B tumors and WT or IFN-γ−/− CAR4 T cells. When indicated, IFN-γ was neutralized using a blocking antibody. 48 hours later, apoptosis in CD19− pro-B tumors (expressing the caspase 3 reporter) was analyzed by flow cytometry. (c, d) Representative FACS plots (C) and dot plots (D) showing that CAR4 T cell-derived IFN-γ produced upon recognition of CD19+ pro-B tumors contributes to the apoptosis of CD19− pro-B tumors. Each dot represents one technical replicate (n = 1 experiment). CFP, Cyan Fluorescent Protein. (e) Experimental setup. Antigen-negative (CD19−) OVCAR3 tumors were co-cultured with antigen-positive (CD19+) OVCAR3 tumors and human anti-CD19 CAR4 T cells. When indicated, IFN-γ and IFN-γ-R1 were neutralized using blocking antibodies. 48 hours later, cell death in OVCAR3 tumors was analyzed by flow cytometry using fixable viability dye (Zombie NIR). (f) Dot plots showing that human CAR4 T cell-derived IFN-γ produced upon recognition of CD19+ OVCAR3 tumors contributes to the killing of antigen-negative OVCAR3 tumors. Each dot represents one technical replicate (n = 1 experiment). (g) In vivo experimental set-up. 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 C57BL/6 mice after sublethal irradiation. Six days later, mice were injected intravenously with WT or IFN-γ−/− CAR4 T cells or left untreated. Seven days after CAR T cell transfer, blood cells were processed using flow cytometry. (h) Percentage of antigen-positive (CD19+) and emerging antigen-negative (CD19−) pro-B tumors tumor cells recovered from the blood (n = 7 untreated, n = 8 WT CAR4 T cell-treated and n = 8 IFN-γ−/− CAR4 T cell-treated mice from 1 experiment). Statistics for antigen-negative (CD19−) pro-B tumors are shown. Two-way ANOVA and Tukey’s multiple comparisons were used for statistical analysis. Data are expressed as mean ± SEM. *P<0.05; ns, not significant.