Extended Data Fig. 3. Kinetics of tumor apoptotic events mediated by CAR4 T cells.

Pro-B-cell tumors were established by intravenous injection of 0.5 × 10⁶ pro-B-DEVD tumor cells in C57BL/6 mice after sublethal irradiation. Six days later, mice were injected intravenously with WT CAR4 T cells and subjected to intravital two-photon imaging of the bone marrow. Apoptotic events (detected by the genetically-encoded FRET-based reporter for caspase 3 activity) were classified as direct killing when a CAR T cell engaged the target cell before FRET loss detection. Indirect events corresponded to tumor cells undergoing FRET loss without any apparent interactions with a CAR T cell. (a) Kinetics of direct CAR4 T cell-mediated killing events. (b) Kinetics of indirect tumor apoptotic events. Each line (n = 21) represents an individual tumor cell undergoing apoptosis during the imaging period. Gray squares represent periods without contact, green squares represent periods during which tumor cells are contacting a CAR4 T cell and blue squares represent periods during which tumor cells appear apoptotic. Data are representative of n = 26 hours of movie analyzed, from n = 3 mice from n = 3 independent experiments.