Intravital imaging of CAR T cell dynamics and cytotoxicity in the bone marrow in mice with established B cell lymphoma. B cell lymphomas were established by i.v. injection of 0.5 × 106 Eµ-myc-DEVD cells in C57BL/6 mice after sublethal irradiation (4 Gy). 7 d later, mice were injected i.v. with 20 × 106 purified CAR T cells or control activated T cells (untransduced). Bone marrow intravital imaging was performed 40 h after T cell transfer. (A) Representative two-photon images of tumor-bearing mice treated with GFP+ control or CAR T cells. T cells are shown in green, live tumor cells in gray, and apoptotic tumor cells in blue. Scale bars represent 50 µm. (B) Quantification of tumor apoptotic events per hour per mm3 of imaging volume. Events in which tumors became apoptotic during the imaging period were recorded. Apoptotic events were considered as direct killing (n = 55) when a CAR T cell engaged the tumor cells before the detection of FRET loss. Indirect events (n = 20) corresponded to tumor cells undergoing FRET loss without any apparent interactions with a CAR T cell during the imaging period. (C and D) Representative time-lapse images showing examples of tumor apoptosis. (C) Representative time-lapse images showing two examples of direct CAR T cell killing. (D) Time-lapse images illustrating tumor apoptosis in the absence of apparent contact with CAR T cells. (E) Example of two adjacent tumor cells undergoing apoptosis sequentially upon CAR T cell contact. CAR T cells (green) are highlighted by red arrowheads, and dotted circles show tumor cells undergoing apoptosis. (F) For direct killing events, we counted the number of CAR T cells seen contacting the tumor cell before apoptosis. For 73% of the direct killing events, a single CAR T cell was seen interacting with the tumor cell during the imaging period. (G and H) Quantification of CAR T cell killing dynamics. For each direct killing event for which CAR T cell attachment and detachment was visualized, we measured time from T cell contact with tumor cells to killing (G) and T cell detachment (H). Mean durations are shown by red horizontal bars. All data shown were representative of 10 movies for control T cells and 17 movies for CAR T cells performed in three independent experiments.
