CAR T cells eradicate established B cell lymphoma from the bone marrow within 5 d. (A–F) Tumor regression during CAR T cell therapy. 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 10–20 × 10⁶ CAR T cells or left untreated. (A) Representative two-photon images of the bone marrow at days 2 and 5 after CAR T cell transfer showing the near-complete eradication of the tumor. Live cells appear in gray, apoptotic cells in blue, and CAR T cells in green. Representative of 12 images from two independent experiments. (B–D) Ex vivo quantification of tumor apoptosis in the bone marrow. Flow cytometry analysis was performed at days 2 and 5 after CAR T cell therapy or in the absence of treatment Representative FACS plots (B) and summary graph (C) showing the percentage of FRET loss in tumor cells. **, P < 0.01. (D) Absolute numbers of tumors cells recovered from the bone marrow of one tibia at the indicated time points. Means ± SEM are shown. *, P < 0.05; ns, not significant. (E) Bar charts showing CAR T cell density in the bone marrow quantified by two-photon imaging on days 2 and 5 after CAR T cell transfer. ***, P < 0.001. All data shown in B–E are representative of two independent experiments (n = 3 mice per group). (F) The graph shows the correlation between the percentage of FRET loss in tumor cells and the percentage of CAR T cells among total CD8⁺ T cells. Data are compiled from several time points from two independent experiments. Spearman’s correlation coefficient is shown (R² = 0.98). (G) The tumor evolution in one bone (tibia) was modeled in the presence or absence of CAR T cells. Simulations were performed considering only the direct CAR T cell cytotoxicity and using different initial tumor growth rates (α = 0.1; α = 0.5; α = 1.5) corresponding to initial tumor doubling time of 168, 16.8, and 11.2 h respectively (see Materials and methods for details). The graphs suggest that direct killing events by CAR T cells primarily account for the rapid tumor clearance observed experimentally. (H) Heat map showing the tumor fate at day 5 when considering a range of killing rates and levels of CAR T cell infiltration in the bone marrow (modeled for one tibia using α = 0.5). The red square indicates the values measured experimentally.