Figure 3.
Figure 3. Functional heterogeneity in CAR T cell–tumor interactions in the bone marrow. (A and B) 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. Bone marrow intravital imaging was performed 40 h after T cell transfer. (A) The graph shows the percentage of CAR T cells involved in a direct killing event during the imaging period. Each dot represents the value calculated in one movie. Data are compiled from three independent experiments. (B) Representative time-lapse images illustrating that CAR T cell–tumor interactions do not necessarily lead to tumor killing. Scale bar represents 20 µm. Representative of 17 movies from three independent experiments. (C) Experimental setup. CAR T cell therapy was performed using CAR T cells expressing the Twitch2B genetically encoded calcium indicator. (D and E) Only a fraction of CAR T cell–tumor interactions trigger calcium signals in the CAR T cell. (D) Representative time-lapse images showing two CAR T cells (arrowheads) interacting with the same tumor cell (dotted circle) but exhibiting a different calcium response. Calcium elevation is reflected by an increase of Twitch2B FRET signal. Low and high calcium concentrations are shown in yellow and red, respectively. Tumor cells are shown in brown. Scale bar represents 20 µm. (E) The tracks and the color-coded calcium response of the two highlighted CAR T cells are shown (upper images). Some of the contacted tumor cells are highlighted in dotted lines. The bottom graphs show the calcium signals over time during the various contacts with tumor cells. Tumor killing is highlighted by the red dotted line. Results are representative of three independent experiments (F and G) CAR T cells do not exhibit a general block in calcium mobilization. Twitch2B-expressing CAR T cells were prepared from CD8+ T cells bearing the OT-I transgenic TCR. During intravital imaging, OVA257–264 peptide (OVAp) was injected i.v. The calcium responses for two representative CAR T cells are depicted, showing that CAR T cells that displayed low/no calcium signals during tumor cell contacts exhibited strong calcium responses when stimulated in vivo through their TCR. Representative of two independent experiments.

Functional heterogeneity in CAR T cell–tumor interactions in the bone marrow. (A and B) 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. Bone marrow intravital imaging was performed 40 h after T cell transfer. (A) The graph shows the percentage of CAR T cells involved in a direct killing event during the imaging period. Each dot represents the value calculated in one movie. Data are compiled from three independent experiments. (B) Representative time-lapse images illustrating that CAR T cell–tumor interactions do not necessarily lead to tumor killing. Scale bar represents 20 µm. Representative of 17 movies from three independent experiments. (C) Experimental setup. CAR T cell therapy was performed using CAR T cells expressing the Twitch2B genetically encoded calcium indicator. (D and E) Only a fraction of CAR T cell–tumor interactions trigger calcium signals in the CAR T cell. (D) Representative time-lapse images showing two CAR T cells (arrowheads) interacting with the same tumor cell (dotted circle) but exhibiting a different calcium response. Calcium elevation is reflected by an increase of Twitch2B FRET signal. Low and high calcium concentrations are shown in yellow and red, respectively. Tumor cells are shown in brown. Scale bar represents 20 µm. (E) The tracks and the color-coded calcium response of the two highlighted CAR T cells are shown (upper images). Some of the contacted tumor cells are highlighted in dotted lines. The bottom graphs show the calcium signals over time during the various contacts with tumor cells. Tumor killing is highlighted by the red dotted line. Results are representative of three independent experiments (F and G) CAR T cells do not exhibit a general block in calcium mobilization. Twitch2B-expressing CAR T cells were prepared from CD8+ T cells bearing the OT-I transgenic TCR. During intravital imaging, OVA257–264 peptide (OVAp) was injected i.v. The calcium responses for two representative CAR T cells are depicted, showing that CAR T cells that displayed low/no calcium signals during tumor cell contacts exhibited strong calcium responses when stimulated in vivo through their TCR. Representative of two independent experiments.

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