Figure 2.
TanCAR7 T cells formed a stable IS and showed faster degranulation than CD19 CAR or CD20 CAR T cells. (A) Representative confocal imaging of synapses formed by CAR T cells. CD19 CAR, CD20 CAR, and TanCAR7 T cells were loaded onto CD19- and CD20-coated lipid bilayers for 10 minutes, fixed, and stained for CAR (green) and F-actin (red). Scale bars, 2 μm. (B) Quantification of IS structures along the lipid bilayer–T-cell focal plane was performed by measuring the synapse area and the mean fluorescence intensity (MFI) of F-actin at 10, 20, and 30 minutes. Three independent experiments were performed, and representative results are shown. Data are mean ± standard deviation (SD). For each group, 7 to 39 random CAR+ cells were imaged and evaluated. P values were calculated with the Kolmogorov-Smirnov test. (C) Representative MTOC confocal imaging of CAR T cells (left panels). Sorted CD19 CAR, CD20 CAR, and TanCAR7 T cells were loaded onto CD19- and CD20-coated lipid bilayers for 10 minutes. Fixed and permeabilized CAR T cells were stained for γ tubulin (green), F-actin (red), and the nucleus (Hoechst 33342, blue). MTOC polarization at the IS. Data are mean ± SD (right panel). For each group, 20 to 30 random cells were imaged and evaluated. Three independent experiments were performed, and representative results are shown. P values were calculated with the Kolmogorov-Smirnov test. Scale bars, 2 μm. (D) CAR T cells assessed by flow cytometry. The cells were labeled with Fluo-4, AM, stimulated with Raji cells or phorbol 12-myristate-13-acetate/ionomycin, and evaluated by flow cytometry. Three independent experiments were performed, and representative results are shown. CD107a (E) and cytokine (F) expression by CAR T cells, as determined by the percentage of positive cells in flow cytometric analyses. Three independent experiments were performed, and representative results are shown. All data are mean ± SD. *P < .05, **P < .01, ***P < .001, ****P < .0001. IFN-γ, interferon-γ; ns, not significant (P > .05); NT, nontransduced; PMA, phorbol 12-myristate-13-acetate; TNF-α, tumor necrosis factor-α.

TanCAR7 T cells formed a stable IS and showed faster degranulation than CD19 CAR or CD20 CAR T cells. (A) Representative confocal imaging of synapses formed by CAR T cells. CD19 CAR, CD20 CAR, and TanCAR7 T cells were loaded onto CD19- and CD20-coated lipid bilayers for 10 minutes, fixed, and stained for CAR (green) and F-actin (red). Scale bars, 2 μm. (B) Quantification of IS structures along the lipid bilayer–T-cell focal plane was performed by measuring the synapse area and the mean fluorescence intensity (MFI) of F-actin at 10, 20, and 30 minutes. Three independent experiments were performed, and representative results are shown. Data are mean ± standard deviation (SD). For each group, 7 to 39 random CAR+ cells were imaged and evaluated. P values were calculated with the Kolmogorov-Smirnov test. (C) Representative MTOC confocal imaging of CAR T cells (left panels). Sorted CD19 CAR, CD20 CAR, and TanCAR7 T cells were loaded onto CD19- and CD20-coated lipid bilayers for 10 minutes. Fixed and permeabilized CAR T cells were stained for γ tubulin (green), F-actin (red), and the nucleus (Hoechst 33342, blue). MTOC polarization at the IS. Data are mean ± SD (right panel). For each group, 20 to 30 random cells were imaged and evaluated. Three independent experiments were performed, and representative results are shown. P values were calculated with the Kolmogorov-Smirnov test. Scale bars, 2 μm. (D) CAR T cells assessed by flow cytometry. The cells were labeled with Fluo-4, AM, stimulated with Raji cells or phorbol 12-myristate-13-acetate/ionomycin, and evaluated by flow cytometry. Three independent experiments were performed, and representative results are shown. CD107a (E) and cytokine (F) expression by CAR T cells, as determined by the percentage of positive cells in flow cytometric analyses. Three independent experiments were performed, and representative results are shown. All data are mean ± SD. *P < .05, **P < .01, ***P < .001, ****P < .0001. IFN-γ, interferon-γ; ns, not significant (P > .05); NT, nontransduced; PMA, phorbol 12-myristate-13-acetate; TNF-α, tumor necrosis factor-α.

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