American Society of Hematology

Figure 2.

$Functional comparison of memory-derived CD19 CAR T cells (CAR-Tm) and CD19 CAR-T. (A) CAR-Tm and CAR-T were coincubated with Jeko-1 at a 3:1 ratio and analyzed by Incucyte (the curves represent number of live Jeko-1 cells in time). Average cytotoxicity of CAR-Tm (n = 4) and CAR T cells (n = 6) (left). Individual measurements of CD19 CAR-T and CD19 CAR-Tm cytotoxicity; CAR-T products were manufactured using T cells isolated from patients #1 and #2 (right). (B) Area under curve (AUC) and cytotoxity from panel A. CD19 CAR-Tm compared with CD19 CAR-T (n = 4 and n = 6, respectively; each dot is the mean value of individual donor). (C) Secretion of interferonγ and tumor necrosis factor-α by CD4+ and CD8+ fractions of CD19 CAT-Tm (n = 2) in comparison with respective subsets of CD19 CAR-T (n = 10). (D) Degranulation of CD19 CAR-Tm (n = 3) and CD19 CAR T (n = 21) cells in CD4+ and CD8+ populations after coincubation with Jeko-1. (E) Sequential killing of Jeko-1 by CD19− CAR T cells (1:5 ratio). Lines represent the mean values for CAR-Tm (n = 4) and CAR-T (n = 6). Control indicates target-cell proliferation in the absence of CAR-T. The number of target cells was analyzed every 3 days. The remaining CAR T cells were mixed with a fresh portion of target cells at the same ratio and incubated for another 3 days. The procedure was repeated several times until day 18. Plots show the number of survived target cells. (F) Sequential killing assay of CD19 CAR T-cells of individual patients from panel E. (G) Transcriptomic analysis of CD19 CAR-Tm (n = 4) and CD19 CAR T (n = 6) cell products. Heatmap represents genes with highest (green) and lowest (red) expression (P < .01, fold change >2). Color key indicates the intensity associated with normalized expression values. Numbers indicate patients. For panels B-D, statistical analysis was performed using paired t tests. For all panels, ∗P < .05, ∗∗P < .01, ∗∗∗P < .005. For panels A and E, statistical analysis was performed using a 2-way analysis of variance with Turkey multiple comparisons test. For all panels, ∗P < .05, ∗∗P < .01, ∗∗∗P < .005.$

Functional comparison of memory-derived CD19 CAR T cells (CAR-Tm) and CD19 CAR-T. (A) CAR-Tm and CAR-T were coincubated with Jeko-1 at a 3:1 ratio and analyzed by Incucyte (the curves represent number of live Jeko-1 cells in time). Average cytotoxicity of CAR-Tm (n = 4) and CAR T cells (n = 6) (left). Individual measurements of CD19 CAR-T and CD19 CAR-Tm cytotoxicity; CAR-T products were manufactured using T cells isolated from patients #1 and #2 (right). (B) Area under curve (AUC) and cytotoxity from panel A. CD19 CAR-Tm compared with CD19 CAR-T (n = 4 and n = 6, respectively; each dot is the mean value of individual donor). (C) Secretion of interferonγ and tumor necrosis factor-α by CD4⁺ and CD8⁺ fractions of CD19 CAT-Tm (n = 2) in comparison with respective subsets of CD19 CAR-T (n = 10). (D) Degranulation of CD19 CAR-Tm (n = 3) and CD19 CAR T (n = 21) cells in CD4⁺ and CD8⁺ populations after coincubation with Jeko-1. (E) Sequential killing of Jeko-1 by CD19⁻ CAR T cells (1:5 ratio). Lines represent the mean values for CAR-Tm (n = 4) and CAR-T (n = 6). Control indicates target-cell proliferation in the absence of CAR-T. The number of target cells was analyzed every 3 days. The remaining CAR T cells were mixed with a fresh portion of target cells at the same ratio and incubated for another 3 days. The procedure was repeated several times until day 18. Plots show the number of survived target cells. (F) Sequential killing assay of CD19 CAR T-cells of individual patients from panel E. (G) Transcriptomic analysis of CD19 CAR-Tm (n = 4) and CD19 CAR T (n = 6) cell products. Heatmap represents genes with highest (green) and lowest (red) expression (P < .01, fold change >2). Color key indicates the intensity associated with normalized expression values. Numbers indicate patients. For panels B-D, statistical analysis was performed using paired t tests. For all panels, ∗P < .05, ∗∗P < .01, ∗∗∗P < .005. For panels A and E, statistical analysis was performed using a 2-way analysis of variance with Turkey multiple comparisons test. For all panels, ∗P < .05, ∗∗P < .01, ∗∗∗P < .005.

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