Figure 4.
In vitro comparison of the T-cell phenotype and cytotoxic activity of CAR19 and “all-in-one” CAR19–TIM-3–Fc T cells. (A) Cartoon depicting the all-in-one bicistronic CAR19–F2A–TIM-3–Fc lentiviral construct used to block galectin-9 binding to TIM-3 on CAR19 T cells. (B) Transduction efficiencies (GFP+) in UT, CAR19, and CAR19–TIM-3–Fc T cells. (C) TIM-3–Fc decoy released into the supernatants of UT, CAR19, and CAR19–TIM-3–Fc T cells. (D) Proliferation rate of UT, CAR19, and CAR19–TIM-3–Fc T cells over time. (E-F) CD4 and CD8 distribution (E) and naïve (CCR7+CD45RA+), central memory (CM; CCR7+CD45RA–), EM (CCR7–CD45RA–) and terminally differentiated effector memory (EMRA; CCR7–CD45RA+) distribution (F) on UT and transduced (pos) and UT (neg) CAR19 and CAR19–TIM-3–Fc T cells. (G-H) Expression of the ICRs TIM-3, LAG-3, and PD-1 (G) and the activation marker CD69 (H) on transduced (GFP+) CAR19 and CAR19–TIM-3–Fc CD4+ and CD8+ T cells 5 days after transduction. (I) Galectin-9 expression by FACS on 4 independent primary B-ALL samples. (J) Representative FACS gating strategy to analyze cytotoxicity assays with UT, CAR19, or CAR19–TIM-3–Fc T cells and primary B-ALL cells. (K) Absolute numbers of blasts, GFP+CAR19, and CAR19–TIM-3–Fc T cells and total T cells as well as interferon gamma release after cytotoxicity assays with 4 different primary B-ALL cells at 1:8 E:T ratio for 48 hours. All data are shown as mean ± standard error of the mean; and 3 independent experiments were performed with T cells from 3 different donors. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗∗P ≤ .0001. FSC-H, forward scatter height; IFN, interferon; MFI, mean fluorescence intensity; neg, negative; PE-Cy7, phycoerythrin-cyanine 7; pos, positive; SSC-A, side scatter area; SSC-H, side scatter height.

In vitro comparison of the T-cell phenotype and cytotoxic activity of CAR19 and “all-in-one” CAR19–TIM-3–Fc T cells. (A) Cartoon depicting the all-in-one bicistronic CAR19–F2A–TIM-3–Fc lentiviral construct used to block galectin-9 binding to TIM-3 on CAR19 T cells. (B) Transduction efficiencies (GFP+) in UT, CAR19, and CAR19–TIM-3–Fc T cells. (C) TIM-3–Fc decoy released into the supernatants of UT, CAR19, and CAR19–TIM-3–Fc T cells. (D) Proliferation rate of UT, CAR19, and CAR19–TIM-3–Fc T cells over time. (E-F) CD4 and CD8 distribution (E) and naïve (CCR7+CD45RA+), central memory (CM; CCR7+CD45RA–), EM (CCR7–CD45RA–) and terminally differentiated effector memory (EMRA; CCR7–CD45RA+) distribution (F) on UT and transduced (pos) and UT (neg) CAR19 and CAR19–TIM-3–Fc T cells. (G-H) Expression of the ICRs TIM-3, LAG-3, and PD-1 (G) and the activation marker CD69 (H) on transduced (GFP+) CAR19 and CAR19–TIM-3–Fc CD4+ and CD8+ T cells 5 days after transduction. (I) Galectin-9 expression by FACS on 4 independent primary B-ALL samples. (J) Representative FACS gating strategy to analyze cytotoxicity assays with UT, CAR19, or CAR19–TIM-3–Fc T cells and primary B-ALL cells. (K) Absolute numbers of blasts, GFP+CAR19, and CAR19–TIM-3–Fc T cells and total T cells as well as interferon gamma release after cytotoxicity assays with 4 different primary B-ALL cells at 1:8 E:T ratio for 48 hours. All data are shown as mean ± standard error of the mean; and 3 independent experiments were performed with T cells from 3 different donors. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗∗P ≤ .0001. FSC-H, forward scatter height; IFN, interferon; MFI, mean fluorescence intensity; neg, negative; PE-Cy7, phycoerythrin-cyanine 7; pos, positive; SSC-A, side scatter area; SSC-H, side scatter height.

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