Figure 3.

CCR4-CAR T cells specifically lyse CCR4-positive T-cell lymphoma cell lines and produce cytokines upon stimulation, whereas some CCR4 CAR constructs display ligand-independent T-cell degranulation. (A) Lytic activity of CAR T cells against tumor cell lines. CAR T cells and tumor cells expressing luciferase were incubated at the indicated E:T ratios. Specific lysis after 16 hours of coculture was determined based on the luminescence. Bars represent the means and standard deviation from technical triplicates. Data are representative of 3 experiments from 3 donors. (B) Degranulation of CCR4-CAR T cells. CAR T cells were stimulated with media, PMA-Iono, or HH cells at a 1:1 R:S ratio with CD107a detecting antibody and incubated for 6 hours. The cells were analyzed by FCM. The upper panels are representative flow plots for CD107a expression, the lower panels are pooled data from 3 experiments, and the 3 donors indicate baseline degranulation of CCR4CART cells derived from h1567 or its derivatives. The numbers in each column indicate the percentage of CD107a-positive populations. Dots and bars represent individual data from each donor and the SEM, respectively. (C) Cytokine production by CCR4-CAR T cells. CAR T cells were stimulated with media, PMA-Iono, or HH cells at a 1:1 R:S ratio and incubated in the presence of monensin for 6 hours. The cells were analyzed for intracellular cytokines using FCM. The upper panels are representative flow plots for IL-2 and TNFa expression, and the lower panels are pooled data from 3 experiments with 3 donors. The numbers in each column indicate the percentage of IL-2 TNF-a positive populations. Dots and bars represent individual data from each donor and SEM, respectively. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; ns, not significant (vs CD19-CAR T group) using 1-way ANOVA with Tukey’s post hoc test.