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. 2020 Nov 6;218(2):e20192203. doi: 10.1084/jem.20192203

Figure 6.

Figure 6.

4G-CAR-T cells coexpressing mIL-15 exhibit robust expansion during culture and enhanced IL-2 secretion and proliferation in response to antigenic stimulation. (A and B) Expansion (A) and viability (B) of 2G- versus 4G-CAR-T cells in the presence of exogenous hIL-7/IL-15 from day 2 after transduction. Results present the mean fold expansion (A) or percentage of viable cells (B) ± SEM of T cells from n = 4 mice pooled from two independent experiments. Experiments in A and B were repeated four times. (C) Expansion of 2G- versus 4G-CAR-T cells in the presence of the indicated cytokines. Graphs present the mean fold expansion ± SEM of T cells from n = 3 mice on days 5 and 9 after transduction. The experiment was repeated three times. (D) Expansion of 2G-CAR-T cells in T cell medium supplemented with the indicated concentrations of hIL-15 (10, 20, 50, and 100 ng/ml) and 10 ng/ml hIL-7 in comparison to 4G-CAR-T cells expanded with 10 ng/ml hIL-7/IL-15. Cytokines were added from day 2 after transduction and replenished every 2–3 d. Shown are the mean fold expansion values ± SEM of T cells from n = 5 mice. (E) Expansion of 4G-CAR-T cells cultured in the indicated concentrations of hIL-15 and 10 ng/ml hIL-7. Shown are the mean values of fold expansion ± SEM of T cells from n = 5 mice. Experiments in D and E were repeated twice. (F) IL-2 production by 2G- versus 4G-CAR-T cells upon co-culture with target cells. Results present the mean concentration (pg/ml) ± SEM in triplicate wells with T cells pooled from n = 4 mice. (G) Measurement of mIL-15 secretion by 2G- and 4G-CAR-T cells at 24 h after co-culture with target cells at a CAR+ T cell to target cell ratio of 2:1. Numbers represent mean concentration (pg/ml) ± SEM in co-culture supernatants with T cells from n = 4 mice. (H) Expansion of 2G- versus 4G-CAR-T cells after co-culture with target cells. Results present the mean number of CD8+ T cells ± SEM for n = 5 mice. (I) Shown are representative flow cytometric examples indicating the numbers of CD8+ T cells in the co-cultures of 2G- and 4G-CAR-T cells with target cells on days 1 and 4 after co-culture. (J and K) Percentage (J) or number (K) of dividing CD8+ T cells upon co-culture with bEnd3 cells. Results shown are mean values ± SEM of T cells from n = 4 mice. (L) Shown is a representative flow cytometric analysis of CTV-stained CAR-T cells co-cultured with bEnd3 or H5V cells. (M) Quantification of target cell apoptosis on day 3 after co-culture with 2G- or 4G-CAR-T cells using Annexin V and 7-AAD staining. The graph presents mean values ± SEM of Annexin V+/7-AAD+ target cells upon co-culture with T cells from n = 3 mice. (N) Representative dot plots of Annexin V+/7-AAD+ target cells following CAR-T cell co-culture. Experiments in F–N were repeated three times. Statistical analyses were performed using a two-tailed unpaired Student’s t test (A and B) and a one-way ANOVA with Tukey post hoc correction test (C–F, H, J, and K): *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.