Figure 3.
In vitro assessment of S-2HG–treated CAR-T cells. (A) Naive CD8 T cells were isolated, activated, and transduced with an anti-CD19-CAR (4-1BB) lentiviral construct on day 1. After activation, cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 15 and analyzed at day 15 by flow cytometry. Proportion of live CD8 CAR+ cells is shown (n = 8 donors, 4 independent experiments). Paired 2-tailed Student t test was used. (B-C) Naive CD8 T cells were isolated, activated, and transduced with an anti-CD19-CAR (4-1BB) lentiviral construct on day 1. After activation, cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 15 and analyzed at day 15 by flow cytometry. (B) Proportion of live CD8 CAR+ TCM (CCR7+/CD45RO+) cells (n = 8 donors, 4 independent experiments). (C) The total cell number of live CD8 CAR+ TCM (CCR7+/CD45RO+) cells (n = 7 donors, 3 independent experiments; mean ± SEM). Paired 2-tailed Student t test was used. (D-E) Naive CD8 T cells were isolated, activated, and transduced with an anti-CD19-CAR (4-1BB) lentiviral construct on day 1. After activation, cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 15 and analyzed at day 15 by flow cytometry. (D) Proportion of live CD8 CAR+ TEM (CCR7−/CD45RO+) cells. (E) Proportion of live CD8 CAR+ TEF (CCR7−/CD45RO−) cells. For both panels: n = 8 donors, 4 independent experiments; paired 2-tailed Student t test was used. (F) Activated naive CD8 T cells were transduced on day 1 with an anti-CD19-CAR (4-1BB) or with tEGFR (vector-control) lentiviral construct. Transduced cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 12. On day 12, the transduced cells (total population) were cocultured with CD19+ Raji-GFP+ cells or CD19− K562 cells at an effector:target (E:T) ratio of 5:1, 10:1, and 20:1. After 24 hours, cells were analyzed by flow cytometry and cytotoxicity was quantified. Results shown are the mean ± SEM (n = 4 donors, 2 independent experiments). A 2-way ANOVA with the Tukey multiple comparison test was used; variance shown is treatment over dilution. (G-I) Activated naive CD8 T cells were transduced on day 1 with an anti-CD19-CAR (4-1BB) or with tEGFR (vector-control) lentiviral construct. Transduced cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 12. On day 12, the transduced cells (total population) were cocultured with CD19+ Raji-GFP+ cells or CD19− K562 cells at an E:T ratio of 1:1, 5:1, and 10:1. After 6 hours, cytokine levels in the supernatant were quantified. IFN-γ levels (G), TNF-α levels (H), and IL-2 levels (I). For panels showing tEGFR (vector-control), the y-axis is set at the same values as the anti-CD19-CAR panels. Results shown are the mean ± SEM (n = 3 donors, 2 independent experiments). A 2-way ANOVA with the Sidak multiple comparisons test was used; variance shown is treatment over dilution. *P ≤ .05; **P ≤ .01; ****P ≤ .0001. ns, not significant.

In vitro assessment of S-2HG–treated CAR-T cells. (A) Naive CD8 T cells were isolated, activated, and transduced with an anti-CD19-CAR (4-1BB) lentiviral construct on day 1. After activation, cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 15 and analyzed at day 15 by flow cytometry. Proportion of live CD8 CAR+ cells is shown (n = 8 donors, 4 independent experiments). Paired 2-tailed Student t test was used. (B-C) Naive CD8 T cells were isolated, activated, and transduced with an anti-CD19-CAR (4-1BB) lentiviral construct on day 1. After activation, cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 15 and analyzed at day 15 by flow cytometry. (B) Proportion of live CD8 CAR+ TCM (CCR7+/CD45RO+) cells (n = 8 donors, 4 independent experiments). (C) The total cell number of live CD8 CAR+ TCM (CCR7+/CD45RO+) cells (n = 7 donors, 3 independent experiments; mean ± SEM). Paired 2-tailed Student t test was used. (D-E) Naive CD8 T cells were isolated, activated, and transduced with an anti-CD19-CAR (4-1BB) lentiviral construct on day 1. After activation, cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 15 and analyzed at day 15 by flow cytometry. (D) Proportion of live CD8 CAR+ TEM (CCR7−/CD45RO+) cells. (E) Proportion of live CD8 CAR+ TEF (CCR7−/CD45RO−) cells. For both panels: n = 8 donors, 4 independent experiments; paired 2-tailed Student t test was used. (F) Activated naive CD8 T cells were transduced on day 1 with an anti-CD19-CAR (4-1BB) or with tEGFR (vector-control) lentiviral construct. Transduced cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 12. On day 12, the transduced cells (total population) were cocultured with CD19+ Raji-GFP+ cells or CD19− K562 cells at an effector:target (E:T) ratio of 5:1, 10:1, and 20:1. After 24 hours, cells were analyzed by flow cytometry and cytotoxicity was quantified. Results shown are the mean ± SEM (n = 4 donors, 2 independent experiments). A 2-way ANOVA with the Tukey multiple comparison test was used; variance shown is treatment over dilution. (G-I) Activated naive CD8 T cells were transduced on day 1 with an anti-CD19-CAR (4-1BB) or with tEGFR (vector-control) lentiviral construct. Transduced cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 12. On day 12, the transduced cells (total population) were cocultured with CD19+ Raji-GFP+ cells or CD19− K562 cells at an E:T ratio of 1:1, 5:1, and 10:1. After 6 hours, cytokine levels in the supernatant were quantified. IFN-γ levels (G), TNF-α levels (H), and IL-2 levels (I). For panels showing tEGFR (vector-control), the y-axis is set at the same values as the anti-CD19-CAR panels. Results shown are the mean ± SEM (n = 3 donors, 2 independent experiments). A 2-way ANOVA with the Sidak multiple comparisons test was used; variance shown is treatment over dilution. *P ≤ .05; **P ≤ .01; ****P ≤ .0001. ns, not significant.

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