Figure 2.
S-2HG treatment increases the CCR7+/CD45RO+(TCM) population in naive CD8 T cells. (A) Flow cytometry plots of CD8 T cells showing surface expression of CCR7 and CD45RO following activation of naive CD8 T cells cultured with IL-2 (30 U/mL). Cells were treated with vehicle (H2O) or increasing concentrations of S-2HG from days 0 to 12 and analyzed at day 12 by flow cytometry. Representative plots of n = 5 is shown. (B) Naive CD8 T cells were isolated from 12 individual donors over 6 independent experiments. The isolated T cells were activated and treated with vehicle (H2O) or S-2HG (0.4 mM) from days 0 to 12 and analyzed at day 12 by flow cytometry. The proportion of CD8 TCM (CCR7+/CD45RO+), CD8 TEM (CCR7−/CD45RO+), and CD8 TEF (CCR7−/CD45RO−) cells is shown. Paired 2-tailed Student t test was used. (C) Total cell number following activation of naive CD8 T cells isolated from 5 individual donors over 3 independent experiments. Cells were activated and treated with vehicle (H2O) or S-2HG (0.4 mM) from days 0 to 12. Cell number was quantified on the specified dates using the ADAM-MC automated cell counter (NanoEnTek). Graph shows mean ± SEM and a 2-way ANOVA was used, variance shown is treatment over time. (D) Naive CD8 T cells were isolated from 5 individual donors over 2 independent experiments. T cells were activated and treated with vehicle (H2O) or S-2HG (0.4 mM) from days 0 to 12 and analyzed at day 12 by flow cytometry. The total cell number of CD8 TCM (CCR7+/CD45RO+) cells was quantified by flow cytometry (mean ± SEM). Paired 2-tailed Student t test was used. (E-F) Naive CD8 T cells were isolated from 3 individual donors over 2 independent experiments, activated and treated with vehicle (H2O) or S-2HG (0.4 mM) from days 0 to 12. The cells were collected on day 12 and RNA-Seq analysis followed. GSEA of Kamminga_EZH2_target (E) and Delpuech_ FOXO3_targets_up (F) in S-2HG–treated vs vehicle-treated CD8 T cells. Net enrichment score (NES) values and FDR are shown. For all panels: *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.

S-2HG treatment increases the CCR7+/CD45RO+(TCM) population in naive CD8 T cells. (A) Flow cytometry plots of CD8 T cells showing surface expression of CCR7 and CD45RO following activation of naive CD8 T cells cultured with IL-2 (30 U/mL). Cells were treated with vehicle (H2O) or increasing concentrations of S-2HG from days 0 to 12 and analyzed at day 12 by flow cytometry. Representative plots of n = 5 is shown. (B) Naive CD8 T cells were isolated from 12 individual donors over 6 independent experiments. The isolated T cells were activated and treated with vehicle (H2O) or S-2HG (0.4 mM) from days 0 to 12 and analyzed at day 12 by flow cytometry. The proportion of CD8 TCM (CCR7+/CD45RO+), CD8 TEM (CCR7−/CD45RO+), and CD8 TEF (CCR7−/CD45RO−) cells is shown. Paired 2-tailed Student t test was used. (C) Total cell number following activation of naive CD8 T cells isolated from 5 individual donors over 3 independent experiments. Cells were activated and treated with vehicle (H2O) or S-2HG (0.4 mM) from days 0 to 12. Cell number was quantified on the specified dates using the ADAM-MC automated cell counter (NanoEnTek). Graph shows mean ± SEM and a 2-way ANOVA was used, variance shown is treatment over time. (D) Naive CD8 T cells were isolated from 5 individual donors over 2 independent experiments. T cells were activated and treated with vehicle (H2O) or S-2HG (0.4 mM) from days 0 to 12 and analyzed at day 12 by flow cytometry. The total cell number of CD8 TCM (CCR7+/CD45RO+) cells was quantified by flow cytometry (mean ± SEM). Paired 2-tailed Student t test was used. (E-F) Naive CD8 T cells were isolated from 3 individual donors over 2 independent experiments, activated and treated with vehicle (H2O) or S-2HG (0.4 mM) from days 0 to 12. The cells were collected on day 12 and RNA-Seq analysis followed. GSEA of Kamminga_EZH2_target (E) and Delpuech_ FOXO3_targets_up (F) in S-2HG–treated vs vehicle-treated CD8 T cells. Net enrichment score (NES) values and FDR are shown. For all panels: *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.

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