Fig. 7: Inhibiting Akt-mediated Ezh2 phosphorylation improves antitumor immunity | Nature Communications

Fig. 7: Inhibiting Akt-mediated Ezh2 phosphorylation improves antitumor immunity

From: Ezh2 phosphorylation state determines its capacity to maintain CD8+ T memory precursors for antitumor immunity

Fig. 7

. a, b Ex vivo-expanded 7 day-Pmel-1 cells (Thy1.1+), MK2206-treated 7 day-Pmel-1 cells (Thy1.1+), or 7 day-Pmel-1 cells infected with MigR1 retrovirus encoding Ezh2S21A (Thy1.1+, 5 × 105 T cells per mouse) were transferred into sublethally irradiated B6 mice that had pre-established B16 melanoma, followed by treatment with IL-2 and gp100-DCs from 0 to 3 days and repeated once more from 18 to 20 days. Tumor size was monitored over time (a). Some recipient mice were sacrificed at 9 days to examine the presence of donor Pmel-1 cells in the spleen using flow cytometric analysis. Graphs show the percentage of indicated total cells and cell subsets (b). c–h B6 mice with pre-established B16 melanoma cells received no adoptive transfer (Non-T cells), transfer of in vitro expanded 7 day-Pmel-1 cells infected with MigR1 encoding GFP, Ezh2 or Ezh2S21A (5 × 105 T cells per mouse), followed by treatment with IL-2 and gp100-DCs from 0-3 days and repeated once more from 18 to 20 days. Tumor size was monitored over time (c). The percentage of total donor Pmel-1 cells (d) and KLRG1hi Pmel-1 cells (e) in circulating PB from each group of mice receiving GFP-, Ezh2- and Ezh2S21A-7 day-Pmel-1 cells over a period of 24 days after transfer. f–h In separate experiments as described in (c–e) above, donor T cells were recovered from the spleen and tumor at 21 days after transfer. f Plots show the percentage of transferred GFP+ cells in tumor and spleen by gating on donor Thy1.1+ Pmel-1 CD8+ cells, with Pmel-1 cells prior to transfer as controls. g Graphs show the number of total donor GFP+Thy1.1+ Pmel-cells and IFN-γ+ Pmel-1 cells in the spleen and tumor. h Plots show the expression of CD62L on the surface of donor GFP+Thy1.1+ T cells derived from the spleen and tumor. *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed unpaired t test). The data are pooled from two experiments (a; GFP, n = 9; Ezh2, n = 9; S21A, n = 10; mean ± SD), or representative of two independent experiments (b; n = 3–5 mice per group in each, mean ± SD; c–h; n = 5 mice per group in each, mean ± SD)

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