Figure 7.
GA potentiates CD8+T cell antitumor immunity via Mettl8 inhibition. (A) Schematic diagram of GA-treated OT-I–transferred model: CD45.2 mice were subcutaneously injected with 2 × 105 EG7-OVA cells, followed by 5 × 105 CD45.1.2 WT or Mettl8−/− OT-I cells transfer at 9 dpi. GA was administered i.p. every 2 days from 11 to 17 dpi. Mice were harvested at 19 dpi. (B–D) Tumor growth (B), tumor weight (C), and absolute number of tumor infiltrating OT-I cells (D) of the mice in A. n = 7–8 per group. (E and F) Representative flow cytometry plots (E) and cumulative data (F) show the frequency and absolute number of Tcf1+ Tim3− TPEX and Tim3+ Tcf1− TEX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (G and H) Representative flow cytometry plots (G) and cumulative data (H) show the frequency and absolute number of CX3CR1+ Tcf1− Int-TEX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (I) Schematic diagram of Mettl8-mutated mouse model: CD45.2 Mettl8fl/flCd4cre mice were subcutaneously injected with 2 × 105 EG7-OVA cells. Mettl8-WT or Mettl8-mutation (Mut) retrovirus were transduced to CD45.1.2 Mettl8−/− OT-I cells. 5 × 105 GFP+ cells were sorted 48 h after transduction and adoptively transferred into the tumor-bearing mice at 9 dpi. GA was administered i.p. every 2 days from 11 to 17 dpi. Mice were harvested at 18 dpi. (J) Tumor growth of the mice in I. n = 4–6 per group. (K and L) Representative flow cytometry plots (K) and cumulative data (L) show the frequency and absolute number of Tcf1+ Tim3− TPEX, Tim3+ Tcf1− TEX, and CX3CR1+ Tcf1− Int-TEX cells gated on tumor-infiltrating OT-I cells. n = 4–6 per group. Data are representative of two independent experiments. P value was calculated by two-way ANOVA (B and J) and two-tailed Student’s t test (C, D, F, H, and L); *P < 0.05; **P < 0.01; ***P < 0.001. Refer to the image caption for details. Panel A shows schematic diagram of G A treatment in O T I transfer tumor model. Panel B shows line graph comparing tumor growth across treatment groups. Panel C shows cumulative data comparing tumor weight between groups. Panel D shows the cumulative data of infiltrating O T I cell numbers. Panel E shows flow cytometry plots of T c f 1 positive T P E X cells. Panel F quantifying T P E X and T E X cell frequencies. Panel G shows flow cytometry plots of C X 3 C R 1 positive intermediate T E X cells. Panel H quantifying intermediate T E X cell frequencies. Panel I shows a schematic diagram of retroviral M e t t l 8 mutation experiment. Panel J shows a line graph comparing tumor growth after retroviral rescue. Panel K shows flow cytometry plots showing T cell subset distributions. Panel L quantifying T cell subset frequencies.

GA potentiates CD8 + T cell antitumor immunity via Mettl8 inhibition. (A) Schematic diagram of GA-treated OT-I–transferred model: CD45.2 mice were subcutaneously injected with 2 × 105 EG7-OVA cells, followed by 5 × 105 CD45.1.2 WT or Mettl8−/− OT-I cells transfer at 9 dpi. GA was administered i.p. every 2 days from 11 to 17 dpi. Mice were harvested at 19 dpi. (B–D) Tumor growth (B), tumor weight (C), and absolute number of tumor infiltrating OT-I cells (D) of the mice in A. n = 7–8 per group. (E and F) Representative flow cytometry plots (E) and cumulative data (F) show the frequency and absolute number of Tcf1+ Tim3− TPEX and Tim3+ Tcf1− TEX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (G and H) Representative flow cytometry plots (G) and cumulative data (H) show the frequency and absolute number of CX3CR1+ Tcf1− Int-TEX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (I) Schematic diagram of Mettl8-mutated mouse model: CD45.2 Mettl8fl/flCd4cre mice were subcutaneously injected with 2 × 105 EG7-OVA cells. Mettl8-WT or Mettl8-mutation (Mut) retrovirus were transduced to CD45.1.2 Mettl8−/− OT-I cells. 5 × 105 GFP+ cells were sorted 48 h after transduction and adoptively transferred into the tumor-bearing mice at 9 dpi. GA was administered i.p. every 2 days from 11 to 17 dpi. Mice were harvested at 18 dpi. (J) Tumor growth of the mice in I. n = 4–6 per group. (K and L) Representative flow cytometry plots (K) and cumulative data (L) show the frequency and absolute number of Tcf1+ Tim3− TPEX, Tim3+ Tcf1− TEX, and CX3CR1+ Tcf1− Int-TEX cells gated on tumor-infiltrating OT-I cells. n = 4–6 per group. Data are representative of two independent experiments. P value was calculated by two-way ANOVA (B and J) and two-tailed Student’s t test (C, D, F, H, and L); *P < 0.05; **P < 0.01; ***P < 0.001.

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