Effects of Piezo2 KO on intratumoral CD8 ⁺ T cell function and stemness after radiation. (A–I) Mice were transplanted subcutaneously with 2 × 10⁶ MC38 cells and then established tumors were treated locally with one fraction of 18-Gy IR. (A) To deplete CD8⁺ T cells, mice were injected with anti-CD8 antibody (200 µg per mouse, intraperitoneally (i.p.) starting on the day receiving IR treatment, every 2 days for a total of three times), RIgG, rat IgG. The tumor growth curve of WT and Piezo2^−/− MC38 tumors in C57BL/6 mice with or without anti-CD8 treatment after IR was represented from two independent experiments (n = 4–6 mice per group). (B) Representative data and quantification of the percentage of CD8⁺ T cells in CD45⁺ cells from WT and Piezo2^−/− MC38 tumors on days 7 and 14 after IR treatment were shown from three independent experiments (n = 3–8 mice per group). (C) Representative data and quantification of the percentage of IFN-γ⁺TNF-α⁺CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 7 after IR were shown from three independent experiments (n = 3–6 mice per group). (D) Quantitation of the percentage of IFN-γ⁺CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on days 7 and 14 after IR was shown from three independent experiments (n = 3–6 mice per group). (E–H) Representative data and quantification of the percentage of Ki-67⁺ (E), TCF-1⁺ (F), CD62L⁺ (G), and TOX⁺ (H) in PD-1⁺CD44⁺TIM3^low CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 7 after IR are shown from three independent experiments (n = 3–6 mice per group). (I) Quantification of the percentage of PD-1⁺ in CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 7 after IR was shown from three independent experiments (n = 4–6 mice per group). (J and K) Mice were transplanted subcutaneously with 2 × 10⁶ MC38-OVA cells, and then established tumors were treated locally with one fraction of 18-Gy IR. On the next day of receiving radiation treatment, 2 × 10⁶ activated OT-I CD8⁺ T cells were adoptively transferred into mice via retroorbital intravenous injection. Representative data and quantification of the percentage of OT-I CD8⁺ T cells (J), and IFN-γ⁺TNF-α⁺ (K) in OT-I CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 11 after IR were shown from two independent experiments (J, n = 5–7 mice per group; K, n = 4–6 mice per group). (L) Quantification of the percentage of TCF-1⁺ in OT-I CD8⁺ T cells from WT and Piezo2^−/− MC38 tumors on day 11 after IR was shown from two independent experiments (n = 4–5 mice per group). (M) Mice were transplanted subcutaneously with 1 × 10⁶ cells WT, Piezo2^−/−, and Piezo2^−/−-RE B16F1 cells, and then established tumors were treated locally with one fraction of 18-Gy IR. The tumor growth curve was represented from two independent experiments (n = 4–6 mice per group). Data were represented as means ± SEM. A and M were analyzed by two-way ANOVA with multiple comparison tests; B–L were performed by one-way ANOVA with multiple comparison tests. *P < 0.05; **P < 0.01, ***P < 0.001, ns, no significant difference.