(A–C) Total cellular ROS levels (A) of splenic Tregs stimulated with TCR alone or together with 1 μM TG for 12 hours in the presence or absence of NAC (N-acetylcysteine) and quantitative analysis of the MFI of ROS (B) and Foxp3 (C) expression (n = 3). (D and E) FCM levels (D) and analysis (E) of the in vitro suppressive assay of purified Tregs from spleens and LNs from Tmed4^fl/fl and Tmed4^ΔTreg mice, as assessed by the proliferation of activated CD4⁺ T cells in the presence of the indicated ratios of Tregs pretreated with DMSO or NAC for 12 hours (n = 3, detected on day 3). (F) Tumor growth curves for CD45.1⁺ mice that were s.c. injected with MC38 cells, together with WT or Tmed4-deficient Tregs. Both types of Tregs were pretreated with α-CD3/α-CD28 antibodies for 12 hours in the presence or absence of NAC, and then i.v. injected into the mice on day 0 and day 7 (n = 4). (G) FCM analysis of the proportions of activated host CD45.1⁺CD4⁺ (G, left) and CD45.1⁺CD8⁺ (G, right) T cells from WT and KO mouse groups in the presence or absence of NAC, respectively (n = 3). (H and I) IFN-γ–producing host CD45.1⁺CD4⁺ (H) and CD45.1⁺CD8⁺ (I) T cells from WT and KO mouse groups in the presence or absence of NAC, respectively (n = 3). (J) IFN-γ– and IL-17–producing donor CD45.2⁺ Tregs from WT and KO mouse groups in the presence or absence of NAC, respectively (n = 3). Data are presented as the mean ± SEM of biologically independent samples. The tumor rescue model represents 2 independent experiments, and others represent 3 independent experiments, each involving 3–4 mice per group. *P < 0.05 or ^#P < 0.05, **P < 0.01 or ^##P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test (F–J) and 2-tailed Student’s t test.