(A) Scatterplot showing global gene expression profiles of Tregs from Tmed4^fl/fl and Tmed4^ΔTreg mice stimulated with α-CD3/α-CD28 antibodies. Transcripts with a |log₂(fold change)| >0.5 and P < 0.05 in Tmed4-deficient Tregs are shown in blue or red. (B and C) Heatmaps of gene clusters of DEGs of effector-like genes (B) and Treg signature profile genes (C) and between WT and Tmed4-deficient Tregs. Red and blue represent relatively higher and lower levels of expression of the indicated genes, respectively. The colors indicate the value of the log₂ fold change (n = 3). (D) GSEA plots showing the enrichment of the “Treg signature profiles” (FDR <0.2) gene set. (E and F) FCM levels (E) and statistical analysis (F) of the in vitro suppressive assay of purified Tregs from spleens from Tmed4^fl/fl and Tmed4^ΔTreg mice, as assessed by the proliferation of activated CD4⁺ T cells in the presence of various ratios (responder T cells/Tregs [Tresp/Treg] = 1:2, 1:1, and 1:0.5) of Tregs (n = 3, detected on day 3). (G) FCM analysis of MFI of CD25 in Tregs from spleens, pLNs, and lungs of Tmed4^fl/fl and Tmed4^ΔTreg mice (n = 3). (H and I) FCM plots (H) and analysis (I) of Foxp3⁺ Treg frequencies in BM chimeric mice (n = 3). FSC-W, forward scatter width. (J and K) FCM analysis of Foxp3 (J), CD25, CTLA4, and GITR (K) MFI in spleens and pLNs from BM chimeric mice (n = 3). (L and M) FCM plots (L) and Foxp3 frequencies (M) of both YFP⁺ and YFP^– Tregs in spleens and pLNs from female WT and chimeric mice (n = 3). (N) Ratio of YFP⁺ Tregs to YFP^– Tregs in spleens and pLNs from female WT and chimeric mice (n = 3). (O) FCM analysis of Foxp3 MFI between YFP⁺ and YFP^– Tregs from female WT and chimeric mice (n = 3). Data are presented as the mean ± SEM of biologically independent samples and represent at least 3 independent experiments, each involving 3 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-tailed Student’s t test.