Single-cell suspensions from spleens of male and female Foxp3^YFP-Cre or Kdm6b^ΔTreg mice (aged 8–12 weeks) were analyzed by flow cytometry at steady state. (A) Frequency of FOXP3⁺ Tregs among CD4⁺ lymphocytes in spleen (n = 10–11 mice/strain, combining 2 separate experiments). (B) FOXP3 expression measured as gMFI (n = 5 mice/strain, representative of 2 separate experiments). (C) Frequency of CD44⁺CD62L^– Tregs in splenic CD4⁺FOXP3⁺ cells (n = 5–6 mice/strain, representative of 2 separate experiments). (D) Immunophenotyping of splenic CD4⁺FOXP3⁺ lymphocytes (n = 5–6 mice/strain, representative of 2 separate experiments). (E) IL-10 production by splenic Tregs after PMA/ionomycin stimulation (n = 5 mice/strain, representative of 2 separate experiments). (F) In vitro suppression of CD4⁺ effector T cell proliferation by splenic Tregs (combined data from 3 independent assays). ^#P < 0.05. (G) H3K27me3 abundance in CD4⁺FOXP3⁺ or CD4⁺FOXP3^– splenocytes (n = 5–6 mice/strain). (H) Experimental schematic of mixed bone-marrow chimeras (n = 6 mice/condition). (I–L) Analysis of CD45.2⁺ donor-derived cells showing Treg frequency (I), FOXP3 expression (J), Ki67⁺ proliferation (K), and CD44⁺CD62L^– effector phenotype (L) in spleen, lymph nodes (LN), and lung. (M–O) Expression (gMFI) of CD25 (M), CTLA4 (N), and glucocorticoid-induced TNFR-related protein (GITR) (O) on donor-derived Tregs across tissues. Data are given as the mean ± SEM. Statistical significance was determined by unpaired 2-tailed t tests or 2-way ANOVA with Holm-Šídák correction, as indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.