Figure 3: Lkb1 stabilizes Foxp3 expression and prevents CNS2 methylation.

(a) Foxp3⁺ cell percentages among CD4⁺ T cells in spleen and lymph nodes from Foxp3^Cre and Foxp3^CreLkb1^f/f mice of different ages (n=10–15). (b) Foxp3 expression in CD4⁺Rosa26-YFP^high (Rosa26-YFP labelled the cells that have experienced the expression of Foxp3, Rosa26-YFP was much brighter than and thus distinguishable from Foxp3-YFP) cells from Foxp3^Cre Rosa26^YFP and Foxp3^CreLkb1^f/f Rosa26^YFP mice. (c) Foxp3 expression in T_reg cells doubly sorted from Foxp3^Cre and Foxp3^CreLkb1^f/f mice and transferred together with T_n cells into Rag1^−/− mice for 3 weeks (n=3). (d) Methylation of the CpG motifs of the −1.5 kb region, promoter, CNS2, exon 1, exon 8 and exon 11 across the Foxp3 locus in T_reg cells from Foxp3^Cre and Foxp3^CreLkb1^f/f mice, determined by bisulfite sequencing. Filled circles represent methylated CpG sites and open circles represent unmethylated CpG sites. Two-way analysis of variance (ANOVA) was used for statistical analyses in a,c, and unpaired two-tailed Student’s t-test was used for statistical analyses in b (**P<0.01); error bars represent s.d.; all data are representative of at least two independent experiments.