Figure 3.

CCR6⁺ IL-17⁺ Il4^R576 T_reg cells exhibit instability and compromised suppressive activity (a) Methylation status of CpG motifs in T_reg cells isolated from lung tissue of OVA–sensitized and challenged mice. Numbers on the left side indicate the position of the respective motifs. (b) Global methylation status of Foxp3 CNS2 in the respective T_reg cell populations (n = 3 mice per group with 7-12 clones per mouse). (c) In vitro suppression of the proliferation of WT responder CD4⁺ T cells (T_eff) by the respective T_reg cell populations (n = 3 replicates per group) (d) Gene expression profiles (volcano plot) of EGFP⁺CCR6⁻ versus EGFP⁺CCR6⁺ T_reg cells isolated by FACS from lung digests of OVA-sensitized and challenged Foxp3^EGFP Il4^R576 mice (n = 3–4 mice). FDR: false discovery rate; Log₂FC: Log₂ fold change. (e) Flow cytometric analysis and frequencies of exT^reg (GFP⁻YFP⁺) cells, plotted as a fraction of exT_reg to total T_reg cells in lung tissue. (f,g) Flow cytometric analysis and frequencies of CCR6 producing (f) and IL-17 and IL-13 producing (g) exT_reg cells in lung tissues. (h) Flow cytometric analysis and frequencies of exT_reg and T_reg cells among CD4⁺IL-17⁺ T_conv cells in lung tissues of the respective mouse groups (n = 6 mice for PBS- and 9 mice for OVA-treated groups for e–h). Data represent means ± s.e.m. from two independent experiments. *P < 0.05, **P < 0.005 and ****P < 0.0001 by one-way ANOVA with Bonferroni posttest analysis. For suppression assay ****P < 0.0001 by repeated measures two-way ANOVA.