Fig 2: Characterization of Tet2/3-deficient Treg cells. a Representative flow cytometry analyses of the percentage of Treg cells (% of Foxp3+ cells in gated CD4+ T cells) in spleen and pLN from WT and Tet2/3fl/flFoxp3Cre DKO mice (13–16 weeks-old). b Quantification of the percentages (left panel) and cell numbers (right panel) of Treg cells in spleen, pLN and mLN from WT and Tet2/3fl/flFoxp3Cre DKO mice (13-16 weeks old, n = 9). c–f Flow cytometry analysis of Tet2/3-deficient Treg cells from spleen and pLN for the expression of CD25 and Nrp1 (c); Helios, ICOS, CD103, GITR, CTLA4, PD1 and CD127 (d); CD62L versus CD44 (e); and CD69 (f). Shown are WT CD4+ T cells (shaded gray); WT Tregs (black line); DKO Tregs (red line). Statistical analysis was performed using two-tailed unpaired student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Error bars show mean ± s.d. from at least three independent experiments

Fig 3: DNA methylation status of TSDRs in WT and Tet2/3fl/flFoxp3Cre DKO Treg cells. a–f DNA methylation status of TSDRs (Treg-specific demethylated regions). Graphs depict the percentage of 5mC + 5hmC determined by BS-seq in peripheral Tregs in: (a) 4 CpGs in Foxp3 CNS1, and (b) 11 CpGs in Foxp3 CNS2, (c) 4 CpGs in Il2ra intron 1a, (d) 7 CpGs in Tnfrsf18 exon 5, (e) 6 CpGs in Ikzf4 intron 1b and (f) 5 CpGs in Ctla4 exon 2. Error bars show mean ± s.d. of thousands of sequencing reads from two independent experiments. Wild type (WT) Treg cells (8–10 weeks-old) are shown in black, CD4Cre mediated Tet2/3 DKO Treg cells (3–4 weeks-old) are shown in red12 and Foxp3Cre mediated Tet2/3 DKO Treg cells (8–10 weeks-old) are shown in blue

Fig 4: In vitro CRC TO-iTreg cells upregulate functional signature genes associated with CRC TI-Treg cells in vivo(A) Volcano plot displays gene expression fold change against statistical significance, highlighting significant upregulation in mTO2-iTreg cells (red) compared TGFβ-iTreg cells (green) (ANOVA, FDR, p ≤ 0.05).(B) Heatmap of the expression of TFs genes associated with Treg cells in mTO2-iTreg cells compared to TGFβ-iTreg cells.(C) Heatmap of the expression of genes associated with in vivo CRC TI-Treg cells in mTO2-iTreg cells compared to TGFβ-iTreg cells.(D) MFI of OX40, GITR, FAS, and CD27 expression in mTO1- and mTO2-iTreg cells compared to TGFβ-iTreg cells.(E and F) Kaplan-Meier curves for CRC patients of the TCGA cohort, using (E) progression-free interval and (F) overall survival with patients split based on the Z score of the identified signature into two groups (low and high gene expression). The numbers included in the curves indicate the number of CRC patients per time point. p values were calculated using one-way ANOVA or unpaired t-test analysis where appropriate. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001. mTO1 and mTO2: CRC murine tumor-organoid lines.