Fig 2: DC-specific deletion of Lkb1 leads to specific enlargement of Treg cell pool. a Flow cytometric analysis of Treg cell frequencies among splenic CD4+ T cells from Lkb1f/f and Cd11cCreLkb1f/f mice. b Quantification of the percentage and absolute number of Treg cells in the spleen and lymph nodes (LNs) from Lkb1f/f (red dots) and Cd11cCreLkb1f/f (blue dots) mice of different ages. c, d Flow cytometric analysis (c) and quantification (d) of Ki67 expression in splenic Treg cells from Lkb1f/f and Cd11cCreLkb1f/f mice. e Flow cytometric analysis of Treg cell frequencies among CD4+ T cells in the blood, bone marrow (BM), lungs, liver, kidneys and brain of Lkb1f/f and Cd11cCreLkb1f/f mice. f Quantification of the percentages of Treg cells among CD4+ T cells in the blood, BM, lungs, liver, kidneys and brain of Lkb1f/f (red dots) and Cd11cCreLkb1f/f (blue dots) mice. g Flow cytometric analysis of CD25, CD44, CD62L, Helios, CD73, ICOS, Nrp1 and CTLA4 expression on splenic Treg cells from Lkb1f/f (red line) and Cd11cCreLkb1f/f (blue line) mice. h Suppression assay in which CFSE-labelled naïve T (Tn) cells were co-cultured with Treg cells from Lkb1f/f and Cd11cCreLkb1f/f mice at a 4:1 ratio. Each symbol (b, f) indicates an individual mouse; the results are presented as the mean ± S.E.M., *P < 0.05, **P < 0.01, ***P < 0.001, by Student's t-test (b, d, f). Data are pooled from b, d, f or are representative of a, c, e, g, h three independent experiments with similar results

Fig 3: E. coli infection induces Treg cell expansion and suppression through depleting Lkb1 in DCs. a Immunoblot analysis of Lkb1 protein in DCs from C57 mice challenged with or without E. coli (1×107) via intraperitoneal injection overnight. b Flow cytometric analysis of splenic Treg cells from C57 mice treated with or without E. coli (1×107) 5 days in advance. c Percentage and absolute number of Treg cells in the spleen and LNs of C57 mice treated with or without E. coli (1×107) 5 days in advance (Ctrl, n = 18; E. coli, n = 17). d Flow cytometric analysis of Nrp1 and Helios expression on splenic Treg cells from C57 mice challenged with or without E. coli (1×107) 5 days in advance. e The survival curve of Lkb1f/f (n = 13 each group) and Lkb1f/fFoxp3DTR mice (n = 8 each group) pre-treated with or without E. coli (1×107, 5 days in advance), treated with DT (50 µg kg-1, 2 days in advance), and then challenged with a lethal dose of E. coli (1×108). f The survival curve of Lkb1f/f mice, Cd11cCreLkb1f/f mice, and Cd11cCreLkb1f/fFoxp3DTR mice treated with DT (50 µg kg-1, 2 days in advance) and challenged with a lethal dose of E. coli (1×108) administered by intraperitoneal injection (n = 19 each group). Each symbol (c) indicates an individual mouse; the results are presented as the mean ± S.E.M., *P < 0.05, **P < 0.01, by Student's t-test (c). Log-rank survival curve analysis was used (e, f). Data are pooled from c, f or are representative of three a, b, d, e independent experiments with similar results

Fig 4: 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 5: Progesterone receptor stimulation drives proliferation and accumulation of maternal FOXP3+ cells(A) FACS plots and summary graphs showing ratio of adoptively transferred PRKO [CD45.2+, CD45.1+] (red) to PRWT [CD45.2+, CD45.1-] (gray) FOXP3+ and FOXP3- maternal CD4+ splenocytes during allogeneic pregnancy (E11.5) sired by BALB/c males compared with virgin control mice.(B) Expression of Ki-67, CD178 (Fas ligand), Bcl-2, Helios, Neuropilin, and percent dead cells identified by staining with a membrane integrity dye by donor PR-sufficient (FOXP3-WT) compared with PR-deficient (FOXP3-PRKO) FOXP3+ cells for mice described in panel A. Data are from at least three independent experiments, each with similar results, with each point representing data from an individual mouse. Bar, mean ± SEM. *p < 0.05, ***p < 0.005, ****p < 0.001.