Fig 2: In vitro differentiation of Maf-deficient CD4+ T cells in ROR?t+ Treg is defective. CD4+ T cells isolated from splenocytes of Maff/f and Maf?Tcells mice were cultured for 5 days in TH1 (IL-12 + anti-IL-4), TH2 (IL-4 + anti-IFN-?), Treg (TGF-ß +IL-2) or TH17 (TGF-ß +IL-6) polarizing conditions. (A) Representative histograms for the expression of master transcription factors associated with each helper subset: TBET (TH1), GATA3 (TH2), FOXP3 (Treg) and ROR?t (TH17 FOXP3+ and FOXP3-) from Maff/f and Maf?Tcells mice. (B) Percentage of each master transcription factor according to the condition of polarization among CD4+ T cells (C) Representative histograms for the expression of MAF in in vitro differentiated CD4+ T cell subsets according to the expression of their master transcription factor from Maff/f and Maf?Tcells mice. MFI is indicated for T cells from Maff/f (grey) or Maf?Tcells (black) mice. (D) Percentage of MAF+ cells among each TH subset according to the expression of their master transcription factor from WT mice. Each symbol represents an individual mouse. Data are representative of 2 independent experiments with at least 2 mice per group. All graphs indicate means. Error bars display SEM.

Fig 3: Differentiation of icCD3e+ cells in the small intestine(A) Lin-CD127+ cells from WT small intestine were first divided into CD45lo and CD45hi. icCD3e levels were determined for each subset. Transcription factor levels in CD45hi KLRG1- cells were further analyzed and the correlation between the percentages of icCD3e+ cells and the levels of GATA3 or ROR?t are plotted on the right.(B) MLN cells from mice with or without Nb infection for 6 days were analyzed. Lin-CD127+ cells were separated into Thy1+icCD3e- and Thy1+icCD3e+ subsets, which were then scored for IL-5 and IL-13 expression. Numbers of cytokine+ cells were calculated. One-way ANOVA was used for determining the statistical significance. *p<0.05. Data are represented as mean ± SD.

Fig 4: Murine carcinogen-induced oral lesions are enriched for CD4+ Treg and conventional T cells6- to 8-week-old C57BL/6 mice were exposed to drinking water containing 100 µg/mL 4-NQO or vehicle control for 20 weeks.(A) Quantification of histopathology. After 20 weeks of treatment with 4-NQO drinking water, tongues were dissected, fixed in 10% formalin, bisected longitudinally, and stained with H&E. The perimeter of each tongue is outlined and categorized base on histology grade: hyperkeratosis (black); dysplasia (blue); or SCC (green). Shown is a representative H&E stain of FFPE tongue after longitudinal bisection of 4-NQO-treated mouse, perimeter traced based on histology grades noted above.(B and C) Summary plots of 4-NQO-induced histopathology, showing the percentage of tongue perimeter defined as indicated histology grade. Each bar (B) or symbol (C) represents an individual tongue from a single mouse. Median is indicated in (C). n = 10 mice.(D) (Top) Representative H&E image of SCC region of tongue epithelium from (A). (Bottom) Representative CD3 (left) and Foxp3 (right) IHC images of adjacent sections of depicted SCC region denoted by white box in H&E image are shown. White arrows denote CD3+ or Foxp3+ cells. Scale bars represent 400 µm (H&E) or 50 µm (IHC).(E) Summary plot of pooled data from CD3 IHC density analysis, showing the number of CD3+ cells per mm2 for each lesion. Each symbol represents an individual lesion. Median is indicated. n = 10 regions from 1 mouse (control); n = 6–40 lesions from 10 mice (4-NQO).(F) Representative flow cytometric analysis of CD8+ and CD4+ T cells isolated from tongues of 4-NQO-treated or vehicle-treated mice. Left plots depict CD4 versus CD8ß expression by TCRß+ cells, whereas the right plots depict CD4 versus Foxp3 expression by CD4+ T cells. The frequency of cells within the indicated gates is denoted.(G–I) Summary plot of pooled data from (F), showing the number of TCRß+, CD8+, CD4+, or Foxp3+ T cells per mg of tissue (G), the ratio of CD4+ to CD8+ T cells (H), or the frequency of Foxp3+ cells among CD4+ T cells (I) isolated from whole tongues of vehicle-treated mice (open circles), whole tongues from 4-NQO-treated mice (black circles), or excised tongue lesions from 4-NQO-treated mice (red circles). Each symbol represents an individual mouse (open and black circles) or an individual lesion (red circles). Mean is indicated. Dotted line represents ratio = 1 (H). n = 5 (control whole tongues), n = 6 (4-NQO whole tongues), and n = 9–21 (4-NQO excised lesions).(J) Representative flow cytometric analysis of Foxp3+ Treg cells isolated from tongue of 4-NQO-treated mouse. Left plot depicts ROR?t versus Gata3 expression, whereas the right plot depicts ROR?t versus T-bet expression. The frequency of cells within the indicated gates is denoted.Data are pooled from multiple independent experiments. DW, drinking water; HK, hyperkeratosis; SCC, squamous cell carcinoma. One-way ANOVA with Dunn’s post-test analysis, comparing all pairs in column (E; adjusted p values from Dunn’s post-test are depicted); two-tailed nonparametric Mann Whitney test (G and I). *p < 0.0.5, **p < 0.01, and ***p < 0.001.