Fig 2: Effector CD4 T Cells constitutively express IL-1R and require IL-1R signaling for cytokine production. a, b Splenic CD4 T cells were gated on naive (Tnaive) and memory (Tem) T cell markers and stained for a IL-6Ra, IL-4Ra and IL-12Rb1 b IL-1R1, IL-18Ra and IL-33R (ST2). c Relative expression of Il1r1 transcripts in Tnaive and Tem cells. Data is normalized to 18s. d-f Tem cells were stimulated with splenic DCs and soluble aCD3 (30 ng ml-1) in the presence or absence of IL-1R antagonist (IL-1Ra) for 48 h d IL-17A, IL-17F, IL-22, e IL-13, IL-4, IL-5, and f IFN? were measured. g CD4 T cells from SI-LP were stained for IL-1R1. h CD4 T cells from SI-LP were stimulated using CD11C + DCs and soluble aCD3 (30 ng ml-1) in the presence or absence of IL-1Ra for 48 h followed by cytokine ELISA. i IL-17A, IL-13 and IFN? levels in supernatants obtained from Tem cell co-cultures with WT or Il1r-/-CD11c + splenic DCs in the presence of aCD3 (30 ng ml-1) after 48 h of stimulation. Control in receptor staining refers to fluorescence minus one control. IL-17A, IL-13 and IFN? in the culture supernatants were measured using paired-antibody ELISA. Error bars indicate SEM; paired t test, c–f, h, i 4–8 mice were pooled per experiment

Fig 3: DCs from PPs Instruct Mincle- and Syk-Dependent Th17 Differentiation(A–C) Naive OT-II T cells were co-cultured for 3 days with dome CD11b+ DCs, CD8a+ DCs, LysoMacs, or LysoDCs from the indicated genotypes, sorted from PPs (1:1 ratio), and loaded with OVA323–339 peptide. (A) IL-17 secretion by ELISA. Each dot represents an independent co-culture where myeloid cells were from different mice from two independent experiments. (B and C) Representative FACS plots of IL-17 (B) and IL-22 (C) intracellular staining after OTII re-stimulation.(D) Il6, Il23a, Tgfb, and Il12b transcripts in PPs of the indicated genotypes by qPCR; they were normalized to Gapdh.(E and F) Analysis of IL-6 (E) and IL-12p40 (F) intracellular staining in CD11c+MHC-II+CD19- cells from PPs in the indicated genotypes. Shown on the left are representative histograms. On the right is MFI of staining.(G) ELISA of IL-6 and IL-23 production by sorted GM-DCs from WT and Mincle-deficient (Clec4e-/-) mice untreated (medium) or stimulated with gut microbiota (10:1 DC ratio) for 12 h.Data represent two independent pooled experiments (A) or one representative experiment of at least two performed (B–G). *p < 0.05, **p < 0.01, ***p < 0.001 (A: one-way ANOVA and Bonferroni post hoc test; D, E, and G: unpaired two-tailed Student’s t test). See also Figure S3.

Fig 4: Commensal Bacteria Are Required for Mincle-Dependent Th17 Generation in PPs(A and B) Frequency of IL-17 production by intracellular staining in re-stimulated CD4+ T cells from PPs of the indicated genotypes after treatment with an antibiotic cocktail (ABX) containing ampicillin, neomycin, metronidazole, and vancomycin (A) or vancomycin only (B) in the drinking water over 4 weeks.(C) Frequency of IL-17 and IL-22 production by intracellular staining of re-stimulated OT-II T cells co-cultured with sorted WT and Mincle-deficient (Clec4e-/-) GM-DCs loaded with OVA323–339 peptide, as in Figures 1D and 1E; the cells were pulsed or not (medium) with mucosal-associated commensals from WT mice treated with ABX during gestation and lactation and gavaged with L. plantarum at weaning as indicated (L. plantarum-enriched mucus; 10:1 DC ratio).(D) ELISA of IL-6 and IL-23 from the co-culture of GM-DC in (C).(E and F) WT littermates and Mincle-deficient (Clec4e-/-) mice were treated with ABX during gestation and lactation and were gavaged with L. plantarum (1 × 106) at weaning (+L. plantarum gavage) or not (WT), as indicated in the figure. The frequency of IL-17- and IL-22-producing re-stimulated CD4+ T cells (E) or CCR6+ ILCs (F) from PPs by intracellular staining in the indicated genotypes and conditions is shown.Data represent one representative experiment of two performed (A and F) or were pooled from at least two independent experiments (B–E). Each symbol represents an individual mouse. The arithmetic mean for each group is indicated. *p < 0.05, **p < 0.01, ***p < 0.001 (A–E: one-way ANOVA and Bonferroni post hoc test; F: unpaired two-tailed Student’s t test). See also Figure S5.

Fig 5: ?dT cells in the colon of AKR1B8 –/– mice. (A) ?dT cell number. Upper panel: Flow cytometry. Lamina propria leukocytes (LPLs) were isolated from colons and subjected to flow cytometry analysis as described in Materials and Methods. Left, flow cytometry images; middle, percentage of ?dT cells in CD3+ T cells; right, number of ?dT cells in 104 CD3+ T cells. Lower panel: Immunofluorescent staining of ?dT cells. Left, FITC-labeled ?dTCR and DAPI staining images, scale bar: 20 µm; right, ?dTCR positive cell number per mm2. (B–D) IFN?, IL17, and IL22-producing ?dT cells. LPLs were isolated from colon and stimulated with PMA and ionomycin in presence of protein transport inhibitor for 5 h, followed by staining for CD3, ?dTCR, and then IFN-?, IL17, or IL22. Left, flow cytometry images; middle, percentage of IFN?, IL17, and IL22-producing ?dT cells in total ?dT cells; right, number of IFN?, IL17, and IL22-producing ?dT in 104 ?dT cells. WT, wild-type; KO, AKR1B8 –/–. Data indicate mean ± of SEM of four mice per group. *P < 0.05; **P < 0.01.