Fig 2: Ddit3 deletion or silencing increases the effect of T cell-based immunotherapy. a Working model of Pmel T cell adoptive transfer. CD90.2+ wild-type mice were injected subcutaneous with B16 tumors, and after 8 days, they received a single dose of cyclophosphamide (CTX). The next day, mice received 1 × 106 primed wild-type or Ddit3-/- or anti-sense oligonucleotide (ASO)-treated CD90.1+CD8+ Pmel T cells. b Tumor growth in B16-bearing mice (black) transferred with wild-type (blue) or Ddit3-/- (red) CD90.1+ CD8+ Pmel T cells. Average kinetics of tumor volume ± s.e.m. in 9 mice/group. c Percentage of IFN?+ cells in CD90.1+ CD8+ tumor-infiltrating T lymphocytes (TILs) from B16 tumors 5 days after wild-type or Ddit3-/- CD8+ Pmel T cell transfer. (n = 7 tumors/group). d ELISpot for interferon-? (IFN?) in the spleens of B16 tumor-bearing mice 5 days after wild-type or Ddit3-/- Pmel T cell transfer exposed to gp10025–33 for 24 h. (n = 6 spleens/group). e T-bet in CD90.1+CD8+ TILs from B16 tumors 5 days after wild-type or Ddit3-/- CD8+ Pmel T cell transfer. Right: representative result; left: merged results from n = 4/group. f Tumor growth in B16-bearing CD90.2+ mice (black) that received wild-type CD90.1+CD8+ Pmel T cells that were non-treated (gray) or pretreated with ASO-Control (blue) or ASO-Ddit3 (red). Average tumor volume kinetics ± s.e.m. of 8 mice/group. g Percentage of IFN?+ in CD90.1+ CD8+ TILs from B16 tumor-bearing mice transferred with control, ASO-Control, or ASO-Ddit3-pretreated CD90.1+ Pmel T cells. (n = 6 tumors/group). h IFN? ELISpot in spleens collected from B16 tumor-bearing mice treated with Pmel T cells or ASO-Control or ASO-Ddit3-pretreated Pmel T cells. Spleens were collected 5 days after T cell transfer and exposed 24 h to gp10025–33. (n = 4). Bar graphs show mean value ± s.e.m. and *p < 0.05, ***p < 0.001 were calculated using two-tailed unpaired Student’s t test

Fig 3: MyD88 contributes to the IFN-?-producing innate lymphoid cell compartment in the small intestine.LP cells were isolated from infected MyD88 WT and KO mice at day 7 post-infection and ILC activity was determined by flow cytometry. (A) Flow gating scheme to identify CD127+ and CD127- ILC. Numbers indicate the percent of cells falling within each gate. (B) CD127+ staining for T-bet (above) and ROR?t (below). Within each population, IFN-? staining is shown in the scattergrams on the right. (C) In the same fashion, markers of ILC1 and ILC3, and expression of IFN-? was assessed in the CD127- population. (D) Frequency of ILC populations and (E) frequency of frequency of IFN-?+ producing ILC1 and ILC3 were determined from gating shown in A-C. Values are the means ± SEM of two independent experiments where each symbol represents a single mouse and open and closed symbols dots delineate between experiments. Unpaired Student’s t test (D, E) was used to determine statistical significance where **p<0.01.

Fig 4: 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 5: Dectin-1 directs polarization of predominantly TH17 cells with high IL10, MAF and PTGDS and low IFNG, TBX21, IL1R1 and CXCL3 mRNA expression.a-c, Real-time PCR analyses of IFNG, TBX21, IL1R1, CXCL3, CCL3, CSF2, IL22, CCL5, IL17A, IL17F, RORC, IL10, MAF and PTGDS normalized mRNA levels in IL-17+ T cells, isolated by flow cytometry-based sorting from peripheral blood of healthy donors (a, n = 4) or active Crohn’s disease patients, either treated (a, n = 4) or acute and untreated (a, n = 4), or after restimulation of T cells, outgrown in vitro by co-culture of either naive (b) or memory (c) CD4+ T cells with immature DCs (iDC) or DCs primed for 48 h with curdlan or C. albicans (c), at day 5 (b, n = 4) or day 11–14 (c, n = 12). In b,c, IL-17+ cells were further separated during sorting based on intracellular IFN-? expression. Results from real-time PCR were quantified using standard curves for all genes and normalized to the expression of reference household gene ACTB. These data are an addendum to the data presented in Fig. 2i (a) and Fig. 2j (b,c). Data represent mean ± s.d. of independent donors. **P < 0.01, *P < 0.05 (Student’s t-test, paired, two-tailed), calculated between IFN-?-IL-17+ and IFN-?-IL-17+ T cells as indicated by brackets (b,c).