a CD25⁻CD44^loCD62^hiCD4⁺ T cells (Naïve CD4+ T) were FACS-sorted and polarized under Th1, Th2 or Th17 condition for 4 days. b, Naïve CD4+ T cells were stimulated with anti-CD3/CD28 Ab in the presence of indicated cytokine for 24 hours before mRNA expression of IL-1R1 was analyzed. c, FACS-sorted naïve CD4 T cells from WT mice or STAT3 KO, RORα KO, or RORα/γ double KO mice were cultured under Th17 condition for 4 days. d, Naive OT-II CD4⁺ T cells were activated with Ova peptide-pulsed splenic APCs under the neutral (anti-IL-4 and anti-IFN-γ) and coinfected with two bicistronic retroviruses expressing RORα-GFP or GFP vector and RORγt-hCD2 or hCD2. GFP⁺hCD2⁺ cells were FACS sorted. In a, c, and d, cells were restimulated with plate-bound anti-CD3 Ab for four hours before mRNA expression was analyzed by real-time PCR. Data were normalized with expression amounts of Actb. e and f, CD4 T cells were sorted from WT or IL-1R1 KO mice and i.v. transferred into Rag1^−/− mice. The recipient mice were induced EAE and disease incidence (e) and score (f) were measured daily and means ± SEM of all mice in each group were shown (f). *, p<0.05; **, p<0.01 in comparison with WT recipients.

Mixed bone marrow chimeric mice were generated and induced EAE. Mononuclear cells in central nervous system (CNS) and spleen were stained with anti-CD45.2 to distinguish WT and IL-1R1 KO compartments and analyzed for CD4+ and CD11b+ cell presence (a), cytokine production profiles in CD4 T cells after PMA/Ionomycin (b and c) or MOG stimulation (b, only for spleen), or Foxp3+ cell in CD4 T cells (d and e). The mice were divided into two groups based on the disease severity (score, 0.5-2 for early phase; 2.5-4.0 for peak phase). *, p<0.05; **, p<0.01 in comparison with WT compartments. Data shown represent two independent experiments with consistent results.

a, WT and IL-1R1 KO mice were subcutaneously immunized with KLH in CFA. Three or seven days later, lymphoid cells from draining lymph nodes were restimulated with KLH overnight and IL-17- or IFN-γ-expressing cells were measured by intracellular staining. b, FACS-sorted naïve OT-II T cells from WT or IL-1R1-deficient OT-II mice were intravenously transferred into congenic (CD45.1) mice. The recipient mice were immunized with OVA_323-339 peptide in CFA. Three days later, lymphoid cells from draining lymph nodes were restimulated with PMA and Ionomycin for 5 hours and IL-17- or IFN-γ-expressing cells were measured by intracellular staining. Data shown are on gated CD45.1⁺ CD4⁺ T cells. c and d, FACS-sorted naïve CD4 T cells from WT or IL-1R1−/− mice were cocultured with BM-derived DC (WT) in the presence of soluble anti-CD3 Ab (0.1μg/ml) and LPS (100 ng/ml) plus TGF-β (1 ng/ml). IL-17- or IFN-γ-expressing cells were measured by intracellular staining (c). At day 4, mRNA expression was assessed by real-time RT-PCR after restimulation of T cells by anti-CD3 for 4 hr (d). Data shown represent two independent experiments and normalized with expression amounts of Actb.

a, Mononuclear cells in EAE-induced mixed bone marrow chimeric mice as described in Figure 2 were analyzed for IL-17 and Foxp3 expression. Data shown are on gated CD4+ Foxp3+ cells. Mean values are shown as horizontal bars. **, p<0.01 in comparison with WT compartments. Data shown represent two independent experiments with consistent results. b and c, FACS-sorted CD4+GFP+ cells from naïve Foxp3-GFP reporter mice were cultured with DC and in the presence of soluble anti-CD3 Ab, LPS and TGF-β plus anti-IL-1R1 Ab or rat IgG as a control. IL-17- or GFP-expressing cells were measured by intracellular staining (b). At day 4, mRNA expression was assessed by real-time RT-PCR after restimulation of T cells by anti-CD3 for 4 hr (c). mRNA expression in fresh FACS-sorted CD4+GFP+ cells, stimulated with anti-CD3 Ab for 4 hr, was used as a control. Data shown represent two independent experiments and normalized with expression amounts of Actb.

FACS-sorted naïve CD4+ T cells from IL-17F-RFP reporter mice were stimulated under Th17 condition (anti-IL-4, anti-IFNγ, IL-6, TGF-β, IL-23). At day 4, RFP+ cells were sorted and the expression of IL-17, IL-17F, IFNγ, Foxp3 or T-bet was analyzed by intracellular staining (a). b-d, The sorted RFP+ cells were labeled with CFSE and cultured with medium alone, IL-23, IL-1 alone, IL-23 plus IL-1, or IL-6 plus TGF-β for additional 3 days. b, IL-17-, IL-17F-, IL-4-, IFNγ-expressing cells were analyzed by intracellular staining after PMA and Ionomycin stimulation for 4 hr. c, Cytokines in the supernatant of 3 day additional culture were measured by ELISA. d, Cells were further restimulated with plate bound anti-CD3 Ab overnight and cytokines in the supernatant were measured by ELISA. ‘Th17’ is the supernatant of the sorted RFP+ cells after anti-CD3 Ab overnight stimuation. Data shown represent four independent experiments.

FACS-sorted naïve CD4+ T cells from WT or IL-1R1 KO mice were stimulated with anti-CD3/CD28 Abs and anti-IL-4/anti-IFNγ Abs in the presence of indicated cytokines for 4 days. a, IL-17-producing cells were analyzed by intracellular staining. Values are the percentage of IL-17+ cells and mean fluorescence intensity (MFI) of the IL-17 staining of the gated cells. b, Foxp3- and T-bet-expressing cells were analyzed by intracellular staining. c, IL-17- and IFNγ-expressing cells were analyzed by intracellular staining. d, At day 4, cells stimulated under the condition described in c were harvested and restimulated with plated bound anti-CD3 Ab overnight and cytokines in the supernatant were measured by ELSIA. Data shown represent at least two independent experiments.

a, FACS-sorted naïve CD4+ T cells from WT mice were stimulated with anti-CD3/CD28 Abs and anti-IL-4/anti-IFNγAbs in the presence of indicated cytokines for 4 days. mRNA expression was assessed by real-time RT-PCR. b, FACS-sorted naïve CD4 T cells from WT or IL-1R1−/− mice were co-cultured with BM-derived DC (WT) in the presence of soluble anti-CD3 Ab and LPS plus TGF-β. At days 2 and 4, mRNA expression was assessed by real-time RT-PCR. Data shown represent two independent experiments and normalized with expression amounts of Actb. c and d, FACS-sorted naïve CD4+ T cells from WT or IL-1R1−/− mice were co-cultured with BM-derived DC (WT) in the presence of soluble anti-CD3 Ab and LPS plus TGF-β and infected with bicistronic retrovirus expressing GFP vector (RV-KM), RORγt-GFP or IRF4-GFP. Four days after infection, IL-17- or IFNγ-expressing cells were assessed by intracellular staining. Data shown are gated on GFP+ or GFP− cells and represent two independent experiments. In d, cells were coinfected with two bicistronic retroviruses expressing IRF4-GFP or GFP vector and RORγt-hCD2 or hCD2. GFP+ and hCD2+ cells were sorted and assessed by intracellular staining.