(a) ELISA of cytokines production in naïve T cells isolated from wild-type or IL-17R KO mice and activated with anti-CD3 and anti-CD28 in the presence of an anti-IFN-γ antibody with or without IL-25 for 4 days, followed by restimulation with anti-CD3 for 24 hrs. (b) ELISA of cytokines production in naïve T cells activated as above in the presence of IL-4 and TGF-β with or without IL-25 or with IL-17. (c) ELISA of cytokines production in naïve T cells activated as above with IL-4 and TGF-β with or without IL-25 in the presence or absence of soluble IL-17RB. Data shown are a representative of at least two independent experiments.

(a) Real-time PCR of cytokines mRNA expression in wild-type and IL-17R KO T cells retrovirally transduced with IL-17RB or control viral vector (RV-GFP) and cultured with IL-25 for 4 days. Sorted GFP+ cells were activated with anti-CD3 for 4 hrs prior to mRNA isolation. (b) Immunoblot analysis of IL-17RA or HA-tagged IL-17RB of lysates from 293T cells transfected with IL-17RA or HA-tagged IL-17RB or both and immunoprecipitated with anti-HA antibody. (c) Real-time PCR of cytokine mRNA expression in wild-type T cells retrovirally transduced with full length IL-17RB (RB) or IL-17RB lacking the cytoplasmic domain (RB-cyt) or control viral vector (E) and activated for 4 days. Data shown are a representative of at least two independent experiments.

(a) ELISA of cytokines production in naïve T cells isolated from CD4-IL-17RB transgenic stimulated with anti-CD3 and anti-CD28 in the presence or absence of IL-25 with the indicated neutralizing antibodies for 4 days, followed by reactivation with anti-CD3 for 24 hrs. (b) Real-time RT-PCR analysis of cytokine expression in wild-type or IL-4-deficient T cells retrovirally transduced with IL-17RB or control vector and treated with IL-25 for 4 days. Sorted GFP+ cells were activated with anti-CD3 for 4 hrs prior to mRNA isolation. Data shown are a representative of at least two independent experiments.

(a) Schematic diagram of CD4-IL-17RB transgenic construct and mRNA expression of IL-17RB in naïve T cells isolated from wild type or different lines of transgenic mice as assessed by real-time RT-PCR. (b) Frequency of IL-5 and IFN-γ-producing cells in naïve T cells isolated from wild-type or CD4-IL-17RB (TG) mice and activated with anti-CD3 and anti-CD28 in the presence or absence of IL-25 for 4 days, followed by 5 hrs of PMA and ionomycin stimulation in the presence of Golgi Stop. ELISA (c) or by real-time PCR (d) of cytokine production in naïve T cells from wild type or CD4-IL-17RB TG mice activated as above for 4 days, followed by restimulation with anti-CD3. (e) Intracellular IL-9 and IL-4 cytokine staining and GFP expression in wild-type naïve T cells retrovirally transduced with IL-17RB or control viral vector (RV-KM) followed by treatment with IL-25 for 4 days. Data shown are a representative of at least three independent experiments.

(a) Total cell counts in BAL from CD4-IL-17RB transgenic mice (TG) or wild-type littermate controls intranasally challenged with A. oryzae and OVA allergens every other day for 4 allergen challenges in total. Four to five mice were analyzed in each group. (b) ELISA of OVA-specific cytokine production in splenocytes from wild-type and CD4-IL-17RB transgenic mice allergen challenged as in a. Cells were re-stimulated for 72 hours with different concentrations of OVA ex vivo. (c) Total cell counts in BAL from CD4-IL-17RB transgenic mice intranasally challenged with A. oryzae and OVA allergens every other day for 4 allergen challenges in total, with anti-IL-9 blocking antibody or a Rat IgG control antibody administered at the time of challenge. Four to five mice were analyzed in each group. (d) ELISA of OVA-specific cytokine production in splenocytes from CD4-IL-17RB transgenic mice allergen challenged as in c. Data are a representative of at least two independent experiments.

(a). Total cell counts in BAL fluid of IL-25KO mice or wild-type littermate controls sensitized with OVA in alum at day 0 and 14, followed by intranasal challenge with OVA at day 14, 25, 26, and 27. ELISA of OVA-specific cytokine production in splenocytes (b) and draining lymph node cells (c) or BAL fluid (d) from wild-type or IL-25KO mice allergen challenged as in a. Cells were re-stimulated for 72 hours with different concentrations of OVA ex vivo,* p < 0.05. (e) Real-time PCR analysis of cytokine expression in lungs harvested from wil-type or IL-25 KO mice. * p< 0.05. Data are a representative of at least three independent experiments.

(a) Real-time RT-PCR of IL-17RB mRNA expression in T cells activated with the indicated cytokines or antibodies (b) Real-time RT-PCR of IL-17RB mRNA expression in T cells activated in the presence or absence of IL-4 and/or TGF-β for 2 days. (c) Real-time RT-PCR of IL-17RB mRNA expression at the indicated time points in naive T cells differentiated under T_H1, T_H2, T_H17, and T_H9 conditions. Fold induction was calculated using Th1 cells as control and normalizing with actin expression. (d) Real-time PCR of IL-17RB mRNA expression in naive T cells differentiated under T_H2 and T_H9 condition for 5 days and restimulated with anti-CD3 for 4 hours. Naïve T cells were used as control. (e) Enzyme-linked immunosorbent assay (ELISA) of IL-9 production in activated naïve T cells cultured with or without IL-25 in the presence or absence of TGF-β or in the presence of IL-4 and TGF-β for 4 days, followed by restimulation with anti-CD3. ELISA (f) intracellular cytokine staining (g) or real-time PCR by setting no treatment as control (h) of cytokine production in naïve T cells stimulated with anti-CD3 and anti-CD28 in the presence of IL-4 and TGF-β with or without IL-25 for 4 days. . Data shown are a representative of at least two independent experiments.