Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A, Naïve CD4+T cells from WT mice were activated with anti-CD3 and anti-CD28 and cultured with IL-4 and anti-IFN-γ (Th2 conditions) or with IL-4, TGF-β and anti-IFN-γ (Th9 conditions). Each day during differentiation, cells were stained for intracellular-phospho-STAT3 and phospho-STAT6. The bar graphs represent percentages of cells (top) and mean fluorescent intensity (MFI, bottom). Data are average ± S.E.M of 6 mice from 3 experiments.B, C. Naïve CD4+T cells from WT and Stat6−/− (top), WT and Stat3CD4−/− (middle), and WT and Parp14−/− mice (bottom) were cultured under Th9 conditions. After 5d cells were harvested and stimulated with PMA and Ionomycin before intracellular staining for cytokines IL-9 and IL-4 was performed (B) or stimulated with anti-CD3 and 24h later cell-free supernatant was collected and IL-9 production was assessed by ELISA (C). Data are representative of 3 experiments with similar results.*, p<0.05. ***, p<0.001.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Cell Culture, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A-C. WT and Il4−/− naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured under Th9 cell conditions for 48h before being transduced with control or GATA3-expressing retroviruses (A), control or c-Maf expressing retroviruses (B), or control or JunB-expressing retroviruses (C). After 5d in culture cells were stimulated with PMA and Ionomycin for intracellular staining of IL-9 and IL-4. Data are representative of 2-3 independent experiments with similar results.*, p<0.05. **, p<0.01.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Cell Culture, Transduction, Expressing, Staining

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A, Naïve CD4+T cells from WT mice were analyzed directly ex vivo (naïve) or cultured under Th1, Th2, Th17, Th9, and Treg cell conditions for 5d. After RNA isolation, quantitative PCR was performed for the indicated genes. Data are representative of 2 experiments with similar results. B, Total cell lysates were prepared from Th1, Th2, and Th9 cells and immunoblot was performed for Runx3, β-actin was used as loading control. C, WT naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured under Th9 cell conditions for 48h before being transduced with control or Runx3-expressing retroviruses and control or T-bet expressing retroviruses. Cells were harvested after 5d in culture and stimulated with PMA and ionomycin before intracellular IL-9 and IL-4 staining. Data are average ± S.D of 2 experiments with 4 mice. D, Naïve CD4+T cells from WT and Tbx21−/− (bottom left) or WT and Runx3fl/fl-CD4-Cre (bottom right) were cultured in Th9 cell conditions. After 5d cells were harvested and stimulated with PMA and Ionomycin before intracellular IL-9 and IL-4 staining. Data are representative of 2-3 experiments with similar results. E, WT and Stat6−/−naïve CD4+T cells were cultured under Th9 cell conditions for 5d. Cells were harvested and quantitative PCR was performed for Runx3and Tbx21.*, p<0.05. **, p<0.01.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Ex Vivo, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Transduction, Expressing, Staining

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: STAT6-dependent regulation of Th9 development

doi: 10.4049/jimmunol.1102840

Figure Lengend Snippet: A, WT naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and were cultured with increasing doses of TGF-β in the presence (Th9) or absence (Treg) of IL-4 for 5d. Cells were then stained for intracellular Foxp3 (top) and IL-9 (middle). Differentiated Th9 cells were stimulated with anti-CD3 and 24h later IL-9 production was assessed by ELISA (bottom). B, Naïve CD4+T cells were cultured under Th9 and Treg cell conditions as in A (left) or cultured with 2 ng/ml TGF-β and increasing doses of IL-4 (Th9, right) for 5d. After differentiation, RNA was isolated and Sfpi1 expression was measured by quantitative PCR. Data are representative of 2 experiments with similar results. C, WT and Stat6−/− naïve CD4+T cells were cultured under Th9 cell conditions for 5d and Foxp3 expression was measured using qPCR. Data are average ± S.D of 4 mice from 2 experiments. D, Naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured in Th9 cell conditions for 48h before being transduced with control or Foxp3-expressing retroviruses. After 5d in culture cells were stimulated with PMA and Ionomycin and intracellularly stained for IL-9 and IL-17A. Data are representative of 2 experiments with similar results.E, Schematic showing a summary of a transcription factor network in Th9 cells.*, p<0.05.

Article Snippet: Cell free supernatant was collected and the amount of IL-9 was assessed by ELISA using anti-IL-9 capture antibody (D8402E8; BD Biosciences) and biotin-labeled anti-IL-9 (D9302C12; Biolegend) as the secondary antibody.

Techniques: Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, Transduction