Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) GIFT9 amino acid sequence. The signal peptides of each part are underscored, IL9 signal peptide serves as the linker of the two parts. ( B ) Western blot of GIFT9 protein in the condition media from 293T cells retrovirally transduced to express GIFT9. Recombinant mouse GMCSF and IL9 were used as controls. ( C ) Western Blot of phospho-STAT1, STAT3, and STAT5 in JawsII cells after GIFT9 stimulation. Total STAT protein was used as a loading control. ( D ) Western Blot of phospho-STAT1, STAT3, and STAT5 in MC/9 cells after GIFT9 stimulation without or with GMCSF-Rα antibody blocking. Total STAT protein was used as a loading control. Normalized STAT1 phosphorylation level is shown, *: P<0.05, **: P<0.01.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Sequencing, Western Blot, Recombinant, Control, Blocking Assay, Phospho-proteomics

Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) JAK1, JAK2, and JAK3 phosphorylation levels remain same after GIFT9 stimulation. Total JAK protein was used as a loading control. Normalized JAK phosphorylation levels in GMCSF+IL9 and GIFT9 treatments are shown. ( B ) Inhibition of lipid rafts did not affect the hyperphosphorylation of STAT1 induced by GIFT9. Total STAT protein was used as a loading control. Normalized STAT1 phosphorylation level is shown.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Phospho-proteomics, Control, Inhibition

Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) GMCSFR βc was co-immunoprecipitated by α-common γc antibody after GIFT9 stimulation. *: P<0.05. ( B ) Common γc was co-immunoprecipitated by α-GMCSF-Rβ antibody after GIFT9 stimulation. *: P<0.05. ( C ) Representative confocal microscopy images of immunofluorescence staining showed the colocalization of GMCSF receptor and IL9 receptor after GIFT9 stimulation. Scale bar: 5 µm. ( D ) Percentages of color pixel colocalization after different cytokine treatment. **: P<0.01.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Immunoprecipitation, Confocal Microscopy, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) JAK2 inhibitor TG101348 treatment abolished hyperphosphorylation of STAT1 after GIFT9 stimulation. Total STAT protein was used as a loading control. ( B ) JAK3 inhibitor CP690550 treatment had a minor effect of STAT1 hyperphosphorylation induced by JAK2. Total STAT protein was used as a loading control. ( C ) Double inhibition of JAK1 and JAK2 by INCB018424 inhibited STAT5 phosphorylation after GMCSF/IL9 but not GIFT9 stimulation. Total STAT protein was used as a loading control. ( D ) Inhibition of all JAKs by INCB018424 and CP690550 depleted STATs phosphorylation. Total STAT protein was used as a loading control. Normalized STAT1 phosphorylation level is shown, *: P<0.05, **: P<0.01.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Control, Inhibition, Phospho-proteomics

Journal: PLoS ONE

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

doi: 10.1371/journal.pone.0069405

Figure Lengend Snippet: ( A ) Flow cytometry analysis of BMMCs. ( B ) MTT assay of BMMCs after 5 days of culture with GMCSF/IL9 or GIFT9. *: P<0.05. ( C ) Model of GMCSF and IL9 receptor clustering and downstream signaling after GIFT9 stimulation. See text for further details.

Article Snippet: Recombinant mouse GMCSF and IL9 were purchased from R&D systems.

Techniques: Flow Cytometry, MTT Assay

Journal:

Article Title: Neutralization of IL-9 ameliorates experimental autoimmune encephalomyelitis by decreasing the effector T cell population

doi: 10.4049/jimmunol.1000986

Figure Lengend Snippet: C57BL/6 mice immunized for EAE induction were treated with anti-IL-9 mAb, or control IgG every other day starting on day -1 p.i. (A). Filled circles indicate the IgG-treated group (n = 10), and open circles indicate the anti-IL-9 mAb-treated group (n = 10). Clinical scores (averages ± SEM) combining three independent experiments are shown. Incidence of EAE in these mice is shown in (B). Statistics were calculated with the Mann-Whitney U test (A) and the χ2 test (B). ***, p<0.001

Article Snippet: CD4 + T cells, purified using magnetic bead separation kits (Miltenyi Biotec, Auburn, CA), were cultured with anti-CD3 (1 μ g/ml) and anti-CD28 (1 μ g/ml) Abs (BD Pharmingen), TGF-β (3 ng/ml), IL-6 (30 ng/ml) and anti-IL-4 (10 μ g/ml) (all from Invitrogen) in the presence or absence of recombinant mouse IL-9 (10 ng/ml) (Akron Biotech), or anti-IL-9 mAb (100 ng/ml).

Techniques: MANN-WHITNEY

Journal:

Article Title: Neutralization of IL-9 ameliorates experimental autoimmune encephalomyelitis by decreasing the effector T cell population

doi: 10.4049/jimmunol.1000986

Figure Lengend Snippet: At disease peak, spinal cords were harvested after extensive perfusion, and 5 μm sections were stained with H&E and Luxol fast blue. Examples of mice receiving control IgG i.p. infiltration (A), demyelination (B and C), anti-IL-9 mAb i.p. infiltration (D), demyelination (E and F) are shown. The differences between control IgG -i.p. and anti-IL-9 mAb i. p groups were significant (all p <0.01). (G) Mean scores of inflammation and demyelination ± SD combining three independent experiments are shown (n =15 each group). **, p <0.01.

Article Snippet: CD4 + T cells, purified using magnetic bead separation kits (Miltenyi Biotec, Auburn, CA), were cultured with anti-CD3 (1 μ g/ml) and anti-CD28 (1 μ g/ml) Abs (BD Pharmingen), TGF-β (3 ng/ml), IL-6 (30 ng/ml) and anti-IL-4 (10 μ g/ml) (all from Invitrogen) in the presence or absence of recombinant mouse IL-9 (10 ng/ml) (Akron Biotech), or anti-IL-9 mAb (100 ng/ml).

Techniques: Staining

Journal:

Article Title: Neutralization of IL-9 ameliorates experimental autoimmune encephalomyelitis by decreasing the effector T cell population

doi: 10.4049/jimmunol.1000986

Figure Lengend Snippet: (A) Analysis of mononuclear cells infiltrating into the spinal cords. Mononuclear cells were recovered from spinal cord at day 19 p.i. Isolated cells were stained with antibodies against CD45, CD4, CD8, B220, and CD11b. CD45 high populations were gated. (B) RNA was made from spinal cord samples; the mean values of mRNA expression relative to β-actin are shown. Filled bars indicate the control IgG-treated group (n = 8), and open bars indicate the anti-IL-9 mAb-treated group (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: CD4 + T cells, purified using magnetic bead separation kits (Miltenyi Biotec, Auburn, CA), were cultured with anti-CD3 (1 μ g/ml) and anti-CD28 (1 μ g/ml) Abs (BD Pharmingen), TGF-β (3 ng/ml), IL-6 (30 ng/ml) and anti-IL-4 (10 μ g/ml) (all from Invitrogen) in the presence or absence of recombinant mouse IL-9 (10 ng/ml) (Akron Biotech), or anti-IL-9 mAb (100 ng/ml).

Techniques: Isolation, Staining, Expressing

Journal:

Article Title: Neutralization of IL-9 ameliorates experimental autoimmune encephalomyelitis by decreasing the effector T cell population

doi: 10.4049/jimmunol.1000986

Figure Lengend Snippet: Intracellular staining of lymphocytes stimulated with MOG35-55 peptide. Inguinal lymph node cells were recovered at day 8 p.i. All plots were gated on CD4+ T cells (A and B). (C) IL-9 blockade suppressed the antigen-specific cytokine production of IL-17 and IFN-γ from lymphocytes. Splenocytes were recovered at the peak of disease after immunization and restimulated with 50 μg/ml MOG35-55 peptide for 72 h. IL-17 and IFN-γ concentrations in the supernatants were determined. (D) IL-9 blockade suppressed the serum level of IL-17. Mice were treated with anti-IL-9 mAb or control IgG, and serum samples were prepared at the peak of EAE after antigen immunization. IL-17 cytokine concentrations in the serum were analyzed. (E) Spleen cells from MOG35-55-immunized mice obtained 8 days p.i. were re-stimulated ex vivo with MOG35-55 peptide (10 μg/ml) or anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml). Proliferation was determined by [3H] thymidine incorporation as described in the Materials and Methods section. n=5 in each group. All p values were determined by using the Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: CD4 + T cells, purified using magnetic bead separation kits (Miltenyi Biotec, Auburn, CA), were cultured with anti-CD3 (1 μ g/ml) and anti-CD28 (1 μ g/ml) Abs (BD Pharmingen), TGF-β (3 ng/ml), IL-6 (30 ng/ml) and anti-IL-4 (10 μ g/ml) (all from Invitrogen) in the presence or absence of recombinant mouse IL-9 (10 ng/ml) (Akron Biotech), or anti-IL-9 mAb (100 ng/ml).

Techniques: Staining, Ex Vivo

Journal:

Article Title: Neutralization of IL-9 ameliorates experimental autoimmune encephalomyelitis by decreasing the effector T cell population

doi: 10.4049/jimmunol.1000986

Figure Lengend Snippet: (A) IL-17 production by naïve CD4+ T cells cultures under optimal Th17 conditions and in the presence of either IL-9 (10 ng/ml), or anti-IL-9 mAb (100 ng/ml). (B) Donor mice were immunized with MOG, IFA, and M. tuberculosis. Immunized mice were treated with either rIL-9 (20 ng/mouse), anti-IL-9 mAb (300 μg/mouse), or PBS. On day 12 post immunization, mice were sacrificed, and total lymph node cells were further primed with MOG35-55 (50 μg/ml), IL-23 (10 ng/ml) for 3 days. A total of 2 × 107 viable MOG-specific Th17 cells were adoptively transferred to naive recipient mice. n=5 in each group. Mice were examined for clinical signs every day until 20 dpt. *, comparison between rIL-9 -treated mice and wildtype mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001. #, comparison between rIL-9 -treated mice and wildtype mice. #, p < 0.05.

Article Snippet: CD4 + T cells, purified using magnetic bead separation kits (Miltenyi Biotec, Auburn, CA), were cultured with anti-CD3 (1 μ g/ml) and anti-CD28 (1 μ g/ml) Abs (BD Pharmingen), TGF-β (3 ng/ml), IL-6 (30 ng/ml) and anti-IL-4 (10 μ g/ml) (all from Invitrogen) in the presence or absence of recombinant mouse IL-9 (10 ng/ml) (Akron Biotech), or anti-IL-9 mAb (100 ng/ml).

Techniques: