Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2, but no other TLR ligands increase TGF-β and IL-4 driven IL-9 cytokine secretion. Naive CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs in the presence of different TLR ligands (1μg/ml) under non-polarizing and TH9 polarizing conditions for 48 h. IL-9 (A) and IFN-γ (B) ELISA were measured by culture supernatants. Means ± SD of three independent experiments are shown. * p < 0.05, ** p < 0.01, NS denotes non-significant.

Article Snippet: IFN-γ was measured by sandwich ELISA (R&D Systems; MIF00) and IL-9 ELISA was done using the DuoSet ELISA kit (R&D Systems; DY409) by following manufactured protocol.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2 engagement on Antigen85B specific CD4+ T cells increases TH9 differentiation driven by TGF-β and IL-4. Naive Ag85B transgenic CD4+ T cells were co-incubated with TLR2 KO BMDM pulsed with Ag85B peptide (1μg/ml) and co-stimulated with or without TLR2 ligand (P3CSK4) under non-polarizing and TH9 polarizing conditions for 48 h. (A) CD4+ T cells were permeabilized and labeled with mAbs to IL-9 and IFN-γ and percentages of IL-9+ or IFN-γ+ cells were determined by flow cytometry. (B, C) IL-9 and IFN-γ were measured in culture supernatants by ELISA. Means ± SD of five independent experiments are shown. * p < 0.05, ** p < 0.01. NS denote non- significant.

Article Snippet: IFN-γ was measured by sandwich ELISA (R&D Systems; MIF00) and IL-9 ELISA was done using the DuoSet ELISA kit (R&D Systems; DY409) by following manufactured protocol.

Techniques: Transgenic Assay, Incubation, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2 engagement on human CD4+ T cells enhances IL9 mRNA and protein expression driven by polyclonal activation and TGF-β and IL-4. Naïve CD4+ T cells from three human donors were stimulated with anti-CD3 (10ug/ml) and anti-CD28 mAbs (1ug/ml) and exogenous TGF-β (5ng/ml) and IL-4 (10ng/ml), with or without P3CSK4 (2μg/ml) for 48h. (A) Relative expression of Il9 mRNA under non-polarizing and polarizing conditions with or without P3CSK4 (n=3) is shown. (B) IL-9 cytokine in culture supernatants were determined by ELISA. Means ± SD of three technical replicates for each donor are shown.

Article Snippet: IFN-γ was measured by sandwich ELISA (R&D Systems; MIF00) and IL-9 ELISA was done using the DuoSet ELISA kit (R&D Systems; DY409) by following manufactured protocol.

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Critical role of IL-33 receptor ST2 in experimental cerebral malaria development.

doi: 10.1002/eji.201445206

Figure Lengend Snippet: Figure 6. ST2 deficiency does not affect lung or spleen inflammation after PbA sporozoite infection. WT- or ST2-deficient mice were infected with 1000 PbA sporozoites. (A) Lung inflammation with alveolar septae thickening with mononuclear cell infiltration, hemorrhage in alveoli, and interstitial edema on day 9 post infection in WT or ST2−/−mice; H&E staining. Magnification 20×; scale bar = 100 μm. Images are representative of two independent experiments. (B) Semiquantitative score of lung pathology, expressed in arbitrary unit (AUs). The bar graphs show the mean ± SEM of n = 9 mice per group and are pooled from two independent experiments. (C–F) PbA-induced spleen inflammation in WT or ST2−/−mice. The absolute numbers of CD8+ (C) and CD4+ (D) T cells, respectively, CD8+CD69+ and CD4+CD69+ T cells, CD8+CXCR3+ and CD4+CXCR3+ T cells per spleen are shown. IFN-γ (E) and IL-10 (F) levels were measured by ELISA in spleen homogenates on days 0 and 9 post infection. (C–F) Data are expressed as mean ± SEM of n = 5–6 mice per condition and are representative of two independent experiments; *p < 0.05; statistical analysis with Kruskal–Wallis test followed by Dunn’s comparison test.

Article Snippet: After centrifugation IL-33, IL-10, and IFN-γ levels were determined in supernatants using ELISA assay kits (Mouse DuoSet, R&D Systems, Minneapolis, USA).

Techniques: Infection, Staining, Enzyme-linked Immunosorbent Assay, Comparison

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