anti gitr agonist dta 1  (Bio X Cell)

Journal: Nature Communications

Article Title: GITR subverts Foxp3 + Tregs to boost Th9 immunity through regulation of histone acetylation

doi: 10.1038/ncomms9266

Figure Lengend Snippet: Naive CD4 + T cells were FACS sorted from WT B6 ( a , b ) and OT-II ( c , d ) Foxp3gfp reporter mice and activated under iTreg-polarizing conditions. Anti-GITR agonistic antibody (DTA-1, 10 μg ml −1 ) or His-GITR Ligand (200 ng ml −1 , along with anti-His mAb) were used to ligate the GITR receptor on activated CD4 + T cells. Three days later, cells were harvested and analysed for Foxp3 and cytokines expression by flow cytometry. ( a , c ) Colour FACS plots depicting Foxp3- and IL-9-producing cells. Numbers in the quadrants indicate the percentage of cells. Data are representative of five independent experiments. ( b , d ) DTA-1 titrations and their impact on induction of Foxp3 + Tregs and Th9 cells, and graphs depict the percentage of cells that express Foxp3 (left) or IL-9 (right). Data are pooled from five independent experiments with triplicate cultures. ( e ) Expression of other cytokines, as indicated in the x axis, by WT naive CD4 + T cells polarized under iTreg conditions for 3 days with or without GITR ligation. Data are representative of three independent experiments.

Article Snippet: To deliver GITR costimulation to activated T cells, an agonist anti-GITR antibody DTA-1 (10 μg ml −1 ) or a His-tagged recombinant mouse GITR Ligand (200 ng ml −1 ) (2177-GL-025, R&D Systems) were added into the cultures, and in the case of His-tagged GITRL, anti-His mAb (10 μg/ml, MAB050, R&D Systems) were used to cross-link surface bound GITRL.

Techniques: Expressing, Flow Cytometry, Ligation

Journal: Nature Communications

Article Title: GITR subverts Foxp3 + Tregs to boost Th9 immunity through regulation of histone acetylation

doi: 10.1038/ncomms9266

Figure Lengend Snippet: ( a ) Immunoblot analysis of total and phosphorylated (p-) Smad2, Smad3 and Stat5 in the cytosol and nucleus in WT B6 naive CD4 + T cells left untreated (d0) or activated for 1 to 3 days under iTreg-polarizing conditions with DTA-1 or control IgG. β-actin and Histone 3 (H3) serve as loading controls, respectively. Plots shown are representative data of 3 experiments. ( b ) ChIP assays for Smad3 in Foxp3 CNS1 and Il9 promoter regions in WT naive CD4 + T cells activated as in a . IgG serves as immunoprecipitation control. Values are presented as relative binding based on normalization to input DNA. Data represent mean values±s.d. ( n =3). ( c , d ) ChIP assays for H3Ac, H3K27Ac, H3K9Ac, H4Ac, H3K9Me2, H3K9Me3, H3K27Me2 and H3K27Me3 modifications in Foxp3 ( c ) and Il9 ( d ) loci in WT naive CD4 + T cells activated as in ( b ) for 2 days. Data represent mean values±s.d. ( n =3). ( e , f ) Flow cytometry analysis of Foxp3 expression in WT naive CD4 + T cells activated as in ( b ) for 3 days in the presence or absence of HDAC inhibitor sodium butyrate (NaB, 250 μg ml −1 ). Numbers in the quadrants indicate the percentage of Foxp3 + cells ( e ). ( f ) The graph represents Mean±s.d. of Foxp3 + T cells from five experiments with triplicate cultures. P values were determined by Student's t -test (* P <0.05).

Article Snippet: To deliver GITR costimulation to activated T cells, an agonist anti-GITR antibody DTA-1 (10 μg ml −1 ) or a His-tagged recombinant mouse GITR Ligand (200 ng ml −1 ) (2177-GL-025, R&D Systems) were added into the cultures, and in the case of His-tagged GITRL, anti-His mAb (10 μg/ml, MAB050, R&D Systems) were used to cross-link surface bound GITRL.

Techniques: Western Blot, Immunoprecipitation, Binding Assay, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: GITR subverts Foxp3 + Tregs to boost Th9 immunity through regulation of histone acetylation

doi: 10.1038/ncomms9266

Figure Lengend Snippet: ( a ) Immunoblot analysis of the induction of the canonical (p50, RelA) and non-canonical (p52, RelB) NF-κB pathways in the cytosol and nucleus in WT B6 naive T cells activated for 1 to 3 days under iTreg-polarizing conditions with DTA-1 or Ctrl IgG. Data are representative of three independent experiments. ( b , c ) Naive CD4 + T cells from WT B6, p50 −/− and p52 −/− mice were activated under iTreg conditions, with or without GITR ligation for 3 days, and induction of Foxp3 + T cells and IL-9+ T cells was analysed by FACS and shown. Numbers in the quadrants indicate the percentage of cells. ( c ) Graphs depict Mean±s.d. of five experiments with triplicate cultures. ( d , e ) ChIP analysis of H3Ac and H4Ac modifications at Foxp3 ( d ) and Il9 ( e ) promoter and CNS regions from naive WT and p50 −/− CD4 + T cells, either left untreated (Unstim) or activated under iTreg-polarizing conditions for 2 days in the presence of DTA-1 or Ctrl IgG. Data represent mean values±s.d. ( n =3). P values were determined by Student's t -test (* P <0.05).

Article Snippet: To deliver GITR costimulation to activated T cells, an agonist anti-GITR antibody DTA-1 (10 μg ml −1 ) or a His-tagged recombinant mouse GITR Ligand (200 ng ml −1 ) (2177-GL-025, R&D Systems) were added into the cultures, and in the case of His-tagged GITRL, anti-His mAb (10 μg/ml, MAB050, R&D Systems) were used to cross-link surface bound GITRL.

Techniques: Western Blot, Ligation

Journal: Nature Communications

Article Title: GITR subverts Foxp3 + Tregs to boost Th9 immunity through regulation of histone acetylation

doi: 10.1038/ncomms9266

Figure Lengend Snippet: ( a ) Schematic diagram depicting Foxp3 locus structure and consensus κB binding sites (boxed) in the Foxp3 promoter and CNS1–2 regions. ( b ) ChIP analysis of p50 enrichment at the Foxp3 promoter and CNS1–2 sites in naive CD4 + cells left untreated (Unstim) or activated under iTreg-polarizing conditions in the presence of DTA-1. Data represent mean values±s.d. ( n =4). ( c ) Co-immunoprecipitation analysis of p50 in naive CD4 + T cells activated as in ( b ). Anti-p50 or control IgG immunoprecipitates (IP) were subjected to immunoblot analysis (IB) using anti-HDAC1 and anti-Sirt1 antibodies. Data are representative of three independent experiments. ( d ) ChIP analysis of HDAC1 binding to the Foxp3 promoter and CNS1–2 sites in naive CD4 + cells stimulated under iTreg-polarizing conditions in the presence of DTA-1 or IgG. Data represent mean values±s.d. ( n =3). ( e , f ) Flow cytometry analysis of Foxp3 expression in naive CD4 + T cells activated as in ( d ) for 3 days in the presence of HDAC1 inhibitor CI-994 (CI, 1 μM) or Sirt1 inhibitor EX-527 ( EX, 0.5 μM). Numbers in the quadrants indicate the percentage of Foxp3 + cells ( e ). ( f ) Plots shows mean±s.d. of Foxp3 + T cells from 3 experiments with duplicate culture. P values were determined by Student's t -test (* P <0.05).

Article Snippet: To deliver GITR costimulation to activated T cells, an agonist anti-GITR antibody DTA-1 (10 μg ml −1 ) or a His-tagged recombinant mouse GITR Ligand (200 ng ml −1 ) (2177-GL-025, R&D Systems) were added into the cultures, and in the case of His-tagged GITRL, anti-His mAb (10 μg/ml, MAB050, R&D Systems) were used to cross-link surface bound GITRL.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: GITR subverts Foxp3 + Tregs to boost Th9 immunity through regulation of histone acetylation

doi: 10.1038/ncomms9266

Figure Lengend Snippet: ( a ) Graph depicting Il9 locus and putative Foxp3 binding sites (red) at the Il9 promoter and CNS regions. ( b ) ChIP analysis of Foxp3 binding to the Il9 gene locus in CD4 + T cells transduced with retrovirus expressing GFP alone (Ctrl) or GFP-Flag-Foxp3 (Foxp3), then cultured under iTreg-polarizing conditions in the presence of DTA-1. GFP + cells were sorted and fixed for ChIP using anti-Flag antibody. Data represent mean values±s.d. ( n =3). ( c , d ) Flow cytometry plots showing IL-9-producing cells in CD4 + T cells transduced with retrovirus expressing GFP alone or Foxp3-GFP, and cultured under iTreg-polarizing conditions for 3 days in the presence of DTA-1. Numbers indicate the percentage of cells in the quadrants ( c ). Graph depicts the percentage of IL-9+ cells in GFP + population ( d ). Data represent mean values±s.d. ( n =6). ( e ) Co-immunoprecipitation of flag-foxp3 in CD4 + T cells transduced with retrovirus expressing flag-foxp3, and cultured under iTreg-polarizing conditions for 2 days. Anti-flag and control IgG immunoprecipitates (IP) were subjected to immunoblot analysis (IB) with anti-HDAC1, anti-HDAC2, anti-Sirt1 and anti-Sirt7 antibodies, and representative plots of 3 experiments are shown. ( f ) ChIP analysis of H3Ac and H4Ac modifications at Il9 promoter and CNS regions in GFP + cells sorted from naive CD4 + T cells transduced and activated as in ( b ) for 2 days. Data represent mean values±s.d. ( n =3). ( g , h ) Flow cytometry analysis of IL-9-producing cells in naive CD4 + T cells transduced and activated as in ( c ) for 3 days in the presence or absence of HDAC inhibitor Trichostatin A (TSA, 2 nM) and Sirt1 inhibitor EX-527 (EX, 0.5 μM). Numbers in the quadrants indicate the percentage of positive cells ( g ). Graph depicts the percentage of IL-9+ cells in GFP + population. Data represent mean values±s.d. ( n =9) ( h ). ( i ) ChIP analysis of H3Ac and H4Ac modifications at Il9 promoter and CNS regions in Foxp3 − GFP + cells sorted from naive CD4 + T cells transduced with retrovirus expressing Foxp3-GFP and cultured as in ( g ) for 2 days. Data represent mean values±s.d. ( n =3). P values were determined by Student's t -test (* P <0.05).

Article Snippet: To deliver GITR costimulation to activated T cells, an agonist anti-GITR antibody DTA-1 (10 μg ml −1 ) or a His-tagged recombinant mouse GITR Ligand (200 ng ml −1 ) (2177-GL-025, R&D Systems) were added into the cultures, and in the case of His-tagged GITRL, anti-His mAb (10 μg/ml, MAB050, R&D Systems) were used to cross-link surface bound GITRL.

Techniques: Binding Assay, Transduction, Expressing, Cell Culture, Flow Cytometry, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: GITR subverts Foxp3 + Tregs to boost Th9 immunity through regulation of histone acetylation

doi: 10.1038/ncomms9266

Figure Lengend Snippet: ( a ) Immunoblot analysis of PU.1, IRF4, BATF, STAT6, and p-STAT6 in WT naive CD4 + T cells activated for 1 to 3 days under iTreg-polarizing conditions with DTA-1 or control IgG. Data shown are representative of two independent experiments. ( b ) Induction of Foxp3 + T cells and IL-9+ T cells from WT B6, Stat6 −/− , Spi1 −/− , Irf4 f/f Cd4-Cre, and Batf −/− CD4 + T cells activated under iTreg conditions for 3 days with or without GITR ligation. Numbers in the quadrants indicate the percentage of cells. Data are representative of five independent experiments. ( c ) Immunoblot analysis of STAT6 and p-STAT6 in WT B6 and p50 −/− naive CD4 + T cells activated for 1 to 2 days under iTreg-polarizing conditions with DTA-1 or Ctrl IgG. Data are representative of two independent experiments.

Article Snippet: To deliver GITR costimulation to activated T cells, an agonist anti-GITR antibody DTA-1 (10 μg ml −1 ) or a His-tagged recombinant mouse GITR Ligand (200 ng ml −1 ) (2177-GL-025, R&D Systems) were added into the cultures, and in the case of His-tagged GITRL, anti-His mAb (10 μg/ml, MAB050, R&D Systems) were used to cross-link surface bound GITRL.

Techniques: Western Blot, Ligation

Journal: Nature Communications

Article Title: GITR subverts Foxp3 + Tregs to boost Th9 immunity through regulation of histone acetylation

doi: 10.1038/ncomms9266

Figure Lengend Snippet: ( a ) Graph showing the Il9 locus and putative STAT6 binding sites at the Il9 promoter and CNS regions. ( b ) ChIP analysis of the binding of STAT6 to Il9 locus in WT B6 and Stat6 −/− naive CD4 + T cells activated under iTreg-polarizing conditions for 2 days in the presence of DTA-1 or control IgG. Data are representative of three independent experiments. ( c ) ChIP analysis of H3Ac and H4Ac modifications in Il9 promoter (top) and CNS regions (bottom) in WT B6 and Stat6 −/− naive CD4 + T cells activated as in ( b ) for 2 days. Data are representative of three independent experiments. ( d ) Co-immunoprecipitation analysis of STAT6 in WT B6 and Stat6 −/− naive CD4 + T cells activated as in ( b ). Anti-STAT6 immunoprecipitates (IP) were subjected to immunoblot analysis (IB) with anti-P300 and anti-STAT6 antibodies. Data are representative of three independent experiments. ( e ) ChIP analysis of p300 binding to the Il9 promoter and CNS in WT B6 and Stat6 −/− naive CD4 + T cells activated as in ( b ) for 2 days. Data are representative of three independent experiments. ( f , g ) Flow cytometry analysis of IL-9 and Foxp3 expression by WT B6 naive CD4 + T cells activated as in for 3 days in the presence or absence of p300/CBP inhibitor 1 (p300i-1, 4 μM) or p300 HAT activator (CTPB, 50 μM). Numbers in the quadrants indicate the percentage of cells ( f ). The graph represents Mean±s.d. of IL-9 + T cells from three independent experiments with triplicate cultures ( g ). P -values were determined by Student's t -test (* P <0.05).

Article Snippet: To deliver GITR costimulation to activated T cells, an agonist anti-GITR antibody DTA-1 (10 μg ml −1 ) or a His-tagged recombinant mouse GITR Ligand (200 ng ml −1 ) (2177-GL-025, R&D Systems) were added into the cultures, and in the case of His-tagged GITRL, anti-His mAb (10 μg/ml, MAB050, R&D Systems) were used to cross-link surface bound GITRL.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: GITR subverts Foxp3 + Tregs to boost Th9 immunity through regulation of histone acetylation

doi: 10.1038/ncomms9266

Figure Lengend Snippet: ( a ) Rag1 −/− mice were injected with B16-OVA melanoma cells together with sorted CD4 + T cells from OT-II mice. The host mice were treated with 3 doses of DTA-1 (0.5 mg, i.p., n =14) or control IgG ( n =14) every five days starting from Day 5 after B16 inoculation. A cohort of anti-GITR treated Rag-1−/− mice ( n =7) was also given a neutralizing anti-IL-9 mAb (0.2 mg, i.p.). All animals were sacrificed on day 18 for analysis. ( b ) Numbers of tumor foci in the lungs from mice treated as in ( a ) (left panel). Representative images of the lungs of mice receiving different treatments (right panel). ( c ) Quantitative RT–PCR analysis of Foxp3, Il9, Il17a, IFNγ and Il4 mRNA from the lung infiltrating CD4 + cells. Data represent mean values±s.d. ( n =6). ( d ) Percentage of Foxp3 + (left) or IL-9 + (right) cells among the lung infiltrating CD4 + cells and in host spleen from control IgG and DTA treated mice as assessed by flow cytometry. Data represent mean values±s.d. ( n =6). P -values were determined by Student's t -test (* P <0.05).

Article Snippet: To deliver GITR costimulation to activated T cells, an agonist anti-GITR antibody DTA-1 (10 μg ml −1 ) or a His-tagged recombinant mouse GITR Ligand (200 ng ml −1 ) (2177-GL-025, R&D Systems) were added into the cultures, and in the case of His-tagged GITRL, anti-His mAb (10 μg/ml, MAB050, R&D Systems) were used to cross-link surface bound GITRL.

Techniques: Injection, Quantitative RT-PCR, Flow Cytometry