ox86 agonist antibody  (Bio X Cell)

Journal: The Journal of Experimental Medicine

Article Title: Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation

doi: 10.1084/jem.20170928

Figure Lengend Snippet: Identification of SEs in Th9 cells induced by OX40 stimulation. (A) The FACS plots depict Th9 cells induced by TGF-β and IL-4 with or without OX40 stimulation. Naive CD4 + T cells were activated by anti-CD3/APCs, and OX40 stimulation was provided by an agonist anti-OX40 mAb (OX86), OX40 ligand-His recombinant protein (OX40L-His), or OX40 ligand transgenic APC (OX40L). An isotype control IgG was used as control. Representative plots from one of five independent experiments. (B) Summary of Th9 cells induced as described in A from five independent experiments. (C) Distribution of SEs based on H3K27Ac tag signals and enhancer ranks in control Th9 (Ctrl) and OX40-induced Th9 cells (OX40L). The number denotes the SEs induced in Th9 cells with or without OX40 costimulation. The red dot shows the positions of Il9 TE and Il9 SE. (D) ChIP-seq profiles showing levels of genome-wide H3K27 acetylation in control Th9 cells (Ctrl) and OX40-induced Th9 cells (OX40L). The position of SE and the Il9 locus are highlighted. (E) Distribution of SE in control Th9 (Ctrl) and OX40-induced Th9 cells (OX40L) based Brd4 tag signals and enhancer ranks. The number denotes the SEs identified in Th9 cells, and the red dot shows the position of Il9 SE. (F) Brd4 ChIP-seq profiles showing enrichment of Brd4 in Th9 cells induced with or without OX40 costimulation. The position of SE and Il9 locus is shown. (G) The Venn diagrams show the number of unique and overlapping SEs induced by OX40 based on the H3K27Ac and Brd4 ChIP-seq signals. (H) The top five SEs induced by OX40 ranked by fold changes of both H3K27Ac and Brd4 ChIP-seq signals. Graphs in B depict mean ± SEM of five experiments, each with triplicate cultures. Statistical differences between groups were determined by the Student’s t test. ***, P < 0.001.

Article Snippet: As an alternative method, OX40 agonist antibody OX86 (5 μg/ml, BioXcell) or recombinant mouse OX40L-His protein (100 ng/ml, along with 10 μg/ml of anti-His mAb; both from R&D Systems) was also used to activate OX40 signaling.

Techniques: Recombinant, Transgenic Assay, Control, ChIP-sequencing, Genome Wide

Journal: The Journal of Experimental Medicine

Article Title: Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation

doi: 10.1084/jem.20170928

Figure Lengend Snippet: Role of the BET inhibitor JQ1 in OX40-mediated induction of Th9 cells. (A) A representative FACS plot showing induction of Th9 cells by OX40 in the presence or absence of JQ1. DMSO was included as a vehicle control. (B) The bar graphs are summaries of Th9 cells induced by OX40 with or without JQ1 from five individual experiments performed as in A. (C) A representative FACS plot showing induction of Th9 cells by TGF-β and IL-4 plus OX40 stimulation where graded doses of JQ1 were added. (D) A summary of Th9 cells induced by OX40 as in C from five individual experiments. (E) FACS analysis of Th9 cells induced by OX40 stimulation from activated CD4 + T cells transduced with Brd4 and Med1 specific shRNAs. A empty vector (sh-Ctrl) was used as a control. Plots shown were gated on GFP + cells. (F) The bar graphs show summary of Th9 cells induced as described in E from five independent experiments. Graphs in B, D, and F depict mean ± SEM of five experiments with triplicate cultures. Statistical differences between groups were determined by the Student’s t test. ***, P < 0.001.

Article Snippet: As an alternative method, OX40 agonist antibody OX86 (5 μg/ml, BioXcell) or recombinant mouse OX40L-His protein (100 ng/ml, along with 10 μg/ml of anti-His mAb; both from R&D Systems) was also used to activate OX40 signaling.

Techniques: Control, Transduction, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation

doi: 10.1084/jem.20170928

Figure Lengend Snippet: Role of JQ1 in expression of Th9 cell–associated transcription factors and their roles in Th9 induction. (A) Quantitative RT-PCR data showing expression of Th9 cell–associated transcription factors in control Th9 cells and OX40-induced Th9 cells with or without JQ1 at 2 d after T cell activation. Data were normalized to GAPDH expression. (B) Induction of Th9 cells from WT B6, Spi1 −/− , Batf −/− , and Irf4 −/− CD4 + T cells with or without OX40 stimulation. The FACS plots depict the percentage of IL-9 + cells among total CD4 + T cells 3 d after Th9 cell induction. (C) Luciferase reporter assay showing Il9 promoter activities induced by Th9 cell–associated transcription factors in HEK293T cells. The Il17a reporter, which is driven by Rorc, was used as a positive control. Graphs in A and C depict mean ± SEM of three independent experiments, each with triplicate cultures. Data in B are representative of five independent experiments. Statistical differences between groups were determined by one-way ANOVA or between two groups by the Student’s t test. ***, P < 0.001.

Article Snippet: As an alternative method, OX40 agonist antibody OX86 (5 μg/ml, BioXcell) or recombinant mouse OX40L-His protein (100 ng/ml, along with 10 μg/ml of anti-His mAb; both from R&D Systems) was also used to activate OX40 signaling.

Techniques: Expressing, Quantitative RT-PCR, Control, Activation Assay, Luciferase, Reporter Assay, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation

doi: 10.1084/jem.20170928

Figure Lengend Snippet: Functional mapping of Il9 SE clusters. (A) Schematic representation of Il9 SE clusters based on H3K27Ac ChIP-seq signals, namely, Il9 SEa, SEb, and SEc, is shown in shaded areas. The location of PCR primer sets designed to cover all three SE clusters is shown at the bottom. (B) Top: shows the locations of sgRNAs designed to target individual SE clusters where CRISPR/Cas9-mediated deletion of SEa, SEb, and SEc occurred. The primer sets used to validate the deletion is indicated. F, forward primer; R, reverse primer. Bottom: PCR-based products and DNA sequencing data showing specific deletion of target SE sequences in activated CD4 + T cells. (C) Induction of Th9 cells by OX40 upon CRISPR/Cas9-mediated deletion of individual SE clusters showing in representative FACS plots and summary of three independent experiments (mean and SD of n = 5). Cas9 transgenic CD4 + T cells were transduced with sgRNA vectors marked by GFP and RFP, and T cells positive for both GFP and RFP were selectively gated for analysis. (D) Expression of eRNA from individual SE clusters in control Th9 cells and OX40-stimulated Th9 cells was quantified by quantitative real-time PCR using position-specific primer sets as indicated in A. Data shown are mean ± SEM of three independent experiments with triplicate cultures. (E) Real-time qPCR data showing eRNA expression in control Th9 cells and OX40-induced Th9 cells with or without addition of JQ1 inhibitor. Data shown are mean ± SEM of three independent experiments. (F) 3C assays showing formation of chromatin loop between Il9 SEa and Il9 promoter regions. DNA from control Th9 cells (Ctrl) and OX40-indcued Th9 cells (OX40L) was digested by DpnII and ligated with T4 DNA ligase. Primer sets covering all potential ligation events were used to detect ligation products between Il9 promoter and Il9 SEa. R8 and C primer sets produced a PCR amplicon corresponding to a specific ligation event, which is confirmed by DNA sequencing. Data from one of three experiments are shown. (G) 3C assays measuring chromatin loop formations between Il9 promoter and Il9 SEa regions in control Th9 cells (Ctrl) and OX40-induced Th9 cells (OX40L) with or without the JQ1 inhibitor. The image shows representative data from one of three independent experiments. ***, P < 0.001.

Article Snippet: As an alternative method, OX40 agonist antibody OX86 (5 μg/ml, BioXcell) or recombinant mouse OX40L-His protein (100 ng/ml, along with 10 μg/ml of anti-His mAb; both from R&D Systems) was also used to activate OX40 signaling.

Techniques: Functional Assay, ChIP-sequencing, CRISPR, DNA Sequencing, Transgenic Assay, Transduction, Expressing, Control, Real-time Polymerase Chain Reaction, Ligation, Produced, Amplification

Journal: The Journal of Experimental Medicine

Article Title: Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation

doi: 10.1084/jem.20170928

Figure Lengend Snippet: Critical role of RelB in SE formation and Th9 cell induction. (A) Representative FACS plots on Th9 cell induction and quantification of Th9 cells from WT B6, Relb −/− , and Rela −/− CD4 + T cells under Th9 cell–inducing conditions with or without OX40 signal. (B) H3K27Ac ChIP assay showing levels of H3K27Ac at the SEa and Il9 coding regions from WT B6 and Relb −/− Th9 cells induced with or without OX40 stimulation. (C) Real-time qPCR assay showing Il9 SEa (a4) eRNA expression by WT B6 and Relb −/− Th9 cells induced with or without OX40 stimulation. (D) Data shown are 3C assays measuring DNA loops between Il9 promoter and Il9 SEa regions in WT B6 and Relb −/− Th9 cells induced with (OX40L) or without OX40 stimulation (Ctrl). Primer sets amplifying the DNA fragment without DpnII sites were used as controls. (E) H3K27Ac ChIP assay showing levels of H3K27 acetylation at the Il9 locus and the SEa region in CD4 + T cells transduced with control vector or with RelB expressing vector and cultured under Th9 cell conditions. (F) Expression of eRNA from Il9 SEa (a4) by CD4 + T cells transduced by control vector and RelB expressing vector and cultured under Th9 cell conditions. (G) 3C assays based on R8 and C primer sets and qPCR measuring DNA loop formations between the Il9 promoter and Il9 SEa regions in CD4 + T cells transduced with Ctrl- or RelB-expressing vector. (H) A representative FACS plot and quantification of Th9 cells induced under Th9 cell conditions from CD4 + T cells transduced with Ctrl- or RelB-expressing vector and treated with JQ1. Graphs are representative images from at least three independent experiments and shown as mean ± SEM of three experiments, each with triplicate cultures. ***, P < 0.001.

Article Snippet: As an alternative method, OX40 agonist antibody OX86 (5 μg/ml, BioXcell) or recombinant mouse OX40L-His protein (100 ng/ml, along with 10 μg/ml of anti-His mAb; both from R&D Systems) was also used to activate OX40 signaling.

Techniques: Expressing, Transduction, Control, Plasmid Preparation, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation

doi: 10.1084/jem.20170928

Figure Lengend Snippet: Formation of SEs requires the histone acetyltransferase p300. (A) ChIP assay showing RelB enrichment at the Il9 coding region and the SEa region in Th9 cells induced with (OX40L) and without OX40 stimulation (Ctrl). (B) Wt B6 CD4 + T cells transduced with retroviral vectors expressing full-length RelB or RelB mutants, with specific deletion of the LZD (ΔLZD), RHD (ΔRHD), or TAD (ΔTAD), and then cultured under Th9 cell conditions for 3 d. The FACS plots depict a representative experiment in Th9 cell induction. The bar graphs on the right side show quantification of Th9 cells from three independent experiments. (C) Coimmunoprecipitation analysis showing RelB and p300 interactions. FLAG-tagged RelB was introduced into CD4 + T cells and cultured under Th9 cell conditions. Left: RelB was immunoprecipitated with anti-FLAG mAb, followed by immunoblotting for p300 using anti-p300 mAb. Right: P300 was immunoprecipitated from control Th9 cells and OX40-induced Th9 cells, followed by immunoblotting for RelB. WCL was included as a positive control. (D) Full-length RelB or RelB with TAD deletion (RelB-ΔTAD) was introduced into 293 T cells, along with p300, and binding of RelB to p300 was determined by Co-IP experiments. The blots show binding of p300 to full length and TAD-deleted RelB. WCL was included as a control. (E) ChIP assay showing binding p300 to the Il9 coding region and the SEa region in WT B6 and Relb −/− Th9 cells induced with or without OX40 stimulation. (F) H3K27Ac ChIP data showing levels of H3K27 acetylation at Il9 locus and the SEa region in control Th9 cells and OX40-induced Th9 cells with or without addition of the p300 inhibitor SGC-CBP30 (p300i). (G) Expression of eRNA from Il9 SEa (a4) by control Th9 cells and OX40-induced Th9 cells treated with the p300 inhibitor or DMSO vehicle control. (H) 3C assay measuring DNA loop formations based PCR products amplified by R8 and C primer sets in control Th9 cells and OX40-induced Th9 cells with or without the p300 inhibitor. (I) A representative FACS plot showing induction of Th9 cells by OX40 in the presence of the p300 inhibitor or DMSO as a control. The bar graphs on the right show summary of Th9 cells with or without the p300i inhibitor. All graphs are representative images from at least three independent experiments, and bar graphs shown are mean ± SEM of three independent experiments with triplicate cultures. ***, P < 0.001.

Article Snippet: As an alternative method, OX40 agonist antibody OX86 (5 μg/ml, BioXcell) or recombinant mouse OX40L-His protein (100 ng/ml, along with 10 μg/ml of anti-His mAb; both from R&D Systems) was also used to activate OX40 signaling.

Techniques: Transduction, Retroviral, Expressing, Cell Culture, Immunoprecipitation, Western Blot, Control, Positive Control, Binding Assay, Co-Immunoprecipitation Assay, Amplification

Journal: The Journal of Experimental Medicine

Article Title: Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation

doi: 10.1084/jem.20170928

Figure Lengend Snippet: Disruption of SEs by the BET inhibitor JQ1 suppresses allergic airway inflammation in vivo. (A) Representative lung histology sections showing tissue inflammation and hyperplasia of airway epithelia in OX40Ltg mice treated with JQ1 (50 mg/kg) or vehicle control DMSO for 2 wk. The square represents areas that are shown under high power (400×). The arrow shows mucin-producing cells in the airway epithelia stained purple-red using PAS analysis. Bars, 100 µm. (B) Frequency of PAS + mucin-producing cells in the airway epithelia in control or JQ1-treated OX40Ltg mice. (C) ELISA analysis of IL-9 in the BAL in OX40Ltg mice treated with or without JQ1. (D) Lung tissue sections from WT B6 mice immunized with OVA in alum, followed by challenges with aerosolized OVA with or without JQ1 treatment. Groups of mice were given OX86 or an isotype control IgG at the time of OVA challenge. Arrows denote hyperplasia of mucin-producing cells in airway epithelia. Bars, 100 µm. (E) Quantitation of PAS + mucin-producing cells in control and JQ1-treated mice. (F) Histological scores based on cell infiltration, epithelial hyperplasia, and tissue damage. (G and H) The number of eosinophils (EOS; G) and IL-9 levels (H) in the BAL in mice treated with JQ1. Mice treated with the vehicle control DMSO were included as controls. Data in A and D are representative of two independent experiments, and data in B, C, and E–H are pooled from two independent experiments with five to seven mice per group in each experiment (mean and SD of n = 10–14). ***, P < 0.001.

Article Snippet: As an alternative method, OX40 agonist antibody OX86 (5 μg/ml, BioXcell) or recombinant mouse OX40L-His protein (100 ng/ml, along with 10 μg/ml of anti-His mAb; both from R&D Systems) was also used to activate OX40 signaling.

Techniques: Disruption, In Vivo, Control, Staining, Enzyme-linked Immunosorbent Assay, Quantitation Assay

Journal: The Journal of Experimental Medicine

Article Title: Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation

doi: 10.1084/jem.20170928

Figure Lengend Snippet: Role of Brd4 knockdown in allergic airway inflammation in an adoptive transfer model. (A) Lung histology showing inflammatory cell infiltration and proliferation of mucin producing cells in the airway epithelia in Rag-1 −/− hosts adoptively transferred with WT OT-II cells and Brd4 knockdown OT-II cells. Arrows show airway epithelia and mucin-producing cells. The picture is representative of two experiments with five mice per group in each experiment. Bars, 100 µm. (B) The bar graph shows the lung histological score in control IgG and OX86-treated mice. (C) Quantitation of PAS + mucin-producing cells in control IgG and OX86-treated mice. (D and E) The eosinophil numbers (EOS; D) and IL-9 levels (E) in the BAL of mice treated with control IgG and OX86. Data in A are representative of two independent experiments, and data in B–E are pooled from two independent experiments with five to six mice per group in each experiment (mean and SD of n = 10–12). *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Article Snippet: As an alternative method, OX40 agonist antibody OX86 (5 μg/ml, BioXcell) or recombinant mouse OX40L-His protein (100 ng/ml, along with 10 μg/ml of anti-His mAb; both from R&D Systems) was also used to activate OX40 signaling.

Techniques: Knockdown, Adoptive Transfer Assay, Control, Quantitation Assay