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human ccl17 tarc kit  (R&D Systems)


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    R&D Systems human ccl17 tarc kit
    Neutralization of TSLP activity by TAVO101 and tezepelumab in functional potency assays. (A) . Neutralization of human TSLP-driven STAT5 reporter gene activation by TAVO101 and tezepelumab. Increasing amounts of TAVO101 or tezepelumab along with 3 ng/mL recombinant human TSLP were applied to HEK293T cells transfected with human TSLP receptor complex and a STAT5-responsive luciferase reporter gene and reporter gene expression was quantitated. The percentages of TSLP activity normalized to the maximum activity driven by 3 ng/mL human TSLP were plotted against the concentrations of testing antibodies (Data expressed as mean ± SEM, n=3). (B) . Neutralization of human TSLP-driven proliferation of BaF3 cells transfected with human TSLP receptor complex by TAVO101. Increasing amounts of TAVO101 along with 0.5 ng/mL recombinant human TSLP were applied to the transfected cells and cell proliferation was quantitated. Luminescence signals reflecting cell proliferation were plotted against the concentrations of TAVO101 (Data expressed as mean ± SEM, n=3). (C) . Neutralization of TSLP-driven <t>CCL17</t> release from activated dendritic cells by TAVO101. CD1c + blood dendritic cells isolated from the PBMC of two healthy donors were treated with 15 ng/mL human TSLP with or without 1 μg/mL TAVO101. The CCL17 releases from the activated dendritic cells were quantitated. The CCL17 levels were plotted against the testing antibodies in the bar graphs as shown. (D) . Neutralization of TSLP-driven proliferation of activated human CD4 + T cells. Human CD4 + T cells isolated from the PBMC of two healthy donors were labelled by Cell Proliferation Dye eFluor 450, activated by plate bound anti-CD3 antibody and treated with 50 ng/mL human TSLP with or without null control antibody, tezepelumab or TAVO101 at the indicated concentrations. The fraction of proliferated human CD4 + T cells were quantitated by flow cytometry as cells stained with diluted dyes. The fold of T cell proliferation over that without TSLP treatment were plotted against the testing antibodies in the bar graphs as shown. (Data expressed as mean ± SEM, n=2).
    Human Ccl17 Tarc Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A novel monoclonal antibody against human thymic stromal lymphopoietin for the treatment of TSLP-mediated diseases"

    Article Title: A novel monoclonal antibody against human thymic stromal lymphopoietin for the treatment of TSLP-mediated diseases

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2024.1442588

    Neutralization of TSLP activity by TAVO101 and tezepelumab in functional potency assays. (A) . Neutralization of human TSLP-driven STAT5 reporter gene activation by TAVO101 and tezepelumab. Increasing amounts of TAVO101 or tezepelumab along with 3 ng/mL recombinant human TSLP were applied to HEK293T cells transfected with human TSLP receptor complex and a STAT5-responsive luciferase reporter gene and reporter gene expression was quantitated. The percentages of TSLP activity normalized to the maximum activity driven by 3 ng/mL human TSLP were plotted against the concentrations of testing antibodies (Data expressed as mean ± SEM, n=3). (B) . Neutralization of human TSLP-driven proliferation of BaF3 cells transfected with human TSLP receptor complex by TAVO101. Increasing amounts of TAVO101 along with 0.5 ng/mL recombinant human TSLP were applied to the transfected cells and cell proliferation was quantitated. Luminescence signals reflecting cell proliferation were plotted against the concentrations of TAVO101 (Data expressed as mean ± SEM, n=3). (C) . Neutralization of TSLP-driven CCL17 release from activated dendritic cells by TAVO101. CD1c + blood dendritic cells isolated from the PBMC of two healthy donors were treated with 15 ng/mL human TSLP with or without 1 μg/mL TAVO101. The CCL17 releases from the activated dendritic cells were quantitated. The CCL17 levels were plotted against the testing antibodies in the bar graphs as shown. (D) . Neutralization of TSLP-driven proliferation of activated human CD4 + T cells. Human CD4 + T cells isolated from the PBMC of two healthy donors were labelled by Cell Proliferation Dye eFluor 450, activated by plate bound anti-CD3 antibody and treated with 50 ng/mL human TSLP with or without null control antibody, tezepelumab or TAVO101 at the indicated concentrations. The fraction of proliferated human CD4 + T cells were quantitated by flow cytometry as cells stained with diluted dyes. The fold of T cell proliferation over that without TSLP treatment were plotted against the testing antibodies in the bar graphs as shown. (Data expressed as mean ± SEM, n=2).
    Figure Legend Snippet: Neutralization of TSLP activity by TAVO101 and tezepelumab in functional potency assays. (A) . Neutralization of human TSLP-driven STAT5 reporter gene activation by TAVO101 and tezepelumab. Increasing amounts of TAVO101 or tezepelumab along with 3 ng/mL recombinant human TSLP were applied to HEK293T cells transfected with human TSLP receptor complex and a STAT5-responsive luciferase reporter gene and reporter gene expression was quantitated. The percentages of TSLP activity normalized to the maximum activity driven by 3 ng/mL human TSLP were plotted against the concentrations of testing antibodies (Data expressed as mean ± SEM, n=3). (B) . Neutralization of human TSLP-driven proliferation of BaF3 cells transfected with human TSLP receptor complex by TAVO101. Increasing amounts of TAVO101 along with 0.5 ng/mL recombinant human TSLP were applied to the transfected cells and cell proliferation was quantitated. Luminescence signals reflecting cell proliferation were plotted against the concentrations of TAVO101 (Data expressed as mean ± SEM, n=3). (C) . Neutralization of TSLP-driven CCL17 release from activated dendritic cells by TAVO101. CD1c + blood dendritic cells isolated from the PBMC of two healthy donors were treated with 15 ng/mL human TSLP with or without 1 μg/mL TAVO101. The CCL17 releases from the activated dendritic cells were quantitated. The CCL17 levels were plotted against the testing antibodies in the bar graphs as shown. (D) . Neutralization of TSLP-driven proliferation of activated human CD4 + T cells. Human CD4 + T cells isolated from the PBMC of two healthy donors were labelled by Cell Proliferation Dye eFluor 450, activated by plate bound anti-CD3 antibody and treated with 50 ng/mL human TSLP with or without null control antibody, tezepelumab or TAVO101 at the indicated concentrations. The fraction of proliferated human CD4 + T cells were quantitated by flow cytometry as cells stained with diluted dyes. The fold of T cell proliferation over that without TSLP treatment were plotted against the testing antibodies in the bar graphs as shown. (Data expressed as mean ± SEM, n=2).

    Techniques Used: Neutralization, Activity Assay, Functional Assay, Activation Assay, Recombinant, Transfection, Luciferase, Expressing, Isolation, Control, Flow Cytometry, Staining

    Efficacy of TAVO101 in TSLP/OVA-induced asthma model using hTSLP/hTSLPR humanized mice. (A) . Dosing regimen and animal grouping in the asthmatic model. Four mice were enrolled in the G1 group while eight mice were enrolled in each of the G2 to G6 groups. (B) . The concentration of mouse total serum IgE and lung tissue CCL17 and IL-13. (C) . Cell counts of mouse CD45 + leukocytes and eosinophils and the associated percentage of eosinophils in CD45 + leukocytes in BALF of asthmatic mice. (D) . Scores of inflammatory cell infiltration and eosinophil infiltration by H&E staining and the positive area proportion of Goblet cells and mucus of asthmatic lungs. Note: Data was represented by mean ± SEM and analyzed by One-way ANOVA with Dunnett’s multiple comparisons test. Comparison between each experimental group and G2 group. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
    Figure Legend Snippet: Efficacy of TAVO101 in TSLP/OVA-induced asthma model using hTSLP/hTSLPR humanized mice. (A) . Dosing regimen and animal grouping in the asthmatic model. Four mice were enrolled in the G1 group while eight mice were enrolled in each of the G2 to G6 groups. (B) . The concentration of mouse total serum IgE and lung tissue CCL17 and IL-13. (C) . Cell counts of mouse CD45 + leukocytes and eosinophils and the associated percentage of eosinophils in CD45 + leukocytes in BALF of asthmatic mice. (D) . Scores of inflammatory cell infiltration and eosinophil infiltration by H&E staining and the positive area proportion of Goblet cells and mucus of asthmatic lungs. Note: Data was represented by mean ± SEM and analyzed by One-way ANOVA with Dunnett’s multiple comparisons test. Comparison between each experimental group and G2 group. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

    Techniques Used: Concentration Assay, Staining, Comparison



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    Neutralization of TSLP activity by TAVO101 and tezepelumab in functional potency assays. (A) . Neutralization of human TSLP-driven STAT5 reporter gene activation by TAVO101 and tezepelumab. Increasing amounts of TAVO101 or tezepelumab along with 3 ng/mL recombinant human TSLP were applied to HEK293T cells transfected with human TSLP receptor complex and a STAT5-responsive luciferase reporter gene and reporter gene expression was quantitated. The percentages of TSLP activity normalized to the maximum activity driven by 3 ng/mL human TSLP were plotted against the concentrations of testing antibodies (Data expressed as mean ± SEM, n=3). (B) . Neutralization of human TSLP-driven proliferation of BaF3 cells transfected with human TSLP receptor complex by TAVO101. Increasing amounts of TAVO101 along with 0.5 ng/mL recombinant human TSLP were applied to the transfected cells and cell proliferation was quantitated. Luminescence signals reflecting cell proliferation were plotted against the concentrations of TAVO101 (Data expressed as mean ± SEM, n=3). (C) . Neutralization of TSLP-driven <t>CCL17</t> release from activated dendritic cells by TAVO101. CD1c + blood dendritic cells isolated from the PBMC of two healthy donors were treated with 15 ng/mL human TSLP with or without 1 μg/mL TAVO101. The CCL17 releases from the activated dendritic cells were quantitated. The CCL17 levels were plotted against the testing antibodies in the bar graphs as shown. (D) . Neutralization of TSLP-driven proliferation of activated human CD4 + T cells. Human CD4 + T cells isolated from the PBMC of two healthy donors were labelled by Cell Proliferation Dye eFluor 450, activated by plate bound anti-CD3 antibody and treated with 50 ng/mL human TSLP with or without null control antibody, tezepelumab or TAVO101 at the indicated concentrations. The fraction of proliferated human CD4 + T cells were quantitated by flow cytometry as cells stained with diluted dyes. The fold of T cell proliferation over that without TSLP treatment were plotted against the testing antibodies in the bar graphs as shown. (Data expressed as mean ± SEM, n=2).
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    Neutralization of TSLP activity by TAVO101 and tezepelumab in functional potency assays. (A) . Neutralization of human TSLP-driven STAT5 reporter gene activation by TAVO101 and tezepelumab. Increasing amounts of TAVO101 or tezepelumab along with 3 ng/mL recombinant human TSLP were applied to HEK293T cells transfected with human TSLP receptor complex and a STAT5-responsive luciferase reporter gene and reporter gene expression was quantitated. The percentages of TSLP activity normalized to the maximum activity driven by 3 ng/mL human TSLP were plotted against the concentrations of testing antibodies (Data expressed as mean ± SEM, n=3). (B) . Neutralization of human TSLP-driven proliferation of BaF3 cells transfected with human TSLP receptor complex by TAVO101. Increasing amounts of TAVO101 along with 0.5 ng/mL recombinant human TSLP were applied to the transfected cells and cell proliferation was quantitated. Luminescence signals reflecting cell proliferation were plotted against the concentrations of TAVO101 (Data expressed as mean ± SEM, n=3). (C) . Neutralization of TSLP-driven CCL17 release from activated dendritic cells by TAVO101. CD1c + blood dendritic cells isolated from the PBMC of two healthy donors were treated with 15 ng/mL human TSLP with or without 1 μg/mL TAVO101. The CCL17 releases from the activated dendritic cells were quantitated. The CCL17 levels were plotted against the testing antibodies in the bar graphs as shown. (D) . Neutralization of TSLP-driven proliferation of activated human CD4 + T cells. Human CD4 + T cells isolated from the PBMC of two healthy donors were labelled by Cell Proliferation Dye eFluor 450, activated by plate bound anti-CD3 antibody and treated with 50 ng/mL human TSLP with or without null control antibody, tezepelumab or TAVO101 at the indicated concentrations. The fraction of proliferated human CD4 + T cells were quantitated by flow cytometry as cells stained with diluted dyes. The fold of T cell proliferation over that without TSLP treatment were plotted against the testing antibodies in the bar graphs as shown. (Data expressed as mean ± SEM, n=2).

    Journal: Frontiers in Immunology

    Article Title: A novel monoclonal antibody against human thymic stromal lymphopoietin for the treatment of TSLP-mediated diseases

    doi: 10.3389/fimmu.2024.1442588

    Figure Lengend Snippet: Neutralization of TSLP activity by TAVO101 and tezepelumab in functional potency assays. (A) . Neutralization of human TSLP-driven STAT5 reporter gene activation by TAVO101 and tezepelumab. Increasing amounts of TAVO101 or tezepelumab along with 3 ng/mL recombinant human TSLP were applied to HEK293T cells transfected with human TSLP receptor complex and a STAT5-responsive luciferase reporter gene and reporter gene expression was quantitated. The percentages of TSLP activity normalized to the maximum activity driven by 3 ng/mL human TSLP were plotted against the concentrations of testing antibodies (Data expressed as mean ± SEM, n=3). (B) . Neutralization of human TSLP-driven proliferation of BaF3 cells transfected with human TSLP receptor complex by TAVO101. Increasing amounts of TAVO101 along with 0.5 ng/mL recombinant human TSLP were applied to the transfected cells and cell proliferation was quantitated. Luminescence signals reflecting cell proliferation were plotted against the concentrations of TAVO101 (Data expressed as mean ± SEM, n=3). (C) . Neutralization of TSLP-driven CCL17 release from activated dendritic cells by TAVO101. CD1c + blood dendritic cells isolated from the PBMC of two healthy donors were treated with 15 ng/mL human TSLP with or without 1 μg/mL TAVO101. The CCL17 releases from the activated dendritic cells were quantitated. The CCL17 levels were plotted against the testing antibodies in the bar graphs as shown. (D) . Neutralization of TSLP-driven proliferation of activated human CD4 + T cells. Human CD4 + T cells isolated from the PBMC of two healthy donors were labelled by Cell Proliferation Dye eFluor 450, activated by plate bound anti-CD3 antibody and treated with 50 ng/mL human TSLP with or without null control antibody, tezepelumab or TAVO101 at the indicated concentrations. The fraction of proliferated human CD4 + T cells were quantitated by flow cytometry as cells stained with diluted dyes. The fold of T cell proliferation over that without TSLP treatment were plotted against the testing antibodies in the bar graphs as shown. (Data expressed as mean ± SEM, n=2).

    Article Snippet: The cell supernatants were collected and the TSLP-driven release of CCL17 protein was quantitated using the human CCL17/TARC kit (R&D Systems, Minneapolis, MN).

    Techniques: Neutralization, Activity Assay, Functional Assay, Activation Assay, Recombinant, Transfection, Luciferase, Expressing, Isolation, Control, Flow Cytometry, Staining

    Efficacy of TAVO101 in TSLP/OVA-induced asthma model using hTSLP/hTSLPR humanized mice. (A) . Dosing regimen and animal grouping in the asthmatic model. Four mice were enrolled in the G1 group while eight mice were enrolled in each of the G2 to G6 groups. (B) . The concentration of mouse total serum IgE and lung tissue CCL17 and IL-13. (C) . Cell counts of mouse CD45 + leukocytes and eosinophils and the associated percentage of eosinophils in CD45 + leukocytes in BALF of asthmatic mice. (D) . Scores of inflammatory cell infiltration and eosinophil infiltration by H&E staining and the positive area proportion of Goblet cells and mucus of asthmatic lungs. Note: Data was represented by mean ± SEM and analyzed by One-way ANOVA with Dunnett’s multiple comparisons test. Comparison between each experimental group and G2 group. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

    Journal: Frontiers in Immunology

    Article Title: A novel monoclonal antibody against human thymic stromal lymphopoietin for the treatment of TSLP-mediated diseases

    doi: 10.3389/fimmu.2024.1442588

    Figure Lengend Snippet: Efficacy of TAVO101 in TSLP/OVA-induced asthma model using hTSLP/hTSLPR humanized mice. (A) . Dosing regimen and animal grouping in the asthmatic model. Four mice were enrolled in the G1 group while eight mice were enrolled in each of the G2 to G6 groups. (B) . The concentration of mouse total serum IgE and lung tissue CCL17 and IL-13. (C) . Cell counts of mouse CD45 + leukocytes and eosinophils and the associated percentage of eosinophils in CD45 + leukocytes in BALF of asthmatic mice. (D) . Scores of inflammatory cell infiltration and eosinophil infiltration by H&E staining and the positive area proportion of Goblet cells and mucus of asthmatic lungs. Note: Data was represented by mean ± SEM and analyzed by One-way ANOVA with Dunnett’s multiple comparisons test. Comparison between each experimental group and G2 group. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

    Article Snippet: The cell supernatants were collected and the TSLP-driven release of CCL17 protein was quantitated using the human CCL17/TARC kit (R&D Systems, Minneapolis, MN).

    Techniques: Concentration Assay, Staining, Comparison

    (A) Lollipop plot summarizing IL4R mutations identified in cHL patients (n=26, top) as compared to PMBL patients (n=16, bottom42). Nonsense (black) and missense mutations (green). (B) Dose-response curves for IL13/IL4 in transduced DEV cells using phospho-STAT6 (flow cytometry) as readout. WT and E684Kfs2: n=3 each. * P<0.05 (t-test, two-sided; P=0.013 at 0.5ng/mL and P=0.028 at 1ng/mL). (C) Phospho-STAT6 levels in unstimulated and IL13-stimulated transduced DEV cells (n=6 each; grey bar: all mutants [all variants, n=48]). Unadjusted Wilcoxon p-values (two-sided) compared to WT are provided. (D) Phospho-STAT6 levels in unstimulated and IL13-stimulated transduced KM-H2 cells (n=4 each; grey bar: all mutants [all variants, n=8]). Unadjusted Wilcoxon p-values (two-sided) compared to WT are provided. (E) Phospho-STAT6 levels in transduced DEV (n=5 each) and KM-H2 (n=4 each) cells under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-antibody (IL4R-Ab) treated as well as IL13-stimulated + STAT6-Inhibitor (STAT6-I.) treated conditions. Unadjusted Wilcoxon p-values (two-sided) compared to IL13 stimulation alone are provided. (F) CCL17 (TARC) concentrations with unadjusted Wilcoxon p-values (two-sided) in supernatant of transduced DEV (n=5 for unstimulated/IL13; n=4 for IL4R-Ab/STAT6-I.) and KM-H2 cells (n=3 each) under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. (G) Volcano plot summarizing differentially expressed genes from the KEGG Cytokine cytokine-receptor interaction gene set between cHL (n=86) and LBCL (n=66) in bulk RNA-Sequencing. (H) Copy-number (CN) z-score (density plot) of the 5q31.1 cytoband harboring IL13 stratified by IL4R mutation status in patients with plasma exome sequencing [IL4R mutant: n=12; IL4R WT: n=107]. 7/12 (58%, IL4R mutant) and 16/107 (15%, IL4R WT) cases were found to have a 5q31.1 amplification when considering a z-score of 1.96 as threshold. The corresponding two-sided Fisher’s exact test p-value is provided. Panels B-F: mean +/− standard error (se).

    Journal: Nature

    Article Title: Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling

    doi: 10.1038/s41586-023-06903-x

    Figure Lengend Snippet: (A) Lollipop plot summarizing IL4R mutations identified in cHL patients (n=26, top) as compared to PMBL patients (n=16, bottom42). Nonsense (black) and missense mutations (green). (B) Dose-response curves for IL13/IL4 in transduced DEV cells using phospho-STAT6 (flow cytometry) as readout. WT and E684Kfs2: n=3 each. * P<0.05 (t-test, two-sided; P=0.013 at 0.5ng/mL and P=0.028 at 1ng/mL). (C) Phospho-STAT6 levels in unstimulated and IL13-stimulated transduced DEV cells (n=6 each; grey bar: all mutants [all variants, n=48]). Unadjusted Wilcoxon p-values (two-sided) compared to WT are provided. (D) Phospho-STAT6 levels in unstimulated and IL13-stimulated transduced KM-H2 cells (n=4 each; grey bar: all mutants [all variants, n=8]). Unadjusted Wilcoxon p-values (two-sided) compared to WT are provided. (E) Phospho-STAT6 levels in transduced DEV (n=5 each) and KM-H2 (n=4 each) cells under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-antibody (IL4R-Ab) treated as well as IL13-stimulated + STAT6-Inhibitor (STAT6-I.) treated conditions. Unadjusted Wilcoxon p-values (two-sided) compared to IL13 stimulation alone are provided. (F) CCL17 (TARC) concentrations with unadjusted Wilcoxon p-values (two-sided) in supernatant of transduced DEV (n=5 for unstimulated/IL13; n=4 for IL4R-Ab/STAT6-I.) and KM-H2 cells (n=3 each) under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. (G) Volcano plot summarizing differentially expressed genes from the KEGG Cytokine cytokine-receptor interaction gene set between cHL (n=86) and LBCL (n=66) in bulk RNA-Sequencing. (H) Copy-number (CN) z-score (density plot) of the 5q31.1 cytoband harboring IL13 stratified by IL4R mutation status in patients with plasma exome sequencing [IL4R mutant: n=12; IL4R WT: n=107]. 7/12 (58%, IL4R mutant) and 16/107 (15%, IL4R WT) cases were found to have a 5q31.1 amplification when considering a z-score of 1.96 as threshold. The corresponding two-sided Fisher’s exact test p-value is provided. Panels B-F: mean +/− standard error (se).

    Article Snippet: Briefly, fresh cell line supernatant was prepared by centrifuging cell cultures and assayed using the Human CCL17/TARC DuoSet ELISA kit (R&D Systems).

    Techniques: Flow Cytometry, RNA Sequencing Assay, Mutagenesis, Sequencing, Amplification

    (A) Immunoblot showing protein levels of IL4R and GAPDH in transduced DEV and KM-H2 cells. (B) Gene set enrichment plots from RNA-Sequencing showing enrichment of canonical KEGG pathways in mutant (E684Kfs2, n=6) vs WT (n=6) expressing DEV cells. Normalized enrichment scores (NES) and adjusted p-values (fgsea R-package) are provided. (C) Scatter plot visualizing base mean expression and log2 fold change comparing gene expression in mutant (E684Kfs2, n=6) vs WT (n=6) expressing DEV cells. Absolute log2 fold changes >1 are highlighted in orange or black, respectively. (D) Phospho-STAT6 levels (flow) in transduced DEV (n=5 each) and KM-H2 (n=4 each) cells under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. Unadjusted Wilcoxon p-values (two-sided) compared to IL13 stimulation alone are provided. (E) Representative phospho-STAT6 flow raw data for KM-H2 Empty, WT, Q666* and Q698* constructs under IL13-stimulated conditions. (F) Phospho-STAT6 levels in WT, E684Kfs2 and I242N (PMBL hotspot) DEV cells under unstimulated, IL4-stimulated, IL4-stimulated + IL4R-Ab treated as well as IL4-stimulated + STAT6-I. treated conditions (n=5 each). Unadjusted Wilcoxon p-values (two-sided) compared to IL4 stimulation alone are provided. (G) Representative immunoblot showing protein levels of pSTAT6, STAT6 and GAPDH in KM-H2 cells as a function on IL4R construct expression under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. All conditions were run on the same gel. Ladders run between IL4R constructs were cropped and are not shown. (H) Immunoblot showing protein levels of pSTAT6, STAT6 and GAPDH in IL4R I242N expressing KM-H2 cells under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. (I-J) CCL17 (TARC) concentrations in supernatant of transduced (I) DEV (n=5 for unstimulated and IL13; n=4 for IL4R-Ab and STAT6-I.) and (J) KM-H2 (n=3 each) cells under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. Unadjusted Wilcoxon p-values (two-sided) are provided. (K) Boxplots and Wilcoxon p-values (two-sided) comparing IL13 and IL4 expression in RNA-Sequencing of primary bulk tumor specimens visualized as normalized counts (n=86 cHL, n=66 LBCL32). (L) Log2 copy number ratio (L2CNR, boxplot and Wilcoxon p-value, two-sided) of the 5q31.1 cytoband harboring IL13 stratified by IL4R mutation status in n=119 patients with plasma exome sequencing [IL4R mutant: n=12; IL4R WT: n=107]. Panels A,G-H: At least 2 independent experiments were performed for each condition. Panels D,F,I,J: mean +/− standard error (se). Panels K-L: each box represents the interquartile range (the range between the 25th and 75th percentile) with the median of the data, whiskers indicate the upper and lower value within 1.5 times the IQR.

    Journal: Nature

    Article Title: Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling

    doi: 10.1038/s41586-023-06903-x

    Figure Lengend Snippet: (A) Immunoblot showing protein levels of IL4R and GAPDH in transduced DEV and KM-H2 cells. (B) Gene set enrichment plots from RNA-Sequencing showing enrichment of canonical KEGG pathways in mutant (E684Kfs2, n=6) vs WT (n=6) expressing DEV cells. Normalized enrichment scores (NES) and adjusted p-values (fgsea R-package) are provided. (C) Scatter plot visualizing base mean expression and log2 fold change comparing gene expression in mutant (E684Kfs2, n=6) vs WT (n=6) expressing DEV cells. Absolute log2 fold changes >1 are highlighted in orange or black, respectively. (D) Phospho-STAT6 levels (flow) in transduced DEV (n=5 each) and KM-H2 (n=4 each) cells under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. Unadjusted Wilcoxon p-values (two-sided) compared to IL13 stimulation alone are provided. (E) Representative phospho-STAT6 flow raw data for KM-H2 Empty, WT, Q666* and Q698* constructs under IL13-stimulated conditions. (F) Phospho-STAT6 levels in WT, E684Kfs2 and I242N (PMBL hotspot) DEV cells under unstimulated, IL4-stimulated, IL4-stimulated + IL4R-Ab treated as well as IL4-stimulated + STAT6-I. treated conditions (n=5 each). Unadjusted Wilcoxon p-values (two-sided) compared to IL4 stimulation alone are provided. (G) Representative immunoblot showing protein levels of pSTAT6, STAT6 and GAPDH in KM-H2 cells as a function on IL4R construct expression under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. All conditions were run on the same gel. Ladders run between IL4R constructs were cropped and are not shown. (H) Immunoblot showing protein levels of pSTAT6, STAT6 and GAPDH in IL4R I242N expressing KM-H2 cells under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. (I-J) CCL17 (TARC) concentrations in supernatant of transduced (I) DEV (n=5 for unstimulated and IL13; n=4 for IL4R-Ab and STAT6-I.) and (J) KM-H2 (n=3 each) cells under unstimulated, IL13-stimulated, IL13-stimulated + IL4R-Ab treated as well as IL13-stimulated + STAT6-I. treated conditions. Unadjusted Wilcoxon p-values (two-sided) are provided. (K) Boxplots and Wilcoxon p-values (two-sided) comparing IL13 and IL4 expression in RNA-Sequencing of primary bulk tumor specimens visualized as normalized counts (n=86 cHL, n=66 LBCL32). (L) Log2 copy number ratio (L2CNR, boxplot and Wilcoxon p-value, two-sided) of the 5q31.1 cytoband harboring IL13 stratified by IL4R mutation status in n=119 patients with plasma exome sequencing [IL4R mutant: n=12; IL4R WT: n=107]. Panels A,G-H: At least 2 independent experiments were performed for each condition. Panels D,F,I,J: mean +/− standard error (se). Panels K-L: each box represents the interquartile range (the range between the 25th and 75th percentile) with the median of the data, whiskers indicate the upper and lower value within 1.5 times the IQR.

    Article Snippet: Briefly, fresh cell line supernatant was prepared by centrifuging cell cultures and assayed using the Human CCL17/TARC DuoSet ELISA kit (R&D Systems).

    Techniques: Western Blot, RNA Sequencing Assay, Mutagenesis, Expressing, Construct, Sequencing

    a Hierarchical plot showing inferred intercellular communication network of CCL17 - CCR4 signaling. b The feature plot and the bar chart showing CCL17 mRNA expression within total skin cells between BP ( n = 5) and HC ( n = 8). c Protein level of CCL17 in serum from BP ( n = 73) and HC ( n = 32) by ELISA. d The feature plot and the bar chart showing CCR4 mRNA expression within total skin cells between BP ( n = 5) and HC ( n = 8). In the box plot b – d : Minima: Lower limit of the whisker. Maxima: Upper limit of the whisker. Center: Median line inside the box. The upper and lower box bounds represent the 25% and 75% percentile of data. e Flow plots of CD3 + T cells from PBMCs showing the expression of CCR4 treated by CCL17 recombinant protein (left panel), and the frequency of CCR4 (right panel) in BP ( n = 30) and HC ( n = 16) groups. f The positive correlation between the level of PLA2G2A and CCL17 in serum from in BP patients ( n = 73). P -value was calculated using two-sided Pearson correlation test. r -value was Pearson correlation coefficient. g The level of CCL17 in PBMC from BP patients ( n = 26) and HC ( n = 7) treated with PLA2G2A recombinant protein. h Effect of CCL17 treatment on the IL-13 secretion from BP patients ( n = 26) and HC ( n = 13). i Effect of PLA2G2A treatment on the IL-13 secretion from BP patients ( n = 20) and HC ( n = 13). j , k ELISA analysis of anti-BP180-NC16A antibody titers in supernatants of CCL17 ( j ) or PLA2G2A ( k ) stimulated PBMCs from BP patients ( n = 26) and HC ( n = 22). P -values in b–d were calculated using two-sided Mann–Whitney U-test. P -values in e and g – k were calculated using paired two-sided Student’s t -test. * P < 0.05, ** P < 0.01, **** P < 0.0001, only P -values < 0.05 are shown. Each sample is represented as one dot.

    Journal: Nature Communications

    Article Title: Single-cell transcriptomics analysis of bullous pemphigoid unveils immune-stromal crosstalk in type 2 inflammatory disease

    doi: 10.1038/s41467-024-50283-3

    Figure Lengend Snippet: a Hierarchical plot showing inferred intercellular communication network of CCL17 - CCR4 signaling. b The feature plot and the bar chart showing CCL17 mRNA expression within total skin cells between BP ( n = 5) and HC ( n = 8). c Protein level of CCL17 in serum from BP ( n = 73) and HC ( n = 32) by ELISA. d The feature plot and the bar chart showing CCR4 mRNA expression within total skin cells between BP ( n = 5) and HC ( n = 8). In the box plot b – d : Minima: Lower limit of the whisker. Maxima: Upper limit of the whisker. Center: Median line inside the box. The upper and lower box bounds represent the 25% and 75% percentile of data. e Flow plots of CD3 + T cells from PBMCs showing the expression of CCR4 treated by CCL17 recombinant protein (left panel), and the frequency of CCR4 (right panel) in BP ( n = 30) and HC ( n = 16) groups. f The positive correlation between the level of PLA2G2A and CCL17 in serum from in BP patients ( n = 73). P -value was calculated using two-sided Pearson correlation test. r -value was Pearson correlation coefficient. g The level of CCL17 in PBMC from BP patients ( n = 26) and HC ( n = 7) treated with PLA2G2A recombinant protein. h Effect of CCL17 treatment on the IL-13 secretion from BP patients ( n = 26) and HC ( n = 13). i Effect of PLA2G2A treatment on the IL-13 secretion from BP patients ( n = 20) and HC ( n = 13). j , k ELISA analysis of anti-BP180-NC16A antibody titers in supernatants of CCL17 ( j ) or PLA2G2A ( k ) stimulated PBMCs from BP patients ( n = 26) and HC ( n = 22). P -values in b–d were calculated using two-sided Mann–Whitney U-test. P -values in e and g – k were calculated using paired two-sided Student’s t -test. * P < 0.05, ** P < 0.01, **** P < 0.0001, only P -values < 0.05 are shown. Each sample is represented as one dot.

    Article Snippet: The serum levels of IL-13 (EK0424), IL-4 (EK0404), IL-5 (EK0407), PLA2G2A (EK1944), and CCL17 (EK0684) in both patients and controls were quantified using ELISA Development Kits from BosterBio, USA, following the manufacturer’s instructions.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Whisker Assay, Recombinant, MANN-WHITNEY

    It illustrates a positive feedback loop where fibroblasts respond to Th2 cells through the IL13RA1 - IL13 pair, leading to increased secretion of PLA2G2A and CXCL12. This, in turn, amplifies the Th2-mediated response. (i) The lesional fibroblasts respond to IL-13 and induce the overexpression of PLA2G2A, which promotes the expression of CXCR4 on the surface of immune cells to recruit the immune cells from peripheral blood into skin lesions. (ii) Fibroblasts-derived PLA2G2A and myeloid cells-derived CCL17 elevate the secretion of IL-13, and fibroblast and myeloid cells further respond to IL-13, forming a positive feedback loop between immune cells and fibroblasts. (iii) IL-13 activates B cells to secrete autoantibodies. (iv) In blister, the secretion of autoantibodies recruits T cells, myeloid cells, mast cells, neutrophils and eosinophils. T cell-derived IL-13 activates eosinophils to secrete various cytokines (including IL-13), which further promotes Th2 polarization and mediates the crosstalk between immune cells.

    Journal: Nature Communications

    Article Title: Single-cell transcriptomics analysis of bullous pemphigoid unveils immune-stromal crosstalk in type 2 inflammatory disease

    doi: 10.1038/s41467-024-50283-3

    Figure Lengend Snippet: It illustrates a positive feedback loop where fibroblasts respond to Th2 cells through the IL13RA1 - IL13 pair, leading to increased secretion of PLA2G2A and CXCL12. This, in turn, amplifies the Th2-mediated response. (i) The lesional fibroblasts respond to IL-13 and induce the overexpression of PLA2G2A, which promotes the expression of CXCR4 on the surface of immune cells to recruit the immune cells from peripheral blood into skin lesions. (ii) Fibroblasts-derived PLA2G2A and myeloid cells-derived CCL17 elevate the secretion of IL-13, and fibroblast and myeloid cells further respond to IL-13, forming a positive feedback loop between immune cells and fibroblasts. (iii) IL-13 activates B cells to secrete autoantibodies. (iv) In blister, the secretion of autoantibodies recruits T cells, myeloid cells, mast cells, neutrophils and eosinophils. T cell-derived IL-13 activates eosinophils to secrete various cytokines (including IL-13), which further promotes Th2 polarization and mediates the crosstalk between immune cells.

    Article Snippet: The serum levels of IL-13 (EK0424), IL-4 (EK0404), IL-5 (EK0407), PLA2G2A (EK1944), and CCL17 (EK0684) in both patients and controls were quantified using ELISA Development Kits from BosterBio, USA, following the manufacturer’s instructions.

    Techniques: Over Expression, Expressing, Derivative Assay