ccl17 protein  (R&D Systems)

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

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

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

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

Journal: bioRxiv

Article Title: The CCR4/CCL17 axis drives intestinal acute Graft versus Host disease after allogeneic bone marrow transplantation

doi: 10.1101/2024.03.02.583093

Figure Lengend Snippet: (A+B) Transwell migration assay of splenic T cells from either C57BL6/N wild-type mice or CCR4-/-animals was measured towards a CCL17 (800 ng/ml) gradient. Cells were counted via FACS for 90 seconds. WT CD3+ T cells ( p=0.0046 ) and CD4+ T cells ( p=0.0035 ) showed significant migration towards CCL17. (C) Naïve CD8+ T cells were isolated from the spleen of C57BL6/N mice. These cells were co-cultured with αCD3/CD28 beads and CCL17 (800ng/ml) when indicated. Proliferation was measured by CFSE distribution 60 hours later. (p p=0.8986 ) (C) Naïve CD4+ T cells were differentiated into Th1, Treg or Th2 cells in the presence (red) or absence of CCL17 (blue). Representative plots are shown. (D) Survival of BALB/c recipient mice after alloHSCT. Survival is not affected by CCL17 of donor bonemarrow or T cells; p=0.11 . Schematic figure created using biorender.com. (E) CCL17-/-recipient mice showed prolonged survival compared to CCL17 +/+ recipients, p=0.0069 . Schematic figure created using biorender.com. The data are pooled from 3 independent experiments with at least 3 mice per group. (F) Samples from gut biopsies of patients with the suspicion of intestinal aGvHD were collected. An experienced pathologist performed GvHD grading and samples were stained for CCL17 expression. Patients with proven aGvHD (n=12) show increased CCL17 positive area in representative areas compared to patients without GvHD (n=9). Representative samples of each group are shown.

Article Snippet: When indicated, CCL17 (800ng/ml, R&D Systems, #529-TR) was added during the differentiation process.

Techniques: Transwell Migration Assay, Migration, Isolation, Cell Culture, Staining, Expressing

Journal: bioRxiv

Article Title: The CCR4/CCL17 axis drives intestinal acute Graft versus Host disease after allogeneic bone marrow transplantation

doi: 10.1101/2024.03.02.583093

Figure Lengend Snippet: (A) CCL17egfp/+ recipient mice underwent alloHSCT. Mice receiving only bone marrow cells (no aGvHD induction) were compared to those receiving additional T cells for induction of aGvHD. Quantification of the CCL17 positive area was higher in the GvHD group p=0.027 . The experiment was performed twice. Representative data from one experiment is shown. Schematic figure created using biorender.com. Representative images of the small intestine stained for CD11c (DC marker) and DAPI are shown. Note the co-localization of CCL17(eGFP) and CD11c(PE). (B) Irradiated BALB/c mice received bone marrow cells and additional T cells from C57BL6/N donors. Mice were fed with ruxolitinib until day 3. Mice were sacrificed and CCL5 ( p=0.3267 ), CXCL10 ( p=0.0622 ) and CCL17 ( p=0.0045 ) expression was determined via qPCR using the ΔΔCT method. Pooled data from 2 independent experiments. Schematic figure created using biorender.com. (C) Splenic DCs were treated with IL-33 for 18 hours, which induced CCL17 secretion ( p<0.0001). (D) The presence of ruxolitinib (1µM) dampend CCL17 levels ( p=0.023 ). Schematic figure created using

Article Snippet: When indicated, CCL17 (800ng/ml, R&D Systems, #529-TR) was added during the differentiation process.

Techniques: Staining, Marker, Irradiation, Expressing