recombinant mouse cxcl16  (R&D Systems)

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) CXCL16 mRNA expression in the kidney during MuPyV infection. Expression is shown as fold change relative to sham infected samples. Data are from three independent experiments (n = 10-11). (B) Expression of NKCC2 [marks the ascending loop of Henle ], CD8, and CXCL16 in epithelia in sham-infected and 8 dpi kidneys; bottom right photomicrographs are merged images. Representative of two independent experiments. (C) CXCL16 expression in sham-infected, 4 dpi, and 8 dpi kidney lysates (left). Western blot image is representative of two independent experiments with each lane indicating protein lysate from kidneys of individual mice. Protein band intensity quantification for sCXCL16 was normalized to β-actin and analyzed by ImageLab and normalized to the loading control (right). Data are combined from two independent experiments (n = 3-5). (D) Experimental design of in vivo CXCL16 mAb administration. Mice were administered 250 µg of CXCL16 mAb or control rat IgG every two days from days 4-14 post infection and euthanized at 15 dpi. Numbers of CD45 mAb i.v.-negative, CD8 + CD44 + D b -LT359 tetramer + T cells and CD4 + CD44 + T cells in kidneys and spleens of infected mice given anti-CXCL16 or control rat IgG. Data were analyzed by one-way ANOVA (A and C) and by multiple Mann-Whitney tests (D).

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Infection, Western Blot, Control, In Vivo, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) Dense lymphoplasmacytic infiltrate surrounding and infiltrating cortical tubules (H&E, x400). (B) Immunohistochemical staining for Large T antigen in nuclei of tubular epithelium (x250). (C) Expression of CXCR6 on infiltrating CD8 + cells. Photomicrographs (right) are enlarged images in the white squares (left); bottom right are merged images. No 1 o , no primary antibody. (D) Log-fold change of CXCL16 and CXCR6 across four independent studies, each comparing KTx biopsies from patients with PVAN and stable graft function. The error bars indicate the 95% confidence interval of log-fold change, with horizontal dashed grey line indicating log-fold change = 0, or no difference. GSE120495 is an RNA-seq study, while remaining studies are microarray based. Given the heterogeneity of the data, a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. The combined p-values for CXCL16 = 0.000337 and CXCR6 = 2.72x10 -05 . ns, non-significant at adjusted p-value > 0.05; *, statistical significance at adjusted p-value ≤ 0.05; **, statistical significance at adjusted p-value ≤ 0.01; ***, statistical significance at adjusted p-value ≤ 0.001.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Immunohistochemical staining, Staining, Expressing, RNA Sequencing, Microarray

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: Summary of differential gene expression of CXCL16 and CXCR6 in four studies of KTx biopsies with PVAN. Because of the heterogeneity of the data (i.e., one RNA-seq, three microarray), a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. P-values significant after false discover rate (FDR) correction are bolded and italicized in the Adj. P-value column. Cauchy combined p-values < 0.05 are bolded and italicized.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Gene Expression, Microarray

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) CXCL16 mRNA expression in the kidney during MuPyV infection. Expression is shown as fold change relative to sham infected samples. Data are from three independent experiments (n = 10-11). (B) Expression of NKCC2 [marks the ascending loop of Henle ], CD8, and CXCL16 in epithelia in sham-infected and 8 dpi kidneys; bottom right photomicrographs are merged images. Representative of two independent experiments. (C) CXCL16 expression in sham-infected, 4 dpi, and 8 dpi kidney lysates (left). Western blot image is representative of two independent experiments with each lane indicating protein lysate from kidneys of individual mice. Protein band intensity quantification for sCXCL16 was normalized to β-actin and analyzed by ImageLab and normalized to the loading control (right). Data are combined from two independent experiments (n = 3-5). (D) Experimental design of in vivo CXCL16 mAb administration. Mice were administered 250 µg of CXCL16 mAb or control rat IgG every two days from days 4-14 post infection and euthanized at 15 dpi. Numbers of CD45 mAb i.v.-negative, CD8 + CD44 + D b -LT359 tetramer + T cells and CD4 + CD44 + T cells in kidneys and spleens of infected mice given anti-CXCL16 or control rat IgG. Data were analyzed by one-way ANOVA (A and C) and by multiple Mann-Whitney tests (D).

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Infection, Western Blot, Control, In Vivo, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) Dense lymphoplasmacytic infiltrate surrounding and infiltrating cortical tubules (H&E, x400). (B) Immunohistochemical staining for Large T antigen in nuclei of tubular epithelium (x250). (C) Expression of CXCR6 on infiltrating CD8 + cells. Photomicrographs (right) are enlarged images in the white squares (left); bottom right are merged images. No 1 o , no primary antibody. (D) Log-fold change of CXCL16 and CXCR6 across four independent studies, each comparing KTx biopsies from patients with PVAN and stable graft function. The error bars indicate the 95% confidence interval of log-fold change, with horizontal dashed grey line indicating log-fold change = 0, or no difference. GSE120495 is an RNA-seq study, while remaining studies are microarray based. Given the heterogeneity of the data, a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. The combined p-values for CXCL16 = 0.000337 and CXCR6 = 2.72x10 -05 . ns, non-significant at adjusted p-value > 0.05; *, statistical significance at adjusted p-value ≤ 0.05; **, statistical significance at adjusted p-value ≤ 0.01; ***, statistical significance at adjusted p-value ≤ 0.001.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Immunohistochemical staining, Staining, Expressing, RNA Sequencing, Microarray

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: Summary of differential gene expression of CXCL16 and CXCR6 in four studies of KTx biopsies with PVAN. Because of the heterogeneity of the data (i.e., one RNA-seq, three microarray), a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. P-values significant after false discover rate (FDR) correction are bolded and italicized in the Adj. P-value column. Cauchy combined p-values < 0.05 are bolded and italicized.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Gene Expression, Microarray

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 1. Circulating CXCL16 concentrations in the patients with sepsis were increased. (A) Soluble levels of serum CXCL16 in 55 septic patients and 30 healthy controls. (B) The dynamics of soluble CXCL16 levels in the serum of patients with sepsis. (C) Soluble CXCL16 levels in the serum collected from patients with septic shock and patients without shock on day of ICU admission. (D) Soluble CXCL16 levels in the serum collected from septic survivors and nonsurvivors. Nonparametric ManneWhitney U test or KruskaleWallis test followed by Dunn's multiple comparisons post test was performed to analyze results between groups. Horizontal bars represent median values, and dots represent individual participants.

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques:

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 2. Soluble serum CXCL16 levels at admission correlated with disease severity in the patients with sepsis. (A) Correlation of soluble CXCL16 levels with Sequential Organ Failure Assessment (SOFA) scores in septic patients. (B) Correlation of soluble CXCL16 levels with white blood cells (WBC) in septic patients. (C) Correlation of soluble CXCL16 levels with C- reactive protein (CRP) levels in septic patients. Spearman's correlation coefficient was performed to analyze the correlation between the levels of CXCL16 and SOFA scores, WBC, or CRP levels. Horizontal bars represent median values, and dots represent individual participants.

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques:

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 3. Receiving operating characteristic (ROC) curve analysis of CXCL16, SOFA score, PCT, and CRP at admission for predicting 28-day mortality in septic patients. Area under the ROC curve: 0.717 ([95% CI] 0.497e0.939) for CXCL16; 0.849 ([95% CI] 0.715e0.982) for SOFA score; 0.617 ([95% CI] 0.429e0.805) for PCT; and 0.521 ([95% CI] 0.338e0.705) for CRP.

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques:

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 4. CXCL16 worsened cecal ligation and puncture (CLP)-induced nonsevere sepsis. (A) Survival of CLP mice (n ¼ 12 per group) after treatment with recombinant CXCL16 protein in the absence or presence of CLP-induced nonsevere sepsis. Murine recombinant CXCL16 protein was administered intraperitoneally at 0.5e1.0 mg/injection immediately after nonsevere CLP, and PBS was used as a control. Comparison between groups was performed using KaplaneMeier analysis followed by log-rank tests. Results are representative of three independent experiments. p < 0.001 when compared with septic mice treated with PBS control. (B) Representative examples of hematoxylin and eosinestained lung, liver, and kidney tissues from nonsevere CLP mice (n ¼ 6) treated with or without recombinant CXCL16 (0.5 mg/injection). (C) Histological scores for lung, liver, and kidney in nonsevere CLP mice (n ¼ 6) treated with or without recombinant CXCL16 (0.5 mg/injection). p < 0.05, p < 0.001when compared with septic mice treated with PBS control (ManneWhitney U test). (D) ALT, AST, LDH, and creatinine levels in nonsevere CLP mice (n ¼ 6), treated with or without recombinant CXCL16 (0.5 mg/injection). p < 0.001 when compared with septic mice treated with PBS control (ManneWhitney U test).

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques: Ligation, Recombinant, Injection, Control, Comparison

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 5. Treatment with recombinant murine CXCL16 enhanced inflammatory response in CLP-induced nonsevere sepsis. (A) Numbers of leukocytes including neutrophils, monocytes/macrophages and lymphocytes in peritoneal lavage fluid (PLF) of septic mice treated with or without recombinant murine CXCL16 (0.5 mg/injection). (B) Cytokine and chemokine levels in PLF and blood from septic mice (n ¼ 5) treated with or without recombinant murine CXCL16 (0.5 mg/injection) at 24 h after nonsevere CLP. p < 0.05, p < 0.01, p < 0.001 when compared with septic mice treated with PBS control (ManneWhitney U test).

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques: Recombinant, Injection, Control

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 6. Anti-CXCL16 antibody ameliorated cecal ligation and puncture (CLP)-induced severe sepsis. C57BL/6 mice were subjected to severe CLP, and then 10 mg of anti-mouse CXCL16 monoclonal antibody was injected intraperitoneally at 2 h or 6 h after severe CLP. IgG2A control antibody was delivered in a similar way. (A) Survival of septic mice (n ¼ 12 per group) following administration with antieCXCL16 antibody (2 h after CLP) or control antibody after severe CLP. Comparison between groups was performed by KaplaneMeier analysis followed by log-rank tests. Results are representative of three independent experiments. p < 0.05 when compared with septic mice treated with IgG control. (B) Sur- vival of septic mice (n ¼ 12 per group) following administration with antieCXCL16 antibody (6 h after CLP) or control antibody after severe CLP. Comparison between groups was performed by KaplaneMeier analysis followed by log-rank tests. Results are representative of three independent experiments. p < 0.05 when compared with septic mice treated with IgG control. (C) Representative examples of hematoxylin and eosinestained lung, liver, and kidney tissues from septic mice (n ¼ 6) treated with or without anti-CXCL16 neutralizing antibodies (2 h after CLP). (D) Histological scores for lung, liver, and kidney in septic mice (n ¼ 6) treated with or without anti-CXCL16 neutralizing antibodies (2 h after CLP). p < 0.05, p < 0.01 when compared with septic mice treated with isotypical IgG control (ManneWhitney U test). (E) ALT, AST, LDH, and creatinine levels in septic mice (n ¼ 6) treated with or without anti- CXCL16 neutralizing antibodies (2 h after CLP). p < 0.05, p < 0.01 when compared with septic mice treated with isotypical IgG control (ManneWhitney U test).

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques: Ligation, Injection, Control, Comparison

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 1. Circulating CXCL16 concentrations in the patients with sepsis were increased. (A) Soluble levels of serum CXCL16 in 55 septic patients and 30 healthy controls. (B) The dynamics of soluble CXCL16 levels in the serum of patients with sepsis. (C) Soluble CXCL16 levels in the serum collected from patients with septic shock and patients without shock on day of ICU admission. (D) Soluble CXCL16 levels in the serum collected from septic survivors and nonsurvivors. Nonparametric ManneWhitney U test or KruskaleWallis test followed by Dunn's multiple comparisons post test was performed to analyze results between groups. Horizontal bars represent median values, and dots represent individual participants.

Article Snippet: Recombinant murine CXCL16 protein (R&D systems, Catalog # 503-CX-025) was administered i.p. into septic C57BL/6 mice immediately after nonsevere CLP.

Techniques:

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 2. Soluble serum CXCL16 levels at admission correlated with disease severity in the patients with sepsis. (A) Correlation of soluble CXCL16 levels with Sequential Organ Failure Assessment (SOFA) scores in septic patients. (B) Correlation of soluble CXCL16 levels with white blood cells (WBC) in septic patients. (C) Correlation of soluble CXCL16 levels with C- reactive protein (CRP) levels in septic patients. Spearman's correlation coefficient was performed to analyze the correlation between the levels of CXCL16 and SOFA scores, WBC, or CRP levels. Horizontal bars represent median values, and dots represent individual participants.

Article Snippet: Recombinant murine CXCL16 protein (R&D systems, Catalog # 503-CX-025) was administered i.p. into septic C57BL/6 mice immediately after nonsevere CLP.

Techniques:

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 3. Receiving operating characteristic (ROC) curve analysis of CXCL16, SOFA score, PCT, and CRP at admission for predicting 28-day mortality in septic patients. Area under the ROC curve: 0.717 ([95% CI] 0.497e0.939) for CXCL16; 0.849 ([95% CI] 0.715e0.982) for SOFA score; 0.617 ([95% CI] 0.429e0.805) for PCT; and 0.521 ([95% CI] 0.338e0.705) for CRP.

Article Snippet: Recombinant murine CXCL16 protein (R&D systems, Catalog # 503-CX-025) was administered i.p. into septic C57BL/6 mice immediately after nonsevere CLP.

Techniques:

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 4. CXCL16 worsened cecal ligation and puncture (CLP)-induced nonsevere sepsis. (A) Survival of CLP mice (n ¼ 12 per group) after treatment with recombinant CXCL16 protein in the absence or presence of CLP-induced nonsevere sepsis. Murine recombinant CXCL16 protein was administered intraperitoneally at 0.5e1.0 mg/injection immediately after nonsevere CLP, and PBS was used as a control. Comparison between groups was performed using KaplaneMeier analysis followed by log-rank tests. Results are representative of three independent experiments. p < 0.001 when compared with septic mice treated with PBS control. (B) Representative examples of hematoxylin and eosinestained lung, liver, and kidney tissues from nonsevere CLP mice (n ¼ 6) treated with or without recombinant CXCL16 (0.5 mg/injection). (C) Histological scores for lung, liver, and kidney in nonsevere CLP mice (n ¼ 6) treated with or without recombinant CXCL16 (0.5 mg/injection). p < 0.05, p < 0.001when compared with septic mice treated with PBS control (ManneWhitney U test). (D) ALT, AST, LDH, and creatinine levels in nonsevere CLP mice (n ¼ 6), treated with or without recombinant CXCL16 (0.5 mg/injection). p < 0.001 when compared with septic mice treated with PBS control (ManneWhitney U test).

Article Snippet: Recombinant murine CXCL16 protein (R&D systems, Catalog # 503-CX-025) was administered i.p. into septic C57BL/6 mice immediately after nonsevere CLP.

Techniques: Ligation, Recombinant, Injection, Control, Comparison

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 5. Treatment with recombinant murine CXCL16 enhanced inflammatory response in CLP-induced nonsevere sepsis. (A) Numbers of leukocytes including neutrophils, monocytes/macrophages and lymphocytes in peritoneal lavage fluid (PLF) of septic mice treated with or without recombinant murine CXCL16 (0.5 mg/injection). (B) Cytokine and chemokine levels in PLF and blood from septic mice (n ¼ 5) treated with or without recombinant murine CXCL16 (0.5 mg/injection) at 24 h after nonsevere CLP. p < 0.05, p < 0.01, p < 0.001 when compared with septic mice treated with PBS control (ManneWhitney U test).

Article Snippet: Recombinant murine CXCL16 protein (R&D systems, Catalog # 503-CX-025) was administered i.p. into septic C57BL/6 mice immediately after nonsevere CLP.

Techniques: Recombinant, Injection, Control

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 6. Anti-CXCL16 antibody ameliorated cecal ligation and puncture (CLP)-induced severe sepsis. C57BL/6 mice were subjected to severe CLP, and then 10 mg of anti-mouse CXCL16 monoclonal antibody was injected intraperitoneally at 2 h or 6 h after severe CLP. IgG2A control antibody was delivered in a similar way. (A) Survival of septic mice (n ¼ 12 per group) following administration with antieCXCL16 antibody (2 h after CLP) or control antibody after severe CLP. Comparison between groups was performed by KaplaneMeier analysis followed by log-rank tests. Results are representative of three independent experiments. p < 0.05 when compared with septic mice treated with IgG control. (B) Sur- vival of septic mice (n ¼ 12 per group) following administration with antieCXCL16 antibody (6 h after CLP) or control antibody after severe CLP. Comparison between groups was performed by KaplaneMeier analysis followed by log-rank tests. Results are representative of three independent experiments. p < 0.05 when compared with septic mice treated with IgG control. (C) Representative examples of hematoxylin and eosinestained lung, liver, and kidney tissues from septic mice (n ¼ 6) treated with or without anti-CXCL16 neutralizing antibodies (2 h after CLP). (D) Histological scores for lung, liver, and kidney in septic mice (n ¼ 6) treated with or without anti-CXCL16 neutralizing antibodies (2 h after CLP). p < 0.05, p < 0.01 when compared with septic mice treated with isotypical IgG control (ManneWhitney U test). (E) ALT, AST, LDH, and creatinine levels in septic mice (n ¼ 6) treated with or without anti- CXCL16 neutralizing antibodies (2 h after CLP). p < 0.05, p < 0.01 when compared with septic mice treated with isotypical IgG control (ManneWhitney U test).

Article Snippet: Recombinant murine CXCL16 protein (R&D systems, Catalog # 503-CX-025) was administered i.p. into septic C57BL/6 mice immediately after nonsevere CLP.

Techniques: Ligation, Injection, Control, Comparison

Journal: Nature biomedical engineering

Article Title: T cells armed with the C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours

doi: 10.1038/s41551-021-00737-6

Figure Lengend Snippet: ( a ) C-X-C chemokine expression profile of Panc02-OVA tumours quantified by qPCR (n = 6). ( b ) ELISA evaluating murine CXCL16 protein concentrations in different organs of Panc02-OVA tumour-bearing mice (n = 12). ( c ) Expression level of murine CXCL16 by Panc02-OVA tumour cells after stimulation with 20 ng/ml IFN-γ, 20 ng/ml TNF-α or a combination of both, quantified using ELISA. ( d ) CXCL16 levels of CXCL16-knockout Panc02-OVA (n = 10) and CRISPR control Panc02-OVA tumours (n = 15) determined using CXCL16 ELISA. ( e ) CD11c - and CD11c + cells were isolated from Panc02-OVA tumour tissue by FACS sorting, and qPCR was used to analyse CXCL16 expression levels of both populations (n = 10 mice). ( f ) Trans-well migration of GFP- and CXCR6-transduced T cells towards descending concentrations of recombinant murine CXCL16. After 3 h the number of migrated T cells was quantified by flow cytometry. ( g ) Target cell lysis of CXCL16-overexpressing Panc02-OVA by CXCR6- and GFP-transduced OT-1 T cells following migration through a permeable membrane . After a migration period of 3 h, migrated T cells and tumour cells were co-cultured for further 1.5 h. ( h ) ELISA revealing time-dependent activation levels of CXCR6- and GFP-transduced OT-1 T cells upon co-culture with Panc02-OVA tumour cells. E:T ratio 5:1. ( i ) Panc02-OVA tumour cells were co-incubated with GFP- and CXCR6-transduced OT-1 T cells, and lysis of tumour cells was measured after 6.5 h. ( j ) Adherence of GFP- or CXCR6-transduced T cells to a CXCL16-coated (9 pmol) or control BSA-coated (9 pmol) surface. As an additional control, T cells were pre-incubated with soluble recombinant CXCL16 (2 μg/ml). ( k ) Membrane expression of CXCR6 upon stimulation with 200 ng recombinant CXCL16 or CCL1 (arrow) indicating intracellular trafficking and receptor recycling. In vitro experiments ( c, d, f, g, h, I, j ) show mean values ± SEM of at least two biological replicates and are representative of three independent experiments (n = 3). p-values are based on two-sided unpaired t-test. Data shown in k are representative for two independent experiments (n = 2). Ex vivo experiments shown are representative of n = 2 ( a, d ) or n = 3 ( b, e ). Data shown in e are comprised of two independent experiments (n = 2). Analyses of differences between groups were performed using unpaired Mann-Whitney test.

Article Snippet: Number C34554, ThermoScientific, Darmstadt) and pre-incubated with or without 9 pmol recombinant mouse CXCL16 (Cat.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Knock-Out, CRISPR, Control, Isolation, Migration, Recombinant, Flow Cytometry, Lysis, Membrane, Cell Culture, Activation Assay, Co-Culture Assay, Incubation, In Vitro, Ex Vivo, MANN-WHITNEY

Journal: Nature biomedical engineering

Article Title: T cells armed with the C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours

doi: 10.1038/s41551-021-00737-6

Figure Lengend Snippet: ( a ) Tumour growth curves of Panc02-OVA-bearing mice with adoptive transfer of 10 7 GFP- or CXCR6-transduced OT-1 T cells (n= 5 mice per group). T cells were transferred when tumours were palpable (day 5). 2 out of 5 mice treated with CXCR6-transduced T cells showed complete response (CR). ( b ) C57BL/6 mice inoculated with CXCL16-knockout Panc02-OVA (clone 55) or ( c ) CRISPR control Panc02-OVA (clone 50) were treated with a single i.v. injection of 10 7 GFP- or CXCR6-transduced OT-1 T cells (n = 5-12 mice per group). ( d ) Tumour growth of subcutaneous E.G7-OVA-CXCL16 tumours following treatment with a single injection of 10 7 mCherry- or CXCR6-transduced OT-1 T cells (n = 4-5 mice per group). ( e ) Tumour growth of subcutaneous Panc02-OVA-pCAM tumour with adoptive transfer of 10 7 T cells transduced with either anti-EpCAM-CAR or anti-EpCAM-CAR-CXCR6 (n = 5 mice per group). ( f ) Tumour growth of subcutaneous Panc02-OVA-EpCAM tumours with adoptive transfer of 10 7 T cells transduced with anti-EpCAM-CAR, anti-EpCAM-CAR-CXCR3, anti-EpCAM-CAR-CXCR6 or anti-EpCAM-CAR-CCR4 (n = 10-14 mice per group). Experiments shown are representative of two ( b, c, d, e, f ) or three independent ( a ) experiments. Analyses of differences between groups were performed using two-way ANOVA with correction for multiple testing by the Bonferroni method.

Article Snippet: Number C34554, ThermoScientific, Darmstadt) and pre-incubated with or without 9 pmol recombinant mouse CXCL16 (Cat.

Techniques: Adoptive Transfer Assay, Knock-Out, CRISPR, Control, Injection, Transduction

Journal: Nature biomedical engineering

Article Title: T cells armed with the C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours

doi: 10.1038/s41551-021-00737-6

Figure Lengend Snippet: ( a ) Secretion of CXCL16 by human pancreatic cancer cells was measured by ELISA. ( b ) Migration capability of GFP- and CXCR6-transduced human T cells toward Capan-1 and MSLN-CXCL16-overexpressing SUIT-2 supernatants. The number of migrated cells was normalized to the medium control condition. ( c, d) Number of sphere-penetrating GFP- and CXCR6-transduced human T cells and infiltration depth into spheres formed by HEK overexpressing human CXCL16. ( e ) In a combined migration-cytotoxicity assay anti-MSLN-CAR and anti-MSLN-CAR-CXCR6-transduced human T cells are compared with regard of specific migratory and cytotoxic efficiency. T cells migrated towards MSLN-CXCL16-overexpressing SUIT-2 tumour cells followed by CAR-mediated cytotoxicity presented by real-time target cell lysis. ( f - i ) In a subcutaneous xenograft model, MSLN-CXCL16-overexpressing SUIT-2 tumour bearing mice were treated with GFP-transduced ( f ), anti-MSLN-CAR-transduced ( g ) or anti-MSLN-CAR-CXCR6 co-transduced T cells ( h ). Tumour growth and survival was measured over 110 days (n = 10 mice per group). One mouse of the anti-MSLN-CAR-CXCR6 treated group was sacrificed on day 103 post tumour injection due to (unclear) neck swelling, presumably unrelated to subcutaneous tumour injection and was therefore censored at the timepoint. ( i ). ( j ) For orthotopic treatment experiments, SUIT-2-MSLN-CXCL16 tumour cells were implanted into the pancreas. Mice were treated with a single i.v. injection of either non-transduced human T cells, anti-MSLN-CAR- or anti-MSLN-CAR-CXCR6-transdcued T cells. Tumour growth and survival was monitored over 115 days (n = 17-20 mice per group). In vitro experiments ( b, e ) show mean values ± SEM of at least two biological replicates and are representative of three independent experiments (n = 3). Data shown in a, c and d are comprised of three independent experiments (n = 3) each with three biological replicates. In vivo experiments ( f – j ) are summarized from two independent experiments. p-values are based on two-sided unpaired t-test or two-way ANOVA with correction for multiple testing by the Bonferroni method. Comparison of survival rates was performed with the Log-rank (Mantel-Cox) test.

Article Snippet: Number C34554, ThermoScientific, Darmstadt) and pre-incubated with or without 9 pmol recombinant mouse CXCL16 (Cat.

Techniques: Enzyme-linked Immunosorbent Assay, Migration, Control, Cytotoxicity Assay, Lysis, Injection, In Vitro, In Vivo, Comparison

Journal: Nature biomedical engineering

Article Title: T cells armed with the C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours

doi: 10.1038/s41551-021-00737-6

Figure Lengend Snippet: ( a ) Gene expression analysis (mRNA) of pancreatic cancer specimens in comparison to healthy pancreatic tissue (n = 36 PDAC patients and n = 12 healthy controls) using NanoString nCounter® System. ( b ) TCGA data analysis comparing CXCL16 expression (mRNA) by PDAC and healthy tissue (n = 178 PDAC patients and n = 165 healthy controls). ( c ) Representative images of PDAC specimens stained for CXCL16. ( d) Quantification of CXCL16 expression by tumour cells and tumour-infiltrating immune cells validated by immunohistochemical staining of CXCL16 in PDAC specimens (n = 399 with three biopsies per patient). ( e ) Single cell RNA (scRNA) sequencing analysis of PDAC and healthy pancreas tissue comparing CXCL16 expression levels. ( f ) CXCL16 levels in plasma of PDAC patients and healthy specimens quantified by ELISA (n = 10 PDAC patients and n = 11 healthy specimens). ( g ) Activation level of human T cells (quantified by IFN-γ concentrations) following co-culture with pancreatic cancer PDO (summarized data from independent co-cultures with 3 different PDO specimens: B34, B54 and B61; n = 3). ( h ) Representative images showing confocal analysis of pancreatic cancer PDO (specimen B61) infiltrated by GFP- or CXCR6-transduced T cells. ( i ) Quantification of GFP- and CXCR6-transduced T cells penetrating into PDO (summarized from specimen B34 and B48; n = 2) in the absence or presence of an anti-CXCL16 neutralization antibody. ( j ) For PDX experiments, PDO (MGH1247) were heterotopically implanted in NCG mice and treated with non-transduced, anti-MSLN-CAR or anti-MSLN-CAR-CXCR6-transduced T cells. Tumour growth was monitored for 35 days post ACT (n = 5 mice per group). ( k ) PDX tumour weight after treatment with either non-transduced, anti-MSLN-CAR or anti-MSLN-CAR-CXCR6-transduced T cells. ( l ) Quantification of GFP- and CXCR6-transduced T cells after penetration into surgical ovarian cancer (OC) specimens of seven patients. Analyses of differences between groups in a , b and f were performed using unpaired Mann-Whitney test. Data shown in g are comprised of three independent experiments (n = 3). Data shown in i are comprised of two independent experiments with mean values ± SEM of at least 10 organoids per condition ( i ; n = 2). p-values are based on two-sided unpaired t-test. Data shown in j, k and l resulted from one single experiment (n = 1). Differences in tumour growth were analysed by using two-way ANOVA with correction for multiple testing by the Bonferroni method and differences in tumour weight were analysed by using unpaired Mann-Whitney test. Data shown in l are mean values ± SEM of seven OC specimen co-cultures with the same T cell donor. p-vales in k are based Wilcoxon signed-rank test.

Article Snippet: Number C34554, ThermoScientific, Darmstadt) and pre-incubated with or without 9 pmol recombinant mouse CXCL16 (Cat.

Techniques: Gene Expression, Comparison, Expressing, Staining, Immunohistochemical staining, Sequencing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Activation Assay, Co-Culture Assay, Neutralization, MANN-WHITNEY