Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions

doi: 10.1136/jitc-2020-001054

Figure Lengend Snippet: Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) protein expression in tumor infiltrating natural killer (NK) cells. (A, B) Identification of CTLA4-expressing NKp46 + cells in patients with non-small cell lung carcinoma (NSCLC) by immunofluorescence (A) or immunohistochemistry (B) double staining. (C, D) CTLA4 protein expression was analyzed in NK cells (CD3 − CD56 + ), conventional T cells (Tconv) (CD3 + CD56 − Foxp3 − ) and regulatory T cells (Tregs) (CD3 + CD56 − Foxp3 + ) by flow cytometry after intracellular or cell surface staining of cells from tumorous (Tum) or non-tumorous (Non-Tum) tissue of patients with NSCLC. Cells from blood were also analyzed for some patients. Representative images of intracellular CTLA4 staining are shown in (C) and the summary of analyzes are shown in (D). Statistical analyses were performed by Wilcoxon method with the GraphPad software. ns: not significant.

Article Snippet: Thereafter, sections were incubated with Protein Bock solution (Dako) for 30 min and incubated with mouse anti-human NKp46 (clone 195314, R&D Systems, 5 μg/mL) and/or goat anti-human CTLA4 mAb (AF-386-PB, R&D Systems, 2.5 μg/mL) for 1 hour at room temperature.

Techniques: Expressing, Immunofluorescence, Immunohistochemistry, Double Staining, Flow Cytometry, Staining, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions

doi: 10.1136/jitc-2020-001054

Figure Lengend Snippet: Intratumoral NK cells phenotype and cytokine secretion. (A) Eomes, NKG2A, CD69, NKp44, CD107a, FS7-associated cell surface antigen (FAS), C-X-C chemokine receptor type 6 (CXCR6), CX3CR1 and sphingosine-1-phosphate receptor 1 (S1PR1) protein expression were analyzed in natural killer (NK) cells (CD3 − CD56 + ) by flow cytometry after cell surface staining of cells from non-tumorous (Non-Tum) or tumorous (Tum) tissue of patients with non-small cell lung carcinoma. Percentages or GeoMean is presented. (B) Co-staining for NKp46/Eomes, NKp46/cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and CTLA4/NKG2A is shown on CD3 − CD56 + intratumoral NK cells. (C) Quantification of cytokines produced by NK cells sorted from Tum or Non-Tum tissue. Statistical analyses were performed by Wilcoxon method with the GraphPad software. ns: not significant. *p<0.05; **p<0.01; ***p<0.001; p<0.0001.

Article Snippet: Thereafter, sections were incubated with Protein Bock solution (Dako) for 30 min and incubated with mouse anti-human NKp46 (clone 195314, R&D Systems, 5 μg/mL) and/or goat anti-human CTLA4 mAb (AF-386-PB, R&D Systems, 2.5 μg/mL) for 1 hour at room temperature.

Techniques: Expressing, Flow Cytometry, Staining, Produced, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions

doi: 10.1136/jitc-2020-001054

Figure Lengend Snippet: Intratumoral natural killer (NK) cells reduces dendritic cell (DC) maturation. (A, B) major histocompatibility complex (MHC)-II and CD86 surface expression was analyzed by flow cytometry on lipopolysaccharide (LPS)-treated CD11c + DC after 2–3 days of co-culture with CD3 − CD56 + intratumoral NK cells. Experimental design of the co-culture experiment is shown in (A). (B) Expression of CD86 or MHC-II expression on LPS-treated DC alone (DC alone) or in co-culture with CD56 + cells (DC +CD56 + cells) was analyzed. Data are represented as a ratio of mean fluorescence intensity of CD86 or MHC-II expression in DC alone (blue dots) versus DC +CD56 + cells co-culture (red dots). (C) The ratio of MHC-II surface protein expression on LPS-treated DC were analyzed by flow cytometry after 3–4 days of culture of DC cells alone (blue dots) or in co-culture with CD56 + cells (red dots) and in the presence of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) blocking antibody (green triangles). Statistical analyses were performed by the Wilcoxon non-parametric test with the GraphPad software. ns: not significant.

Article Snippet: Thereafter, sections were incubated with Protein Bock solution (Dako) for 30 min and incubated with mouse anti-human NKp46 (clone 195314, R&D Systems, 5 μg/mL) and/or goat anti-human CTLA4 mAb (AF-386-PB, R&D Systems, 2.5 μg/mL) for 1 hour at room temperature.

Techniques: Immunopeptidomics, Expressing, Flow Cytometry, Co-Culture Assay, Fluorescence, Blocking Assay, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions

doi: 10.1136/jitc-2020-001054

Figure Lengend Snippet: Correlation between cytotoxic T-lymphocyte-associated protein 4 (CTLA4) expression and gene signature expression in tumor versus non-tumorous NK cells Sorted NK cells from non-tumorous distant tissue (Non-Tum NK) and tumorous (Tum NK) for 47 patients were extracted and total RNA was analyzed. CTLA4 mRNA expression was then compared with Ct values of the 14 genes previously identified. The correlation between both values was assessed in non-tumorous and tumorous NK cells using Pearson correlation test with the GraphPad software.

Article Snippet: Thereafter, sections were incubated with Protein Bock solution (Dako) for 30 min and incubated with mouse anti-human NKp46 (clone 195314, R&D Systems, 5 μg/mL) and/or goat anti-human CTLA4 mAb (AF-386-PB, R&D Systems, 2.5 μg/mL) for 1 hour at room temperature.

Techniques: Expressing, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions

doi: 10.1136/jitc-2020-001054

Figure Lengend Snippet: Clinical impact of NKp46 + cells in patients with non-small cell lung carcinoma. (A, B) Patients from the retrospective cohort (cohort 3) were splitted into two groups according to the density of intratumoral NKp46 + cells (A) or CD8 + cells (B). Separation was done by median and their overall survival was analyzed. (C) Patients with low density (CD8 + cells Low ) or high density (CD8 + cells High ) of CD8 + cells were splitted into two groups according to their number of intratumoral Nkp46 + cells and the overall survival were done in each group. Separation was done by median. (D, E) CD8 density of patients from the validation cohort (cohort 2) was assessed by immunohistochemistry on paraffin-embedded slides and the correlation with cytotoxic T-lymphocyte-associated protein 4 (CTLA4) mRNA expression in natural killer (NK) cells was calculated using Pearson correlation test with the GraphPad software (D). (E) Patients from validation cohort (cohort 2) were split in two groups according to the density of intratumoral CD8 + cells (using median) and the expression of CTLA4 mRNA was analyzed in each group. Statistical analyses were performed by the Mann-Whitney non-parametric test with the GraphPad software.

Article Snippet: Thereafter, sections were incubated with Protein Bock solution (Dako) for 30 min and incubated with mouse anti-human NKp46 (clone 195314, R&D Systems, 5 μg/mL) and/or goat anti-human CTLA4 mAb (AF-386-PB, R&D Systems, 2.5 μg/mL) for 1 hour at room temperature.

Techniques: Biomarker Discovery, Immunohistochemistry, Expressing, Software, MANN-WHITNEY