anti nectin2 neutralizing antibody  (R&D Systems)

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts serve as decoys to suppress NK cell anti-cancer cytotoxicity

doi: 10.1101/2023.11.23.568355

Figure Lengend Snippet: (A-B) NK cells from BALB/c or C57BL/6 mice were activated for 24 hours, and then incubated with NMFs, pCAFs, or sCAFs at a 5:1 NK:fibroblast ratio to determine cytotoxicity. NK cells were treated with either 50μg/ml IgG control or combinations of 50μg/ml α-NKG2D/DNAM1 antibodies to determine dependency on either receptor for cytotoxicity of fibroblast targets. The relative cytotoxicity (normalized to cytotoxicity of NK cells activated with IgG control) is presented (N=3 biologically independent experiments, data is presented as mean ± SEM). (C) NK cells from BALB/c mice were activated for 24 hours alone or in the presence of pCAFs that were pre-incubated with either 50μg/ml IgG control or combinations of 50μg/ml α-CD155, Nectin2, and Rae-1 antibodies. Subsequently, NK cells were subjected to FACS staining for NKG2D and DNAM-1. Quantification of the FACS experiments is shown in the top panel. N=5 biologically independent experiments, data is presented as mean ± SEM. The representative histograms (bottom) were normalized to the modal value. (D) NK cells from BALB/c mice were activated for 24 hours alone or as in (C), and then incubated with 4T1 cancer cells at a 5:1 NK:cancer ratio to determine cytotoxicity. Cytotoxicity (normalized to cytotoxicity of NK cells activated alone) is presented. N=6 biologically independent experiments, data is presented as mean ± SEM.

Article Snippet: For CAF ligand blockade experiments, CD155, Nectin2, and Pan-Rae-1 expressed on CAFs were blocked utilizing 25 μg/ml of anti-mouse pan Rae-1 (R&D,AF1136), Ultra-LEAFTM anti-mouse CD155 (Biolegend, 942103), anti-mouse Nectin2 (R&D, MAB3869), or Mouse IgG1 Isotype Control control (R&D, MAB002) for 15 minutes at room temperature prior to culture with NK cells.

Techniques: Incubation, Control, Staining

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts serve as decoys to suppress NK cell anti-cancer cytotoxicity

doi: 10.1101/2023.11.23.568355

Figure Lengend Snippet: (A) Representative IHC staining of NK cells (NKp46) in TMAs of TNBC patient cohorts. (B) Quantification of IHC images was conducted on the proportion of NK cells in stromal areas compared to cancer specified regions (Exact Wilcoxon rank sum test p−value = 0.00005) (C-D) Representative immunofluorescence images of 2 patient TMAs. Staining was conducted on DAPI, cytokeratin (cancer cells), CD45 (immune cells), and NK cell ligands NECTIN2, PVR, and MICA/B. (E) Kaplan-Meir survival analysis of patients stained in (C-D). Analysis was conducted on the ratio of CAFs expressing NECTIN2 out of the total CAFs, compared to the ratio of positive cancer cells out of total cancer cells, with groups stratified above (high) or below (low) the median value. Statistical analysis was conducted using two-sided log-rank test. (F) Scheme of proposed model. Reduction of NK cell receptors following engagement with CAFs may result in impaired cytolysis of cancer cells by NK cells in the TME

Article Snippet: For CAF ligand blockade experiments, CD155, Nectin2, and Pan-Rae-1 expressed on CAFs were blocked utilizing 25 μg/ml of anti-mouse pan Rae-1 (R&D,AF1136), Ultra-LEAFTM anti-mouse CD155 (Biolegend, 942103), anti-mouse Nectin2 (R&D, MAB3869), or Mouse IgG1 Isotype Control control (R&D, MAB002) for 15 minutes at room temperature prior to culture with NK cells.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Expressing

Journal: Nature Communications

Article Title: Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma

doi: 10.1038/s41467-021-24010-1

Figure Lengend Snippet: a We examined the immune checkpoint interactions between lymphocytes and APCs (tumor cells and TAMs) and identified the prominent interaction via the TIGIT – NECTIN2 axis (circle size indicates the statistical significance and circle color indicates the level of interaction). The empirical P value was estimated by 1000 imputations. b The expression of TIGIT and NECTIN2 was respectively enriched in T cells and APCs. c Upregulation of NECTIN2 was detected in HCC tumors, as compared to non-tumorous livers in both in-house and TCGA datasets. Student’s t test (2-sided). Source data are provided as a Source Data file.

Article Snippet: In the coculturing experiment of T and parental Hepa1–6 cells, 15 μg/mL anti-Nectin2 neutralizing antibody (MAB3869, R&D Systems, MN, USA) (Supplementary Table ) was added.

Techniques: Expressing

Journal: Nature Communications

Article Title: Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma

doi: 10.1038/s41467-021-24010-1

Figure Lengend Snippet: a CellTrace Violet (CTV)-labeled mouse splenic T cells were isolated and co-cultured with Hepa1–6 cells in the presence or absence of anti-Nectin2 neutralizing antibody (15 µg/mL). Mean ± SD is presented. b CTV-labeled T cells were cocultured with Hepa1–6 (WT), - Nectin2 -KO1, - Nectin2 -KO2, - Nectin2 -KO3 cells. Mean ± SD is presented. c Representative picture and weight of Nectin2 WT ( Nectin2 WT: Tp53 KO: c-Myc OE), and Nectin2 KO ( Nectin2 KO: Tp53 KO: c-Myc OE) HCC tumors. Scale bar = 1 cm. d – f Numbers of tumor-infiltrating lymphocytes were analyzed by flow cytometry. g , h Representative pictures, and quantification of CD4 + T cells and CD8 + T cells in HCC tumors by IHC staining. Scale bar = 100μm in IHC representative pictures. a – h Student’s t test. The experiment was performed with a variable number of biologically independent samples ( n number) ( a n = 6, n = 3, and n = 3 for T cells only Ctrl and anti-Nectin2 respectively in CD4+ cells, and n = 3 for all groups in CD8+ cells; b n = 10, n = 6, n = 4, and n = 4 for WT, Nectin2 -KO1, Nectin2 -KO2, and Nectin2 -KO3, respectively in both CD4+ and CD8+ cells; c – e , g : n = 7 per group; f , h : n = 21 per group). Source data are provided as a Source Data file.

Article Snippet: In the coculturing experiment of T and parental Hepa1–6 cells, 15 μg/mL anti-Nectin2 neutralizing antibody (MAB3869, R&D Systems, MN, USA) (Supplementary Table ) was added.

Techniques: Labeling, Isolation, Cell Culture, Flow Cytometry, Immunohistochemistry