Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed in BM-DCs and LECs and supports DC transmigration. ( A ) Flow cytometry analysis of immature (−LPS) and LPS-matured (+LPS) BM-DCs (gated on live/single cells). ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression of 11 independent experiments. ( C – F ) FACS analysis of CD112 expression in ( C ) LPS-matured BM-DCs and ( E ) primary LN-LECs, derived from WT and CD112 KO mice. ( D , F ) Summary of the ∆MFI values of CD112 expression of 4–6 independent experiments. Data points of the same experiment in ( B , D , F ) are connected by a line, and the mean ΔMFI values are indicated by horizontal lines. ( G ) Set up of the transmigration experiments to investigate the transmigration of BM-DCs (WT or KO) across an LEC monolayer (WT or KO). ( H ) Impact of ICAM-1 blockade on transmigration of WT BM-DCs. ( I,J ) Impact of loss of CD112 in either ( I ) LECs or ( J ) BM-DCs on transmigration. ( K ) Impact of simultaneous loss of CD112 in LECs and BM-DCs on transmigration. For each condition in ( H – K ), one representative experiment with n = 3 technical replicates is shown on the left, and a summary of the averages of 4 independent experiments (biological replicates, each experiment in a different color) is shown on the right. Data points of the same experiment are connected by a line. ( L ) Adhesion assay of WT and KO BM-DCs to WT or KO lymphatic endothelium. The pool of two independent experiments with three replicates per condition is shown (each dot represents a sample). # BM-DCs: number of BM-DCs. Data in all graphs show mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Transmigration Assay, Flow Cytometry, Staining, Expressing, Derivative Assay, Cell Adhesion Assay

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 expression is high in LECs but low in DCs present in murine skin. ( A , B ) FACS analysis was performed to detect CD112 expression in dermal LECs and BECs. ( A ) Depiction of the gating strategy in one representative experiment. ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression observed in 5 independent experiments. ( C – G ) Impact of TPA-induced skin inflammation on the expression of CD112 in LECs. ( C ) Schematic depiction of the experiment: Inflammation was induced in the murine ear skin by topical application of TPA and the ear skin and draining auricular LNs analyzed 24 h later. ( D – G ) FACS analyses were performed to quantify CD112 expression levels in LECs present in control or inflamed tissues. ( D , E ) Analysis of murine ear skin and ( F , G ) auricular LN single-cell suspensions. ( E , G ) The summary of ∆ MFI values was recorded in 5–6 different experiments performed in one control (CTL) and one TPA-inflamed (TPA) ear skin. ( H , I ) FACS gating and quantification of CD112 expression in DCs present in CTL and TPA-inflamed ear skin. ( H ) Gating strategy and ( I ) summary of ∆MFI values recorded in 3 different experiments. ( J – P ) Crawl-out experiments. ( J ) Schematic depiction of the experiment performed to evaluate CD112 expression in ( K – M ) DCs that had emigrated from murine ear skin into the culture medium or in ( N – P ) DCs that had remained in the cultured ear skin at the end of the experiment. Representative ( K , N ) FACS dot plots (gating on single/live cells), identifying DCs as MHCII + CD11c + cells. ( L , O ) Representative histogram plots showing CD112 expression in WT and KO DCs as well as the corresponding fluorescence minus one (FMO) control. ( M , P ) Summary of ∆MFI values (defined as specific staining—FMO) recorded in 4 different experiments performed with one WT and one KO mouse each. Data points in ( B , E , G , I , M , P ) of the same experiment are connected by a line.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Expressing, Staining, Control, Cell Culture, Fluorescence

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Loss of CD112 does not impact the in vivo migration of adoptively transferred or endogenous DCs to dLNs. ( A – D ) Adoptive transfer experiment. ( A ) Scheme of the experiment. ( B ) Gating strategy to identify fluorescently labeled adoptively transferred BM-DCs in popliteal LNs. ( C ) The ratio of KO–WT DCs recovered from popliteal LNs draining control (CTL) or CHS-inflamed (CHS) footpads of WT or KO mice. ( D – J ) FITC painting experiment. ( D ) Scheme of the experiment. ( E ) ΔEar thickness, defined as the difference between the ear thickness measured at the start and at the end of the experiment. ( F ) Cellularity and ( G ) weight of the ear-draining auricular LN at the end of the experiment. ( H ) Gating strategy to identify and quantify the number (#) of ( I ) all CD11c + MHCII hi migratory DCs (mDCs) and ( J ) FITC + mDCs. Summaries of three ( A – D ) and two ( D – J ) independent experiments, each with 2–7 mice per condition, are shown. Each dot represents one mouse. Mann–Whitney t -test was used. Red bars in all graphs show the mean. ns: not significant.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: In Vivo, Migration, Adoptive Transfer Assay, Labeling, Control, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Blockade of CD112 decreases in vitro transmigration of human moDCs across human dermal LEC monolayers. ( A – C ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in in vitro-differentiated ( A ) immature (−LPS) and ( B ) LPS-matured (+LPS) human moDCs. LPS was added 24 h prior to FACS analysis. Representative FACS plots are shown in ( A , B ). ( C ) Summary of the delta mean fluorescent intensity (∆MFI; defined as specific-isotype staining) values recorded for each corresponding marker in 3–6 independent experiments (biological replicates). Data points of the same experiment are connected by a line, and the means of the ΔMFI values are indicated by horizontal red lines. ( D , E ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in primary human dermal LECs. ( D ) Representative FACS histograms recorded upon gating on CD31 + podoplanin + cells, and ( E ) summary of the MFI values recorded for all markers and corresponding isotype controls in 4–5 independent experiments performed on LECs from two different donors. Data points of the same experiment are connected by a line, and the means of the MFI values are indicated by horizontal red lines. ( F – I ) Transmigration experiments involving human moDCs and human dermal LECs, performed in the presence/absence of ( F , G ) αICAM-1 or of ( H , I ) αCD112 or the corresponding isotype controls; ( F – I ) The number of transmigrated DCs (# DCs) was assessed. ( F , H ) show representative results from one representative experiment with n = 6 technical replicates per condition. ( G , I ) show the summaries of four independent experiments (i.e., different biological replicates, shown with different colors) with 3–6 replicates per condition. The averages from each experiment are connected by a line. The standard error of the mean (SEM) is shown; the Mann–Whitney t -test was used. * p < 0.05; ** p < 0.01.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: In Vitro, Transmigration Assay, Expressing, Staining, Marker, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed by DCs and LECs in human skin. ( A – D ) FACS-based analysis of CD112 expression in endothelial cells and DCs present in human skin. ( A , C ) Gating strategy used to detect CD112 expression in ( A ) BECs and LECs and ( C ) DCs. ( B , D ) Summary of mean fluorescent intensity (MFI) values of CD112 expression in ( B ) LEC and BECs or ( D ) HLA-DR + CD86 + DCs in 2 independent experiments (i.e., different biological replicates) was analyzed. Data points of the same experiment are connected by a line. ( E , F ) Confocal images of human skin sections depicting ( E ) CD112 expression (white) by dendritic cells (examples indicated by white arrows), identified as HLA-DR + (green) and CD11c + (red). Scale bar = 100 μm ( F ) CD112 expression (white) by lymphatic vessels, LYVE-1 (green) and PLVAP (red). Scale bar = 100 μm. ( G ) Top: Gating strategy and Bottom: representative histogram plot showing CD112 expression on DCs that had emigrated from a human breast skin punch biopsy. ( H ) Crawl-out experiments from punch biopsies derived from either breast or abdominal skin were performed in the presence of a CD112-blocking antibody or media/isotype control (CTL) in the culture medium. Top: Representative FACS gating plot from abdominal skin. Bottom: Quantification of emigrated HLA-DR+CD86 + DCs. Pooled data from 5 independent experiments with 4–10 punches per condition are shown. ( I ) Crawl-out experiment from abdominal skin punch biopsies to verify the expression of CD112-binding partners DNAM-1, TIGIT and CD113 on human DCs, identified as live, HLA-DR + cells. Representative stainings from one out of three independent experiments are shown. The mean and standard deviation (SD) are shown in (H). Mann–Whitney t -test was used. ** p < 0.01.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Expressing, Derivative Assay, Blocking Assay, Control, Binding Assay, Standard Deviation, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Extracellular CIRP Induces Novel Nectin-2+ (CD112+) Neutrophils to Promote Th1 Differentiation in Sepsis.

doi: 10.4049/jimmunol.2200308

Figure Lengend Snippet: FIGURE 1. eCIRP increases the expression of CD112 in neutrophils. (A) A total of 3 × 106 BMDNs were sorted by negative selection using a magnetic column and treated with rmCIRP (1 mg/ml) for 2 h, and mRNA levels of CD112 were assessed by real-time PCR. (B) A total of 2 × 106 BMDNs were treated with rmCIRP at differ- ent time points, and the protein levels of CD112 were assessed by Western blotting. (C) Purified 1 × 106 BMDNs were treated with rmCIRP at different doses and time points, and CD112 protein expression on the surface of BMDNs was assessed using flow cytometry. A representative gating strategy is shown in PBS-treated sample. Total BMDNs were gated for the single granulocyte population based on FSC and SSC. Single granulocytes were subsequently gated for the Ly6G-AF488-positive cell popula- tion and further gated into those staining for CD112-BV421-positive cells in the PBS group. (C and E) Representative histograms of CD112-BV421-positive neutrophil pop- ulation and (D and F) their corresponding bar diagrams representing the mean frequency of CD112 expression are shown at different doses and different time points. n 5 510 samples/group obtained from at least four mice. Experiments were performed two or three times, and all data were used for analysis. Data are expressed as mean ± SEM and compared by Student t test for (A) mRNA and (BF) by one-way ANOVA and Student-Newman-Keuls method for protein expression. *p < 0.05 versus PBS.

Article Snippet: Anti-CD112 Ab (catalog no. MAB3869) was purchased from R&D Systems (Minneapolis, MN), and anti-b-actin Ab (catalog no. A5441) was purchased from SigmaAldrich (St. Louis, MO).

Techniques: Expressing, Selection, Real-time Polymerase Chain Reaction, Western Blot, Purification, Flow Cytometry, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Extracellular CIRP Induces Novel Nectin-2+ (CD112+) Neutrophils to Promote Th1 Differentiation in Sepsis.

doi: 10.4049/jimmunol.2200308

Figure Lengend Snippet: FIGURE 2. eCIRP induces CD112 expression on neutrophils through the TLR4 pathway. (A and B) Purified BMDNs from WT or TLR4−/−mice were treated with 1 mg/ml of rmCIRP for 2 h, and CD112 expression on the BMDNs was assessed using flow cytometry. (C and D) BMDNs were pretreated with 10 mg/ml of anti-TLR4-neutralizing Ab or isotype IgG Ab for 30 min and then stimulated with 1 mg/ml of rmCIRP for 2 h. (A and C) Representative histo- grams indicating CD112-BV421-positive neutrophils using the previous gating and (B and D) the corresponding bar diagrams of the frequency of CD1121

Article Snippet: Anti-CD112 Ab (catalog no. MAB3869) was purchased from R&D Systems (Minneapolis, MN), and anti-b-actin Ab (catalog no. A5441) was purchased from SigmaAldrich (St. Louis, MO).

Techniques: Expressing, Purification, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Extracellular CIRP Induces Novel Nectin-2+ (CD112+) Neutrophils to Promote Th1 Differentiation in Sepsis.

doi: 10.4049/jimmunol.2200308

Figure Lengend Snippet: FIGURE 3. In vivo administration of rmCIRP increases frequencies of CD1121 neutrophils in blood and spleen. After 4 h of rmCIRP i.p. injection, the blood and spleen were collected. (A) A representative sample shows the flow cytometry gating strategy in blood. Blood cells were gated for the single granulocytes based on FSC and SSC characteristics and then gated into the Ly6G-AF488- and CD112-BV421-positive cell population. Representative his- tograms of CD112 expression on neutrophils are shown. (B) Flow cytometric analysis representing the frequencies of CD1121 neutrophils in blood is shown. (C) Representative samples providing flow cytometry gating strategy and (D) their flow cytometric analysis of CD1121 neutrophils in spleen are shown. n 5 7 samples/group. Experiments were performed twice, and all data were used for analysis. Data are expressed as mean ± SEM and compared by Student t test. *p < 0.05 versus vehicle.

Article Snippet: Anti-CD112 Ab (catalog no. MAB3869) was purchased from R&D Systems (Minneapolis, MN), and anti-b-actin Ab (catalog no. A5441) was purchased from SigmaAldrich (St. Louis, MO).

Techniques: In Vivo, Injection, Flow Cytometry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Extracellular CIRP Induces Novel Nectin-2+ (CD112+) Neutrophils to Promote Th1 Differentiation in Sepsis.

doi: 10.4049/jimmunol.2200308

Figure Lengend Snippet: FIGURE 4. CIRP−/−mice contain decreased amounts of CD1121 neutrophils in sepsis. WT and CIRP−/−mice underwent CLP or sham operation, and the spleen and blood were collected 20 h later. Representative histograms of CD112 expression on neutrophils in (A) blood and (C) spleen are shown. Flow cyto- metric analysis representing the frequencies of CD1121 neutrophils in (B) blood and (D) spleen are shown. n 5 78 samples/group. Experiments were per- formed three times, and all data were used for analysis. Data are expressed as mean ± SEM and compared by one-way ANOVA and Student-Newman-Keuls test. *p < 0.05 versus WT sham; #p < 0.05 versus WT CLP.

Article Snippet: Anti-CD112 Ab (catalog no. MAB3869) was purchased from R&D Systems (Minneapolis, MN), and anti-b-actin Ab (catalog no. A5441) was purchased from SigmaAldrich (St. Louis, MO).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Extracellular CIRP Induces Novel Nectin-2+ (CD112+) Neutrophils to Promote Th1 Differentiation in Sepsis.

doi: 10.4049/jimmunol.2200308

Figure Lengend Snippet: FIGURE 5. CD1121 neutrophils promote Th1 differentiation. A total of 5 × 105 naive CD41 T cells were cocultured with 5 × 105 BMDNs pretreated with PBS or rmCIRP (1 mg/ml) for 2 h in the presence of CD112 or isotype IgG Abs. After 20 h of coculture, the cells were washed with complete RPMI medium and restimulated with 5 mg/ml of brefeldin A and cell activation mixture for 5 h. IFN-g/IL-4 expression in T cells was assessed by flow cytometry. (A) A representative sample showing the flow cytometry gating strategy. Total cells were gated for the single lymphocyte population based on FSC and SSC characteristics. Single lymphocytes were subsequently gated for the CD4-FITC-positive cell population and further gated into those staining for IFN-g- BV421- and IL-4-PE-positive cells. (B) IFN-g production and (C) IL-4 production in CD4 T cells cocultured with rmCIRP-treated neutrophils in the presence of anti-CD112 Ab. n 5 58 samples/group obtained from at least five mice. Experiments were performed three times, and all data were used for analysis. Data are expressed as mean ± SEM and compared by one-way ANOVA and Student-Newman-Keuls test. *p < 0.05 versus PMA/In. activation and untreated neutrophil group; #p < 0.05 versus PMA/In. rmCIRP-treated neutrophil and isotype IgG groups. In., ionomycin.

Article Snippet: Anti-CD112 Ab (catalog no. MAB3869) was purchased from R&D Systems (Minneapolis, MN), and anti-b-actin Ab (catalog no. A5441) was purchased from SigmaAldrich (St. Louis, MO).

Techniques: Activation Assay, Expressing, Flow Cytometry, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Extracellular CIRP Induces Novel Nectin-2+ (CD112+) Neutrophils to Promote Th1 Differentiation in Sepsis.

doi: 10.4049/jimmunol.2200308

Figure Lengend Snippet: FIGURE 8. Summary of findings. eCIRP increases CD112 mRNA and protein expression in neutrophils via TLR4 in sepsis. eCIRP-induced CD1121

Article Snippet: Anti-CD112 Ab (catalog no. MAB3869) was purchased from R&D Systems (Minneapolis, MN), and anti-b-actin Ab (catalog no. A5441) was purchased from SigmaAldrich (St. Louis, MO).

Techniques: Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: TIGIT but not DNAM1, CD112R or CD96—interacts with Nectin4. (A) FACS staining of IL-2 activated primary NK cells with Nectin4-Ig. Gray filled histogram represents background staining with secondary antibody only; black line histogram represents specific binding as indicated. (B–F) FACS staining of Raji cells transfected either with an empty vector as control (gray histograms) or with Nectin4 (black histograms). Cells were stained with commercial anti-Nectin4 mAb (B), TIGIT-Ig (C), DNAM1-Ig (D), CD112-Ig (E) or CD96-Ig (F). Figures show one representative experiment out of three performed. Graph depicting the mean fluorescence intensity values of the stainings appears in . (G) Direct binding of Nectin4 to TIGIT. The binding of fluorophore-labeled TIGIT-Ig and its ligands PVR-Ig (red) and Nectin4-Ig (green) was determined using MST. Measurements were repeated with at least three independent protein preparations. FACS, fluorescence-activated cell sorting; IL-2, interleukin-2; MST, microscale thermophoresis; NK, natural killer.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Staining, Binding Assay, Transfection, Plasmid Preparation, Control, Fluorescence, Labeling, FACS, Microscale Thermophoresis

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: Nectin4 inhibits NK cytotoxicity via TIGIT. (A, C) IL2 secretion by parental BW (A, C), (A) BW-TIGIT and (C) BW-DNAM1 cells. IL2 secretion was determined by ELISA (od 650 nm) following incubation with control anti-TIGIT or anti-DNAM1 antibodies (left in a and C) or with PVR expressing cells (right in a and C). (B, D) IL2 secretion of parental BW (B, D), (B) BW-TIGIT and (D) BW-DNAM1 cells. IL2 secretion was determined by ELISA (od 650 nm) following incubation with Raji cells transfected either with an empty vector (Raji E) as a control, or with Nectin4 (Raji N4). Figure shows one representative experiment out of 3 performed. *P<0.05. (E) FACS staining of Raji cells overexpressing Nectin4 with TIGIT-Ig. TIGIT-Ig was preincubated with no antibody (left), with a control mAb (anti-CD99 mAb clone 12E7, middle) or with anti-TIGIT blocking antibody (mAb #4 generated as described previously, right). Black line histograms represent TIGIT-Ig binding. Gray filled histograms represent background staining of the secondary antibody only. (F) Mean fluorescence intensity (MFI) values of the TIGIT-Ig staining shown in (E) relative to NO antibody staining, *p<2×10 -4 . (G) [ 35 S] methionine-labeled Raji cells transfected either with an empty vector as control (Raji empty—gray) or with Nectin4 (Raji Nectin4—black), were incubated for 5 hours with NK cells. NK cells were preincubated with no antibody (left), with a control antibody (anti-CD99 mAb clone 12E7, middle) or with an anti-TIGIT antibody (mAb #4 generated as described previously, right). The effector to target (E:T) ratios are indicated on the x-axis. Figure shows one representative experiment out of three performed. Shown is the relative average killing ±SD, *p<0.05. FACS, fluorescence-activated cell sorting; IL2, interleukin-2; NK, natural killer; NS, not significant.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Control, Expressing, Transfection, Plasmid Preparation, Staining, Blocking Assay, Generated, Binding Assay, Fluorescence, Labeling, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: Novel checkpoint inhibitor anti-Nectin4 antibodies block TIGIT binding. (A, B) FACS staining of Raji cells transfected either with an empty vector (gray histograms) or with Nectin4 over-expression (black histograms). Gray filled histograms represent background staining of secondary antibody only. Cells were stained with anti-Nectin4 mAb clone .01 (A) or clone .05 (B). (C, D) FACS staining with TIGIT-Ig of Raji overexpressing Nectin4 with (black histograms) or without (gray histograms) preincubation with anti-Nectin4 mAb clone .01 (C) or clone .05 (D). Gray, filled histograms represent background staining. Figures show one representative experiment out of three performed. Graph depicting the mean fluorescence intensity values of the stainings appears in . (E) Avidity quantification between Nectin4-Ig and anti-Nectin4 mAb clone .01 and clone .05 using MST. Measurements were repeated with at least three independent protein preparations. FACS, fluorescence-activated cell sorting; MST, microscale thermophoresis

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Blocking Assay, Binding Assay, Staining, Transfection, Plasmid Preparation, Over Expression, Fluorescence, FACS, Microscale Thermophoresis

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: Anti-Nectin4 antibodies increase NK cytotoxicity. (A) [ 35 S] methionine-labeled Raji cells transfected with Nectin4 were incubated with 1 µg/well of either mouse IgG1 as control Ab (green) or with clone .01 (purple) or clone .05 (blue) for 1 hour and then incubated with activated human NK cells for 5 hours. The effector to target (E:T) ratios are indicated on the X-axis. Figure shows one representative experiment out of three performed. Shown is the relative average killing ±SD., *p<0.05 (significance between the clones’ blocking and control antibody blocking). (B–E) FACS staining of cell lines stained with commercial anti-Nectin4 antibody (black line histograms). Gray filled histograms are background staining with secondary antibody only. Cell lines used are MDA-MB-453 (B), SK-BR-3 (C), T47D (D) and LNCaP (E). Figures show one representative experiment out of three performed. Graph depicting the mean fluorescence intensity values of the stainings appears in . (F–I) [ 35 S] methionine-labeled MDA-MB-453 (F), SK-BR-3 (G), T47D (H), and LNCaP (I) cells were incubated with either mouse IgG1 as control mAb (green) or with clone .01 (purple) or clone .05 (blue) for 1 hour and then incubated with activated human NK cell cultures for 5 hours. The E:T ratios are indicated on the X-axis. Figures show one representative experiment out of three performed. Shown is the relative average killing ±SD., *p<0.05 (significance between the clones’ blocking and the control antibody). FACS, fluorescence-activated cell sorting; NK, natural killer.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Labeling, Transfection, Incubation, Control, Clone Assay, Blocking Assay, Staining, Fluorescence, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: Murine Nectin4 does not bind murine TIGIT. (A) Overexpression of murine Nectin4 (indicated as mNectin4) on Raji cells. Western blots were performed with antimurine Nectin4 AB and expression was compared with Raji cells expressing empty vector (indicated as empty). Staining for GAPDH was used as a loading control. (B) FACS staining of Raji cells transfected either with an empty vector as control (gray histograms), or with murine Nectin4 (black histograms). Cells were stained with murine TIGIT-Ig. Figures show one representative experiment out of three performed. Graph depicting the mean fluorescence intensity values of the stainings appears in FACS, fluorescence-activated cell sorting.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Over Expression, Western Blot, Expressing, Plasmid Preparation, Staining, Control, Transfection, Fluorescence, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: In vivo effect of anti-Nectin4 mAb on tumors overexpressing Nectin4. (A–D) SCID-beige mice were subcutaneously implanted with 5×10 6 Raji cells either overexpressing Nectin4 (N4 oe) or empty vector (EV) either alone (A, B) or with 1×10 6 NK cells (C, D). (E, F) SCID-beige mice were subcutaneously implanted with 5×10 6 Raji cells overexpressing Nectin4 (N4 oe) and 1×10 6 NK cells. Mice were then treated with either anti-Nectin4 clone .05 mAb (clone .05) and or a control Ab. (A, C, E) Tumor growth was followed with standard caliper. Starting day of antibody treatment is marked by a black arrow. Tumor volumes were calculated by the formula: length×width 2 ×0.5. (B, D, F) Tumors ware harvested and weighed on day 21 (B, D) or 27 (F) post tumor injection. For all murine experiments n=7. *p<0.05. NK, natural killer.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: In Vivo, Plasmid Preparation, Control, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: The checkpoint inhibitor anti-Nectin4 mAb enhances NK killing of tumors expressing all ligands of TIGIT in vivo. (A–D) SCID-beige mice were subcutaneously implanted with 5×10 6 MDA-MB-453 cells either alone (A, B) or with 7×10 5 NK cells (C, D). Half of each group was treated with anti-Nectin4 clone .05 mAb (clone.05) or a control Ab. (A, C) Tumor growth was followed with standard caliper. starting day of antibody treatment is marked by a black arrow. Tumor volumes were calculated by the formula: length×width 2 ×0.5. (B, D) Tumors ware harvested and weighed on day 23 post-tumor injection. For all murine experiments n=7. *P<0.05. (E) Kaplan-Meier curves of lung adenocarcinoma patients (left, Geo dataset ID: GSE36471) and colon cancer patients (right, Geo dataset ID: GSE17538) after stratification by Nectin4 expression level. *P<0.05. Data were obtained from DRUGSURV. NK, natural killer

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Expressing, In Vivo, Control, Injection