Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) and ( B ) A series of PVRL4 deletion constructs were designed and their expression confirmed by Western blot. ( C ) PVRL4 mutants from ( A ) were tested for their ability to induce anchorage-independent colony formation in triplicate (error bars ± SD). ( D ) Cells with full-length PVRL4 or the cytoplasmic region deletion mutant were assayed for viability under conditions of anchorage deprivation by measuring total ATP content in cells cultured on ultra-low attachment plates for 72 hr. Values were normalized to an empty vector-transduced sample. Assays were performed in triplicate (error bars ± SD). ( E ) and ( F ) TL-HMECs expressing empty vector, full-length PVRL4 or cytoplasmic region deletion mutant containing cells were cultured on tissue culture-treated (adherent) or ultra-low attachment (suspension) dishes for 72 hr. RNA was isolated and mRNA levels for TGM1 ( E ) and KRT6A and IVL ( F ) were measured by RT-qPCR. Transcript levels were normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.004

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Construct, Expressing, Western Blot, Mutagenesis, Cell Culture, Plasmid Preparation, Suspension, Isolation, Quantitative RT-PCR

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) and ( B ) PVRL4 promotes cell clustering of TL-HMECs. Cells were dissociated off the tissue culture surface with trypsin-free cell dissociation buffer and kept in suspension for 1 hr. Small (3–5 cells) and large (>5 cells) cell clusters per field of view were counted, n = 3 (error bars ± SD). ( C ) GFP-labeled PVRL4-expressing TL-HMECs were allowed to aggregate with dsRed-labeled cells expressing either a PVRL1-targeting shRNA or a control shRNA. Representative phase-contrast and fluorescent images (red and green channels superimposed) are shown. ( D ) PVRL4 was co-expressed with the indicated shRNAs and anchorage-independent colony formation in TL-HMECs was assayed. Values were normalized to an empty vector-transduced sample. Assays were performed in triplicate (error bars ± SD). ( E ) PVRL4-expressing TL-HMECs were assayed for clustering in the presence of the indicated antibodies or isotype controls. Cell clusters were quantified as before. ( F ) Anchorage-independent growth induced by PVRL4 or an shRNA against PTEN was assayed in the presence of PVRL4-targeting antibody or control IgG. Colony numbers were normalized to the control sample. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.005

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Suspension, Labeling, Expressing, shRNA, Control, Plasmid Preparation

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4 expressing TL-HMECs were stably transduced with dsRed or GFP and mixed in equal proportions, followed by (i) immediate plating into semi-solid medium, or (ii) co-culturing on an adherent surface for 2 d, followed by plating into semi-solid medium. Resulting colonies were visualized under a fluorescent microscope and each colony was assessed for the presence of red and green fluorescence. Representative phase-contrast and fluorescent images (red and green channels) are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.006

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing, Stable Transfection, Transduction, Microscopy, Fluorescence

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: The colony formation efficiency of TL-HMECs transduced with empty vector or vector expressing PVRL4 was compared between unfiltered cell suspensions and cell suspensions that were filtered through a 35 μm nylon mesh strainer prior to plating into methylcellulose. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.007

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Transduction, Plasmid Preparation, Expressing

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4-expressing TL-HMECs were allowed to aggregate in the presence of the indicated antibodies or isotype controls. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.009

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4-expressing TL-HMECs were assayed for clustering in the presence of the indicated antibodies or isotype controls. Cell clusters were quantified as before. DOI: http://dx.doi.org/10.7554/eLife.00358.010

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Schematics of chimeric constructs containing extracellular domains of PVRL4 or an shRNA-resistant version of PVRL1 fused to the transmembrane domain of CD8 (blue). ( B ) and ( C ) TL-HMECs were stably transduced with the indicated combinations of expression constructs and assayed for anchorage-independent growth ( B ) and clustering ( C ). Colony numbers were normalized to the control sample. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD). ( D ) Expression levels of endogenous and chimeric proteins were verified by Western blot. DOI: http://dx.doi.org/10.7554/eLife.00358.011

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Construct, shRNA, Stable Transfection, Transduction, Expressing, Control, Western Blot

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: TL-HMEC cells infected with PVRL4-CD8tm or empty vector were fixed with methanol and stained with goat polyclonal anti-PVRL4 antibody followed by anti-goat Alexa Fluor 488 secondary antibody. DOI: http://dx.doi.org/10.7554/eLife.00358.012

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Infection, Plasmid Preparation, Staining

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Clustering assays were performed with TL-HMECs expressing the following transgenes: (i) empty vector/control shRNA; (ii) PVRL4-CD8tm/control shRNA; (iii) PVRL4-CD8tm/anti-PVRL1 shRNA; and (iv) a 1:1 mixture of (iii) and (i). DOI: http://dx.doi.org/10.7554/eLife.00358.013

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing, Plasmid Preparation, Control, shRNA

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Cell surface-localized proteins interacting with HA/FLAG-tagged PVRL4, but not with HA/FLAG-tagged GFP, as determined by mass spectrometry. ( B ) TL-HMECs expressing HA/FLAG tagged PVRL4 or HA/FLAG-tagged GFP were detached from the adherent surface with the enzyme-free cell dissociation buffer and incubated in suspension for 1 hr. Immunoprecipitations were performed with HA beads, followed by Western blot with FLAG and integrin β4 antibody. ( C ) TL-HMECs expressing vector control, PVRL4, or myr-PI3K were stably transduced with the indicated shRNA constructs and integrin β4 levels were assayed by Western blot. ( D ) TL-HMECs from ( C ) were assayed for anchorage-independent colony formation. Colony numbers were normalized to the vector control sample. Assays were performed in triplicate (error bars ± SD). ( E ) TL-HMECs stably transduced with the indicated constructs were detached from the adherent surface with enzyme-free cell dissociation buffer and incubated in 0.5% methylcellulose in suspension for 6 hr or cultured on an adherent surface for 48 hr. Levels of pY416-SFK (Src family kinases), total SFK, and vinculin loading control were measured by Western blot. Band intensity was measured with ImageJ software. ( F ) PVRL4-expressing TL-HMEC cells transduced with control or anti-PVRL1 shRNA were incubated in suspension in the conditions indicated. Levels of pY416-SFK (Src family kinases) and tubulin loading control were measured by Western blot. Band intensity was measured with ImageJ software. ( G ) TL-HMECs stably expressing PVRL4 or control vector were assayed for anchorage-independent colony formation in the presence of PP2 or vehicle control. Colony numbers were normalized to the vector sample. Assays were performed in triplicate (error bars ± SD). ( H ) TL-HMECs expressing PVRL4 were stably transduced with the indicated shRNA constructs and assayed for anchorage-independent colony formation. Colony numbers were normalized to the vector control sample. Assays were performed in triplicate (error bars ± SD). ( I ) SHP-2 levels were assayed by Western blot in TL-HMEC lysates from ( H ). DOI: http://dx.doi.org/10.7554/eLife.00358.019

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Mass Spectrometry, Expressing, Incubation, Suspension, Western Blot, Plasmid Preparation, Control, Stable Transfection, Transduction, shRNA, Construct, Cell Culture, Software

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Anti-HA beads were used to immunoprecipitate HA/FLAG-PVRL4 from the indicated lysates. Immunoprecipitates were blotted with anti-integrin β4 and anti-FLAG antibodies. ( B ) Immunoprecipitations with anti-integrin β4 antibodies or with a control IgG were performed from TL-HMEC lysates expressing either full-length PVRL4 or its cytoplasmic deletion mutant. Immunoprecipitates and input lysates were blotted with anti-integrin β4 and anti-PVRL4 antibodies. Asterisks denote heavy chains of control and anti-integrin β4 antibodies, which cross-react with an anti-goat secondary antibody. DOI: http://dx.doi.org/10.7554/eLife.00358.020

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Expressing, Mutagenesis

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) A view from the integrated Genome Viewer program showing focal amplification of the PVRL4 locus in SUM190 cells. The degree of amplification is denoted by the intensity of the color. ( B ) PVRL4 mRNA was stably depleted from SUM190 cells by four independent shRNAs. Transcript levels were measured by RT-qPCR and normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). PVRL4-depleted and control cells were assayed for clonogenic survival and anchorage-independent colony formation. Assays were performed in triplicate (error bars ± SD). All values were normalized to the uninfected control sample. ECM: extracellular matrix. ( C ) The PVRL4-CD8 chimeric construct was used to rescue the defect in clonogenic survival observed with RNAi-mediated PVRL4 depletion. Assays were performed in triplicate (error bars ± SD). Colony numbers were normalized to the control shRNA sample. ( D ) Expression levels of endogenous and chimeric proteins were verified by Western blot. DOI: http://dx.doi.org/10.7554/eLife.00358.021

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Amplification, Stable Transfection, Quantitative RT-PCR, Control, Construct, shRNA, Expressing, Western Blot

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4 transcript levels were measured by RT-qPCR and normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.026

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: The clonogenic potential of the indicated cell lines in the presence of control or PVRL4 shRNA constructs was assessed. Assays were performed in triplicate (error bars ± SD). Colony numbers were normalized to the control shRNA sample. DOI: http://dx.doi.org/10.7554/eLife.00358.027

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, shRNA, Construct

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Cell line growth in methylcellulose-containing media on an ultra-low attachment surface was assessed in the presence of control or PVRL4 shRNA constructs. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.028

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, shRNA, Construct

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) and ( B ) Female nude mice were injected into their mammary fat pads with SUM190 cells expressing PVRL4-targeted or control shRNA (n = 10 per group, error bars ± SEM). The resulting tumors were excised, scaled ( A ), and photographed ( B ). ( C ) SUM185 cells were stably transduced with PVRL4-targeted or control shRNA and injected into the mammary fat pads of female nude mice (n = 10 per group, error bars ± SEM). Tumor volume was measured with calipers at the indicated time points. ( D ) Female nude mice with ∼50 mm 3 SUM190-eGFP xenografts were randomized into two cohorts (n = 7 per group) and injected with anti-PVRL4 monoclonal antibodies or control IgG on the indicated days. Tumor volume was measured with calipers (error bars ± SEM). ( E ) Levels of PVRL4 protein were measured in tumor lysates from anti-PVRL4 antibody or control-treated mice, 7 days after the last treatment. ( F ) Tumor sections from control IgG ( A – C ) or anti-PVRL4 antibody-treated ( D – F ) mice were stained with hematoxylin/eosin and photographed. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.029

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Injection, Expressing, Control, shRNA, Stable Transfection, Transduction, Bioprocessing, Staining

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Freshly explanted tumors from control IgG or anti-PVRL4 antibody-treated mice (N = 3 per group) were visualized using a two-photon confocal microscope. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.031

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Microscopy

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Europium-labeled SUM190 cells were incubated with fresh human NK cells in the presence of an isotype control or anti-PVRL4 antibody, and the degree of lysis was measured by the DELFIA europium assay. The maximum signal was determined by a complete lysis of labeled SUM190 cells in DELFIA lysis buffer. As a positive control, hMB humanized mouse lymphoma cells were mixed with effector cells in the presence of ADCC-competent anti-CD52 antibody. ADCC: antibody-dependent cytotoxicity. DOI: http://dx.doi.org/10.7554/eLife.00358.032

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Labeling, Incubation, Control, Lysis, Positive Control

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Paraffin-embedded sections of SUM190 xenografts from mice treated with either control IgG or anti-PVRL4 antibody were stained with anti-mouse F4/80 antibody. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.033

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Staining

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Tumor lysates from control antibody- or anti-PVRL4 antibody-treated mice were blotted for E-cadherin and vimentin. ( B ) PVRL4 was stably depleted by two independent shRNAs in SUM190 cells and lysates were blotted for E-cadherin and vimentin. DOI: http://dx.doi.org/10.7554/eLife.00358.034

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Stable Transfection

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: 293T cells were transfected with empty pQCXIN (green line), pQCXIN-human PVRL4 (blue line), or pCMV-SPORT6-mouse PVRL4 (magenta line). Live-cell FACS was performed with mouse anti-human PVRL4 antibody followed by anti-mouse secondary antibody conjugated to Alexa Fluor 488 fluorophore. The FITC-A fluorescent signal for three labeled cell populations is shown. DOI: http://dx.doi.org/10.7554/eLife.00358.035

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Transfection, Labeling

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