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: Extracellular fluorescence-activated cell sorting (FACS) staining for Nectin4 (MAB2659, R&D Systems), Nectin2 (BLG-337402, BioLegend), PVR (MAB25301, R&D Systems) DNAM1 (BLG-338302, BioLegend) and TIGIT (MAB7898, R&D Systems) was performed with 0.2 μg antibody per 100 000 cells.

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: Extracellular fluorescence-activated cell sorting (FACS) staining for Nectin4 (MAB2659, R&D Systems), Nectin2 (BLG-337402, BioLegend), PVR (MAB25301, R&D Systems) DNAM1 (BLG-338302, BioLegend) and TIGIT (MAB7898, R&D Systems) was performed with 0.2 μg antibody per 100 000 cells.

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

Journal: Science Advances

Article Title: Human stem cell-derived β cells expressing an optimized CD155 reduce cytotoxic immune cell function for application in type 1 diabetes

doi: 10.1126/sciadv.adx9755

Figure Lengend Snippet: ( A ) Schematic of parental Mel1 INS-GFP (blue) and engineered WT (red) or Mut CD155 (green) hPSC lines [Created in BioRender. Russ, H. (2025); https://BioRender.com/r06dcp3 ]. ( B ) Immunofluorescence staining for OCT3/4 (green), NANOG (red), and 4′,6-diamidino-2-phenylindole (white) in hPSC. Scale bar represents 100 µm. ( C ) Flow cytometry for expression of CD155 in hPSC. ( D to F ) hPSC were treated with fluorescently labeled TIGIT-Ig to compare binding efficiency between the parental Mel1 (blue), CD155 WT–expressing (red), and CD155 Mut–expressing (green) lines. (D) Representative histograms of cell-bound Ig after treatment at the 25,000 ng/ml condition. (E) Differences in Ig binding efficiency characterized across a range of 0 to 25,000 ng/ml, with additional comparisons made using (F) TIGIT-Ig selectivity area under the curve (AUC) values for each binding curve, where the bound TIGIT-Ig gMFI value for each line was normalized to the Mel1-bound TIGIT-Ig gMFI at each concentration. Data reflect biological n = 3 per condition for TIGIT-Ig sBC. Significant P values are reported for one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons of AUC values between paired samples. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: CD155 plasmid template was purchased from OriGene (RC202254), followed by transformation, amplification, and purification in New England Biolabs (NEB) 5-alpha competent Escherichia coli.

Techniques: Immunofluorescence, Staining, Flow Cytometry, Expressing, Labeling, Binding Assay, Concentration Assay

Journal: Science Advances

Article Title: Human stem cell-derived β cells expressing an optimized CD155 reduce cytotoxic immune cell function for application in type 1 diabetes

doi: 10.1126/sciadv.adx9755

Figure Lengend Snippet: ( A ) Schematic of differentiation from hPSC to sBC [Created in BioRender. Russ, H. (2025); https://BioRender.com/0d823q8 ]. ( B ) Representative bright field and pINS.GFP reporter live images of differentiating clusters at subsequent time points. ×4 or ×10 magnification images as denoted. ( C to H ) Flow cytometry analysis at day 23 of n = 6 independent differentiations of Mel1, CD155 WT, and CD155 Mutant sBC for frequency of (C) C-peptide + cells, (D) C-peptide + NKX6.1 + double-positive cells, (E) glucagon + cells, (F) total CD155 + cells or (G) CD155 + C-peptide + populations, and (H) geometric mean fluorescence intensity (gMFI) of CD155 within C-peptide + cells. ( I ) Immunofluorescence staining of day 23 sBC for insulin (green), NKX6.1 (magenta), and DAPI (white). Scale bar represents 200 µm. ( J ) Dynamic glucose stimulated insulin secretion data from perfusion study as percent of total insulin content. n = 3 independent differentiations. Significant P-values are reported for one-way ANOVA with Bonferroni’s multiple comparisons. **** P < 0.0001. ns, not significant; IBMX, 3-isobutyl-1-methylxanthine (IBMX); PP, pancreatic progenitors; DE, definitive endoderm.

Article Snippet: CD155 plasmid template was purchased from OriGene (RC202254), followed by transformation, amplification, and purification in New England Biolabs (NEB) 5-alpha competent Escherichia coli.

Techniques: Flow Cytometry, Mutagenesis, Fluorescence, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Human stem cell-derived β cells expressing an optimized CD155 reduce cytotoxic immune cell function for application in type 1 diabetes

doi: 10.1126/sciadv.adx9755

Figure Lengend Snippet: sBC were treated with fluorescently labeled ( A to C ) TIGIT-Ig or ( D to F ) CD226-Ig to compare binding efficiency between the parental Mel1 (blue), CD155 WT–expressing (red), and CD155 Mut–expressing (green) lines. [(A) and (D)] Representative histograms show cell-bound Ig after treatment at the 25,000 ng/ml condition. [(B) and (E)] Differences in Ig binding efficiency were characterized across a range of 0 to 25,000 ng/ml, with additional comparisons made using [(C) and (F)] AUC values for each binding curve. Data reflect biological n = 3 per condition for TIGIT-Ig sBC and n = 4 per condition for CD226-Ig. Significant P values are reported for one-way ANOVA with Bonferroni’s multiple comparisons of AUC values between paired samples. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: CD155 plasmid template was purchased from OriGene (RC202254), followed by transformation, amplification, and purification in New England Biolabs (NEB) 5-alpha competent Escherichia coli.

Techniques: Labeling, Binding Assay, Expressing

Journal: Science Advances

Article Title: Human stem cell-derived β cells expressing an optimized CD155 reduce cytotoxic immune cell function for application in type 1 diabetes

doi: 10.1126/sciadv.adx9755

Figure Lengend Snippet: The expression of ( A and B ) HLA-A2; ( C and D ) HLA-A,B,C; ( E and F ) PD-L1; ( G and H ) CD112; and ( I and J ) CD155 was evaluated on INS-GFP + sBC following 48 hours with (orange) or without (blue) IFN-γ treatment. [(A), (C), (E), (G), and (I)] Representative histograms show staining of Mel1, CD155-WT, and CD155-Mut sBC lines relative to fluorescence-minus one (FMO) controls (black). [(B), (D), (F), (H), and (J)] Paired dot plots show gMFI values by sBC line and treatment condition. Data reflect biological n = 6 per condition. Significant P values are reported for two-way ANOVA with Bonferroni’s multiple comparisons between paired samples. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: CD155 plasmid template was purchased from OriGene (RC202254), followed by transformation, amplification, and purification in New England Biolabs (NEB) 5-alpha competent Escherichia coli.

Techniques: Expressing, Staining, Fluorescence

Journal: Science Advances

Article Title: Human stem cell-derived β cells expressing an optimized CD155 reduce cytotoxic immune cell function for application in type 1 diabetes

doi: 10.1126/sciadv.adx9755

Figure Lengend Snippet: ( A ) Experimental scheme depicting activation assay to assess the immunogenicity of Mel1 (blue), CD155 WT–expressing (red), and CD155 Mut–expressing (green) sBC by MART- or PPI-reactive avatars, as measured by flow immunophenotyping of T cells [Created in BioRender. Brown, M. (2025); https://BioRender.com/sdjn41g ]. Plots show differences in expression of the T cell activation markers, ( B to E ) CD96, ( F to I ) CD226, and ( J to M ) TIGIT on [(B) and (C), (F) and (G), and (J) and (K)] MART-1– or [(D) and (E), (H) and (I), and (L) and (M)] PPI-reactive avatars between sBC lines at the 10:1 E:T ratio compared to no sBC (purple) and no dye (black) controls. Data reflect biological n = 8 per condition. Significant P values are reported for one-way ANOVA with Bonferroni correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: CD155 plasmid template was purchased from OriGene (RC202254), followed by transformation, amplification, and purification in New England Biolabs (NEB) 5-alpha competent Escherichia coli.

Techniques: Activation Assay, Immunopeptidomics, Expressing

Journal: Science Advances

Article Title: Human stem cell-derived β cells expressing an optimized CD155 reduce cytotoxic immune cell function for application in type 1 diabetes

doi: 10.1126/sciadv.adx9755

Figure Lengend Snippet: ( A ) Experimental scheme depicting proliferation assay used to characterize the immunogenicity of sBC lines when cultured with allogenic naïve CD8 + T cells [Created in BioRender. Brown, M. (2025); https://BioRender.com/5fgeu0g ]. ( B ) Representative dye dilution plots depict the proliferation of naïve CD8 + T cells following 6 days of coculture with Mel1 (blue), CD155 WT–expressing (red), and CD155 Mut–expressing (green) sBC compared to no sBC (purple) and no dye (black) controls, with ( C ) violin plots showing proliferation indices for each condition (biological n = 4 per condition). Significant P values are reported for paired samples using one-way ANOVA with Bonferroni correction for multiple comparisons. *** P < 0.001 and **** P < 0.0001.

Article Snippet: CD155 plasmid template was purchased from OriGene (RC202254), followed by transformation, amplification, and purification in New England Biolabs (NEB) 5-alpha competent Escherichia coli.

Techniques: Proliferation Assay, Immunopeptidomics, Cell Culture, Expressing

Journal: Science Advances

Article Title: Human stem cell-derived β cells expressing an optimized CD155 reduce cytotoxic immune cell function for application in type 1 diabetes

doi: 10.1126/sciadv.adx9755

Figure Lengend Snippet: ( A ) Experimental scheme depicting CML assay to assess the immunogenicity of Mel1 (blue), CD155 WT–expressing (red), and CD155 Mut–expressing (green) sBC by MART- or PPI-reactive avatars, as measured by 51 Cr release, with T cell cytokine production assessed by LEGENDplex [Created in BioRender. Brown, M. (2025); https://BioRender.com/nehgyik ]. Plots show differences in cell culture supernatant concentrations following coculture with ( B to E ) MART-1– or ( F to I ) PPI-reactive avatars of [(B) and (F)] FasL, [(C) and (G)] granzyme B, [(D) and (H)] perforin, and [(E) and (I)] granulysin between sBC lines at each E:T ratio. Data reflect biological n = 8 per condition. Significant P values are reported for two-way ANOVA with Bonferroni correction for multiple comparisons between CD155 WT and CD155 Mut conditions. ( J and K ) Violin plots show percent-specific lysis of sBC by (J) MART-1– or (K) PPI-reactive avatars at each E:T ratio. Data reflect biological n = 5 per condition. Significant P values are reported for two-way ANOVA with Bonferroni correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: CD155 plasmid template was purchased from OriGene (RC202254), followed by transformation, amplification, and purification in New England Biolabs (NEB) 5-alpha competent Escherichia coli.

Techniques: Immunopeptidomics, Expressing, Cell Culture, Lysis

Journal: Science Advances

Article Title: Human stem cell-derived β cells expressing an optimized CD155 reduce cytotoxic immune cell function for application in type 1 diabetes

doi: 10.1126/sciadv.adx9755

Figure Lengend Snippet: ( A ) Experimental scheme depicting CML assay to assess the effect of TIGIT blockade (pink), relative to an isotype control (black) on CML of parental Mel1 and CD155 Mut–expressing sBC lines by PPI-reactive avatars, as measured by 51 Cr release, with T cell cytokine production assessed by LEGENDplex [Created in BioRender. Brown, M. (2025); https://BioRender.com/3l30agq ]. Plots show differences in cell culture supernatant concentrations following coculture with Mel1 or CD155 Mut sBC of ( B ) FasL, ( C ) granzyme B, ( D ) perforin, and ( E ) granulysin between anti (α)–TIGIT and isotype-treated PPI-reactive avatars at each E:T ratio. Data reflect biological n = 4 per condition. ( F ) Violin plots show percent-specific lysis of Mel1 (dark gray and dark pink) or CD155 Mut (light gray and light pink) sBC at each E:T ratio with or without anti-TIGIT blockade. Data reflect biological n = 4 per condition. Significant P values are reported for three-way ANOVA with Bonferroni correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: CD155 plasmid template was purchased from OriGene (RC202254), followed by transformation, amplification, and purification in New England Biolabs (NEB) 5-alpha competent Escherichia coli.

Techniques: Control, Expressing, Cell Culture, Lysis

Journal: Science Advances

Article Title: Human stem cell-derived β cells expressing an optimized CD155 reduce cytotoxic immune cell function for application in type 1 diabetes

doi: 10.1126/sciadv.adx9755

Figure Lengend Snippet: ( A ) Experimental scheme depicting CML assay to assess the effect of TIGIT blockade (pink), relative to an isotype control (black) on CML of Mel1 (blue), CD155 WT–expressing (red), and CD155 Mut–expressing (green) sBC by NK cells, as measured by flow immunophenotyping of NK cells and 51 Cr release assays [Created in BioRender. Brown, M. (2025); https://BioRender.com/2cr4baw ]. ( B to I ) Violin plots show differences in expression of the NK cell activation markers [(B) and (C)] CD27, [(D) and (E)] CD69, [(F) and (G)] NKG2D, and [(H) and (I)] HLA-DR between sBC lines at the 1:1 E:T ratio compared to no sBC (purple) and no dye (black) controls. Data reflect biological n = 8 per condition. Significant P values are reported for one-way ANOVA with Bonferroni correction for multiple comparisons. ( J and K ) Violin plots show percent-specific lysis of (J) Mel1, CD155 WT, and CD155 Mut sBC at each E:T ratio in isotype control cultures and (K) Mel1 (dark gray and dark pink) or CD155 Mut (light gray and light pink) sBC at each E:T ratio with or without anti-TIGIT blockade. Data reflect biological n = 6 per condition. Significant P values are reported for three-way ANOVA with Bonferroni correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. mAb, monoclonal antibody.

Article Snippet: CD155 plasmid template was purchased from OriGene (RC202254), followed by transformation, amplification, and purification in New England Biolabs (NEB) 5-alpha competent Escherichia coli.

Techniques: Control, Expressing, Activation Assay, Lysis