Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Flow cytometry profiles of B16, B16-mβ6, B16-mβ8, A549 and A549-hβ6 cells. Green and red histograms show β6 and β8 specific staining, respectively, and grey histograms show background staining using an isotype control. Numbers indicate MFI values of specific or control antibodies. (B, C) Virus binding and dependency on divalent ions. Detached mouse (B) and human cells (C) were incubated with control medium (not containing virus) or medium containing the indicated viruses for 1 h on ice, followed by washing and staining with primary rabbit anti-FK-M3 antibodies and secondary fluorescently labeled antibodies for flow cytometry analysis. Incubation/washing buffers were adjusted to contain either Mg 2+ /Ca 2+ , 1 mM each, 1/0.2 mM Mn 2+ /Ca 2+ , or EDTA 2.5 mM. (D) Virus-receptor antibody competition experiment. Detached cells were first incubated on ice with control medium or the indicated viruses, followed by washing and incubation with either the anti-αvβ6 antibody (CMT-93 and B16-mβ6 cells), or the αvβ8 antibody (M000216 cells). Subsequently the cells were stained with secondary fluorescently labeled antibodies for flow cytometry analysis. (E) The indicated mouse and human cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (MFI) was determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. (F) For analysis of virus progeny, CMT-93, B16, B16-mβ6 and B16-mβ8 cells were infected with M1-/M3-IX-G using an MOI of 1.5. After 14 h, the cells were thoroughly washed, trypsinized and re-seeded. Virus-containing supernatant samples were collected 48 (d2) and 72 h (d3) pi and used for titration analyses. Based on the virus input, fold increases of progeny virus were calculated. For B16 cells no measurable levels of viruses were detected, which translated to a virus progeny production of less than a factor of 0.01, based on the sensitivity level of this assay. Data in (B) to (F) represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Flow Cytometry, Staining, Control, Virus, Binding Assay, Incubation, Labeling, Infection, Recombinant, Titration, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Virus binding interference in CMT-93 and M000216 cells by β6/-β8 function blocking antibodies. Detached cells were sequentially incubated for 1 h on ice with control antibody, or the anti-β6/-β8 antibodies, followed by incubation with control medium or medium containing the indicated viruses at an MOI of 4, the rabbit anti-FK-M3 antibodies, and finally the secondary PE-conjugate antibodies. Incubation and washing buffer contained either Mg 2+ /Ca 2+ , 1 mM each, or 1/0.2 mM Mn 2+ /Ca 2+ . (B-C) Virus infection interference in CMT-93 and M000216 cells by β6- and β8-specific antibodies. CMT-93 (B) and M000216 cells (C) were pre-incubated for 1 h on ice using 5-fold dilution series of the specific β6- or β8-integrin antibodies, respectively, starting with 800 ng/ml as highest concentration, followed by addition of the different indicated GFP-expressing viruses and transfer to 37°C for 48 h. An MOI of 1 was used for CMT-93 cells and MOI of 3 for M000216 cells in all experiments shown in this figure. GFP analysis was performed 48 h pi, and expression index was normalized to a control antibody. IC 50 values determined in this experiment are summarized in . (D, E) Infection blocking assays by sITGs. M1-IX-G virus was incubated for 1 h at RT with 5-fold serial dilutions of the indicated sITGs starting from 800 ng/ml to 6.4 ng/ml, followed by addition to CMT-93 cells (D) and M00216 cells (E) and cultivated and further processed as above. (F-H) Infection blocking assays by peptides. (F) The 20-mer peptides tested for virus infection inhibition included peptides A20FMDV2 derived from the VP1 coat protein of FMDV2, A20FMDV2-E containing a D to E mutation in the critical RGD motif, A20M1 and A20M3 derived from M1-/M3-FK, respectively, as compared to LAP-hTGFβ1, all containing the critical αvβ6/αvβ8-binding RGDLXX(L/I) motif. (G, H) Cells were pre-incubated on ice with 5-fold serial dilutions of peptides resulting in final concentrations from 5,000 to 0.32 nM. Subsequently, M1-IX-G virus was added to CMT-93 cells (G) or M000216 cells (H), followed by processing as described above. (I) Comparative flow cytometry profiles of αvβ8 expression in 3T6 cells. Blue and red show β8-specific staining in 3T6-sgNT and 3T6-sgItgβ8 cells, respectively, and grey histogram shows background staining of 3T6-sgItgβ8 cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (J, K) Transduction of 3T6-sgNT and 3T6-sgItgβ8 cells using M1-/M3-IX-G, the fiber-chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 and control H5-ΔE3B-CG at an MOI of 9. Cells were processed as described in . (L) Comparative flow cytometry MFI αvβ6 expression values in control CMT-93-sgNT versus β6 integrin shRNA knock down CMT-93-sgItgβ6 cells. (M-O) Infection of control CMT-93-sgNT and CMT-93-sgItgβ6 cells using M1-IX-G (M), M3-IX-G (N) and H5-ΔE3B-CG (O) at an MOI of 1. Cells were processed as described above. Except for the representative flow cytometry histogram in (I), data represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Virus, Binding Assay, Blocking Assay, Incubation, Control, Infection, Concentration Assay, Expressing, Inhibition, Derivative Assay, Mutagenesis, Flow Cytometry, Staining, Transduction, shRNA, Knockdown, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A, B) Sensor chips containing immobilized biotinylated FK-M1 and FK-M3 were probed with mouse sITG αvβ6. Following consecutive analyte injections over 120 s, dissociation was monitored for 600 s (black). Sensorgrams were fitted with a 1:1 kinetic model (red). (C-E) FK saturation cell binding assays using cells with defined αvβ6/αvβ8 expression included FK-M1 binding to B16-mβ6 (C), FK-M3 binding to B16-mβ6 (D) and FK-M3 binding to B16-mβ8 (E). Parental B16 cells were included in order to subtract background levels when calculating equilibrium dissociation constant K D values by Scatchard plot analyses.

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Binding Assay, Expressing

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Overview of αvβ6 complexed with the 20mer A20M3 peptide. αv (light green), β6 (pink) and A20M3 (grey) chains are shown as cartoon traces. Side chains of the RGD motif are shown as sticks and spheres. (B) Superposition of αvβ6 in surface representation in complex with A20M1 (yellow carbons), A20M3 (grey carbons) and A20FMDV2 (salmon carbons). The position of manganese ions (cyan spheres) shown superimposed were derived from the human αvβ6/TGFβ1 complex (PDB ID: 5ffo). Labeled aa refer to the A20M3 sequence. (C) Superposition of the αvβ6/A20M3 complex onto the αvβ8 structure (blue carbons). Note that the A20M3 peptide is not shown here. Residue numbering refers to the Uniprot entry Q9Z0T9 (mouse integrin subunit β6) and the A20M3 peptide sequence (Arg16).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Derivative Assay, Labeling, Sequencing, Residue

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human integrin αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.

Article Snippet: For the viral binding inhibition assay, SAFV-3 (1 × 10 6 PFU/well) was pretreated with either 1 μg or 10 μg of heparin sodium salt (17513-96, Nacalai Tesque) or recombinant human integrin αVβ8 (4135-AV, R&D Systems).

Techniques: Transformation Assay, Infection, Software, Expressing, Staining, Flow Cytometry, Incubation

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a Western blot analysis of integrin αV (left panel) and integrin β8 (right panel) expression in BHK-21 cells. BHK-21 cells that were lentivirally transduced with either human integrin αV (BHK + human AV) or hamster integrin β8 (BHK + hamster B8) were used as positive controls. The anti-integrin αV antibody cross-reacted with both human and hamster integrin αV. Actin served as the loading control. b Expression of HS, integrin αV, and β8 in BHK-21 derivatives. BHK-21 cells were stained with anti-HS antibody (upper left panel). BHK-21 cells stably expressing human integrin αV and/or β8 (BHK + human AV, BHK + human B8, BHK + human AVB8), as well as the control cells, were stained with anti-integrin αV or anti-integrin αVβ8 antibodies. The cells were analyzed by flow cytometry. c Susceptibility analysis using SAF/UnaG in BHK-21 cells expressing human integrin αV and/or β8. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. The percentage of infected cells was determined by examining at least 1000 cells. Data are representative of two independent experiments. d One-step growth kinetics of SAFV-3 in BHK + human AV, BHK + human B8, BHK + human AVB8, and control cells. The cells were infected with SAFV-3 and incubated for up to 24 h. The dotted line indicates the limit of detection. e Western blot analysis of exogenous integrin β8 expression in BHK-21 cells lentivirally transduced with either mouse or hamster integrin β8. The anti-integrin β8 antibody cross-reacted with both mouse and hamster integrin β8. Actin served as the loading control. f Susceptibility analysis using SAF/UnaG in mouse and hamster integrin β8 expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were captured at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments. Data in ( c and d ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test ( c ) and the two-sided Welch’s t -test ( d ). **, P < 0.01, *, P < 0.05, n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: For the viral binding inhibition assay, SAFV-3 (1 × 10 6 PFU/well) was pretreated with either 1 μg or 10 μg of heparin sodium salt (17513-96, Nacalai Tesque) or recombinant human integrin αVβ8 (4135-AV, R&D Systems).

Techniques: Western Blot, Expressing, Transduction, Control, Staining, Stable Transfection, Flow Cytometry, Infection, Incubation, Comparison

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a Pull-down assay of SAFV-3 using heparin (left panel) and integrin αVβ8 (right panel). Heparin and Fc chimera of extracellular domains of integrin αVβ8, αVβ3, or the signal sequence (ss) of integrin αV (negative control) were prepared as complexes with magnetic beads. These complexes were incubated with SAFV-3, followed by western blot analysis of the bound virus using anti-SAFV-3 antiserum (left and right upper panels). The bottom right panel shows an image of the integrin-Fc complex on magnetic beads used for pulldown, detected using an anti-mouse IgG antibody. b Cell surface attachment assay for SAFV-3. HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 were incubated with SAFV-3, followed by RT-qPCR analysis of the bound virus. c Expression of human integrin β8 in HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells. To compare the expression levels of integrin αVβ8 on the cell surface, HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells were stained with anti-integrin αVβ8 antibodies and analyzed by flow cytometry. d , e Inhibition of SAFV-3 attachment to the cell surface by soluble heparin ( d ) or recombinant integrin αVβ8 ( e ). HeLaN-WT cells ( d ) or HeLaN-∆SLC + human AVB8 cells ( e ) were incubated with SAFV-3 pretreated with 1 or 10 μg of soluble heparin or recombinant integrin αVβ8, respectively. Recombinant integrin αVβ3 was the negative control. After incubation at 4 °C for 2 h, bound virus was analyzed using RT-qPCR. f HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of SAFV-3, and viable cells were stained with crystal violet to assess infection levels. All data are representative of two independent experiments. Data in ( b , d , and e ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Asterisks directly placed on bars indicate statistically significant differences compared to WT or untreated samples, while asterisks placed on the lines connecting bars denote statistically significant differences between those bars. Source data are provided as a Source Data file. Ag antigen, Fc fragment crystallizable region.

Article Snippet: For the viral binding inhibition assay, SAFV-3 (1 × 10 6 PFU/well) was pretreated with either 1 μg or 10 μg of heparin sodium salt (17513-96, Nacalai Tesque) or recombinant human integrin αVβ8 (4135-AV, R&D Systems).

Techniques: Pull Down Assay, Sequencing, Negative Control, Magnetic Beads, Incubation, Western Blot, Virus, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Inhibition, Recombinant, Infection, Comparison

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: a Expression of HS and human integrin β8 in BHK-WT, BHK-∆SLC, BHK + human AVB8, BHK-∆SLC + human AVB8, and revertant cells expressing human SLC35B2 (BHK-∆SLC + human AVB8 + SLC). The cells were stained with anti-HS or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. b Cell surface attachment assay for SAFV-3. BHK-WT and BHK-∆SLC cells were incubated with SAFV-3 at 4 °C for 2 h. Viral binding to HS was assessed by RT-qPCR quantification of cell-bound virus ( n = 3). c Susceptibility analysis using SAF/UnaG in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells. The cells used in this experiment were sorted to equalize the surface expression levels of integrin αVβ8 between BHK + human AVB8 and BHK-∆SLC + human AVB8 cells. UnaG-positive cells (green) and nuclei stained with Hoechst (blue) were imaged at 16 h post-infection. The percentage of infected cells was determined by examining at least 800 cells/well ( n = 4). Scale bar, 200 μm. d Cell surface attachment assay for SAFV-3 in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells ( n = 3). Bar graphs in ( b– d ) are presented as means with s.d. Statistical significance was determined using the two-sided Welch’s t -test ( b ) and a one-way ANOVA with Dunnett’s multiple comparison test ( c , d ). **, P < 0.01, *, P < 0.05. All data are representative of two independent experiments. Source data are provided as a Source Data file.

Article Snippet: For the viral binding inhibition assay, SAFV-3 (1 × 10 6 PFU/well) was pretreated with either 1 μg or 10 μg of heparin sodium salt (17513-96, Nacalai Tesque) or recombinant human integrin αVβ8 (4135-AV, R&D Systems).

Techniques: Expressing, Staining, Flow Cytometry, Incubation, Binding Assay, Quantitative RT-PCR, Virus, Infection, Comparison

Journal: Nature Communications

Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

doi: 10.1038/s41467-025-67236-z

Figure Lengend Snippet: Sulfated GAGs and integrin αVβ8 function as interconnected dual receptors for SAFV infection in HeLa-N cells. SAFV can directly bind to either sulfated GAGs or integrin αVβ8, while a portion of viruses bound to sulfated GAGs subsequently interact with integrin αVβ8. In addition, the data suggest the existence of a downstream molecule (factor X) required for an unspecified step in the viral entry process following sulfated GAGs binding.

Article Snippet: For the viral binding inhibition assay, SAFV-3 (1 × 10 6 PFU/well) was pretreated with either 1 μg or 10 μg of heparin sodium salt (17513-96, Nacalai Tesque) or recombinant human integrin αVβ8 (4135-AV, R&D Systems).

Techniques: Infection, Binding Assay

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of αvβ6-dependent activation of chimeric G115 γδ TCRs. ( A ) Analysis of integrin expression on A375 puro and A375-β6 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between A375 puro ( B ) and A375-β6 ( C ) tumour cells and untransduced (untrans) or transduced T-cell populations at an effector-to-target ratio of 1:1 for 72 h. Residual tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001. ( D ) Supernatants collected from co-cultures described in ( B , C ) were analysed for IFN-γ by ELISA (mean ± SEM; n = 21–45 from 4–6 independent donors). Statistical analysis was performed using one-way ANOVA; * p < 0.05; ** p < 0.01.

Article Snippet: Integrin expression was detected by flow cytometry using the following antibodies: αvβ3 integrin (FAB3050A, R&D Systems, Minneapolis, MN, USA), αvβ5 integrin (FAB2528A, R&D systems), αvβ6 integrin (FAB4155A, R&D Systems) and αvβ8 integrin (FAB4775A, R&D systems).

Techniques: Activation Assay, Expressing, Control, MTT Assay, Enzyme-linked Immunosorbent Assay

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of PAg-dependent activation of chimeric G115 γδ TCRs by ffLuc-expressing K562 cells. ( A ) Analysis of αvβ6 integrin expression on ffLuc + K562 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. ( B ) Firefly luciferase-expressing K562 cells were pre-incubated with the indicated Zol concentration for 24 h prior to the establishment of co-cultures with untrans(duced) or transduced T-cell populations at an effector to target ratio 1:1 for 72 h. Data show mean ± SEM of residual K562 viability (n = 6–11 from 4 independent donors), as determined by luciferase assay. Statistical analysis was performed using two-way ANOVA; *** p < 0.001; **** p < 0.0001. ( C ) Supernatants collected from co-cultures described in B were analysed for IFN-γ by ELISA (mean ± SEM; n = 19 from 3 independent donors). Statistical analysis was performed using two-way ANOVA; *** p < 0.001; **** p < 0.0001.

Article Snippet: Integrin expression was detected by flow cytometry using the following antibodies: αvβ3 integrin (FAB3050A, R&D Systems, Minneapolis, MN, USA), αvβ5 integrin (FAB2528A, R&D systems), αvβ6 integrin (FAB4155A, R&D Systems) and αvβ8 integrin (FAB4775A, R&D systems).

Techniques: Activation Assay, Expressing, Control, Luciferase, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of the anti-tumour activity of chimeric G115 γδ TCRs against BxPC3 pancreatic tumour cells. ( A ) Analysis of integrin expression on BxPC3 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between BxPC3 tumour cells (No Zol; B ) or Zol-sensitised BxPC3 tumour cells (+Zol; C ) and untrans(duced) or transduced T-cell populations at an effector to target ratio of 1:1 for 72 h. Tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001, * p < 0.05, N/S–not significant.

Article Snippet: Integrin expression was detected by flow cytometry using the following antibodies: αvβ3 integrin (FAB3050A, R&D Systems, Minneapolis, MN, USA), αvβ5 integrin (FAB2528A, R&D systems), αvβ6 integrin (FAB4155A, R&D Systems) and αvβ8 integrin (FAB4775A, R&D systems).

Techniques: Activity Assay, Expressing, Control, MTT Assay

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of the anti-tumour activity of chimeric G115 γδ TCRs against Panc1 pancreatic tumour cells. ( A ) Analysis of integrin expression on Panc1 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between Panc1 tumour cells (No Zol; B ) or Zol-sensitised Panc1 tumour cells (+ Zol; C ) and untransduced (untrans) or transduced T-cell populations at an effector to target ratio of 1:1 for 72 h. Tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001, ** p < 0.01, N/S–not significant.

Article Snippet: Integrin expression was detected by flow cytometry using the following antibodies: αvβ3 integrin (FAB3050A, R&D Systems, Minneapolis, MN, USA), αvβ5 integrin (FAB2528A, R&D systems), αvβ6 integrin (FAB4155A, R&D Systems) and αvβ8 integrin (FAB4775A, R&D systems).

Techniques: Activity Assay, Expressing, Control, MTT Assay

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Flow cytometry profiles of B16, B16-mβ6, B16-mβ8, A549 and A549-hβ6 cells. Green and red histograms show β6 and β8 specific staining, respectively, and grey histograms show background staining using an isotype control. Numbers indicate MFI values of specific or control antibodies. (B, C) Virus binding and dependency on divalent ions. Detached mouse (B) and human cells (C) were incubated with control medium (not containing virus) or medium containing the indicated viruses for 1 h on ice, followed by washing and staining with primary rabbit anti-FK-M3 antibodies and secondary fluorescently labeled antibodies for flow cytometry analysis. Incubation/washing buffers were adjusted to contain either Mg 2+ /Ca 2+ , 1 mM each, 1/0.2 mM Mn 2+ /Ca 2+ , or EDTA 2.5 mM. (D) Virus-receptor antibody competition experiment. Detached cells were first incubated on ice with control medium or the indicated viruses, followed by washing and incubation with either the anti-αvβ6 antibody (CMT-93 and B16-mβ6 cells), or the αvβ8 antibody (M000216 cells). Subsequently the cells were stained with secondary fluorescently labeled antibodies for flow cytometry analysis. (E) The indicated mouse and human cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (MFI) was determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. (F) For analysis of virus progeny, CMT-93, B16, B16-mβ6 and B16-mβ8 cells were infected with M1-/M3-IX-G using an MOI of 1.5. After 14 h, the cells were thoroughly washed, trypsinized and re-seeded. Virus-containing supernatant samples were collected 48 (d2) and 72 h (d3) pi and used for titration analyses. Based on the virus input, fold increases of progeny virus were calculated. For B16 cells no measurable levels of viruses were detected, which translated to a virus progeny production of less than a factor of 0.01, based on the sensitivity level of this assay. Data in (B) to (F) represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Flow Cytometry, Staining, Control, Virus, Binding Assay, Incubation, Labeling, Infection, Recombinant, Titration, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Virus binding interference in CMT-93 and M000216 cells by β6/-β8 function blocking antibodies. Detached cells were sequentially incubated for 1 h on ice with control antibody, or the anti-β6/-β8 antibodies, followed by incubation with control medium or medium containing the indicated viruses at an MOI of 4, the rabbit anti-FK-M3 antibodies, and finally the secondary PE-conjugate antibodies. Incubation and washing buffer contained either Mg 2+ /Ca 2+ , 1 mM each, or 1/0.2 mM Mn 2+ /Ca 2+ . (B-C) Virus infection interference in CMT-93 and M000216 cells by β6- and β8-specific antibodies. CMT-93 (B) and M000216 cells (C) were pre-incubated for 1 h on ice using 5-fold dilution series of the specific β6- or β8-integrin antibodies, respectively, starting with 800 ng/ml as highest concentration, followed by addition of the different indicated GFP-expressing viruses and transfer to 37°C for 48 h. An MOI of 1 was used for CMT-93 cells and MOI of 3 for M000216 cells in all experiments shown in this figure. GFP analysis was performed 48 h pi, and expression index was normalized to a control antibody. IC 50 values determined in this experiment are summarized in . (D, E) Infection blocking assays by sITGs. M1-IX-G virus was incubated for 1 h at RT with 5-fold serial dilutions of the indicated sITGs starting from 800 ng/ml to 6.4 ng/ml, followed by addition to CMT-93 cells (D) and M00216 cells (E) and cultivated and further processed as above. (F-H) Infection blocking assays by peptides. (F) The 20-mer peptides tested for virus infection inhibition included peptides A20FMDV2 derived from the VP1 coat protein of FMDV2, A20FMDV2-E containing a D to E mutation in the critical RGD motif, A20M1 and A20M3 derived from M1-/M3-FK, respectively, as compared to LAP-hTGFβ1, all containing the critical αvβ6/αvβ8-binding RGDLXX(L/I) motif. (G, H) Cells were pre-incubated on ice with 5-fold serial dilutions of peptides resulting in final concentrations from 5,000 to 0.32 nM. Subsequently, M1-IX-G virus was added to CMT-93 cells (G) or M000216 cells (H), followed by processing as described above. (I) Comparative flow cytometry profiles of αvβ8 expression in 3T6 cells. Blue and red show β8-specific staining in 3T6-sgNT and 3T6-sgItgβ8 cells, respectively, and grey histogram shows background staining of 3T6-sgItgβ8 cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (J, K) Transduction of 3T6-sgNT and 3T6-sgItgβ8 cells using M1-/M3-IX-G, the fiber-chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 and control H5-ΔE3B-CG at an MOI of 9. Cells were processed as described in . (L) Comparative flow cytometry MFI αvβ6 expression values in control CMT-93-sgNT versus β6 integrin shRNA knock down CMT-93-sgItgβ6 cells. (M-O) Infection of control CMT-93-sgNT and CMT-93-sgItgβ6 cells using M1-IX-G (M), M3-IX-G (N) and H5-ΔE3B-CG (O) at an MOI of 1. Cells were processed as described above. Except for the representative flow cytometry histogram in (I), data represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Virus, Binding Assay, Blocking Assay, Incubation, Control, Infection, Concentration Assay, Expressing, Inhibition, Derivative Assay, Mutagenesis, Flow Cytometry, Staining, Transduction, shRNA, Knockdown, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A, B) Sensor chips containing immobilized biotinylated FK-M1 and FK-M3 were probed with mouse sITG αvβ6. Following consecutive analyte injections over 120 s, dissociation was monitored for 600 s (black). Sensorgrams were fitted with a 1:1 kinetic model (red). (C-E) FK saturation cell binding assays using cells with defined αvβ6/αvβ8 expression included FK-M1 binding to B16-mβ6 (C), FK-M3 binding to B16-mβ6 (D) and FK-M3 binding to B16-mβ8 (E). Parental B16 cells were included in order to subtract background levels when calculating equilibrium dissociation constant K D values by Scatchard plot analyses.

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Binding Assay, Expressing

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Overview of αvβ6 complexed with the 20mer A20M3 peptide. αv (light green), β6 (pink) and A20M3 (grey) chains are shown as cartoon traces. Side chains of the RGD motif are shown as sticks and spheres. (B) Superposition of αvβ6 in surface representation in complex with A20M1 (yellow carbons), A20M3 (grey carbons) and A20FMDV2 (salmon carbons). The position of manganese ions (cyan spheres) shown superimposed were derived from the human αvβ6/TGFβ1 complex (PDB ID: 5ffo). Labeled aa refer to the A20M3 sequence. (C) Superposition of the αvβ6/A20M3 complex onto the αvβ8 structure (blue carbons). Note that the A20M3 peptide is not shown here. Residue numbering refers to the Uniprot entry Q9Z0T9 (mouse integrin subunit β6) and the A20M3 peptide sequence (Arg16).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Derivative Assay, Labeling, Sequencing, Residue