recombinant human integrin αvβ8  (R&D Systems)

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: bioRxiv

Article Title: Overcoming Immune Checkpoint Inhibitor Resistance via Potent and Selective Dual αvβ6/8 Inhibitors Based on Engineered Lasso Peptides

doi: 10.1101/2025.01.28.635346

Figure Lengend Snippet:

Article Snippet: To each well, 50 mL of integrin (0.5 μg/ml αvβ6 (R&D Systems, cat. no. 3817-AV-050) or αvβ8 (R&D Systems, cat. no. 4135-AV-050)), that was preincubated with serially diluted inhibitor (from 0.001 nM to 10 μM), or with MK-0429 positive control, for at least 30 minutes in TSB buffer, was added and incubated for 1 hour at room temperature.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Overcoming Immune Checkpoint Inhibitor Resistance via Potent and Selective Dual αvβ6/8 Inhibitors Based on Engineered Lasso Peptides

doi: 10.1101/2025.01.28.635346

Figure Lengend Snippet: Conjugation studies involving lassotides 36 - 38 . A. General structures of lassotides 36 - 38 , showing sites of conjugation as yellow balls, as well as acylating agent (Acyl 50 ) and amidating agent (Amine 51 ), each bearing a PEG2 linker. B . Sequences and IC50 data for lassotide conjugates 47 - 49 vs αvβ6 and αvβ8.

Article Snippet: To each well, 50 mL of integrin (0.5 μg/ml αvβ6 (R&D Systems, cat. no. 3817-AV-050) or αvβ8 (R&D Systems, cat. no. 4135-AV-050)), that was preincubated with serially diluted inhibitor (from 0.001 nM to 10 μM), or with MK-0429 positive control, for at least 30 minutes in TSB buffer, was added and incubated for 1 hour at room temperature.

Techniques: Conjugation Assay

Journal: bioRxiv

Article Title: Overcoming Immune Checkpoint Inhibitor Resistance via Potent and Selective Dual αvβ6/8 Inhibitors Based on Engineered Lasso Peptides

doi: 10.1101/2025.01.28.635346

Figure Lengend Snippet: Reaction Biology expression data showing high expression levels of integrins αvβ6 and αvβ8 in ETM6 tumors.

Article Snippet: To each well, 50 mL of integrin (0.5 μg/ml αvβ6 (R&D Systems, cat. no. 3817-AV-050) or αvβ8 (R&D Systems, cat. no. 4135-AV-050)), that was preincubated with serially diluted inhibitor (from 0.001 nM to 10 μM), or with MK-0429 positive control, for at least 30 minutes in TSB buffer, was added and incubated for 1 hour at room temperature.

Techniques: Expressing

Journal: bioRxiv

Article Title: Overcoming Immune Checkpoint Inhibitor Resistance via Potent and Selective Dual αvβ6/8 Inhibitors Based on Engineered Lasso Peptides

doi: 10.1101/2025.01.28.635346

Figure Lengend Snippet:

Article Snippet: Human integrins αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, and αIIbβ3 were purchased from R&D Systems (Minneapolis, MN) or Acro Biosystems (Newark, DE, USA).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Specifically blocking αvβ8-mediated TGF-β signaling to reverse immunosuppression by modulating macrophage polarization

doi: 10.1186/s13046-024-03250-1

Figure Lengend Snippet: Integrin αvβ8 expression in KIRC and HNSCC as revealed by scRNA-seq analysis. (A) Ten freshly collected primary KIRC tissues were subjected to scRNA-seq analysis. (B) UMAP lots of KIRC patient cells, colored to depict the 12 identified clusters. (C) Distribution of ITGB8 -expressing cells within each cluster of KIRC. (D) Point plot illustrating the distribution of ITGB8 -expressing cells across various immune cell types in KIRC. (E) Analysis of 52 HNSCC tumor samples from individual patients as referenced in the literature. (F) UMAP plots of HNSCC patient cells, colored to show the 9 identified clusters. (G) Distribution of ITGB8 -expressing cells within each cluster of HNSCC. ( H) Point plot illustrating the distribution of ITGB8 -expressing cells across various types of immune cells of HNSCC

Article Snippet: Aliquots (3 μL) of 2 mg/mL mixtures of the human integrin αvβ8 (ACROBiosystems) in complex with excess Fab fragments of mAb 130H2 were prepared.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Specifically blocking αvβ8-mediated TGF-β signaling to reverse immunosuppression by modulating macrophage polarization

doi: 10.1186/s13046-024-03250-1

Figure Lengend Snippet: 130H2 regulates macrophage polarization. (A) Volcano plot illustrating differentially expressed genes between ITGB8 + and ITGB8 − macrophages. Differential gene expression was calculated using the FindMarkers function with the Wilcoxon Rank Sum test in the Seurat package. The filtering criteria were set as: adjusted p-value (P-adj) < 0.05 and log 2 (fold change) ≥ 0.25. (B) The αvβ8 expression on monocyte isolated from PBMC by CD14 positive selection kit was detected by flow cytometry. NC represents the absence of staining. (C) Monocytes were differentiated to M1, M2 macrophages, and DCs stimulated by GM-CSF, M-CSF or GM-CSF/IL-4, respectively. The expression of αvβ8 on these myeloid cells was detected by FACS (left). Mean fluorescence intensity (MFI) values were quantified and presented as bar graphs (right), with statistical significance indicated. (D-E) CD14 + monocytes were stimulated by M-CSF for 4 days, followed by adding 130H2, TGFβRII trap or an isotype control for an additional 3 days. Cytokine levels of IL-6 and IL-10 were quantified by HTRF technology. (F-I) Feature plots showing the distribution of Treg subpopulations ( F and H ) and ITGB8 expression in Treg cells ( G , I ) derived from Figure . (J) Treg cells isolated from human PBMCs were stimulated with a CD3/CD28 activator and IL-2. CD4 + CD25 + FoxP3 + Tregs were detected using APC-labeled 130H2 or an APC-labeled isotype control. Representative data are shown. The data in figure C, D, E was shown as mean ± SEM. **P < 0.005, ***P < 0.0005, **** P < 0.0001 was determined by Student’s t-test

Article Snippet: Aliquots (3 μL) of 2 mg/mL mixtures of the human integrin αvβ8 (ACROBiosystems) in complex with excess Fab fragments of mAb 130H2 were prepared.

Techniques: Gene Expression, Expressing, Isolation, Selection, Flow Cytometry, Staining, Fluorescence, Control, Derivative Assay, Labeling

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Specifically blocking αvβ8-mediated TGF-β signaling to reverse immunosuppression by modulating macrophage polarization

doi: 10.1186/s13046-024-03250-1

Figure Lengend Snippet: High expression of αvβ8 significantly enhanced the in vivo efficacy of 130H2. (A) mRNA expression of αvβ8 was compared across 9 distinct tumors (red) and corresponding normal tissues (blue) from the TCGA and GTEx projects. (B) LA795 cells bearing Kunming mice ( n = 7) were intravenously treated with 10 mg/kg 130H2 or isotype control every four days for three times. Tumor volume was measured and shown as mean ± SEM. The expression of αvβ8 on LA795 was detected by FACS and the histogram was embedded beside the tumor growth curve. (C) Kunming mice ( n = 7 per group) bearing human αvβ8 overexpressed LA795 cells were intravenously treated with 5 mg/kg 130H2, 130H2-LALA, isotype control or isotype-LALA every four days for three times. The overexpression of αvβ8 on LA795-hαvβ8 is shown at the right of tumor growth curve. Tumor volume was recorded and shown as mean ± SEM. P value of 130H2 verse isotype was determined by two-way ANOVA, **** P < 0.0001. (D) Timeline graphical representation of PK and safety evaluation in two cynomolgus monkeys, which were administrated 10 mg/kg 130H2 via intravenous infusion. (E) The concentration of 130H2 in serum was measured by ELISA

Article Snippet: Aliquots (3 μL) of 2 mg/mL mixtures of the human integrin αvβ8 (ACROBiosystems) in complex with excess Fab fragments of mAb 130H2 were prepared.

Techniques: Expressing, In Vivo, Control, Over Expression, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Specifically blocking αvβ8-mediated TGF-β signaling to reverse immunosuppression by modulating macrophage polarization

doi: 10.1186/s13046-024-03250-1

Figure Lengend Snippet: In vitro characterization of the anti-αvβ8 antibody 130H2. ( A - C ) 130H2 demonstrates robust binding capacity to serially diluted (concentration ranging from 200 to 3.125 nM) human αvβ8 protein ( A ), cynomolgus αvβ8 protein ( B ), and mouse αvβ8 protein ( C ) as determined by Biacore system. (D-E) Flow cytometry-based binding curves of threefold diluted 130H2 starting from 200 nM to native U251MG cells ( D ) and OVCAR3 cells ( E ) detected by APC labeled secondary antibody. The curves were fitted using sigmoidal four parameter logistic (4PL) model. (F) Schematic illustration of the protein-cell interaction system used in Fig. 2G. The TGF-β luciferase reporter HEK293T cell line, co-transfected with GARP, latent TGF-β1, and TGF-βRII, was used as effector cells. (G) The blocking activity of 130H2 was evaluated by reporter-based assay. Concentration dependent inhibition curves of 130H2 compared with TGFβRII trap were generated by incubating them with αvβ8 and HEK293T/GARP/latent-TGFβ1/TGFβRII-luciferase cells for 5 h and detected by luciferase substrate. (H) Schematic illustration of the cell-cell interaction system used in Fig. 2I. U251MG cells, which natively express both integrin αvβ8 and latent TGF-β1, were used as target cells. The TGF-β luciferase reporter HEK293T cell line, co-transfected with GARP, latent TGF-β1, and TGF-βRII, was used as effector cells. (I) The blocking activity of 130H2 was determined by U251MG-effector cell interaction system. Human HEK293T/GARP/latent-TGFβ1/TGFβRII-luciferase cells were incubated with U251MG cells (E: T = 2: 1) for 5 h in the presence of serial dilutions of 130H2 or TGFβRII trap and the blocking reaction was detected by measuring luminescence units

Article Snippet: Aliquots (3 μL) of 2 mg/mL mixtures of the human integrin αvβ8 (ACROBiosystems) in complex with excess Fab fragments of mAb 130H2 were prepared.

Techniques: In Vitro, Binding Assay, Concentration Assay, Flow Cytometry, Labeling, Luciferase, Transfection, Blocking Assay, Activity Assay, Reporter Assay, Inhibition, Generated, Incubation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Specifically blocking αvβ8-mediated TGF-β signaling to reverse immunosuppression by modulating macrophage polarization

doi: 10.1186/s13046-024-03250-1

Figure Lengend Snippet: Cryo-EM structure of human integrin αvβ8 ectodomain in complex with 130H2-Fab. (A) The domain-colored density maps of αvβ8 in complex with 130H2-Fab. Light blue and dark blue represent the light and heavy chain of 130H2-Fab, yellow represents β8 βI domain, and pink represents αvβ-propeller, respectively. (B) The established atomic model of αvβ8 bound with 130H2-Fv, represented by cartoon style. (C) Superimposition of αvβ8-130H2 and αvβ8-L-TGF-β1 (PDB ID: 6UJA) illustrates the steric hindrance between 130H2 and L-TGF-β1 (green). (D) The footprint of 130H2 on αvβ8. The αvβ8 is shown as a gray surface representation, with residues involved in 130H2 interaction depicted as a transparent surface and sticks. ( E) The interaction network between the αvβ8 and 130H2. The heavy chain and light chain mediate a network of hydrogen bonds (black dashed lines) and a salt bridge (green dashed lines). The side chains of key residues involved in hydrogen bonds or salt bridges are labeled and shown as sticks. (F) The binding of 130H2 induces a conformational change in the SDL2 of the βI domain on αvβ8. Key residues were pointed by arrows. Yellow SDL2 represent structure obtained in this study, purple and green SDL2 are from PDB ID 7Y1T and 6UJA, respectively. (G) The triggered conformational change (right) is to prevent the original steric clashes (left) between the key residues N167 and D175, with 130H2

Article Snippet: Aliquots (3 μL) of 2 mg/mL mixtures of the human integrin αvβ8 (ACROBiosystems) in complex with excess Fab fragments of mAb 130H2 were prepared.

Techniques: Cryo-EM Sample Prep, Labeling, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Specifically blocking αvβ8-mediated TGF-β signaling to reverse immunosuppression by modulating macrophage polarization

doi: 10.1186/s13046-024-03250-1

Figure Lengend Snippet: 130H2 inhibited tumor growth by increasing immune cells infiltration. (A) Experimental schematics of the workflow for in vivo efficacy and TILs analysis. (B and J) BALB/c mice ( n = 7) bearing EMT6 cells (B) and CT26 cells ( J ) were intraperitoneally treated with 10 mg/kg 130H2 or isotype antibody every four days on day 5, 9, 13 post inoculation, three times in total. Significance, * P < 0.05 by Student’s t-test, ** P < 0.01 by Student’s t-test. The expression of αvβ8 in the EMT6 cell line and CT26 cell line before inoculation, visualized by staining with 130H2 antibody, is shown in the right. (C-I) BALB/c mice ( n = 7 every time in each group) were sacrificed on day 12 and 17 after tumor inoculation and EMT6 tumors were dissociated to analyze tumor-infiltrating immune cells using FACS. Absolute number of cells per gram tumor tissue were calculated for populations of DCs ( C ) and NK cells ( D ). Proportion of CD4 + T cell ( E ) and CD8 + T cell ( F ) populations were determined by CD3 + CD4 + CD8- and CD3 + CD4-CD8 + markers. Myeloid cell populations including MDSC/myeloid ratio ( G ), M1/macrophage ( H ), and M2/macrophage ( I ) were evaluated. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 were analyzed by Student’s t-test. P < 0.05 means statistical significance. (K) BALB/c mice ( n = 6) bearing EMT6 cells were intraperitoneally administrated with the following treatments: 10 mg/kg isotype-hIgG1(day 5, 12) plus 10 mg/kg isotype-rat IgG2a (day 5, 9, 13), 10 mg/kg 130H2 (day 5, 12) plus 10 mg/kg isotype-rat IgG2a (day 5, 9, 13), 10 mg/kg PD-1 antibody (day 5, 9, 13) plus 10 mg/kg isotype-hIgG1 (day 5, 12), 10 mg/kg 130H2 (day 5, 12) plus 10 mg/kg PD-1 antibody (day 5, 9, 13). P < 0.0001 was indicated as ****, P < 0.05 as *, and statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. (L-O) BALB/c mice ( n = 6 in each group) were sacrificed on day 15 post-tumor inoculation, and EMT6 tumors were dissociated for analysis of tumor-infiltrating immune cells using FACS. The proportions of CD45 + cell, CD3 + T cell, macrophages, and M1 macrophages were quantified. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons. Data in figures B-O are presented as mean ± SEM

Article Snippet: Aliquots (3 μL) of 2 mg/mL mixtures of the human integrin αvβ8 (ACROBiosystems) in complex with excess Fab fragments of mAb 130H2 were prepared.

Techniques: In Vivo, Expressing, Staining

Journal: Cell death & disease

Article Title: Humanin activates integrin αV-TGFβ axis and leads to glioblastoma progression.

doi: 10.1038/s41419-024-06790-8

Figure Lengend Snippet: Fig. 7 Hypothetical model of humanin function. Humanin treatment induces cell attachment in GSCs via integrin αV (upper). The activation of humanin-induced integrin signaling further triggered filopodia formation via Rac1/CDC42 and canonical TGFβ signaling pathway, which eventually supported cell migration and angiogenesis for the more aggressive GBM phenotype (lower).

Article Snippet: Briefly, 500 ng of integrin αvβ8 (R&D System, Cat# 4135- AV-050), fibronectin (Thermo, Cat# PHE0023), and humanin and scrambled humanin (Anygen, Cat# AGP-8245) were blotted onto nitrocellulose membranes (BioTrace, Cat# 66485).

Techniques: Cell Attachment Assay, Activation Assay, Migration

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