Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a Schematic presentation of different fractions collected for neutrophil (PMN) quantification (left) and model of CD47 regulation of CD11b/CD18-dependent PMN transepithelial migration (TEpM) across intestinal mucosa (right). b Analysis of PMN TEpM in vivo using an ileal loop model reveals that Cd47 −/− mice have significantly reduced numbers of PMN that migrated to the intestinal lumen 1 hr after intraluminal instillation of leukotriene B 4 (LTB 4 ; 1 nM) compared with WT mice. Dots represent individual mice. Data are Means ± SEM of three independent experiments, 14 mice/group (*p ≤ 0.05) as determined by Mann-Whitney U test. c, d After luminal content collection, sections of intestinal loop were enzymatically digested to obtain a Lamina propria (LP)-enriched fraction or fixed and paraffin-embedded for histological analysis. c Quantification of leukocytes by flow cytometry shows comparable numbers of PMN within the LP from WT and Cd47 −/− mice. Data are Means ± SEM of 2 independent experiments, 8 mice/group. d Representative images of immunohistochemical staining depicting Ly6G + PMN. 20x (top) and 40x (bottom) objectives. Scale bars: 100 μm and 50 μm, respectively.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Migration, In Vivo, MANN-WHITNEY, Flow Cytometry, Immunohistochemical staining, Staining

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a CD47 expression on bone marrow neutrophils (BMN) by western blotting and b densitometry analysis of three experiments reveal that nearly 80% of CD47 is selectively depleted on PMN from mice with specific deletion of CD47 on PMN ( MRP8-Cre;Cd47 fl/fl ) compared to Cd47 fl/fl control mice. c Reduced surface expression of CD47 was corroborated by flow cytometry on BMN from MRP8-Cre;Cd47 fl/fl compared to Cd47 fl/fl mice. d Analysis of PMN TEpM in vivo using a murine ileal loop model reveals significant reduction of transmigrated PMN into the intestinal lumen 1 hr after intraluminal instillation of LTB 4 (1 nM) in MRP8-Cre;Cd47 fl/fl compared with Cd47 fl/fl mice. Data are Means ± SEM of at least three independent experiments, 16-18 mice/group (**p≤0.01) as determined by Mann-Whitney U test. e PMN TEpM in vivo in mice with specific deletion of CD47 on IECs ( Villin-Cre;Cd47 fl/fl ) revealed similar numbers of PMN in the intestinal lumen 1 hr after intraluminal instillation of LTB 4 (1 nM) compared with Cd47 fl/fl control littermates. Data are Means ± SEM of 2 independent experiments, 6-9 mice/group.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Control, Flow Cytometry, In Vivo, MANN-WHITNEY

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a PMN TEpM in vivo in response to LTB 4 (1 nM) was determined in ileal loops of WT and Cd47 −/− mice after intraluminal instillation of blocking mAb against CD11b (clone M1/70; 20 μg/mL), or rat IgG 2b isotype control (20 μg/mL). WT PMN migration was significantly reduced by blocking CD11b in the intestinal lumen compared to mice treated with isotype control. CD47 deficiency also resulted in a significant reduced PMN migration in Cd47 −/− mice compared with WT mice. Importantly, blocking CD11b on Cd47 −/− mice did not further decrease PMN migration compared to Cd47 −/− mice treated with isotype control. Data are Means ± SEM of 3 independent experiments, 12-14 mice/group (**p≤0.01) as determined by two-way ANOVA analysis. b After luminal content collection, intestinal sections were enzymatically digested for quantification of PMN in LP-enriched fraction by flow cytometry, or fixed and paraffin-embedded for histological analysis. Numbers of PMN within the LP fraction were similar between different treatments. Data are Means ± SEM of 2 independent experiments, 8-14 mice/group. c Representative images of immunohistochemical staining including higher magnification insets, depicting Ly6G+ PMN. 40x objective. Scale bars: 50 μm.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: In Vivo, Blocking Assay, Control, Migration, Flow Cytometry, Immunohistochemical staining, Staining

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a Schematic representation of BMN migration in vitro in response to a chemoattractant gradient of LTB 4 (10 nM) for 2 hr at 37°C. b CD47 cell surface expression on BMN isolated from WT and Cd47 −/− mice by flow cytometry. c WT and Cd47 −/− BMN migration in response to LTB 4 in presence of blocking mAb against CD11b, CD47 or isotype control (20 μg/mL in each case). WT BMN migration was significantly reduced by addition of inhibitory CD11b or CD47 mAb. Loss of CD47 on Cd47 −/− BMN resulted in a significant reduced migration compared to WT BMN. Addition of blocking mAbs against CD11b or CD47 did not further reduce Cd47 −/− BMN migration in comparison to Cd47 −/− BMN migration in the presence of isotype mAb. Data are Means ± SEM of three independent experiments. *p ≤ 0.01 as determined by two-way ANOVA analysis.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Migration, In Vitro, Expressing, Isolation, Flow Cytometry, Blocking Assay, Control, Comparison

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a, b Murine CD18, CD11a, CD11b and CD47 were immunoprecipitated from non-stimulated WT and Cd47 −/− BMN lysates and immunoblotted for CD47 as detailed in the methods. a Representative western blot confirming the expression of CD47 in total lysates from WT but CD47 loss in Cd47 −/− BMN. b CD47 was co-immunoprecipitated with CD11b and CD18, and, to a lesser extent, with CD11a on WT BMN. Cd47 −/− BMN were used as negative control. c The association between CD47 and CD11b on unstimulated, non-permeabilized BMN was confirmed by in situ proximal ligation assay (PLA) using a combination of CD47 and CD11b Abs as detailed in the methods. The positive fluorescent staining (white dots) indicates close proximity (<40 nm) between CD47 and CD11b targets. Positive controls include use of CD11b-CD18 Abs as a known heterodimeric interaction. Furthermore, as a negative control, CD47-CD11b interaction was probed on BMN from Cd47 −/− mice and no signal was detected. Nuclei were counter-stained with Hoechst 33342. 100x objective. Scale bars: 50 μm.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Western Blot, Expressing, Negative Control, In Situ, Ligation, Staining

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a WT and Cd47 −/− BMN express comparable surface levels of CD11a and CD11b integrins. b-d BMN were drawn across coverslips coated with immobilized ICAM-1-Fc, VCAM-1-Fc or E-selectin-Fc adhesion molecules at decreasing levels of shear stress ranging from 1.5 to 0.75 dynes/cm 2 , and BMN adhesion was measured as described in methods. In some experiments, BMN were pre-treated with function blocking mAbs anti-CD11a (M1/70; 20 μg/mL) or anti-CD11b (M1/70; 20 μg/mL). Data are Means ± SEM of at least three independent experiments. *p≤0.05, **p≤0.01, ***p≤0.001 as measured by ANOVA analysis followed by Tukey’s multiple comparison test.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Shear, Blocking Assay, Comparison

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: CD47 was knocked down by CRISPR/Cas9 in the human promyelocitic cell line (HL60). HL60 and CD47-null HL60 (HL60/E2) were then differentiated into a neutrophil-like phenotype as described in the methods. a, b CD11b expression on resting, differentiated HL60 was not significantly altered by CD47 knockdown: a cell surface expression of CD11b on differentiated PMN was quantified by flow cytometry and b total CD11b content was determined by western blotting. Image shows a representative western blot and graph represents a densitometry analysis of three different assays. c CD47 and CD11b association in HL60 was assessed by PLA. Positive fluorescent signals were elicited between CD47 and CD11b Abs, as well as between CD11b and CD18 Abs indicating close association of these proteins. CD47 and CD11b Abs did not elicit fluorescent signals on HL60/E2 (CD47 null). Nuclei were counterstained with Hoechst 33342. 100x objective. Scale bars: 50 μm. d Differentiated HL60 and HL60/E2 were stimulated with fMLF (1 μM) for different times and CD11b surface expression was determined by flow cytometry. Increased surface expression of total CD11b after fMLF stimulation was not different between HL60 and HL60/E2. e, f CD11b and CD18 activation on HL60 was determined by using activation reporter mAbs, CBRM1/5 and m24 respectively. fMLF stimulation resulted in activation of CD11b and CD18 on HL60 control cells, but the magnitude was significantly reduced in HL60/E2. Data are Means ± SEM of at least three independent experiments. *p≤0.05, **p≤0.01 as determined by Two-way ANOVA analysis. No Tx (no fMLF treatment).

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: CRISPR, Expressing, Knockdown, Flow Cytometry, Western Blot, Activation Assay, Control

Journal: Frontiers in Oncology

Article Title: YB-1-based oncolytic virotherapy in combination with CD47 blockade enhances phagocytosis of pediatric sarcoma cells

doi: 10.3389/fonc.2024.1304374

Figure Lengend Snippet: Infection with XVir-N-31 (XVir) increases both calreticulin (CALR) and CD47 surface expression on pediatric sarcoma cell lines. (A) Analysis of CALR (‘eat-me’) and CD47 surface expression (‘don’t-eat-me’) surface expression of pediatric sarcoma cell lines A673, SKNMC, and U2OS 48 hours post infection (hpi) at indicated multiplicity of infection (MOI) assessed by FACs analysis after dead cell exclusion via DAPI. Y-axis depicts the fold change of expression compared to controls (ctrl) using frequency of parent minus isotype (IT). (B) Analysis MOI/dose-dependency of CD47 surface expression at 48hpi using indicated MOI. Statistical analysis was performed using the unpaired student’s t-test in (A) and one way ANOVA with multiple comparison and Tukey correction in (B) . Plotted is the mean with SD. Each dot represents one biological replicate. Experiments were repeated at least twice to ensure reproducibility. Levels of significance are indicated as asterisks *p<0,0332; **p<0,0021; ***p<0,0002; ****p<0,0001.

Article Snippet: Following antibodies were used: Human CD47 Antibody, (#AF4670, R&D Systems, 1:500), Adenovirus-Hexon (#AB1056, Merck Millipore, 1:1000), GAPDH (#2118S, Cell Signaling Technology, 1:2000), Mouse anti-rabbit IgG HRP (#Sc-2357, Santa Cruz Biotech, 1:1000), anti-sheep IgG HRP (#HAF016, R&D Systems, 1:1000).

Techniques: Infection, Expressing, Comparison

Journal: Frontiers in Oncology

Article Title: YB-1-based oncolytic virotherapy in combination with CD47 blockade enhances phagocytosis of pediatric sarcoma cells

doi: 10.3389/fonc.2024.1304374

Figure Lengend Snippet: The combination (combo) of XVir-N-31 (XVir) and the CD47-inhbitor (CD47i) B6H12.2 shows the highest levels of phagocytosis for all tested phagocytes and cell lines. Phagocytosis by THP-1 macrophages (A) , THP-1 imDCs (B) , and healthy donor-derived monocytic imDCs (C) was assessed at indicated MOI (48hpi) and time (y-axis) for A673 (left panels) and U2OS (right panels). CD47i was added when starting phagocytosis. Each dot represents one biological replicate. Experiments were repeated at least three times. Plotted is the normalized phagocytosis compared to ctrl as mean and SD. One way ANOVA with multiple comparison and Tukey correction was used for statistical analysis. Levels of significance are indicated as asterisks *p<0,0332; **p<0,0021; ***p<0,0002; ****p<0,0001.

Article Snippet: Following antibodies were used: Human CD47 Antibody, (#AF4670, R&D Systems, 1:500), Adenovirus-Hexon (#AB1056, Merck Millipore, 1:1000), GAPDH (#2118S, Cell Signaling Technology, 1:2000), Mouse anti-rabbit IgG HRP (#Sc-2357, Santa Cruz Biotech, 1:1000), anti-sheep IgG HRP (#HAF016, R&D Systems, 1:1000).

Techniques: Derivative Assay, Comparison

Journal: Frontiers in Immunology

Article Title: IL-27 Derived From Macrophages Facilitates IL-15 Production and T Cell Maintenance Following Allergic Hypersensitivity Responses

doi: 10.3389/fimmu.2021.713304

Figure Lengend Snippet: CD3, CD47, and CD172a expression in human ACD clusters. (A–C) Immunofluorescence staining of CD3 (green), CD47 (red), CD172a (purple), CD14 [green, a serial slide section with the staining of CD172a (purple)], and Hoechst (blue) in human donor-matched patch-test negative control and patch-test (+) ACD skin. White dashed lines mark the epidermal-dermal junction. Data are representative of 3 patient samples per tested condition. (A) Scale bars are 200 µm (left) and 100 µm (right). (B, C) Scale bars are 20 µm.

Article Snippet: Mouse IgG1 isotype control (MOPC-21) (Tonbo Biosciences), Goat IgG isotype control (R&D Systems), Sheep IgG isotype control (R&D Systems), Rabbit isotype control (Southern Biotech, Birmingham, AL), anti-human CD14 (61D3, Tonbo Biosciences), anti-human iNOS (polyclonal, Thermo Fisher Scientific), anti-human CD8 (MCD8, Santa Cruz Biotechnology, Dallas, TX), and IL27R (polyclonal, R&D Systems), anti-human IL-27 (polyclonal, R&D Systems), anti-human CD86 (IT2.2, Biolegend), anti-human CD3 (SP7, Abcam, Cambridge, England), anti-human CD47 (polyclonal, R&D Systems), anti-human SIRP alpha (CD172a) (OTI7B3, Origene), anti-human IL-15 (polyclonal, R&D systems), anti-human BCL2 (clone 100, BioLegend), anti-mouse CD3 (17A2, Tonbo Biosciences), and anti-mouse CD8 (YTS 105.18, Novus Biologicals, Littleton, CO) followed by reaction with Cy3, Alexa Fluor 555, Alexa Fluor 647, Alexa Fluor 488, or FITC-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Immunofluorescence, Staining, Negative Control