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: Immuno-Oncology Technology

Article Title: CD47–SIRPα-targeted therapeutics: status and prospects

doi: 10.1016/j.iotech.2022.100070

Figure Lengend Snippet: Ongoing and recruiting trials of anti-CD47 and anti-SIRPα agents (by estimated study completion date, grouped by agent)

Article Snippet: TG-1801 (NI-1701) (CD47 and CD19 antibody) , TG Therapeutics, Inc. , TG-1801 OR TG-1801 + ublituximab , B-cell lymphoma , 16 , December 2022 , NCT03804996 , I , Recruiting.

Techniques: Recombinant, Transformation Assay, Mutagenesis

Journal: Nature Communications

Article Title: Cancer cell-expressed SLAMF7 is not required for CD47-mediated phagocytosis

doi: 10.1038/s41467-018-08013-z

Figure Lengend Snippet: Tumor-expressing SLAMF7 is not required for induction of phagocytosis upon CD47-targeting treatment in DLBCL cells. a Percentage of phagocytosis of DLBCL cell lines by allogeneic human macrophages primed with LPS/IFN-γ upon 3 h treatment with F(ab′)2 of anti-CD47 antibody inhibrix (CD47 F(ab′)2) vs. untreated cells ( n = 3–5). b Representative microscopy pictures of phagocytosis of tumor cells by macrophages primed with LPS/IFN-γ upon 3 h treatment with CD47 F(ab′)2 (left, MØ + V450-labeled OCIly3 cells, right, MØ + pHrodogreen-labeled SUDHL5 cells). Scale bar = 20 µm. c Representative graphs of flow cytometric analysis for phagocytosis of tumor cells by macrophages with LPS/IFN-γ upon 3 h treatment with CD47 F(ab′)2 (left, MØ + SUDHL5, right, MØ + SUDHL10). d Quantification of phagocytosis of DLBCL cell lines by flow cytometric analysis. Experimental setting is the same as in ( a ) ( n = 3–4). e Percentage of different types of macrophages from cibersort fraction of DLBCL biopsies ( n = 1804). f Percentage of phagocytosis of DLBCL cell lines by allogeneic type 0 human macrophages upon 3 h treatment with F(ab′)2 of anti-CD47 antibody inhibrix (CD47 F(ab′)2) vs. untreated cells ( n = 4–6). g Percentage of phagocytosis of DLBCL cell lines by allogeneic human macrophages primed with IL-10 upon 3 h treatment with F(ab′)2 of anti-CD47 antibody inhibrix (CD47 F(ab′)2) vs. untreated cells ( n = 4–6). Statistics was performed using paired Student’s t -test. n.s. = not significant, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Error bars stand for standard deviation (SD)

Article Snippet: APC-labeled anti-CD3, FITC-labeled anti-CD19 and APC-labeled anti-CD47 were purchased from Immunotools (Germany).

Techniques: Expressing, Microscopy, Labeling, Standard Deviation

Journal: Nature Communications

Article Title: Cancer cell-expressed SLAMF7 is not required for CD47-mediated phagocytosis

doi: 10.1038/s41467-018-08013-z

Figure Lengend Snippet: Efficacy of CD47-targeting antibodies in B-cell malignant cells does not correlate with SLAMF7 expression. a Experimental phagocytosis of DLBCL lines by macrophages either upon CD47 F(ab′)2 treatment or inhibrix treatment ( n = 3). Box plot contains center line representing median and whiskers representing 5–95%. b Representative microscopy pictures of phagocytosis of primary DLBCL cells by autologous macrophages upon 3 h treatment with inhibrix. Quantification of phagocytosis of primary DLBCL and MCL cells by autologous macrophages. c Quantification of surface SLAMF7 expression on five NHL lines ( n = 3). d Percentage of phagocytosis of NHL cell lines by allogeneic human macrophages primed with LPS/IFN-γ upon 3 h treatment with F(ab′)2 of anti-CD47 antibody inhibrix (CD47 F(ab′)2) vs. untreated cells ( n = 3–4). e Correlation between SLAMF7 expression and the percentage of experimental phagocytosis induced by CD47 F(ab′)2 in NHL and DLBCL cell panel ( n = 3). f Correlation between SLAMF7 expression and the percentage of experimental phagocytosis induced by anti-CD47 antibody inhibrix in DLBCL cell panel ( n = 3–4). g Correlation between SLAMF7 expression and the percentage of experimental phagocytosis induced by Rituximab in NHL and DLBCL cell panel ( n = 3). h Correlation between SLAMF7 expression and the percentage of experimental phagocytosis induced by the combinatory treatment of Rituximab and Inhibrix in DLBCL cell panel ( n = 3). i Experimental phagocytosis of tumor cells by macrophages upon RTX treatment or combination treatment with inhibrix ( n = 3). Experiments with primary patient-derived samples were performed in triplicates. Statistics was performed using paired Student’s t -test. n.s. = not significant, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Error bars stand for standard deviation (SD)

Article Snippet: APC-labeled anti-CD3, FITC-labeled anti-CD19 and APC-labeled anti-CD47 were purchased from Immunotools (Germany).

Techniques: Expressing, Microscopy, Derivative Assay, Standard Deviation

Journal: Nature Communications

Article Title: Cancer cell-expressed SLAMF7 is not required for CD47-mediated phagocytosis

doi: 10.1038/s41467-018-08013-z

Figure Lengend Snippet: mRNA expression of SLAMF7 does not, but of CD47 does, associate with survival in DLBCL patients. a Kaplan–Meijer curve analysis of survival of DLBCL patients within high and low SLAMF7-expressing quartiles. b Kaplan–Meijer curve analysis of survival of DLBCL patients within high and low CD47-expressing quartiles. c Kaplan–Meijer curve analysis of survival of DLBCL patients within high and low SLAMF7-expressing quartiles additionally sorted on high and low expression of CD47. d Kaplan–Meijer curve analysis of survival of DLBCL patients within high and low CD47-expressing quartiles additionally sorted on high and low expression of SLAMF7

Article Snippet: APC-labeled anti-CD3, FITC-labeled anti-CD19 and APC-labeled anti-CD47 were purchased from Immunotools (Germany).

Techniques: Expressing

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from . P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from ). P-values were determined by two-sided Student’s t-test.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Infection, Quantitative Proteomics, Control, Virus, Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 aging” (A) and “CD47 hypertension” (B). C) Overview figure of the data derived from the literature searches. Age-related increased CD47 levels may contribute to pathogenic conditions associated with severe COVID-19.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay, Infection