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

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Surface CD47 (A) and ecto-CRT protein expression (B) shown as geometric MFI in a panel of six different NSCLC cell lines. Each histogram represents the mean (± SD) of three to five independent experiments. Comparisons made by ANOVA with Fisher's post hoc multiple comparison analysis. ### p < 0.03, ## p < 0.01, # p < 0.0005. Below each histogram, a matrix table where all p values resulting from post hoc analysis are reported. Expression levels of CD47 (C) and CRT mRNA (D) in 226 untreated primary NSCL adenocarcinomas (GEO accession number GSE31210 ). Middle lines in box plots represent the medians and whiskers represent 5–95% CI ( ### p < 0.03, Kruskal-Wallis test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Comparison

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Modulation by gefitinib of surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Flow cytometric profiles of surface CD47 (A) and ecto-CRT expression (B) on DMSO-treated (CTRL, gray lines) and gefitinib-treated (GEF, red lines) NSCLC cells. Histograms show the mean (± SD) of fold changes of CD47 (C) and ecto-CRT (D) geometric MFI, relative to DMSO-treated controls ( N = 3–5, * p < 0.05, ** p < 0.01 paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Gefitinib-induced CD47 down-regulation promotes tumor cell phagocytosis by dendritic cells. Representative flow cytometric analyses and mean ± SD ( N = 4 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells (see Methods) against PC9 (A,B) , HCC827 (C,D) , and H1975 cells (E,F) treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Cancer cells exposed to the drug for 48 h were labeled with DiO tracer and then co-cultured with dendritic cells for 2 h at a 1:1 ratio. Phagocytosis assays were also run at 4°C as controls. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (* p < 0.05, n.s., not significant, paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Activity Assay, Derivative Assay, Labeling, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Blocking of CD47 on tumor cells induces phagocytosis by dendritic cells. Dendritic cells were co-cultured with DiO tracer-labeled HCC827 (A) and H1975 (B) cancer cells in the presence of IgG isotype control or anti-CD47 mAb as indicated. Shown is the mean (± SD, N = 3 independent healthy donors) percentage increase of CD11c/DiO tracer double positive cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( # p < 0.05, ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Blocking Assay, Cell Culture, Labeling, Control

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Expression levels of surface CD47 increase in cancer cells acquiring resistance to gefitinib and inhibit tumor cell phagocytosis by dendritic cells. Surface CD47 (A) and ecto-CRT expression (B) in gefitinib-sensitive PC9 and HCC827 (gray lines) and resistant PC9GR and HCC827GR (green lines) cell lines. Representative flow cytometric histograms (left) and mean (± SD, N = 3–5) fold changes of treatment-resistant over sensitive cells (right). (C) Representative flow cytometric histogram plots (left) and mean (± SD) fold changes (right) of surface CD47 levels in resistant cell lines treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Acquisition of resistance to gefitinib abolished drug-induced CD47 down-regulation in PC9GR (* p < 0.05, ** p < 0.01, n.s., not significant, paired two-tailed Student's t -test). (D) Mean ± SD ( N = 3 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells against PC9GR cells in the absence or presence of gefitinib treatment, performed at 4°C as control and at 37°C. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (paired two-tailed Student's t -test. n.s., not significant). (E) Dendritic cells were co-cultured with gefitinib-treated, DiO tracer-labeled PC9GR cells in the presence of IgG isotype control or anti-CD47 mAb. Shown is the mean ± SD ( N = 3 independent healthy donors) percent change of CD11c + /DiO + tracer double positive dendritic cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Two Tailed Test, Activity Assay, Derivative Assay, Control, Cell Culture, Labeling

Journal: PNAS Nexus

Article Title: Immunotherapy of endometrial cancer via CD47 blockade-mediated macrophage phagocytosis

doi: 10.1093/pnasnexus/pgaf143

Figure Lengend Snippet: Up-regulation of CD47 expression in hECCs. A) IF staining of CD47 on human uterine tissue sections with (EC, n = 42) or without (PM, n = 23) EC. A stronger green fluorescence signal of CD47 staining on the surface of hECCs was detected compared with control PM endometrial epithelia with a basal level of fluorescence signal (PM). EpCAM was applied to label carcinoma cancer cells in EC or normal epithelial cells in PM shown as magenta. The cell proliferation was assessed by KI67 antibody staining as red. The quantification of cell proliferation is shown in Fig. . Blue DAPI staining was used to label cell nuclei and to assess gross cell morphology. B) Fluorescence intensity of CD47 staining in EC ( n = 42) and PM ( n = 23). ImageJ was used to measure the fluorescence staining signal of CD47. Thirty stained spots in EC or PM epithelium per human uterine sample were randomly captured for analysis. AU, arbitrary units. C) Level of CD47 protein in EC ( n = 34) or PM ( n = 16). ELISA was used to measure the CD47 protein level in EC tissue homogenate or in control endometrial tissue homogenate of PM. Thirty micrograms of total protein per human endometrial sample were used. Each dot in B) and C) represents a uterine tissue sample from a different patient. Data were analyzed by t test using Prism 10. * P < 0.05; **** P < 0.0001. Scale bar: 100 μm.

Article Snippet: Per instruction of Human CD47 DuoSet ELISA kit (catalog #: DY4670-05, R&D Systems), human endometrial tissue homogenate was used for CD47 protein measurement.

Techniques: Expressing, Staining, Fluorescence, Control, Enzyme-linked Immunosorbent Assay

Journal: PNAS Nexus

Article Title: Immunotherapy of endometrial cancer via CD47 blockade-mediated macrophage phagocytosis

doi: 10.1093/pnasnexus/pgaf143

Figure Lengend Snippet: CD47 protects hECCs from phagocytosis by macrophages. Phagocytosis assay using primary hECCs and healthy human macrophages. CFSE-labeled green hECCs (green) were incubated with macrophages derived from human peripheral blood monocytes in the presence of IgG1 isotype control (A) or an anti-CD47 (clone B6H12) mAb antibody (B), and their phagocytic indices were calculated (C). Phagocytosis assay using primary hECCs and normal mouse macrophages. CFSE-labeled hECCs (green) were incubated with RFP-labeled mouse bone marrow-derived macrophages in the presence of IgG1 isotype control (D) or an anti-CD47 (clone B6H12) mAb (E), and their phagocytic indices were calculated (F). The human macrophages were derived from one healthy human sample. The primary EC cells isolated from three independent human EC samples (EC#01, EC#02, and EC#03) were used. Each experiment was repeated for three times. More macrophages containing ingested green cancer cells were found in the anti-CD47 antibody treatment groups. Two-way ANOVA was performed with GraphPad Prism 10 for statistical analysis. **** P < 0.0001. Scale bar: 100 μm in human macrophage + human EC; 30 μm in murine macrophage + human EC.

Article Snippet: Per instruction of Human CD47 DuoSet ELISA kit (catalog #: DY4670-05, R&D Systems), human endometrial tissue homogenate was used for CD47 protein measurement.

Techniques: Phagocytosis Assay, Labeling, Incubation, Derivative Assay, Control, Isolation

Journal: PNAS Nexus

Article Title: Immunotherapy of endometrial cancer via CD47 blockade-mediated macrophage phagocytosis

doi: 10.1093/pnasnexus/pgaf143

Figure Lengend Snippet: Systemic treatment with an anti-CD47 mAb reduces EC burden. A) A schematic of systemic treatment with a CD47 blocking antibody. Anti-CD47 mAb ( n = 7 mice) or control IgG ( n = 8 mice) was given via IP injection to 1-month-old PR-cre +/− ;Pten flox/flox female mice twice per week for 8 weeks at 100 μg in 100 μL PBS per injection each mouse. B) Morphology of uteri posttreatment. Representative uteri from either anti-CD47 mAb or control IgG treatment groups were dissected out and imaged. C) Histology analysis of uteri posttreatments. H&E staining of uterine cross tissue sections shows a thin endometrium in the anti-CD47 mAb treatment group. IF staining of EpCAM was applied for labeling EC cells shown as green. D) Assessment of CD4 + helper T cells or CD8 + cytotoxic T cells by IF staining in uterus tissue from anti-CD47 mAb or control IgG treatment groups. E) Weight of uteri posttreatment with an anti-CD47 mAb or control IgG. F) Weight of bodies posttreatment with an anti-CD47 mAb or control IgG. G) EpCAM + epithelia tumor area in uterus tissue from anti-CD47 mAb or control IgG treatment groups. H) Quantification of CD4 + helper T cells in uterus tissue from either anti-CD47 mAb or control IgG treatment groups. I) Quantification of CD8 + cytotoxic T cells in uterus tissue from either anti-CD47 mAb or control IgG treatment groups. Blue DAPI staining was used to label cell nuclei and to assess gross cell morphology. Each dot in E) and F) represents an individual animal. The size of the green EpCAM + area of fluorescence was measured using ImageJ software to evaluate tumor size, at least five cross-sectional images per uterus for analysis, three uteri per treatment. The immune cells within a tissue area of 0.29 mm 2 under 40× magnification were counted based on the red fluorescence signal using a Leica DM5500 B automated upright microscope. At least nine tissue areas in each mouse uterus were randomly captured for analysis, three uteri per treatment. Data were analyzed by t test using Prism 10. *P < 0.05; **** P < 0.0001; ns P > 0.05. Scale bar: 1,000 μm in C) and 100 μm in D).

Article Snippet: Per instruction of Human CD47 DuoSet ELISA kit (catalog #: DY4670-05, R&D Systems), human endometrial tissue homogenate was used for CD47 protein measurement.

Techniques: Blocking Assay, Control, Injection, Staining, Labeling, Fluorescence, Software, Microscopy

Journal: PNAS Nexus

Article Title: Immunotherapy of endometrial cancer via CD47 blockade-mediated macrophage phagocytosis

doi: 10.1093/pnasnexus/pgaf143

Figure Lengend Snippet: In situ treatment with an anti-CD47 mAb reduces EC burden. A) A schematic of in situ treatment with a CD47 blocking antibody. Anti-CD47 mAb ( n = 10 mice) or control IgG ( n = 5 mice) was given daily via TV delivery to 1-month-old PR-cre +/− ;Pten flox/flox female mouse uterine lumen for 8 weeks at 10 μL of 25 μg per delivery each mouse. B) Morphology of uteri posttreatment. Representative uteri from anti-CD47 mAb or control IgG treatment groups were dissected out and imaged. C) Histology analysis of uteri posttreatments. H&E staining of uterine cross tissue sections shows a thin endometrium in the anti-CD47 mAb treatment group. IF staining of EpCAM was applied for labeling EC cells shown as green. D) Assessment of CD4 + helper T cells or CD8 + cytotoxic T cells by IF staining in uterus tissue from anti-CD47 mAb or control IgG treatment groups. E) Weight of uteri posttreatment with an anti-CD47 mAb or control IgG. F) Weight of bodies posttreatment with an anti-CD47 mAb or control IgG. G) EpCAM + epithelia tumor area in uterus tissue from anti-CD47 mAb or control IgG treatment groups. H) Quantification of CD4 + helper T cells in in uterus tissue from either anti-CD47 mAb or control IgG treatment groups. I) Quantification of CD8 + cytotoxic T cells in uterus tissue from either anti-CD47 mAb or control IgG treatment groups. Blue DAPI staining was used to label cell nuclei and to assess gross cell morphology. Each dot in E) and F) represents an individual animal. The size of the green EpCAM + area of fluorescence was measured using ImageJ software to evaluate tumor size, at least five cross-sectional images per uterus for analysis, three uteri per treatment. The immune cells within a tissue area of 0.29 mm 2 under 40× magnification were counted based on the red fluorescence signal using a Leica DM5500 B automated upright microscope. At least nine tissue areas in each mouse uterus were randomly captured for analysis, three uteri per treatment. Data were analyzed by t test using Prism 10. *P < 0.05; **** P < 0.0001; ns P > 0.05. Scale bar: 1,000 μm in C) and 100 μm in D).

Article Snippet: Per instruction of Human CD47 DuoSet ELISA kit (catalog #: DY4670-05, R&D Systems), human endometrial tissue homogenate was used for CD47 protein measurement.

Techniques: In Situ, Blocking Assay, Control, Staining, Labeling, Fluorescence, Software, Microscopy

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