Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: HBECs were infected with ( + IAV ) or without ( Mock ) influenza A virus. a Immunoblot analysis of junction protein ZO-1 and surface protein CD47 at 1 day post-infection (dpi). Normalized CD47 and ZO-1 protein levels are presented as bar graphs (Mock, n = 4; + IAV, n = 4). b Representative whole-mount images of ZO-1 (white) and CD47 (red) at 1 dpi. The area where ZO-1 disconnection occurred are presented as bar graphs (Mock, n = 6; + IAV, n = 6). c Quantitative PCR (qPCR) analysis of ZO-1 and CD47 mRNAs at 1 and 3 dpi [Mock, n = 3; + IAV (1 dpi), n = 3; + IAV (3 dpi), n = 3]. d HBECs were treated with or without 10 μM NF-κB inhibitor caffeic acid phenethyl ester (CAPE) for 1 h before influenza virus infection. Immunoblot analysis of ICAM-1 and CD47 at 1 dpi. Normalized ICAM-1 and CD47 protein levels are presented as bar graphs (DMSO + Mock, n = 4; DMSO + IAV, n = 4; CAPE + Mock, n = 4; CAPE + IAV, n = 4). e Whole-mount images of influenza virus-infected HBECs. Co-staining of CD47 (red) and ciliated cell-specific marker protein Ac-α-tubulin (green, n = 4) or goblet cell-specific marker protein MUC5AC (cyan, n = 3). Percentages of CD47-positive cells are presented as bar graphs. Data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed Student’s t test or one-way ANOVA with Tukey’s multiple comparisons test. n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Infection, Virus, Western Blot, Real-time Polymerase Chain Reaction, Staining, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: For CD47 knock-down, HBECs were transfected with scrambled shRNA ( sc-shRNA ) or shRNA targeting CD47 ( CD47 shRNA ) using a lentiviral delivery system. For CD47 neutralization, HBECs were treated with either IgG1 (MOPC-21) or α-hCD47 (B6H12.2) antibodies. a Gene expression of CD47 was analyzed using qRT-PCR (normalized by GAPDH mRNA) (sc-shRNA, n = 4; CD47 shRNA, n = 4). b Protein expression of CD47 was analyzed using immunoblotting (sc-shRNA, n = 5; CD47 shRNA, n = 5). c Paracellular FITC-dextran permeability was measured in Mock ( n = 16), Virus only (IAV, n = 16), Bacteria only ( S. aureus , n = 16), sc-shRNA + Super-infection ( n = 12), CD47 shRNA + Super-infection ( n = 6), IgG1 + Super-infection ( n = 6), and α-hCD47 antibodies (Ab) + Super-infection ( n = 8). d Trans-epithelial resistance was measured in Mock ( n = 15), IAV ( n = 14), S. aureus ( n = 13), sc-shRNA + Super-infection ( n = 13), CD47 shRNA + Super-infection ( n = 6), IgG1 + Super-infection ( n = 6), and α-hCD47 Ab + Super-infection ( n = 8). e Microscopic images of HBECs at 5 dpi. The percentage of the damaged area is presented as bar graphs (Mock, n = 3; Virus only, n = 3; Bacteria only, n = 3; Super-infection, n = 4; IgG1 + Super-infection, n = 4; α-hCD47 Ab + Super-infection, n = 4). Data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed Student’s t test or one-way ANOVA with Tukey’s multiple comparisons test. N.D. not determined. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Knockdown, Transfection, shRNA, Neutralization, Gene Expression, Quantitative RT-PCR, Expressing, Western Blot, Permeability, Virus, Bacteria, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: a , b Influenza virus-infected HBECs ( IAV ) infected with live S. aureus or S. aureus -derived samples ( S , supernatant of S. aureus -cultured media; U , UV-killed S. aureus ; H , heat-killed S. aureus ). Paracellular FITC-dextran permeability ( a ) and trans-epithelial electrical resistance ( b ) of 7 groups: i ) Mock ( n = 3), ii ) Virus only ( n = 3), iii ) Bacteria only ( n = 3), iv ) Super-infection ( n = 3), v ) Virus with S ( n = 3), vi ) Virus with U ( n = 3), and vii ) Virus with H ( n = 3). c Whole mount image of CD47 (red) and S. aureus (green) in the influenza virus-infected HBECs at 1 dpi. An open arrow head indicates CD47 + cell without S. aureus and closed arrow heads indicate CD47 + cells with S. aureus . Co-localization of CD47 + cells and S. aureus are presented as violin plots (Mock, n = 6; + IAV, n = 6). d – g Bacterial adhesion assay. Colonization of S. aureus was assessed in HBECs inoculated with the virus (MOI 1) for 2 h before treatment with IgG1 control antibodies or α-hCD47 neutralizing antibodies (2 h), followed by S. aureus (MOI 3) infection for 3 h (IgG1 + S. aureus , n = 4; α-hCD47 Ab + S. aureus , n = 4; IgG1 + Super-infection, n = 4; α-hCD47 Ab+ Super-infection, n = 4) ( d ). Adherence of S. aureus WT (FnBP A+/B+, n = 4) and three mutant strains (FnBP A+/B–, n = 4; FnBP A–/B+, n = 4; FnBP A–/B–, n = 4) was assessed in HBECs inoculated with the virus (MOI 1) for 2 h, followed by S. aureus (MOI 3) infection for 3 h ( e ). Colonization of S. aureus WT (FnBP A+/B+) ( f ) and double deletion mutant (FnBP A–/B–) ( g ) was assessed in HBECs inoculated with the virus (MOI 1) for 2 h before treatment with IgG1 control antibodies ( n = 4) or α-hCD47 neutralizing antibodies (1, 5, and 10 μg/mL for FnBP A+/B+, and 10 μg/mL for FnBP A–/B–, n = 4 each) for 2 h, followed by S. aureus (MOI 3) infection for 3 h. h Pull-down assay using His-tagged hCD47 recombinant protein. Bacterial plating [FnBP A+/B+ and FnBP A–/B– ( n = 3 each in the absence or in the presence of His-tagged hCD47)] and immunoblot analysis were performed using supernatants and pellets after separation with α-His-Dynabeads™/DynaMag™−2 system. The graphs present the percentage of colony numbers grown in the culture of the supernatants or the pellets. Data are presented as mean values ± SEM. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test or unpaired two-tailed Student’s t test. n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Virus, Infection, Derivative Assay, Cell Culture, Permeability, Bacteria, Cell Adhesion Assay, Control, Mutagenesis, Pull Down Assay, Recombinant, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: 6–8-weeks-old (18–21 g of body weight) FoxJ1 -Cre;floxed ( CD47 Foxj1 ), LysM -Cre;floxed ( CD47 LysM ), and control floxed ( CD47 f/f ) mice were infected with 100 PFU of influenza virus on day 0, and 1 ×10 8 CFU of S. aureus on day 7. a , b , k , l Body weight loss ( a , k ) and survival rates ( b , l ) were monitored for 29 days. The dotted line indicates the body weight exclusion cut-off. A mantel cox survival analysis was used to compare the survival rates between groups. The numbers in parenthesis represent the count of surviving mice. c , m Representative hematoxylin and eosin (H&E) staining of lung sections. The dotted lines indicate lymphocytic infiltration and arrows indicate alveolar hemorrhage. Lung injury scores are presented as violin plots in CD47 Foxj1 mice ( CD47 f/f , n = 8; CD47 Foxj1 n = 5) ( c ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 8) ( m ). d – j , n – t Tissue injury parameters were measured at 24 h after bacterial infection; total cell number in BAL fluids (BALF) of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 5) ( d ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 10) ( n ); bacterial adherence in the lung of CD47 Foxj1 mice ( CD47 f/f , n = 10; CD47 Foxj1 , n = 11) ( e ) and CD47 LysM mice ( CD47 f/f , n = 13; CD47 LysM , n = 15) ( o ); bacterial invasion in the lung of CD47 Foxj1 mice ( CD47 f/f , n = 7; CD47 Foxj1 , n = 7) ( f ) and CD47 LysM mice ( CD47 f/f , n = 5; CD47 LysM , n = 5) ( p ); and bacterial burden in the spleen of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 6) ( g ) and CD47 LysM mice ( CD47 f/f , n = 5; CD47 LysM , n = 5) ( q ); total protein concentrations in BALF of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 5) ( h ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 10) ( r ); and inflammatory cytokines TNF-α in BALF of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 5) ( i ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 10) ( s ), and IL-6 in BALF of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 5) ( j ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 10) ( t ). Data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Control, Infection, Virus, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: a – j 6–8-weeks-old (18–21 g of body weight) FoxJ1 -Cre; floxed ( Cd47 Foxj1 ) and control floxed ( Cd47 f/f ) mice were infected with 100 PFU of influenza virus on day 0, and 1 ×10 8 CFU of S. aureus WT (FnBP A+/B+) and double deletion mutant (FnBP A–/B–) on day 7 ( Cd47 f/f + FnBP A+/B+, n = 9; Cd47 f/f + FnBP A–/B–, n = 11; Cd47 Foxj1 + FnBP A+/B+, n = 12; Cd47 Foxj1 +FnBP A–/B–, n = 10). Body weight loss ( a ) and survival rates ( b ) were monitored for 29 days. The dotted line indicates the body weight exclusion cut-off. A mantel cox survival analysis was used to compare the survival rates between groups. The numbers within circles or squares represent the count of surviving mice. Representative hematoxylin and eosin (H&E) staining of lung sections ( c ). Lung injury scores are presented as violin plots ( Cd47 f/f + FnBP A+/B+, n = 4; Cd47 f/f + FnBP A–/B–, n = 5; Cd47 Foxj1 + FnBP A+/B+, n = 5; Cd47 Foxj1 +FnBP A–/B–, n = 5). Tissue injury parameters were measured at 24 h after bacterial infection ( Cd47 f/f + FnBP A+/B+, n = 4; Cd47 f/f + FnBP A–/B–, n = 5; Cd47 Foxj1 + FnBP A+/B+, n = 5; Cd47 Foxj1 +FnBP A–/B–, n = 5); total cell number in BAL fluids (BALF) ( d ), bacterial adherence ( e ) and invasion ( f ) in the lung, and bacterial burden in the spleen ( g ), total protein concentrations in BALF ( h ), and inflammatory cytokines TNF-α ( i ) and IL-6 ( j ) in BALF. k Proposed model of viral infection-induced CD47-mediated secondary bacterial infection. Data are presented as mean values ± SEM. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Control, Infection, Virus, Mutagenesis, Staining

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Histograms depicting cell surface expression of CD24 and CD47 by flow cytometry on mouse cancer cell lines. B, Correlation of CD24 and CD47 surface expression of cell lines shown in A by geometric MFI. Data shown as mean ± SD of 3 technical replicates. Simple linear regression was performed to assess correlation. C, Representative plots showing quantification of CD45+ phagocytic primary mouse macrophages co-cultured with CFSE+ KPCA.C. Co-cultures were exposed to vehicle control (PBS) or 10 ug/ml of monoclonal antibodies against mouse CD47, CD24, or the combination for 2 hours. Phagocytosis is represented as CD45+ macrophages that had engulfed CFSE+ KPCA.C cells as a percentage of the total macrophage population. D, Quantification of phagocytosis for cell lines in A . Cell lines are organized based on expression levels of each surface marker. Data represent mean ± SD of 3 technical replicates. E, Correlation of cell surface expression levels of CD47 and CD24 compared to phagocytosis upon treatment with the corresponding antibodies for each cell line. Data points depict mean ± SD from 3 replicates for each experiment. Correlation was assessed by simple linear regression. F, Representative microscopy images of GFP+ KPCA.C cells when co-cultured with primary mouse macrophages upon treatment with vehicle control (PBS), 10 ug/mL anti-CD47, 10 ug/mL anti-CD24, or the combination for 6.5 days. Top row depicts raw images of GFP+ fluorescence. Bottom row depicts purple GFP+ mask for above images used for quantification of cancer cell growth. Scale bar, 800 µm. G, Quantification of fluorescent well area from co-culture experiments for multiple cell lines after 6.5 days, organized by surface expression of CD24. Cancer cells were quantified by either green (KPCA.C, 3LL ΔNRAS, MC38) or red (238N1) fluorescent area based on their fluorophore expression. Data and means shown from one (3LL ΔNRAS, MC38) or two (238N1, KPCA.C) independent experiments with 3 technical replicates per experiment. D,G, statistical significance ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 determined by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Bioprocessing, Marker, Microscopy, Fluorescence, Co-Culture Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Representative histograms demonstrating cell surface expression of CD47 and CD24 on knockouts of KPCA.C and knockdowns of 238N1 by flow cytometry. B, Representative gating of phagocytic APC CD45+ mouse macrophages when co-cultured with the indicated CFSE+ KPCA.C knockouts treated with vehicle control (PBS) for 2 hours. Phagocytic macrophages are calculated as CD45+ cells that have engulfed CFSE+ cancer cells after 2 hours as a percent of all macrophages. C,D, Quantification of phagocytosis as a percentage of the maximum phagocytic response of macrophages using KPCA.C knockout cells ( C ) or 238N1 knockdown cells ( D ) treated with vehicle control (PBS), anti-mouse CD47 antibody, anti-mouse CD24 antibody, or the combination. Data represents mean ± SD of 3 technical replicates. E,F, Quantification of fluorescent well area as a measure of GFP+ KPCA.C knockout cells ( E ) or mCherry+ 238N1 knockdown cells ( F ) growth after co-culture with primary mouse macrophages and the indicated antibodies on day 6.5. Data represents mean ± SD from two independent experiments of 3 technical replicates each. G,H, Quantification of phagocytosis using CFSE+ MC38 ( G ) or 3LL ΔNRAS ( H ) cancer cells that overexpress CD24 after co-culture with primary mouse macrophages and the indicated antibodies. Data represent mean ± SD from 3 individual experiments each containing 3 technical replicates. I, Quantification of phagocytosis using StayGold+ KPCA.C cancer cells treated with vehicle control (PBS), or anti-mouse CD24 antibody, in the absence or presence of FcR blocking reagents (Fc1, anti-mouse Truestain clone 93; Fc2, anti-mouse CD16/CD32 clone 2.4G2). Data represents mean ± SD of 3 technical replicates. ( C-H ) ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Knock-Out, Knockdown, Co-Culture Assay, Blocking Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: Results of scRNA-seq of sorted CD45+ immune cells from experiments using CD24 or CD47 knockout tumors. ( A,C,E ) Comparison of CD47- tumors (KPCA.C CD47 knockout, 238N1 CD47 knockout) to wild-type tumors (KPCA.C control, 238N1 control). A, Relative frequencies of immune cells from CD47- versus wild-type tumors. C, UMAP showing identified cell clusters. E, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways. ( B,D,F ) Comparison of CD24- tumors (KPCA.C CD24 knockout, 238N1 CD24 knockout) to wild-type tumors (KPCA.C control, 238N1 control). B, Relative frequencies of immune cells from CD24- versus wild-type tumors. D, UMAP showing identified cell clusters. F, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Knock-Out, Comparison, Control

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Diagram showing process for high-throughput development and functional evaluation of bispecific antibodies targeting macrophage immune checkpoints. Antibody sequences were transformed into scFvs and cloned into a knob-into-hole format using a human IgG1 Fc. Constructs targeting macrophage immune checkpoints (CD47, CD24, SIRPa, PD-1) were cloned into knob formats and crossed with tumor-binding constructs in a hole format. Bispecific antibodies (n = 77) were expressed in Expi293F cells and used for downstream biochemical and functional analysis. B, Growth of StayGold+ DLD-1 cells in co-culture with human macrophages and each bispecific antibody. Each curve represents the mean for an individual bispecific antibody from 4 replicates. Black curve with hashed lines represents mean and 95% CI of control wells . C, Anti-tumor efficacy of bispecific antibodies at approximately t = 6.5 days as evaluated by macrophage checkpoint category. *p<0.05, ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. D-F, Growth curves for each of the WTa2d1 constructs ( D ), CD24-3 constructs ( E ), or CV1 constructs ( F ). G, Representative whole-well imaging of co-cultures treated with different bispecific antibodies at approximately t = 6.5 day. Green signal depicts growth of StayGold+ DLD-1 cells. Rows contain different macrophage checkpoint arms, while columns contain different tumor-binding arms. H, Scatter plot showing binding of each bispecific antibody to human neutrophils versus red blood cells. I, Representative histograms showing binding of the indicated bispecific antibodies to human neutrophils and red blood cells.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: High Throughput Screening Assay, Functional Assay, Transformation Assay, Clone Assay, Construct, Binding Assay, Co-Culture Assay, Control, Imaging

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: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Preparation and characterization of anti‐CD47‐PCM@NP. A) Hydrodynamic size and zeta potential of CM vesicles, PLGA cores (NP), PCM@NP, and anti‐CD47‐PCM@NP. Data are means ± SD ( n = 3). B) Colocalization of NP/C6 (green) with DiD‐PCM (red), and the colocalization of FITC‐antibody (green) with DiD‐PCM@NP (red), both assessed by confocal laser scanning microscope (CLSM) (scale bar = 5 µm). C) Transmission electron micrographs of (a) NP, (b) CM vesicle, (c) PCM@NP, (d) Anti‐CD47‐PCM@NP, and (e) multiple anti‐CD47‐PCM@NP. All scale bars = 100 nm. D) SDS‐PAGE protein analysis of NP, PCM@NP, CM vesicles, and cancer cell lysate. Samples were tested at equal protein concentrations. CD47 protein and membrane‐specific protein on the cancer cell membrane were efficiently retained on the extracted membrane vesicles and the PCM@NP, detected by western blot. E) Determination of the antibody labeled by PE loaded on the surface of anti‐CD47‐PCM@NP by flow nanoanalyzer. F) The binding affinity of the antibody to the CM vesicles by surface plasmon resonance (SPR).

Article Snippet: Streptavidin Conjugation Kit‐Lightning‐Link (Abcam, ab102921). siRNAs (sense 5’‐3’ CACCGAAGAAAUGUUUGUGAATT; sense 5’‐3’ CCAUACGAAUAAGAGAAUCAUTT) were purchased from Shanghai Sangon Biotechnology Co., Ltd. Quantum R‐PE MESF Medium Level (FCSC827B) was from Bio‐Rad Laboratories, Inc. PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D) was purchased from Elabscience (Wuhan, China).

Techniques: Zeta Potential Analyzer, Laser-Scanning Microscopy, Transmission Assay, SDS Page, Membrane, Western Blot, Labeling, Binding Assay, SPR Assay

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Anti‐CD47‐PCM@NP effectively distinguished target cells from CD47‐expressing nontarget cells in vitro through indirect active targeting. A) Anti‐CD47‐PCM@NP avoided the blocking of CD47 on RBC and subsequent phagocytosis thus circumvented the on‐target toxicity of free anti‐CD47 towards RBC through the INTACT strategy. Scale bar = 50 µm. B,C) PCM@NP and anti‐CD47‐PCM@NP efficiently escaped the capture by B) macrophages with enhanced and parallel cellular uptake by C) target 4T1 cells, measured by flow cytometry. The antibody selectively dissociated from the carrier PCM@NP at the presence of 4T1 cells with high expression of D) CD47 in contrast to E) CD47 −/− 4T1 cells, shown by colocalization images and distribution map of PE‐antibody (red) and PCM@NP/C6 (green). Scale bar = 20 µm (multi‐cell images), 2 µm (single‐cell images). F) The schematic diagram of the microfluidic device. The tumor cells were cultured in the cavity of the microfluidic chip till adherence, and then exposed to flowing anti‐CD47 or anti‐CD47‐PCM@NP, and fluorescent images were captured at predetermined time points. G) Free anti‐CD47 sufficiently bound to the surface of 4T1 cells with high expression of CD47 (a). The antibody dissociated from PCM@NP at the presence of 4T1 cells (b), in contrast with CD47 −/− 4T1 group (c) (antibody labeled with FITC, green. PCM@NP labeled with DiD, red). Scale bar = 10 µm. Original movies are shown in Movie S1 (Supporting Information) (a), Movie S2 (Supporting Information) (b), and Movie S3 (Supporting Information) (c), respectively. Data are presented as mean ± SD ( n = 3). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: Streptavidin Conjugation Kit‐Lightning‐Link (Abcam, ab102921). siRNAs (sense 5’‐3’ CACCGAAGAAAUGUUUGUGAATT; sense 5’‐3’ CCAUACGAAUAAGAGAAUCAUTT) were purchased from Shanghai Sangon Biotechnology Co., Ltd. Quantum R‐PE MESF Medium Level (FCSC827B) was from Bio‐Rad Laboratories, Inc. PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D) was purchased from Elabscience (Wuhan, China).

Techniques: Expressing, In Vitro, Blocking Assay, Flow Cytometry, Cell Culture, Labeling

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Evaluation of the biological functions of anti‐CD47‐PCM@NP in vivo. A) In vivo and ex vivo targeting ability of anti‐CD47‐PCM@NP and anti‐CD47 in tumor‐bearing mice models determined by live imaging. B) The semiquantitative analysis of the ratio of fluorescence intensity (tumor/liver) of ex vivo imaging. C) In vivo biodistribution of coumarin 6 (C6)‐labeled formulations in tumor‐bearing mice models at (a) 2 h, (b) 4 h, (c) 8 h, and (d) 12 h after i.v. injection of NP/C6, PCM@NP/C6 and anti‐CD47‐PCM@NP/C6. Data are presented as mean ± SD ( n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: Streptavidin Conjugation Kit‐Lightning‐Link (Abcam, ab102921). siRNAs (sense 5’‐3’ CACCGAAGAAAUGUUUGUGAATT; sense 5’‐3’ CCAUACGAAUAAGAGAAUCAUTT) were purchased from Shanghai Sangon Biotechnology Co., Ltd. Quantum R‐PE MESF Medium Level (FCSC827B) was from Bio‐Rad Laboratories, Inc. PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D) was purchased from Elabscience (Wuhan, China).

Techniques: In Vivo, Ex Vivo, Imaging, Fluorescence, Labeling, Injection

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Antitumor efficacy of anti‐CD47‐PCM@NP and mechanistic investigation by CyTOF analysis. A) Representative images and phagocytic index of C57BL/6 bone marrow‐derived macrophages (BMDM) phagocytosing tumor cells following treatment with PCM@NP, anti‐CD47, and anti‐CD47‐PCM@NP. Scale bar = 50 µm. B) Timeline of the anti‐tumor efficacy study on tumor‐bearing mice (red arrows indicate intravenous administrations), and average tumor growth curves and picture of tumor tissues after the treatment. C) Individual tumor growth curves in each group. D) viSNE plot of intratumoral cells in tumor tissues after treatment with saline, PCM@NP, anti‐CD47, anti‐CD47‐PCM@NP and all groups merged. E) Heat map of the surface molecule and functional molecule expression of different subsets of immune cells in tumor tissues from all groups merged. F) tSNE visualization of all samples with the expression of CD4 and CD8a respectively. G) Percentage of cells in each cluster after treatment from each group. Data represented as mean ± SD ( n = 6). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents nonsignificance).

Article Snippet: Streptavidin Conjugation Kit‐Lightning‐Link (Abcam, ab102921). siRNAs (sense 5’‐3’ CACCGAAGAAAUGUUUGUGAATT; sense 5’‐3’ CCAUACGAAUAAGAGAAUCAUTT) were purchased from Shanghai Sangon Biotechnology Co., Ltd. Quantum R‐PE MESF Medium Level (FCSC827B) was from Bio‐Rad Laboratories, Inc. PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D) was purchased from Elabscience (Wuhan, China).

Techniques: Derivative Assay, Saline, Functional Assay, Expressing

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: The INTACT strategy efficiently delivers antibodies to tumors with reduced in vivo toxicity. A) Anti‐CD47‐PCM@NP exhibited no significant influence on red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and platelet (PLT). Data represented as mean ± SD ( n = 3). B–D) Anti‐CD47‐PCM@NP relieved the occurrence of fungal infection during antitumor treatment. B) Experimental timeline and treatments in tumor‐bearing mice (arrows indicate intravenous administrations). At day 14, mice were infected with C. albicans via tail vein injection. C) Colony‐forming units (CFU) on day 7 in the kidneys of infected mouse models ( n = 6). D) The survival rates of infected mice with different treatments ( n = 12). Data represented as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: Streptavidin Conjugation Kit‐Lightning‐Link (Abcam, ab102921). siRNAs (sense 5’‐3’ CACCGAAGAAAUGUUUGUGAATT; sense 5’‐3’ CCAUACGAAUAAGAGAAUCAUTT) were purchased from Shanghai Sangon Biotechnology Co., Ltd. Quantum R‐PE MESF Medium Level (FCSC827B) was from Bio‐Rad Laboratories, Inc. PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D) was purchased from Elabscience (Wuhan, China).

Techniques: In Vivo, Infection, Injection

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: The INTACT strategy is adaptive to multiple antibody‐based systems. A) Relative tumor volume growth with anti‐CD47‐PCM@NP/PTX treatment ( n = 6). B–G) The INTACT therapy refined the targeting precision of ADC. B) The diagram of ADC construction: Anti‐CD47 was modified with streptavidin and conjugated with DM1 via the crosslinker BMCC‐biotin. C) The conjugation of ADC was confirmed with SDS‐PAGE. D) Experimental timeline for the anti‐tumor efficacy study and hematology assessments of ADC‐PCM@NP (red arrows indicate intravenous administrations). E) Average tumor growth curves, and picture of the tumor tissues after the treatment ( n = 6). F) Individual tumor growth curves in each group ( n = 6). G) Hematology assessments of red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and platelet (PLT) ( n = 3). Data represented as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: Streptavidin Conjugation Kit‐Lightning‐Link (Abcam, ab102921). siRNAs (sense 5’‐3’ CACCGAAGAAAUGUUUGUGAATT; sense 5’‐3’ CCAUACGAAUAAGAGAAUCAUTT) were purchased from Shanghai Sangon Biotechnology Co., Ltd. Quantum R‐PE MESF Medium Level (FCSC827B) was from Bio‐Rad Laboratories, Inc. PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D) was purchased from Elabscience (Wuhan, China).

Techniques: Modification, Conjugation Assay, SDS Page

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