Journal: International Journal of Cardiology. Heart & Vasculature

Article Title: CD47 blockade enhances phagocytosis of cardiac cell debris by neutrophils

doi: 10.1016/j.ijcha.2023.101269

Figure Lengend Snippet: Effects of CD47 inhibition on infarct size in vivo and ex vivo . (A) Left: Graphical abstract of in vivo ischemia/reperfusion injury procedure. Middle: Exemplary cardiac TTC Evan’s blue staining of mice treated with anti-CD47 antibody (CD47 Ab, bottom row) or IgG isotype control (IgG Ab, top row) after 45 min of ischemia and 24 h of reperfusion. Right: Size comparison of area at risk (AAR, normalized to left ventricle (LV)) and infarct area (normalized to AAR) (n = 6–7, Student’s t -test with Welch correction, *: p < 0.05). (B) Left: Schematic illustration of Langendorff setup. Middle: Exemplary heart slices of wild-type (WT, top row) and CD47 −/− knockout mice (bottom row) after ex vivo ischemia/reperfusion. Right: Comparison of infarct size of WT andCD47 −/− mice after ex vivo ischemia/reperfusion (n = 5–7, Student’s t -test with Welch correction). (C) Left/Middle: Exemplary pictures of human cardiomyocytes (hCM) under normoxia or after in vitro hypoxia/reoxygenation with either anti-CD47 treatment (CD47 Ab) or IgG isotype control (IgG Ab) respectively. Right: Comparison of cell viability (normalized to viability of cells treated with IgG isotype control under normoxia) between anti-CD47 treated cells and control group under normoxia or after hypoxia/reoxygenation (n = 3, ANOVA with Bonferroni post-hoc test, ***: p < 0.001). ns: not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On each plate, three wells were chosen at random for incubation with human anti-CD47 antibody (clone: B6H12) or IgG control (clone: MOPC-21; both BioXCell, Lebanon, NH, USA), both at 1 μg/ml final concentration, which were added before the start of hypoxia.

Techniques: Inhibition, In Vivo, Ex Vivo, Staining, Control, Comparison, Knock-Out, In Vitro

Journal: International Journal of Cardiology. Heart & Vasculature

Article Title: CD47 blockade enhances phagocytosis of cardiac cell debris by neutrophils

doi: 10.1016/j.ijcha.2023.101269

Figure Lengend Snippet: Effects of CD47 inhibition on cardiac immune cell infiltration after repAMI. (A) Left: Exemplary pseudo-color dot plots for separation of immune cell populations in heart tissue using flow cytometry. Right: Number of neutrophils (CD45 + , Ly6G + , and CD11b + ), macrophages (CD45 + , Ly6G − , CD11b + and F4/80 + ), monocytes (CD45 + , Ly6G − , CD11b + and F4/80 − ) and lymphocytes (CD45 + and CD45R/CD3 + ) in heart tissue of mice treated either with anti-CD47 or IgG isotype control antibody (n = 5, Student’s t -test with Welch correction). (B) Exemplary three-dimensional (3D) visualization of CD31 neg volume and Ly6G + spots. (C) Analysis of LSFM-based measurements of myocardial injury volume (CD31 neg volume) and neutrophil count (Ly6G + spots) in mice after CD47 inhibition and control animals. Top left: Quantitative size comparison of CD31 neg volume per heart (n = 5, Student’s t -test with Welch correction, *: p < 0.05). Top right: Number of Ly6G + spots per heart (n = 5, Student’s t -test with Welch correction). Bottom left: Normalization of Ly6G + spots count to CD31 neg volume (n = 5, Student’s t -test with Welch correction, *: p < 0.05). Bottom right: Correlation of Ly6G + spots with CD31 neg volume per heart (n = 5, r - and p -values depicted). (D) Local density of Ly6G + spots associated to myocardial injury volume within 50 µm of CD31 neg border (n = 5, Student’s t -test with Welch correction). ns = not significant.

Article Snippet: On each plate, three wells were chosen at random for incubation with human anti-CD47 antibody (clone: B6H12) or IgG control (clone: MOPC-21; both BioXCell, Lebanon, NH, USA), both at 1 μg/ml final concentration, which were added before the start of hypoxia.

Techniques: Inhibition, Flow Cytometry, Control, Comparison

Journal: International Journal of Cardiology. Heart & Vasculature

Article Title: CD47 blockade enhances phagocytosis of cardiac cell debris by neutrophils

doi: 10.1016/j.ijcha.2023.101269

Figure Lengend Snippet: Baseline expression of CD47 on cardiac cells and effects of anti-CD47 treatment on phagocytotic activity of neutrophils. (A) Measurement of CD47 expression in baseline murine hearts. Left: Exemplary pseudo-color dot plots for separation of cell populations in heart tissue using flow cytometry. Right: Mean fluorescence intensity (MFI) corrected to isotype control of endothelial cells (CD45 − and CD31 + ), fibroblasts (CD45 − , CD31 − and CD140a + ) and cardiomyocytes (CD45 − , CD31 − and CD140a − ) in murine baseline heart tissue depicted as fold change relative to fibroblasts (n = 4, ANOVA with Bonferroni post-hoc test, ***: p < 0.001). (B) Left: Western blot micrographs of CD47 of whole cardiac tissue lysates from baseline and ischemia/reperfusion (I/R) hearts with corresponding whole protein stain. Right: Quantified expression of CD47 from micrographs on the left normalized to baseline group (n = 6, Student’s t -test with Welch correction, **: p < 0.01). (C) Representative data from immunofluorescence imaging in mouse hearts 24 h following I/R. The endothelial cell marker CD31 is shown in green, nuclei (DAPI) in blue and CD47 in red. The infarct zone is identified by a decrease in CD31 expression (white edging). (D) Left: Graphical abstract of in vitro neutrophil phagocytosis assay. Middle: Exemplary pseudo-color dot plots of Calcein AM + neutrophils after co-incubation with Calcein AM-labeled apoptotic cardiomyocytes using flow cytometry. Right: Quantification of percent phagocytosis of Calcein-AM labeled apoptotic cardiomyocytes by neutrophils (n = 5–6, Student’s t -test with Welch correction, *: p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On each plate, three wells were chosen at random for incubation with human anti-CD47 antibody (clone: B6H12) or IgG control (clone: MOPC-21; both BioXCell, Lebanon, NH, USA), both at 1 μg/ml final concentration, which were added before the start of hypoxia.

Techniques: Expressing, Activity Assay, Flow Cytometry, Fluorescence, Control, Western Blot, Staining, Immunofluorescence, Imaging, Marker, In Vitro, Phagocytosis Assay, Incubation, Labeling

Journal: bioRxiv

Article Title: CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals

doi: 10.1101/2021.10.11.463903

Figure Lengend Snippet: (A) PAOC/CD47 p cells and PAOC/CD47 p/h2 or PAOC/CD47 p/h4 cells were cultured alone or cocultured for 24h, and hCD47 cross-dressing on gated PAOC/CD47 p cells was assessed by FACS using anti-h/pCD47-PE mAb (clone CC2C6, reacting with both human and pig CD47). Shown are representative histogram profiles (Left; the numbers in the figure indicate the MFI of gated PAOC/CD47 p cells), and average MFI (Right; mean ± SDs; n = 6 replicates per group) of gated PAOC/CD47 p cells in the indicated cell cultures. ****, p < 0.0001 (two-tailed unpaired t-test). Results shown are representative of 3 independent experiments. (B) PAOC/CD47 null and PAOC/CD47 h2 or PAOC/CD47 h4 were cultured alone or cocultured for 24h, and analyzed for hCD47 cross-dressing on gated PAOC/CD47 null cells by FACS using anti-h/pCD47-PE mAb ( top ) or anti-hCD47-BV786 mAb ( bottom ). Shown are representative histogram profiles (Left panel; the numbers in the figure indicate the MFI of gated PAOC/CD47 null cells), and average MFI (Right panel; mean ± SDs; n = 3 replicates per group) of gated PAOC/CD47 null cells in the indicated cell cultures. **, p < 0.01; ****, p < 0.0001 (two-tailed unpaired t-test). Results shown are representative of 3 independent experiments. (C) Celltrace violet labeled PAOC/CD47 null (Left panel) or PAOC/CD47 h2 (Right panel) were cultured alone (Violet CD47 null or Violet CD47 h2 ) or cocultured with unlabeled PAOC/CD47 h2 (Violet CD47 null + CD47 h2 ) or PAOC/CD47 null (CD47 null or Violet CD47 h2 ) respectively, then the cells were stained by anti-h/pCD47-PE mAb. Shown are representative FACS profiles (n=3 replicates). Results shown are representative of 2 independent experiments. (D) Pig LCL and hCD47-tg LCL (LCL/CD47 p/h ) cells were cultured alone or cocultured for 24h, and analyzed for hCD47 cross-dressing on gated LCL cells (the numbers in the figure indicate the MFI of gated LCL cells). The staining control of “mixed at staining” indicates the two types of cells were cultured separately and mixed immediately prior anti-CD47 staining. Two independent experiments were performed, and each experiment had 2 replicates per group. Representative FACS profiles are shown. (E) PAOC47 null cells were cocultured with bone marrow cells from hCD47-tg miniature swine for 2 days, and analyzed for hCD47 cross-dressing on gated PAOC47 null cells by FACS using anti-h/pCD47-PE mAb (the numbers in the figure indicate the MFI of gated PAOC/CD47 null cells). Two independent experiments were performed, and each experiment had 2 replicates per group. Representative FACS profiles are shown.

Article Snippet: GFP + cells were sorted 3 days after lentivirus transduction, then sorted GFP + cells were expanded and assessed for CD47 expression by staining with BV786-conjugated anti-hCD47 mAb B6H12 (BD Bioscience) and PE-conjugated anti-hCD47 mAb CC2C6 (Biolegend).

Techniques: Cell Culture, Two Tailed Test, Labeling, Staining

Journal: bioRxiv

Article Title: CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals

doi: 10.1101/2021.10.11.463903

Figure Lengend Snippet: (A-B) CD47KO Jurkat cells (A) or pig LCL cells (B) were cultured in the absence ( left ) or presence ( right ) of EVs ( top ) or Exos ( bottom )prepared from PAOC/CD47 h2 cell culture supernatants for 2h or 6h, and analyzed for hCD47 cross-dressing by FACS using anti-hCD47-BV786 mAb. Representative FACS profiles of 3 independent experiments were shown. (C) Celltrace violet labeled PAOC/CD47 null cells were cultured in the absence ( left ) or presence ( right ) of EVs prepared from PAOC/CD47 h2 cells for 18h or 42h, and analyzed for hCD47 cross-dressing by FACS using anti-hCD47-BV786 mAb.

Article Snippet: GFP + cells were sorted 3 days after lentivirus transduction, then sorted GFP + cells were expanded and assessed for CD47 expression by staining with BV786-conjugated anti-hCD47 mAb B6H12 (BD Bioscience) and PE-conjugated anti-hCD47 mAb CC2C6 (Biolegend).

Techniques: Cell Culture, Labeling

Journal: bioRxiv

Article Title: CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals

doi: 10.1101/2021.10.11.463903

Figure Lengend Snippet: (A) CD47KO Jurkat cells were cocultured without ( left ) or with ( right ) EVs from PAOC/CD47 h2 cells at 37°C for 5h, then washed and incubated with recombinant human SIRPα-Fc chimera at 37°C for 1h. The biding of SIRPα-Fc proteins to CD47KO Jurkat cells were visualized by staining with APC-conjugated mouse anti-human IgG Fc mAb. Representative FACS profiles of 2 independent experiments are shown. (B) PAOC47 null and PAOC47 h2 cells were cultured alone ( left and middle ) or together ( right ) for 48h, and stained using anti hCD47-BV786 mAb, then PAOC47 null (R1), PAOC47 h2 (R3), and hCD47 + (i.e., hCD47 cross-dressed) PAOC47 null (R2) cells sorted from cocultures were used immediately for phagocytic assay. (C) PAOC47 null (R1), PAOC47 h2 (R3), or sorted hCD47 cross-dressed PAOC47 null (R2) cells were labeled with Celltrace violet, and incubated with human macrophages for 2 h, then phagocytosis was determined by FACS using anti-human CD14 mAb. Shown are representative FACS profiles (left) and levels (right; mean ± SDs; n = 3) of phagocytosis (i.e., percentages of human macrophages that have engulfed violet+ target cells (CD14 + violet + ) in human CD14 + macrophages). Representative results of 3 independent experiments are shown. (D) CD47KO Jurkat, WT Jurkat and CD47KO Jurkat cells pre-incubated with EVs from PAOC/CD47 h2 cells were labeled by Celltrace violet, and cocultured with human macrophages for 2h, then phagocytosis was analyzed by FACS. Shown are representative FACS profiles (left) and levels (right; mean ± SDs; n = 3) of phagocytosis (i.e., percentages of hCD14 + violet + in total hCD14 + macrophages). Representative results of 2 independent experiments are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant (two-tailed unpaired t-test).

Article Snippet: GFP + cells were sorted 3 days after lentivirus transduction, then sorted GFP + cells were expanded and assessed for CD47 expression by staining with BV786-conjugated anti-hCD47 mAb B6H12 (BD Bioscience) and PE-conjugated anti-hCD47 mAb CC2C6 (Biolegend).

Techniques: Incubation, Recombinant, Staining, Cell Culture, Labeling, Two Tailed Test