Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A) Schematic depicts the supported lipid bilayer system used to study phagocytosis. Silica beads are coated with a supported lipid bilayer containing fluorescent atto647 lipids. Anti-biotin IgG binds to biotinylated lipids in the bilayer. 10xHis-tagged CD47 extracellular domain is attached to beads via a Ni 2+ -chelating DGS-NTA lipid, so that the SIRPα binding domain is positioned outwards. (B) Beads (magenta) conjugated with IgG or IgG+CD47 were incubated with mouse bone marrow derived macrophages (BMDMs) expressing membrane tethered GFP (GFP-CAAX; green) for 30 minutes then imaged with confocal microscopy. The average number of beads engulfed per macrophage was counted and normalized to the maximum average number of beads engulfed per macrophage for that experiment to control for batch to batch variability in macrophage appetite. (C) Schematic depicts the stages of phagocytosis: target particle binding, initiation of phagocytosis, and completion (phagocytic cup closure). (D) Representative images of each stage. BMDMs are expressing GFP-CAAX (green, top; greyscale, bottom) and the bead supported lipid bilayer is labeled with atto647 (magenta, top). Box in the top panel shows area of inset below. Timelapse confocal microscopy was used to quantify the fraction of beads bound to a macrophage (E); the time from binding to initiation of phagocytosis, if it occurred (F); the fraction of bound beads that proceed to the initiation step (G); the engulfment time, which is the time from initiation to completion, if it completed (H); and the fraction of beads that proceeded from the initiation to completion (I). For (F) and (H), the large filled data points represent the mean of an independent replicate, while the smaller unfilled data points of the same shape indicate individual cells quantified on the same day for that replicate. For (B) each dot represents an independent replicate composed of at least 100 macrophages per condition; data was compared using an unpaired t test. For (E)-(I) each independent replicate composed of at least 15 engulfment events. The means from four independent replicates were compared using an unpaired t test. In all graphs, bars denote the mean ± SEM. * denotes p<0.05, ** denotes p<0.005, *** denotes p<0.0005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Binding Assay, Incubation, Derivative Assay, Expressing, Membrane, Confocal Microscopy, Control, Labeling

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A) Timelapse images show reaching phagocytosis of an IgG bead target by a BMDM. White arrowheads point to extension of phagocyte membrane (GFP-CAAX, green) out around the target (atto647 lipid; magenta). White arrow denotes subsequent closure of the phagocytic cup while the bead remains outside the phagocyte cortex. Images correspond to Video S1 (B) Timelapse images show sinking phagocytosis of IgG+CD47 bead. Yellow arrows show closure of the phagocytic cup when the bead is within the macrophage. Images correspond to Video S2. (C) Graph depicts the fraction of successful phagocytosis that occurred via sinking phagocytosis based on the apparent morphology in timelapse confocal microscopy data. (D) Graph depicts the position of the bead centroid relative to the phagocyte cortex when phagocytosis was completed. (E) Graph depicts the fraction of retracted reaching cups out of the total number of initiated reaching cups. Retracted cups were defined as cases in our timelapse data set where membrane extensions grew around the target but were subsequently disassembled. (F) Images show an example of failed reaching phagocytosis of an IgG+CD47 bead. White arrowheads highlight extension of the macrophage membrane, which is subsequently retracted. Images correspond to Video S3. For (C) and (E), the averages from four independent experiments were plotted and compared using an unpaired t test. Bars represent the mean ± SEM. For (D), each data point represents an individual bead ( n =60 targets per condition from 4 independent experiments), and data was compared using an unpaired t test. ** denotes p<0.005, *** denotes p<0.0005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Membrane, Confocal Microscopy

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A, B) BMDMs were incubated with IgG or IgG+CD47 beads then fixed and stained with 488 phalloidin (greyscale) to visualize F-actin during phagocytosis. Enrichment of F-actin at late-stage (>50% completed) phagocytic cup rims was measured by comparing the mean fluorescent intensity (MFI) of phalloidin at the cup rim (red dot) to the MFI of phalloidin at the cell cortex (blue line). Bead position is indicated with a dashed yellow line. (B) Graph depicts data quantification described in (A). The large filled data points represent the mean of an independent replicate, while the smaller unfilled data points of the same shape indicate individual cells collected on that day for that replicate. Dashed line at 1 corresponds to no F-actin enrichment at cup rims. (C) BMDMs were treated with pharmacological inhibitors of Rac and Cdc42 (NSC23766 and MBQ-167) or Rho (C3 transferase) for 24 hours, then incubated with IgG, IgG+CD47, or unopsonized supported lipid bilayer coated beads. Phagocytosis was measured by confocal microscopy. The average number of beads phagocytosed was normalized to the maximum phagocytosis in that replicate. (D) BMDMs were treated with pharmacological inhibitors of Rac and Cdc42 (NSC23766 and MBQ-167) or Rho (C3 transferase) for 24 hours, then the inhibitors were removed and replaced with fresh media. WT or CD47 KO mouse L1210 leukemia cells were dyed with CellTrace Far Red then opsonized with an anti-murine CD20 monoclonal antibody and added to the BMDMs. Phagocytosis was monitored for 10 hours via timelapse microscopy. The percent of BMDMs that engulfed was quantified, and data was normalized to maximum for that experiment. For (B), the means of 4 independent experiments were compared using an unpaired t test. For (C) and (D), each data point represents an independent experiment including quantification of at least 100 macrophages. Data was compared using one-way ANOVA with Holm Sidak multiple comparison test. In all graphs, bars represent the mean ± SEM. * denotes p<0.05, ** denotes p<0.005, *** denotes p<0.0005, **** denotes p<0.00005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Incubation, Staining, Confocal Microscopy, Microscopy, Comparison

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A-E) WT or CD47 KO mouse L1210 leukemia cells were dyed with CellTrace Far Red (magenta) then opsonized with an anti-murine CD20 monoclonal antibody and added to the BMDMs (green) from WT or Rac2 E62K/+ mice. (A) Stills from a representative timelapse showing WT BMDMs incubated with CD47 KO L1210 cells. Yellow arrow indicates phagocytosis. (B) Stills from a representative timelapse showing Rac2 E62K/+ BMDMs incubated with CD47 KO L1210 cells. Yellow arrow indicates phagocytosis. (C) Stills from a representative timelapse showing WT BMDMs incubated with CD47-positive WT L1210 cells. (D) Stills from a representative timelapse showing Rac2 E62K/+ BMDMs incubated with CD47-positive WT L1210 cells. Yellow arrow indicates phagocytosis. (E) Graphs show the phagocytosis of L1210 cells during a 10 hour timelapse, normalized to the maximum phagocytosis on each day. Data compared using one-way ANOVA with Holm Sidak multiple comparison test. Line and bars denote mean and SEM. * denotes p<0.05, ** denotes p<0.005, *** denotes p<0.0005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Incubation, Comparison

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A) BMDMs were incubated with unopsonized (--), IgG, or IgG+CD47 beads for 10 minutes, then lysed directly in 2X Laemmli sample buffer. Whole cell lysates were immunoblotted with the indicated antibodies. Representative of n = 3 independent replicates. (B, C) The ratio of phosphorylated Syk to total Syk or phosphorylated Vav1 to total Vav1 was quantified and normalized to the IgG condition for that replicate. (D) BMDMs were incubated with unopsonized (--), IgG, or IgG+CD47 beads for 10 minutes, then lysed in LB1 lysis buffer. Vav1 was immunoprecipitated, then probed for tyrosine phosphorylation. Representative of n = 2 independent immunoprecipitations. For (B) and (C), data was compared with one-way ANOVA with Holm Sidak multiple comparison test. Error bars denote SEM. * denotes p<0.05.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Incubation, Lysis, Immunoprecipitation, Comparison

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A) TIRF images show IgG (AlexaFluor 488-IgG, green) and mCherry-Vav1 (magenta) as BMDMs interact with an IgG (top) or IgG+CD47 (bottom) bilayer. The linescan shows the fluorescent intensity of AlexaFluor 488-IgG and mCherry-Vav1 at the indicated position (white arrow in images). Intensity was normalized so that 1 is the highest observed intensity and 0 is the lowest observed intensity. (B) The mean fluorescent intensity of mCherry-Vav1 at IgG clusters (left) and the area of the IgG clusters (right) was measured for n =30 cells landing on IgG or IgG+CD47 bilayers, pooled from 3 independent experiments. (C) Images from TIRF microscopy timelapse show IgG (black) clustering as BMDMs land and spread on a bilayer with IgG (top) or IgG+CD47 (bottom). Graph depicts the average area of contact from n ≥16 cells pooled from 3 separate experiments. (D) The Pearson’s Correlation Coefficient was calculated for mCherry-Vav1 and AlexaFluor 488-IgG in the footprint of n ≥38 cells from 3 separate experiments landing on IgG or IgG+CD47 bilayers. (E) BMDMs expressing membrane-tethered mCherry (mCherry-CAAX) or a hyperactive Vav1 mutant (mCherry-Vav3F) were incubated with unopsonized (--), IgG, or IgG+CD47 beads for 30 minutes, then visualized via confocal microscopy. The average number of beads engulfed per macrophage was quantified and normalized to the highest average for that experiment. Points denote averages from each of the 3 replicates comprising at least 100 macrophages. (F) Diagram shows the proposed pathway for CD47 signaling. Data was compared using an unpaired t test (B, D) or one way ANOVA with Holm Sidak multiple comparison test. (E). Line and error bars denote mean and SEM.* denotes p<0.05, ** denotes p<0.005, *** denotes p<0.0005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Microscopy, Expressing, Membrane, Mutagenesis, Incubation, Confocal Microscopy, Comparison

Journal: International immunology

Article Title: CD47 promotes T-cell lymphoma metastasis by up-regulating AKAP13-mediated RhoA activation.

doi: 10.1093/intimm/dxab002

Figure Lengend Snippet: Fig. 1. Generation of CD47-deficient EG7 cells. (A) Expression of Cd47 mRNA in various murine tissues and cell lines was measured by qPCR (n = 3). The mean values and standard errors are depicted. (B) Schematic representation of CD47-targeted sgRNA sequences. (C) Genomic sequences from CD47-deficient EG7 cells. (D) FACS analysis of CD47 expression in EG7 cells.

Article Snippet: To construct the CD47-targeted sgRNA-expressing vectors, the oligo DNA from murine CD47 exon 2 (5′-GGATAAGCGC GATGCCATGG-3′) was inserted into the BbsI site of pX330U6-Chimeric_BB-CBh-hSpCas9 (Addgene).

Techniques: Expressing, Genomic Sequencing

Journal: International immunology

Article Title: CD47 promotes T-cell lymphoma metastasis by up-regulating AKAP13-mediated RhoA activation.

doi: 10.1093/intimm/dxab002

Figure Lengend Snippet: Fig. 2. CD47 deficiency decreases tumor formation. (A) EG7 cells were subcutaneously injected into the right flank of C57BL/6 mice and the tumor volume was measured. (B) The rate of EG7 tumor establishment by subcutaneous injection. (C, D) EG7 cells were intravenously injected into the tail vein of C57BL/6 mice. The survival rates of the mice and representative images are shown. Arrows indicate EG7 tumors in peritoneal cavity. (E) EG7 cells were injected in the same manner as in (C), and then the paraffin-embedded mouse liver was stained by hematoxylin and eosin at day 30. (F) CD47 KO EG7 cells were electroporated with CD47 expression vector and then recovery of CD47 expression was confirmed by FACS. (G) CD47 expression-recovered EG7 cells were intravenously injected to C57BL/6 mice and the survival rates of the mice are shown. (H, I) Macrophages in the C57BL/6 mice were depleted by intra-peritoneal injection of clodronate-containing liposomes, followed by intravenous injection of EG7 cells. Macrophage depletion in splenocytes was validated by FACS. The survival rates of the mice are shown (n = 5). (J) CD47 expression in L5178Y cells was knocked down and confirmed by FACS. (K) CD47-knockdown L5178Y cells were intra-peritoneally injected to BALB/c mice and the survival rates of the mice are shown (n = 5). (L) The expression of immune checkpoint molecules in CD47 KO EG7 cells was confirmed by FACS. The mean values and standard errors are depicted. *P < 0.05 (paired Student’s t-test).

Article Snippet: To construct the CD47-targeted sgRNA-expressing vectors, the oligo DNA from murine CD47 exon 2 (5′-GGATAAGCGC GATGCCATGG-3′) was inserted into the BbsI site of pX330U6-Chimeric_BB-CBh-hSpCas9 (Addgene).

Techniques: Injection, Staining, Expressing, Plasmid Preparation, Liposomes, Knockdown

Journal: International immunology

Article Title: CD47 promotes T-cell lymphoma metastasis by up-regulating AKAP13-mediated RhoA activation.

doi: 10.1093/intimm/dxab002

Figure Lengend Snippet: Fig. 3. Engulfment of EG7 cells by macrophages is independent of CD47. (A) Wild-type and CD47-deficient EG7 cells were cultured for the indicated periods, and then the cell proliferation was evaluated by WST assay. (B) EG7 cells were cultured in 1 μg ml−1 SDF-1α with a Transwell plate, and then the migrated cells were quantified by WST assay. (C) Expression of α4, αv, β1 and β3 integrin in the wild-type and CD47 KO EG7 cells was detected by FACS. The red line represents the results for the negative control, and the blue line represents the results for stained samples. (D) EG7 cells were cultured in fibronectin-coated dishes, and then the attached cells were quantified by crystal violet staining. (E, F) CMTMR-stained EG7 cells were co-cultured with BMMs for 2 h, and then the CMTMR+ cells among CD11b+-gated cells were quantified by FACS. (G, H) EG7 cells were co-cultured with GMDCs in the same manner as described above, and then the CMTMR+ cells among CD11c+- gated cells were quantified by FACS. (I, J) CMTMR-stained L5178Y cells were co-cultured with BALB/c-derived BMMs and then the BMMs were analyzed as in (E). (K) CFSE-stained BMMs (green) were co-cultured with CMTMR-stained EG7 cells (red) for 30 min, and fluorescence was detected by confocal microscopy (n = 3); mean values and standard errors are depicted. N.S. = not significant (paired Student’s t-test).

Article Snippet: To construct the CD47-targeted sgRNA-expressing vectors, the oligo DNA from murine CD47 exon 2 (5′-GGATAAGCGC GATGCCATGG-3′) was inserted into the BbsI site of pX330U6-Chimeric_BB-CBh-hSpCas9 (Addgene).

Techniques: Cell Culture, WST Assay, Expressing, Negative Control, Staining, Derivative Assay, Fluorescence, Confocal Microscopy

Journal: International immunology

Article Title: CD47 promotes T-cell lymphoma metastasis by up-regulating AKAP13-mediated RhoA activation.

doi: 10.1093/intimm/dxab002

Figure Lengend Snippet: Fig. 4. CD47 is required for basal RhoA activity. (A) RhoA-GTP in wild-type and CD47 KO EG7 cells was pulled down using GST-PBD and gluta- thione beads, and then immunoblotted. (B) Band intensity of the pulled-down RhoA/total RhoA was calculated using ImageJ software (n = 4). (C) Rac1-GTP in wild-type and CD47 KO EG7 cells was pulled down and (D) their band intensities were calculated as described above (n = 6). (E, F) Wild-type and CD47 KO EG7 cells were stimulated with 200 ng ml−1 TSP-1 for the indicated times, and then activated RhoA and Rac1 were pulled down and blotted. (G, H) RhoA G14V-stably-expressing EG7 cells were intravenously injected into the tail vein of C57BL/6 mice, and then the survival of the mice was evaluated (n = 5); the mean values and standard errors are depicted. *P < 0.05 (paired Student’s t-test).

Article Snippet: To construct the CD47-targeted sgRNA-expressing vectors, the oligo DNA from murine CD47 exon 2 (5′-GGATAAGCGC GATGCCATGG-3′) was inserted into the BbsI site of pX330U6-Chimeric_BB-CBh-hSpCas9 (Addgene).

Techniques: Activity Assay, Software, Stable Transfection, Expressing, Injection

Journal: International immunology

Article Title: CD47 promotes T-cell lymphoma metastasis by up-regulating AKAP13-mediated RhoA activation.

doi: 10.1093/intimm/dxab002

Figure Lengend Snippet: Fig. 5. CD47 is required for AKAP13-mediated RhoA activation and associates with the AKAP13-RhoA complex. (A) HeLa cells were transiently transfected with CD47 and AKAP13 siRNA, and then activated RhoA in the lysates was pulled down using GST-PBD and glutathione beads and blotted at 64 h post-transfection. (B) HEK293T cells were transiently transfected with an expression vector of AKAP13-Flag. The lysates were incubated with recombinant GST or GST-CD47 cytosol and then immunoprecipitated. (C) Recombinant DHPH, DH and PH domain of AKAP13 were incubated with recombinant GST-CD47 cytosol and then pulled down using Ni-NTA agarose beads and blotted. (D) AKAP13- Flag was stably expressed in wild-type and CD47 KO EG7 cells, and then the AKAP13-RhoA complex was immunoprecipitated and blotted. (E) The lysates of EG7 cells were incubated with recombinant GST-CD47 cytosol and recombinant AKAP13 DHPH for 1 h at 4°C. The AKAP13 DHPH and GST-CD47 cytosol complex was pulled down using Ni-NTA beads following glutathione beads and then blotted. (F) HEK293T cells were transiently transfected with an expression vector of AKAP13-Flag, Flag-PDZ and Flag-p115. The lysates were incubated with recombinant GST-CD47 cytosol and then immunoprecipitated. (G) CD47 KO EG7 cells were transiently transfected with EGFRΔcytosol and EGFRΔcytosol/ CD47 cytosol by electroporation and then activated RhoA in the lysates was pulled down and blotted as in (A) at 48 h post-transfection. (H) Wild-type and CD47 KO EG7 cells were stably expressed AKAP13-Flag, and activated RhoA in the lysates was pulled down and blotted. (I, J) shCtrl- and shCD47-expressing HeLa cells were transiently transfected with the expression vectors of Flag-RhoA G14V and AKAP13-Flag and then fixed and stained with Rhodamine-Phalloidin (red) and Hoechst 33342 (blue) at 48 h post-transfection. The images were obtained by confocal microscopy and the population of stress fiber-positive cells per 50 cells was counted from the image. (K) Akap13 mRNA levels in AKAP13-knockdown EG7 cells were quantified by qPCR. (L) CD47 expression in AKAP13-knockdown EG7 cells was measured by FACS. (M) AKAP13-knockdown EG7 cells were intravenously injected into the tail vein of C57BL/6 mice. The survival rates of the mice are shown (n = 5). n = 3; mean values and standard errors are depicted. *P < 0.05 , **P < 0.01 (paired Student’s t-test).

Article Snippet: To construct the CD47-targeted sgRNA-expressing vectors, the oligo DNA from murine CD47 exon 2 (5′-GGATAAGCGC GATGCCATGG-3′) was inserted into the BbsI site of pX330U6-Chimeric_BB-CBh-hSpCas9 (Addgene).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Recombinant, Immunoprecipitation, Stable Transfection, Electroporation, Staining, Confocal Microscopy, Knockdown, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: Preclinical characterization of the novel anti-SIRPα antibody BR105 that targets the myeloid immune checkpoint

doi: 10.1136/jitc-2021-004054

Figure Lengend Snippet: BR105 blocks CD47 binding to various SIRPα variants. (A, B) Blocking soluble CD47 binding to human SIRPα variants (V1, V2 and V8) was assessed by competition ELISA assay. Inhibiting of CD47 binding to SIRPα-expressing U937 (C) or macrophages (D) by BR105 was determined by flow cytometry analysis. Data represent mean ± SEM; representative of n=3 is shown. *indicate statistical differences compared with the respective isotype control group: **p<0.01.

Article Snippet: Then cells were incubated with 10 μg/mL of murine IgG2a Fc-tagged human CD47 (ACROBiosystems) in the absence or presence of BR105.

Techniques: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Control