CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital, and Institute of Immunology, Jilin University, China
- Columbia Center for Translational Immunology, Columbia University Medical Center, United States
- Lung Biotechnology PBC, United States
- International Center of Future Science, Jilin University, China
Figures
Figure 1 with 2 supplements
Transgenic hCD47 cross-dressing on pig cells.
(A) Porcine aortic cell (PAOC)/CD47p cells and PAOC/CD47p/h2 or PAOC/CD47p/h4 cells were cultured alone or cocultured for 24 hr, and hCD47 cross-dressing on gated PAOC/CD47p cells was assessed by flow cytometry 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 median fluorescent intensity [MFI] of gated PAOC/CD47p cells), and average MFI (right; mean ± SDs; n=6 replicates per group) of gated PAOC/CD47p cells in the indicated cell cultures. ****, p<0.0001 (two-tailed unpaired t-test). Results shown are representative of three independent experiments. (B) PAOC/CD47null, and PAOC/CD47h2 or PAOC/CD47h4 were cultured alone or cocultured for 24 hr, and analyzed for hCD47 cross-dressing on gated PAOC/CD47null cells by flow cytometry 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/CD47null cells), and average MFI (right panel; mean ± SDs; n=3 replicates per group) of gated PAOC/CD47null cells in the indicated cell cultures. **, p<0.01; ****, p<0.0001 (two-tailed unpaired t-test). Results shown are representative of three independent experiments. (C) Celltrace violet-labeled PAOC/CD47null (left panel) or PAOC/CD47h2 (right panel) was cultured alone (Violet CD47null or Violet CD47h2) or cocultured with unlabeled PAOC/CD47h2 (Violet CD47null + CD47h2) or PAOC/CD47null (CD47null or Violet CD47h2), respectively, then the cells were stained by anti-h/pCD47-PE mAb. Shown are representative flow cytometry profiles (n=3 replicates). Results shown are representative of two independent experiments. (D) Pig lymphoma cell line (LCL) and hCD47-tg LCL (LCL/CD47p/h) cells were cultured alone or cocultured for 24 hr, 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 two replicates per group. Representative flow cytometry profiles are shown. (E) PAOC47null cells were cocultured with bone marrow cells from hCD47-tg miniature swine for 2 days and analyzed for hCD47 cross-dressing on gated PAOC47null cells by flow cytometry using anti-h/pCD47-PE mAb (the numbers in the figure indicate the MFI of gated PAOC/CD47null cells). Two independent experiments were performed, and each experiment had two replicates per group. Representative flow cytometry profiles are shown.
Figure 1—figure supplement 1
Generation of CD47-deficient and hCD47-trangeneic porcine aortic cell (PAOC) cell lines.
(A) Schematic of pig CD47 loci and guide RNA sequence targeting the exon 2 of pig CD47 gene used for generating POAC/CD47null cells. (B) Schematics of human CD47 isoform 2 (with 16aa intracellular domain (ID); top) and isoform 4 (with 34aa ID; bottom) plasmids, which were used for making POAC/CD47h2 and POAC/CD47h4 cells, respectively. (C) flow cytometry profiles showing staining of the indicated PAOC cell lines with anti-h/p CD47 (top) and anti-hCD47 (bottom) antibodies.
Figure 1—figure supplement 2
SIRPα expression on pig cells.
Shown are representative flow cytometry profiles of porcine aortic cell (PAOC) CD47null (left) and pig lymphoma cell line (LCL; right) cells stained with anti-pig SIRPα.
Figure 2 with 3 supplements
CD47 expression and CD47 cross-dressing on human T cell leukemia Jurkat cells.
(A) CD47 expression on Jurkat cells and normal human CD3+, CD19+, and CD14+ peripheral blood cells (the numbers indicate median fluorescent intensity [MFI] of human CD47 staining). (B) CD47 expression on wild-type (WT) Jurkat cells, pig lymphoma cell line (LCL)/CD47p/h cells, CD47KO Jurkat cells, CD47KO cells mixed with WT Jurkat cells (mixed at the time of staining), CD47KO Jurkat cells cocultured (24 hr) with WT Jurkat or pig LCL/CD47p/h cells, pig LCL cells, and LCL cells cocultured (24 hr) with WT Jurkat cells. (C) PKH67-labeled pig LCL cells were cocultured with PHK26-labeled LCL/CD47p/h (left) or WT Jurkat (right) cells for 1 day, PHK67-labeled pig LCL cells are sorted and treated with mitomycin C (2 µg/ml) for 30 min (to stop cell division), then analyzed for hCD47 staining on pig LCL cells immediately (Day 0) and at the indicated times after cultured in media. The numbers in the figure indicate MFI of CD47 staining on gated cells.
Figure 2—figure supplement 1
Generation of CD47-deficient Jurkat cells.
(A) Schematic of human CD47 loci and guide RNA sequence targeting the exon 2 of human CD47 gene used for generating CD47KO Jurkat cells. (B) Flow cytometry profiles showing anti-CD47 staining of wild-type (WT) and CD47KO Jurkat cells.
Figure 2—figure supplement 2
Multiple membrane proteins are cross-dressed during cell coculture.
PKH67-labeled pig lymphoma cell line (LCL) cells were cocultured with PKH26 labeled-wild-type (WT) Jurkat cells for 20 hr and analyzed for human CD47, CD45, CD90, and HLA-ABC staining on pig LCL cells. Shown are representative histogram, and MFI (mean ± SDs; n = 3 replicates in the co-cultured group) of pig LCL cells co-cultured with WT Jurkat cells. Results shown are representative of three independent experiments.
Figure 2—figure supplement 3
A functional cytoskeleton is not absolutely required for but may facilitate hCD47 cross-dressing.
Untreated or cytochalasin D-pretreated pig lymphoma cell line (LCL) cells were cocultured with PKH26-labeled wild-type Jurkat cells (A) or extracellular vesicles (prepared from LCL/CD47p/h) (B) for 20 hr, and analyzed for PKH26 fluorescence on pig LCL cells by flow cytometry. MFI: median fluorescent intensity. Results shown are representative of two independent experiments.
Figure 3 with 2 supplements
CD47 cross-dressing by extracellular vesicles (EVs) and exosomes (Exos).
(A–B) CD47KO Jurkat cells (A) or pig lymphoma cell line (LCL) cells (B) were cultured in the absence (left) or presence (right) of EVs (top) or Exos (bottom) prepared from PAOC/CD47h2 cell culture supernatants for 2 hr or 6 hr, and analyzed for hCD47 cross-dressing by flow cytometry using anti-hCD47-BV786 mAb. Representative flow cytometry profiles of three independent experiments were shown. (C) Celltrace violet-labeled PAOC/CD47null cells were cultured in the absence (left) or presence (right) of EVs prepared from PAOC/CD47h2 cells for 18 hr or 42 hr, and analyzed for hCD47 cross-dressing by flow cytometry using anti-hCD47-BV786 mAb.
Figure 3—figure supplement 1
Rapid CD47 cross-dressing on pig lymphoma cell line (LCL) cells incubated with CD47-expressing extracellular vesicles (EVs).
Pig LCL cells were cultured with EVs from hCD47-tg pig LCL (A) or wild-type Jurkat cells (B), and analyzed for CD47 cross-dressing by flow cytometry at 1 and 24 hr after culture. Pig LCL cells that were not cultured with EVs were used as the staining control. Results shown are representative of three independent experiments.
Figure 3—figure supplement 2
Cross-dressing involves membrane fusion.
Wild-type Jurkat cells were labeled with PKH26 and cocultured with PKH67-labeled pig lymphoma cell line (LCL) cells for 24 hr, washed with PBS with or without trypsin/EDTA, then analyzed PKH26 fluorescence on LCL cells by flow cytometry. Shown are staining profiles (left) and median fluorescent intensity (MFI; mean ± SDs) of cocultured LCL cells that were not (Control, n=3) or treated with trypsin/EDTA (EDTA,n=3), ns, not significant (two-tailed unpaired t-test); LCL cells not cocultured with Jurkat cells were used as staining control (LCL). Results shown are representative of two independent experiments.
Figure 4
Protection against phagocytosis by cross-dressed CD47.
(A) CD47KO Jurkat cells were cocultured without (left) or with (right) extracellular vesicles (EVs) from PAOC/CD47h2 cells at 37°C for 5 hr, then washed and incubated with recombinant human SIRPα-Fc chimera at 37°C for 1 hr. The binding of SIRPα-Fc proteins to CD47KO Jurkat cells was visualized by staining with APC-conjugated mouse anti-human IgG Fc mAb. Representative flow cytometry profiles of two independent experiments are shown. (B) PAOC47null and PAOC47h2 cells were cultured alone (left and middle) or together (right) for 48 hr and stained using anti-hCD47-BV786 mAb, then PAOC47null (R1), PAOC47h2 (R3), and hCD47+ (i.e. hCD47 cross-dressed) PAOC47null (R2) cells sorted from cocultures were used immediately for phagocytic assay. (C) PAOC47null (R1), PAOC47h2 (R3), or sorted hCD47 cross-dressed PAOC47null (R2) cells were labeled with Celltrace violet and incubated with human macrophages for 2 hr, then phagocytosis was determined by flow cytometry using anti-human CD14 mAb. Shown are representative flow cytometry 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 three independent experiments are shown. (D) CD47KO Jurkat, wild-type (WT) Jurkat, and CD47KO Jurkat cells pre-incubated with EVs from PAOC/CD47h2 cells were labeled by Celltrace violet and cocultured with human macrophages for 2 hr, then phagocytosis was analyzed by flow cytometry. Shown are representative flow cytometry profiles (left) and levels (right; mean ± SDs; n=3) of phagocytosis (i.e. percentages of hCD14+violet+ in total hCD14+ macrophages). Representative results of two independent experiments are shown. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant (two-tailed unpaired t-test).
Figure 5 with 3 supplements
Ligation of autogenous but not cross-dressed CD47 induces cell death.
(A–B) 5×104 wild-type (WT) Jurkat cells (GFP−) were incubated in the presence (A) or absence (B) of 2.5 µg/ml hSIRPα-Fc proteins at 37°C for 1 hr and stained with anti-hCD47 mAb and Propidium Iodide (PI). Representative flow cytometry profiles show dead cells (PI+) in hCD47+ WT Jurkat population. (C–D) CD47KO Jurkat (GFP+) and WT Jurkat cells were mixed (at 1:1 ratio; 5×104 each) and cocultured in the presence of 2.5 µg/ml human SIRPα-Fc for 1 hr, then stained with anti-hCD47 mAb and PI. Shown are representative flow cytometry profiles (C) and percentages (D; mean ± SDs) of PI+ dead cells in gated CD47KO (R1, CD47-GFP+) and WT (R2, hCD47+GFP−) Jurkat cells. (E–F) CD47KO Jurkat (GFP+) cells were incubated for 2 hr with PAOC/CD47h2 extracellular vesicles (EVs), then mixed with WT Jurkat cells (at 1:1 ratio; 5×104 each) and cocultured in the presence of 2.5 µg/ml human SIRPα-Fc for 1 hr. The cells were stained with anti-hCD47 mAb and PI. Shown are representative flow cytometry profiles (E) and percentages (F; mean ± SDs) of PI+ dead cells in gated EV-treated CD47KO (R1, CD47lowGFP+) and WT (R2, hCD47+GFP-) Jurkat cells. ****, p<0.0001 (two-tailed unpaired t-test). Of note, the cells cultured with hSIRPα-Fc proteins (A, C and E) showed reduced hCD47 staining (as detailed in Figure 5—figure supplement 2). Results shown are representative of three independent experiments.
Figure 5—figure supplement 1
SIRPα-Fc-induced cell death in wild-type (WT) and CD47KO Jurkat cells.
1×105 CD47KO or WT Jurkat cells were incubated in with hSIRPα-Fc proteins at the indicated concentrations for 1 hr at 37°C, then stained with PI to identify dead cells. (A) Representative flow cytometry profiles. (B) Percentages of dead (PI+) cells. Representative results of two independent experiments are shown.
Figure 5—figure supplement 2
Prior incubation with hSIRPα-Fc proteins partially blocks subsequent staining by anti-hCD47 mAb.
(A) Anti-hCD47 staining of wild-type (WT) Jurkat cells with or without prior incubation with hSIRPα-Fc. (B) Anti-hCD47 staining of CD47KO Jurkat cells with or without prior incubation with hSIRPα-Fc. (C) Anti-hCD47 staining of PAOC/CD47h2 extracellular vesicle (EV)-treated (for 2 hr) CD47KO Jurkat cells with or without prior incubation with hSIRPα-Fc. The numbers in the figures indicate median fluorescent intensity levels. Results shown are representative of three independent experiments.
Figure 5—figure supplement 3
CD47-transfected CD47KO Jurkat cells are sensitive to apoptosis induced by CD47 agonists.
(A) CD47 expression on wild-type (WT), transfected CD47KO (KO/CD47tg), and control CD47KO Jurkat cells. (B) Apoptosis induced by SIRPa-Fc proteins. (C) Apoptosis induced by agonistic anti-CD47 antibody CC2C6 (5 µg/ml). Results shown are representative of two independent experiments.
Author response image 1
CD47 cross-dressing is significantly blocked at 4 °C. EVs from hCD47-LCL cells were labeled with PKH26, and recipient WT pig LCL cells were labeled with PKH67, EVs and recipient cells were cocultured for 6h and washed, then analyzed for PKH26 (A) and hCD47 (B) cross-dressing on recipient cells by flow cytometry using anti-hCD47-AF647 mAb.Results shown are representative of three independent experiments.
Author response image 2
EVs from pig LCL/CD47p/h cells contain both hCD47 DNA and RNA, and GAPDH RNA.
DNA and total RNA are extracted from hCD47 transgenic pig LCL and their MVs, and WT pig LCL cells respectively, then cDNA was synthesized from total RNA using a reverse transcription kit, and hCD47 and GAPDH sequences are amplified by PCR. Shown are Agarose gel electrophoresis of hCD47 genomic DNA (A), hCD47 cDNA (B) and GAPDH cDNA PCR products..
Author response image 3
Colocalization of CD47 proteins and EVs on pig LCL cells.
PKH26-labeled EVs from LCL/CD47p/h cells were co-cultured with pig LCL cells for 6h, then stained by anti-huCD47-AF647 antibody and analyzed by confocal microscope. Shown are CD47 staining (blue; left), EVs (PKH26 red fluorescence, mid) and overlayed (right) images.
Author response image 4
Florescence dye cross-dressing.
PKH26-labeled hCD47-LCL (A) or PKH26labeled WT Jurkat (B) as donor cells were co-cultured, respectively, with PKH67-labeled pig LCL for 24h, and then analyzed for fluorescent dye cross-dressing by flow cytometry using anti-hCD47AF647 mAb. Two types of cells mixed immediately prior to flow cytometry analysis (without co-culture) were used as negative controls. Shown are flow cytometry profiles (left panels) and median fluorescence intensities (right panels). Results shown are representative of two independent experiments.
Author response image 5
Gating strategy for analyzing phagocytosis by macrophages.
All PI+ cells are excluded from the first gating, then doublets are excluded by gating on FSC-A vs FSC-H. Macrophages were gated on FSC-A vs SSC-A, then by CD45 and CD14 expression.
Author response image 6
Confocal microscopic analysis of phagocytosis.
CFSE-labeled CD47KO Jurkat cells (green) were cocultured with human macrophages for 2h, floating cells were washed out by PBS, then macrophages were harvested by trypsin/EDTA digestion and stained by anti-huSIRPα-APC (red) antibody and DAPI (blue). The samples were subjected to confocal laser scanning microscopy, and the results confirmed that target cells were engulfed by human macrophages. Shown are representative confocal images.
Author response image 7
Anti-human CD14 antibody does not cross-react with PAOC cells.
PAOC cells were cocultured with human macrophages and analyzed for human CD14 and CD45 expression. Representative flow cytometry profiles showing staining of anti-hCD14 and anti-hCD45. The results conformed that after coculture, POAC cells and human macrophages could be clearly separated by staining with antihuman CD14 and CD45 antibodies on flow cytometry.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/73677/elife-73677-mdarchecklist1-v1.docx
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Source data 1
CD47 crossdressing source data E-life R1.
- https://cdn.elifesciences.org/articles/73677/elife-73677-data1-v1.xlsx
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CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals
eLife 11:e73677.
https://doi.org/10.7554/eLife.73677
