CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals
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Curated by eLife
Evaluation Summary:
The manuscript shows that CD47, which provides inhibitory signals to myeloid cells via SIRP1alpha, is released in exosomal form by cells expressing it, and these exosomes can stick stably to other cells, resulting in CD47-mediated protection of these cells from phagocytosis by myeloid cells. The data are striking, and would be of interest to researchers working in both mechanistic and tranlational aspects of transplant-related immunoregulation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Transgenic CD47 overexpression is an encouraging approach to ameliorating xenograft rejection and alloresponses to pluripotent stem cells, and the efficacy correlates with the level of CD47 expression. However, CD47, upon ligation, also transmits signals leading to cell dysfunction or death, raising a concern that overexpressing CD47 could be harmful. Here, we unveiled an alternative source of cell surface CD47. We showed that extracellular vesicles, including exosomes, released from normal or tumor cells overexpressing CD47 (transgenic or native) can induce efficient CD47 cross-dressing on pig or human cells. Like the autogenous CD47, CD47 cross-dressed on cell surfaces is capable of interacting with SIRPα to inhibit phagocytosis. However, ligation of the autogenous, but not cross-dressed, CD47 induced cell death. Thus, CD47 cross-dressing provides an alternative source of cell surface CD47 that may elicit its anti-phagocytic function without transmitting harmful signals to the cells. CD47 cross-dressing also suggests a previously unidentified mechanism for tumor-induced immunosuppression. Our findings should help to further optimize the CD47 transgenic approach that may improve outcomes by minimizing the harmful effects of CD47 overexpression.
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Evaluation Summary:
The manuscript shows that CD47, which provides inhibitory signals to myeloid cells via SIRP1alpha, is released in exosomal form by cells expressing it, and these exosomes can stick stably to other cells, resulting in CD47-mediated protection of these cells from phagocytosis by myeloid cells. The data are striking, and would be of interest to researchers working in both mechanistic and tranlational aspects of transplant-related immunoregulation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
A) Main findings:
The authors report that extravesicles (EVs) including exosomes (Exos) provide a new source for membrane expression of CD47 in a process termed as "cross-dressing".
B) Strengths:
The experiments reported provide compelling evidence supporting:
- the existence of CD47 cross dressing in vitro.
- the functionality of CD47 (in phagocytosis inhibition assay) when transferred after cross dressing.C) Weaknesses:
In vitro settings. The persistence of cross-dressed CD47 at the surface of recipient cells is not addressed.
The cellular mechanisms underpinning the transfer of CD47-containing membranes is not fully described nor mechanistically explained.
The lack of pro-apoptotic signalling is not explained on a mechanistic basis. For instance, does this implies that cross-dressing does not involve …
Reviewer #1 (Public Review):
A) Main findings:
The authors report that extravesicles (EVs) including exosomes (Exos) provide a new source for membrane expression of CD47 in a process termed as "cross-dressing".
B) Strengths:
The experiments reported provide compelling evidence supporting:
- the existence of CD47 cross dressing in vitro.
- the functionality of CD47 (in phagocytosis inhibition assay) when transferred after cross dressing.C) Weaknesses:
In vitro settings. The persistence of cross-dressed CD47 at the surface of recipient cells is not addressed.
The cellular mechanisms underpinning the transfer of CD47-containing membranes is not fully described nor mechanistically explained.
The lack of pro-apoptotic signalling is not explained on a mechanistic basis. For instance, does this implies that cross-dressing does not involve membrane fusion thereby preventing the access of CD47 ICD to intracellular machineries and signal transduction within host cells? Determining whether EVs/Exos tether on membranes of cross dressed cells or provide integral membrane proteins to the target cell membrane (post membrane fusion) is a key point that is not addressed at all within the manuscript.
The role of exosome release in providing membranes for CD47 is not assessed in physiological settings (exosomes released by cells in vitro or in vivo e.g.). Instead preparation of concentrated EVs/exosomes are used and it is questionable if this recapitulates physiologically relevant concentration of exosomes/EVs.
D) Conclusions:
The conclusions raised from the experiments provided are sound.
E) Impact & significance:
Inhibitory pathways activated upon the SIRPa signalling downstream CD47 ligation are of key relevance for the regulation of xenorejection and allorejection at both innate (phagocytosis) and adaptive (antigen presentation by DCs).
The prospect of uncoupling the inhibition of phagocytosis from apoptosis induction at the level of target cells is attractive. However, one might wonder if engineering of CD47 intracellular domain might not achieve the same goal more directly. Are cells expressing a tail-less CD47 would be protected from SIRPa-mediated apoptosis as well?
Also, it is a little bit unclear how this process unveiled in this study can be practically implemented in transplantation and regenerative medicine, especially taking in account the persistence of cross-dressed CD47 on recipient cells which is not assessed in this study.
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Reviewer #2 (Public Review):
In cell culture, membrane is known to be shed as vesicles from living and dying cells, and such vesicles have the potential to fuse with other cells and confer novel function. CD47 and SIRPa are integral membrane proteins expressed at low to medium levels on many cell types, and if they are shed and properly fuse with another membrane, then they could be expected to affect phagocytosis of the recipient cell. Such in vitro observations made here are potentially impactful for the CD47 field, and although many mechanistic aspects remain unclear as are in vivo effects, the results do extend past work that has already shown shed membrane particles have functional CD47 that - by design - tends to integrate into recipient membrane (PMID: 28053997). However, a number of concerns temper enthusiasm for the present …
Reviewer #2 (Public Review):
In cell culture, membrane is known to be shed as vesicles from living and dying cells, and such vesicles have the potential to fuse with other cells and confer novel function. CD47 and SIRPa are integral membrane proteins expressed at low to medium levels on many cell types, and if they are shed and properly fuse with another membrane, then they could be expected to affect phagocytosis of the recipient cell. Such in vitro observations made here are potentially impactful for the CD47 field, and although many mechanistic aspects remain unclear as are in vivo effects, the results do extend past work that has already shown shed membrane particles have functional CD47 that - by design - tends to integrate into recipient membrane (PMID: 28053997). However, a number of concerns temper enthusiasm for the present submission.
1. Recent studies with particles shed from hCD47-overexpressing HEK cells, as a mix of encapsulated virus and simpler vesicles, showed that non-phagocytic cells (HEX cells, A549 lung cells) take up such particles -- with membrane fusion being the common pathway, and they also take up more when SIRPa is present and not blocked (Fig.2b,e,f in PMID: 28053997). This is counter to this submission's claim that "hCD47 cross-dressing is SIRPα-independent".
2. This study does not provide clear mechanism for many key observations, even though demonstrations of mechanism(s) are usually foundational to the reproducibility and rigor of results. For examples: (A) It is unclear what the basis is for the observation that "ligation of the autogenous, but not cross-dressed, CD47 induced cell death." (B) Given the claimed role for hCD47 transfer by extracellular vesicles and/or exosomes, it is unclear whether the process involves any transfer of hCD47 mRNA or even DNA (e.g. plasmid, even episomal). (C) It is unclear why there is a very broad distribution of intensities on cells that supposedly have 'all' received hCD47; indeed, some cells have little to no hCD47 (e.g. Fig.1c). (D) It is unclear how hCD47 transfers from "florescence Celltrace violet" cells to null cells (Fig.1c) without also transferring some of the dye. (E) It is unclear why results for h2 and h4 spliceforms are sometimes the same (e.g. Fig.1a) versus sometimes different (Fig.1b).
3. Microscopy images always provide more rigorous and reproducible evidence of phagocytosis than flow cytometry assays -- especially because of the use of dyes, given the concerns mentioned above. A further concern with Fig.4c,d is that the null or KO cells with hCD47 inhibit phagocytosis to an equal extent as control, even though the transferred levels of hCD47 tend to be lower and also heterogeneous; those cells showing very low transfer levels (e.g. Fig.1,2b,S5c, etc.) are expected to be engulfed more.
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Reviewer #3 (Public Review):
Here Li et. al. investigates the effects of cross-dressing pig cells with human CD47 in the cocultures of human monocytes and pig cells. Using hCD47 transgenic cell lines, Jurkat cells that constitutively express high levels of CD47, the authors have shown that pig cells can be cross-dressed with hCD47 molecules via a transfer of extracellular vesicles. This transfer can occur in the absence of SIRP-a. While the in vitro evidence presented here may provide a new strategy for minimizing anti-xenograft immune responses by macrophages/ monocytes, the study would immensely benefit from in vivo evidence that addresses whether cross-dressing can indeed be sufficient to protect xenogeneic cells in the host and that the CD47 molecules that land on xenogeneic cells remain on the surface of acceptor cells stably …
Reviewer #3 (Public Review):
Here Li et. al. investigates the effects of cross-dressing pig cells with human CD47 in the cocultures of human monocytes and pig cells. Using hCD47 transgenic cell lines, Jurkat cells that constitutively express high levels of CD47, the authors have shown that pig cells can be cross-dressed with hCD47 molecules via a transfer of extracellular vesicles. This transfer can occur in the absence of SIRP-a. While the in vitro evidence presented here may provide a new strategy for minimizing anti-xenograft immune responses by macrophages/ monocytes, the study would immensely benefit from in vivo evidence that addresses whether cross-dressing can indeed be sufficient to protect xenogeneic cells in the host and that the CD47 molecules that land on xenogeneic cells remain on the surface of acceptor cells stably enough. Once the functional relevance of CD47 cross-dressing is carefully addressed and substantiated, the study would provide valuable knowledge to transplantation immunology field.
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