Cellular & Molecular Immunology (2011) 8, 285–288 ß 2011 CSI and USTC. All rights reserved 1672-7681/11 $32.00 www.nature.com/cmi

REVIEW

CD47: a new player in and xenograft rejection

Nalu Navarro-Alvarez and Yong-Guang Yang

Organ transplantation is limited by the availability of human donor organs. The transplantation of organs and tissues from other species () would supply an unlimited number of organs and offer many other advantages for which the pig has been identified as the most suitable source. However, the robust immune responses to xenografts remain a major obstacle to clinical application of xenotransplantation. The more vigorous xenograft rejection relative to allograft rejection is largely accounted for by the extensive genetic disparities between the donor and recipient. Xenografts activate host immunity not only by expressing immunogenic xenoantigens that provide the targets for immune recognition and rejection, but also by lacking ligands for the host immune inhibitory receptors. This review is focused on recent findings regarding the role of CD47, a ligand of an immune inhibitory receptor, signal regulatory alpha (SIRPa), in phagocytosis and xenograft rejection. Cellular & Molecular Immunology (2011) 8, 285–288; doi:10.1038/cmi.2010.83; published online 24 January 2011

Keywords: CD47; ; signal-regulatory protein alpha; xenotransplantation

INTRODUCTION summarizes recent data regarding the role of CD47 in phagocytosis A persistent shortage of human organs and inexhaustible waiting lists and xenograft rejection. continue to result in many people dying while awaiting transplanta- 1,2 tion which has made it necessary to search for new alternatives. CD47 AND SIRPa: AN IMPORTANT INTERCELLULAR Cross-species transplantation (xenotransplantation) has an enormous COMMUNICATION SYSTEM potential to solve the significant need for organs, tissues and cells for The cells in the body have the need to communicate with each other in clinical transplantation. Although transplants across discordant spe- order to regulate all their functions and events that go from the cies barriers are subjected to vigorous immunological rejection development of a cell to death. There are several different commun- because of the greater phylogenetic differences, the increasing avail- ication systems that the body uses in its different organs and systems. ability of genetically engineered pigs is enabling progress to be made in a 3–5 The immune system for instance has made use of ‘The CD47–SIRP pig-to-non-human experimental models. Hyperacute communication system’ among others, for a number of functions it rejection caused by the presence of natural antibodies against Gal- has, such as phagocytosis, and recognition etc.14,15 a1-3-Gal (a1,3Gal) can now be effectively prevented by using a1,3Gal transferase knockout (KO) pigs.3–7 However, non-a1,3Gal- specific anti-pig antibodies produced either prior to (natural antibod- -ASSOCIATED PROTEIN CD47 ies) or after transplantation present an additional barrier to acceptance CD47 is a member of the immunoglobulin (Ig) superfamily and it is a of transplanted organs from a1,3Gal transferase KO pigs by pri- cell surface glycoprotein ubiquitously expressed. It has a highly glyco- mates.8,9 Moreover, coagulation dysfunction between the pig and sylated transmembrane protein with a V-type Ig-like extracellular primate is proving to be another major problem, and this can result domain in its N-terminus, a hydrophobic five putative membrane- in life-threatening consumptive coagulopathy.10 This complication is spanning segments and a short cytoplasmic tail.16 CD47 was originally unlikely to be overcome until pigs expressing a human ‘antithrombo- identified for its interaction with the avb3, aIIbb3 and a2b1 , tic’ or ‘anticoagulant’ , such as , regulating integrin function and cellular responses to the – pathway inhibitor or CD39, become available.10 –-containing extracellular matrix , and The cellular innate immune responses, including those mediated by thus named integrin-associated protein.17 The Ig domain of CD47 is natural killer cells11–13 and , have also been shown to required for both functional and physical interaction not only with its contribute to the rejection of porcine xenografts. The interspecies associated integrins, but also with its ligands and incompatibility of CD47, a ligand for signal regulatory protein SIRPa. CD47 also has the ability to transduce signals into the cell after alpha (SIRPa), has been implicated in xenograft rejection. This review interaction with thrombospondin or integrins; hence engagement can

Columbia Center for Translational Immunology, Columbia University Medical Center, New York, NY, USA Correspondence: Dr YG Yang, Columbia Center for Translational Immunology, Columbia University College of Physicians and Surgeons, 630 W 168th Str., New York, NY 10032, USA. E-mail: [email protected] Received 23 November 2010; accepted 17 December 2010 CD47 in xenotransplantation N Navarro-Alvarez and Y-G Yang 286

potentially give two-way signaling leading to biological activities such phagocytes after the cells have died, in an efficient and immunologi- as cell spreading, migration or even apoptosis.17–19 cally silent manner. This process has to be accurate enough to spare neighboring healthy cells that are around. The cell surface protein SIRPa CD47 on healthy cells and its engagement of a phagocyte receptor, SIRPa (also known as SHPS-1, BIT, CD172a or p84) belongs to a family SIRPa, appears to constitute a key ‘don’t eat me signal’, being this one of membrane proteins involved in the regulation of leukocyte function of the different systems used by the immune cells namely phagocytes and is a ligand of CD4718 whose interaction has been shown to mediate to discriminate healthy from the unwanted/aged/dying cells. Although cell–.20 SIRPa is expressed on myeloid cells, including the relationship between apoptosis and CD47 is incompletely defined, macrophages, granulocytes, myeloid dendritic cells (DCs), mast cells several studies suggest that the progression of apoptosis is linked to 21,22 the reduction of cellular CD47 levels independent of the apoptotic and their precursors, including hematopoietic stem cells. 29 SIRPa has three Ig-like domains in its extracellular region and two stimulus. Although the mechanism underlying the reduction of CD47 on the surface of apoptotic cells is unknown, it is believed that immunoreceptor inhibitory motifs in its cytoplasmic region, 30 which upon ligand binding become phosphorylated and mediate protease cleavage could be a possibility; as a result, the apoptotic recruitment and activation of the dual Src homology region 2 cells will no longer provide an inhibitory signal for phagocytosis via domain-containing phosphatase (SHP)-2 and SHP-1 to the mem- SIRPa which will facilitate their uptake by macrophages. brane, and thereby negatively modulates intracellular signaling However, the role of SIRPa may be more complex than previously initiated by various cell surface receptors that promote tyrosine kinase anticipated and the molecule may not only provide negative signals. activity or from growth-factor receptors.23 SHP-1 and -2 can, in turn, For instance, it has been shown that engagement of SIRPa by CD47 in also dephosphorylate specific protein substrates and thereby regulate macrophages can promote the production of via the 31 cellular functions, generally in a negative fashion. SIRPa-associated Janus kinase. In addition to the regulatory func- tions in macrophages, interactions between CD47 on T cells and SIRPa on DCs have been shown to downregulate, in a bidirectional CD47 AND SIRPa REGULATING PHAGOCYTOSIS fashion, DC and T-cell activation.32 Macrophages are essential to both innate and adaptive immune responses. They initiate the innate by recogniz- ing pathogens via a variety of receptors with specificity for pattern- THE LEVEL OF CD47 REGULATES CELL CLEARANCE recognition molecules.24,25 Macrophages also express inhibitory CD47 increases transiently in hematopoietic cells (returning to baseline receptors that control macrophage activity, including phagocytosis levels within days) during mobilization or after a strong inflammatory 33 as is the case of SIRPa,23,26 which recognizes CD47 on the surface of stimulus. It was suggested that this transient upregulation is necessary the cell as a ‘marker of self’ and upon binding phagocytosis is inhib- to prevent the clearance of normal, healthy hematopoietic cells during ited.14 Being this one of the reasons why macrophages mainly engulf their recirculation from the bone marrow to blood or extramedullary foreign cells such as transplanted cells, but not self. sites during a stress response, since only low CD47 levels are thought to 33,34 Evidence of this phenomenon came from the observations that CD47- be sufficient to prevent phagocytosis. during a steady state. However, deficient circulating red blood cells were cleared rapidly by splenic macro- during an infection for example in where LPS come into play, macro- phages get activated probably due to downregulation of SIRPa,leadingto phages because this inhibitory signal was lacking, and binding of the 35 blood cells to the macrophages was sufficient to trigger a phagocytic a decreased threshold for phagocytosis. Hence as a protective mech- signal.14 This elimination was abrogated when macrophages were anism, mobilizing cytokines and inflammatory stimuli cause CD47 to be transiently upregulated just prior to and during their migratory phase, in removed by treatment with macrophage-toxic clodronate liposomes. In 33 addition, the use of blocking antibodies to SIRPa resulted in unopsonized order to avoid engulfment by those avidly activated macrophages. CD471 erythrocytes phagocytosed to the same extent as CD472/2 ery- Therefore, it has been hypothesized that the level of CD47 on the cells throcytes. The use of mutant mice for SIRPa supported these findings.27 determines the probability that they are engulfed in vivo. This effect was not only observed in erythrocytes but also in platelets. Recent studies have shown that mouse and human myeloid leukemia Studies have shown that mice expressing a SIRPa mutant that lacks most stem cells upregulate CD47 expression as an escape mechanism, which of the cytoplasmic domain manifest thrombocytopenia, which appar- was associated with worse overall survival prognostic in patients, con- ently results from an increased rate of clearance of circulating platelets. tributing to the pathogenesis and progression of the disease due to Furthermore, peritoneal macrophages from these mice exhibit an engagement of CD47 with SIRPa leading to inhibition and evasion of 36,37 enhanced phagocytic response.28 Because of the rapid clearance of donor phagocytosis. On the other hand, a body of evidence has demon- lymphoid cells, CD472/2 mouse T cells fails to induce graft-versus-host strated the usefulness of monoclonal antibodies directed against CD47 disease in a fully major histocompatibility complex-mismatched allo- in different types of hematopoietic and solid malignancies in vivo and in geneic hematopoietic cell transplantation model, in which injection of vitro, promoting phagocytosis or apoptosis of the desire population and 36,38–42 a similar number of CD471/1 T cells induces lethal graft-versus-host thus effective elimination of the tumorigenic cells. disease.15 It was found that DCs are more efficient than macrophages in engulfing CD472/2 T cells.15 These observations provide strong evid- CD47 AND XENOGRAFT REJECTION ence that CD47 expression is a critical indicator for determining whether Macrophages have been shown to engulf porcine red blood cells in an lymphohematopoietic cells will survive or be cleared. antibody- and complement-independent manner,43 and to mediate Phagocytes have also the ability to discriminate between viable/ the rejection of porcine islet xenografts in both rodents44–46 and pri- healthy and apoptotic/foreign/abnormal cells. The human body has mates.47 Although host macrophages can be activated by cytokines to somehow find the way to get rid of a different array of cells, produced by xenoreactive T cells, these powerful effects of xenogeneic including those produced in excess, altered, damaged or aged cells macrophages could also be accounted for by the combined ability of in order to keep homeostasis. One of such mechanisms is apoptosis certain xenogeneic to activate macrophages,48,49 while where cellular changes mark these cells for further removal by important inhibitory interactions are not effective.50,51

Cellular & Molecular Immunology CD47 in xenotransplantation N Navarro-Alvarez and Y-G Yang 287

The CD47–SIRPa interaction which is species specific has been On the other hand, human SIRPa has a Ser at this site. The ligand- implicated as a critical regulator of xenotransplantation rejection binding activity was destroyed by site-directed mutagenesis of Ser to in several cross-species transplants.50 The rapid rejection of CD47- Asp in this position of human SIRPa/SHPS-1, confirming the critical deficient hematopoietic cells by macrophages in syngeneic wild-type role of this site.57 Thus, it is likely that the residue equivalent to Ala65 (WT) mice demonstrated a critical role for CD47 as a self-protection of mSIRPa is critical in determining the species, strain and/or isoform (or ‘don’t eat me’ marker). This finding also suggested that the rapid specificity in ligand recognition. These results suggest that the variable rejection of xenogeneic cells by recipient macrophages could have complementarity-determining region-like loop structures in the bind- resulted from the inability of donor CD47 to interact with SIRPa on ing surface of SIRPa are generally required for ligand recognition in a the recipient macrophages. This question was first addressed in a pig- manner similar to that of antigen receptors, which may explain the to-mouse xenogeneic combination,50 in which macrophages were diverse ligand-binding specificities of SIRP family receptors. found to mediate robust rejection of porcine hematopoietic cells.52 Most of the previous studies regarding CD47-SIRPa interspecies It was found that porcine CD47 could not induce SIRPa tyrosine incompatibility and xenograft rejection have focused on hematopoi- phosphorylation in mouse macrophages and that blocking SIRPa with etic grafts, and little is known about its role in non-hematopoietic anti-mouse SIRPa (P84) did not affect the grafts. We have recently shown that at least for thymic epithelial cells engulfment of porcine cells by mouse macrophages. These findings CD47 is not an impediment for graft survival. Fetal thymus from demonstrated the lack of cross-reaction between porcine CD47 and syngeneic WT or CD47 KO donors were transplanted in thymecto- mouse SIRPa.51 Importantly, transgenic expression of mouse CD47 mized, -depleted WT and CD47 KO mice. Transplantation of on porcine cells was found to elicit strong protection against pha- CD47 KO mouse thymus led to T-cell recovery in WT mice, showing a gocytosis by mouse macrophages both in vitro and in vivo.50 normal distribution of thymocyte subsets and long-term survival of It has been reported that recombinant human SIRPa proteins mouse thymic epithelial cells. These results demonstrate that, unlike bound to porcine CD47.53 However, the protein-binding assay does hematopoietic cells, CD47 KO mouse thymus can survive and func- not necessarily reveal whether porcine CD47 could deliver inhibitory tion in WT mice.58 However, it is possible that CD47 may still play an signaling via SIRPa to human macrophages. In fact, the demonstrated important role in the rejection of non-hematopoietic cellular xeno- inability of porcine red blood cells to stimulate SIRPa tyrosine phos- grafts. In support of this possibility, we have recently observed that phorylation in human macrophages indicated a lack of functional liver parenchymal cells lacking CD47 do not survive as their WT interaction between porcine CD47 and human SIRPa.51 In agreement counterpart does (Navarro-Alvarez and Yang, manuscript in prepara- with this possibility, porcine cells transfected with human CD47 tion). Thus, the role of CD47 in xenograft rejection may differ for showed markedly reduced sensitivity to in vitro phagocytosis by different types of xenografts. human macrophages.51 CONCLUDING REMARKS SIRPa POLYMORPHISMS The ubiquitously expressed cell surface molecule CD47 serves as an It has been shown that immunodeficient mice on the non-obese dia- important ‘marker of self’ for macrophages and DCs, and its signaling betic background allowed better hematopoietic engraftment than did through SIRPa prevents inappropriate self-phagocytosis. The lack other strains with equivalent immunodeficiency-related mutations. of cross-species interaction in the CD47–SIRPa pathway largely The mouse SIRPa gene exhibits significant coding region polymorph- accounts for the vigorous rejection of xenogeneic hematopoietic cells ism. This variation takes place mainly in the CD47-interacting V-like by macrophages. However, the role for CD47 in non-hematopoietic Ig domain, which in turn results in a differential binding capacity of xenograft rejection has been less clear. While CD47 expression is not human CD47 with the non-obese diabetic SIRPa polymorphic variant required for survival or function of thymic epithelial cells when grafted 58 showing strong binding.54 In contrast to SIRPa, CD47 seems to not be as a tissue, lacking CD47 on donor liver parenchymal cells induces subjected to much polymorphic variation. rapid activation of innate immune cells and promotes graft rejection In order to understand the ligand, species and/or strain specificity of after hepatocyte transplantation (Navarro Alvarez et al., manuscript in SIRP family receptors, studies have determined the crystal structure of preparation). Thus, the role of CD47 in xenograft rejection seems to the ligand-binding domain of murine SIRPa (mSIRPa LBD), for the differ between different types of xenografts, and further studies are specific CD47 recognition. Folding topology revealed that mSIRPa needed to confirm this hypothesis. LBD adopts an I2-set Ig fold, but its overall structure resembles IgV domains of antigen receptors, but with an extended loop structure ACKNOWLEDGEMENTS (C’E loop), forming a dimer interface in the crystal, consistent with The authors thank Dr Emmanuel Zorn for critical reading of the manuscript. the human SIRPa LBD structure55 with the exception that human The work from the authors’ laboratory discussed in this review was supported by grants from NIH (RO1 AI064569 and PO1 AI045897), JDRF (1-2005-72) SIRPa has an additional beta-strand D. and ROTRF (#848155553). 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