NK cells are a subset of lymphocytes of the innate immune system. NK cell infiltrates were found in pig organs perfused with human blood ex vivo 38 , 39 and in pig-to-NHP xenografts 40 , 41 , suggesting that NK cells participate in xenograft rejection. Subsequently the molecular mechanisms involved in human NK cell—porcine endothelial cell interactions have been studied extensively [review in 42 ].
Xenograft rejection is mediated by NK cells through direct NK cytotoxicity or by antibody-dependent cellular cytotoxicity mechanisms Figure 2A. In the direct NK cytotoxicity pathway, through interaction of activating receptors and ligand, NK cells release lytic granules, leading to the lysis of the donor endothelial cell 43 , The direct cytotoxicity of NK cell is tightly regulated by the balance between activating and inhibiting signal pathways mediated by a variety of NK cell receptors The destruction of pig endothelial cells occurs by the recognition of receptors on NK cells.
Interaction between FcRs and antibodies causes cytotoxic granules to release from NK cells and, in turn, to trigger target cell apoptosis In addition to xenoantibodies bound on the endothelium, the induced antiswine leukocyte antigen anti-SLA class I antibodies are recognized by NK cells, also leading to antibody-dependent cellular cytotoxicity Cellular-mediated rejection. A Natural killer NK cells-mediated rejection. Xenoantibodies bind to donor endothelial cells with their Fab portion. The Fc fraction of the antibody is recognized by FcRs located on the surface of NK cells, triggering the signaling cascade that leads to NK cell destruction.
The release of lytic granules marked as dark spots leads to pig endothelial cells lysis. The activating NK cell receptors recognize their ligands on the donor cells and trigger lytic granule release.
The inability of swine leukocyte antigen SLA class I to interact with human inhibitory NK-cell receptors makes porcine cells highly susceptible to human NK-cell-mediated lysis. B Macrophages-mediated rejection. Macrophages can be activated by cytokines [e. Macrophages secrete proinflammatory cytokines [e. C T-cell response in xenograft rejection. The direct pathway refers to the recognition of antigens presented by pig antigen-presenting cells APCs by recipient T cells.
This interaction results in T-cell-mediated cytotoxicity that is directed against the xenograft vascular endothelium. The indirect pathway refers to the recognition of donor-derived peptides on recipient APCs by recipient T cells.
The interaction between primate TCRs and major histocompatibility complex MHC and porcine peptide complexes leads to primate T-cell response, including cytokines production and induction of B-cell activation. The role of NK cells in xenotransplantation still must be fully elucidated. The majority of knowledge on NK in xenotransplantation was generated for in vitro studies and in pig to rodent models.
Further in vivo studies on NHPs are required for a better understanding of the role of NK cells in the rejection of porcine cellular and organ xenografts. Macrophages have been found to be involved in the rejection of both organ grafts and cellular grafts A dense macrophage infiltrate was identified in all the rejected xenografts through histologic analysis Macrophage contribute to xenograft rejection by their activity of modulation adaptive immunity and direct cytotoxicity Macrophages activity can be result from xenoreactive T cells.
T cells recruit and activate macrophages, causing infiltration, and the destruction of xenografts by macrophages. This process, in turn, leads to T-cell response amplification 55 , In addition, macrophages can be active by direct interaction between donor endothelial antigens and receptors on the surface of macrophage Figure 2B Macrophages perform direct toxic effects mediated by the production of proinflammatory cytokines [e. Therefore, the regulation of macrophage activation should improve xenograft survival.
A number of inhibitory receptors have been reported to inhibit phagocytic activity. Other inhibitory molecule, such as CD 63 , immunoglobulin-like transcript 3 64 , and Ig-like receptor B 65 , have been reported involved in macrophage function. However, whether incompatibility between these molecules on pig cells and their receptors on primate macrophages promotes macrophage activation in xenogeneic immune responses requires further evaluation. T lymphocytes are likely important mediators of acute cellular rejection.
Similar to allotransplantation, T cells are activated through both direct and indirect pathways after xenotransplantation Figure 2C In the direct pathway, pig antigen-presenting cells APCs directly active primate T cells.
The interaction between primate T-cell receptors and SLA class I and II peptide complexes results in T-cell-mediated cytotoxicity against the xenograft vascular endothelium.
In the indirect pathway, T cells activation occurs through donor-derived peptides presented by recipient APCs. The cytotoxicity of NK cells and macrophages also can be substantially augmented by cytokines produced by xenoantigen-activated T cells Although similar immunological mechanisms can be observed in allotransplantation and xenotransplantation, T-cell responses against pig antigen, especially in indirect responses, are stronger than responses against alloantigen Surprisingly, acute cellular rejection, as seen in the majority of allotransplants, is rarely documented after pig-to-NHP organ xenotransplantation.
There are two possible reasons for this result: either humoral rejection is so strong that we cannot observe cell rejection following xenotransplantation, or current immunosuppression therapy is sufficient to control T-cell-mediated response in xenotransplantation The compatibility of cross-species adhesion and costimulation molecules is a critical issue in a xenogeneic context.
The strategies to alleviate T-cell rejection in xenografts rely mainly on promoting costimulation and downregulation of MHC expression in porcine cells. In , costimulation blockade-based immunosuppressive therapy was introduced into xenotransplantation by Buhler et al.
Unfortunately, anti-CDmAb was found to be thrombogenic and is currently not available for clinical use Taken together, the blockade of the CD40—CD pathway is a critical component of immunosuppressive agents in the control of xenogeneic T-cell response.
When HAR, AHXR, and T-cell response are prevented, coagulation dysregulation becomes more obvious following xenograft transplantation and is considered another major barrier to prolonged xenograft survival in NHPs Coagulation dysregulation results in the development of thrombotic microangiopathy in the graft.
Features of thrombotic microangiopathy include fibrin deposition and platelet aggregation resulting in thrombosis within the vessels of the graft and eventual ischemic injury 77 , With the development of coagulation dysregulation, systemic consumptive coagulopathy may be observed in the recipient and lead to the recipient's death, but this phenotype does not occur in all xenograft organs Coagulation is a complex pathway that involves interactions with inflammation and innate immunity Normally, coagulation occurs continuously within the bloodstream but is restrained by anticoagulants, thus maintaining coagulation balance When endothelial cells are injured, tissue factor TF is liberated into circulation, triggering the extrinsic coagulation pathway.
The coagulation cascade related to xenotransplantation. A Coagulation cascade in primates. Black arrows designate cascade amplification steps.
The coagulation cascade is initiated by tissue factor TF extrinsic pathway or negatively charged surface contact intrinsic pathway. TF is expressed by vascular subendothelial cells. Factor Xa converts prothrombin to thrombin. Thrombin then cleaves fibrinogen into fibrin monomers and activates factor XIII, which cross-links fibrin monomers into an insoluble clot. In response to shear stress, von Willebrand Factor vWF binds to glycoprotein 1b GPIb on platelets leading to platelet activation and adhesion Activated platelet bind to fibrinogen to mediate platelet aggregation and endothelial adherence.
Red lines show the natural inhibitors of coagulation. These processes consequently prevent the formation of thrombin B Dysregulated coagulation in pig-to-primate xenotransplantation. Red and black arrows designate incompatibility between pig and primates. When pig endothelium is activated, pig TF is expressed and released into the circulation.
After interaction with the pig endothelium, recipient platelets and peripheral blood mononuclear cells PBMCs express primate tissue factor hTF. The porcine TF pTF pathway inhibitor is an ineffective inhibitor of the human Xa factor and may ineffectively shut down the activation of the major TF.
Pig TBM pTBM binds only weakly to primate thrombin, leading to levels of activated PC that are insufficient to inhibit coagulation, resulting in thrombotic microangiopathy in pig grafts within a matter of weeks Porcine vWF spontaneously could aggregate primate platelets through GPIb receptors even in the absence of shear stress Small vessels in the graft become occluded by fibrin and platelet aggregation. In the context of xenotransplantation, the assault by antibodies and complement-activated pig endothelial cells converts endothelial cells from an anticoagulant phenotype to a procoagulant state, leading to vascular destruction, and infiltration by various immune cells Both recipient- and donor-derived TF contribute to activation of the extrinsic coagulation cascade 85 , The molecular incompatibilities between primate and pig coagulation—anticoagulation systems exaggerate this process Figure 3B.
Porcine TBM also fails to regulate primate thrombin. Even in the absence of shear stress, pvWF spontaneously aggregates primate platelets through GPIb receptors Activated platelets develop thrombosis after being recruited to the place of the endothelial cells' injury, which leads to widespread activation of the coagulation system Above all, recent advances in the field of xenotransplantation have enabled a better understanding of the immune mechanisms underlying the failure of porcine xenografts.
It is vital for xenotransplantation be introduced into clinic. However, many molecular mechanisms underlying xenograft rejection needed further elucidation, especially in pig-to-NHP models. As a consequence, diverse strategies are required to overcome the various immunological barriers involved in the rejection of various forms of xenotransplantation procedures.
According to studies on immunological rejection and coagulation dysregulation, plenty of genetically modified pigs were generated to bridge cross-species molecular incompatibilities. Since , most of the advances that have been made in the field of xenotransplantation because of the production of genetically engineered pigs. In this section, we summarize current genetically modified pigs available for xenotransplantation Table 1.
Complement activation is a clearly detriment factor in contributing to xenograft failure. One approach is administering an agent to inhibit complement, but such treatment only had a temporary effect and enhanced the risk of infection 15 , Another approach is engineering genetically modified pigs to overcome immunological rejection.
Pigs possess complement regulatory proteins CRPs that are similar to those of humans, but pig CRPs are not sufficient to protect pig epithelium cells from human complement-mediated injury. In the s, two independent research groups first proposed the suggestion that production of transgenic pigs expressing the human CRPs CD59 and CD55 to protect from hyperacute xenograft rejection. These advances introduced the possibility of genetic modification of the organ-source pig for xenotransplantation.
Today, many pigs expressing hCPRs have been produced [reviewed in ]. Researches have also demonstrated that expression of hCRPs can inhibit complement-mediated graft injury and prolong xenograft survival time , Furthermore, studies have also demonstrated that a combination of hCRPs offers greater protection than the expression of just one hCRP , Rejection of anti-Gal antibodies can be prevented through plasmapheresis or using immunoaffinity columns However, these approaches have demonstrated only partial success because the graft is lost when antibody levels recover.
The production of GTKO donor pigs is a milestone of xenotransplantation field. These results suggest that the deletion of Neu5Gc epitope in pigs is crucial for increasing xenograft survival time. In vitro evidence has also suggested that inactivation of the B4GalNT2 gene reduce human antibody binding 34 , Therefore, this animal is the preferred candidate model for evaluating the effect of the Neu5Gc deletion xenograft in NHP models.
Although expression of hCRP alone does not enable make graft long-term survival, even in the GTKO pigs, the complement system is still activated by ischemia—reperfusion injury.
Herein, the deletion of identified xenoantigens with expression of one or more hCRPs in pigs would form the foundation for future clinical trial. In vitro , porcine cells expressing hCD47 can reduce their phagocytosis by human macrophages In vivo , hCD47 expression increased xenogeneic hematopoietic engraftment chimerism in the murine model and prolonged the survival porcine skin grafts in baboons These findings collectively suggest the beneficial role of hCD47 expression in xenografts.
However, hCD47 expression did not completely prevent phagocytosis from primate macrophages; therefore, the pathway of xenoantigen-activated macrophages may also need to be suppressed.
Martin et al. The beneficial effect of hCTL4-Ig expression extended xenograft survival time in a rat skin transplantation model and a NHP neuronal transplantation model These in vivo evidence also suggested that the expression of hCTLA4-Ig alone could not prevent xenograft rejection, which is consistent with the result blocking costimulatory pathway against B7-CD28 only.
However, these pigs were susceptible to infection because of high levels of pCTLA4-Ig expression in the blood.
Therefore, the expression of this agent only in specific target cells of the pig is favorable. However, the role of these modified genes in protecting xenograft from rejection response requires further evaluation in NHPs. However, both thrombotic microangiopathy and systemic consumptive coagulopathy are increasingly recognized in xenograft and NHP recipients. Therefore, coagulation dysregulation becomes a non-negligible barrier to successful xenotransplantation.
The graft vascular endothelial cells enter into a procoagulant state, which cannot be successfully controlled by the pig's anticoagulant factors, resulting in coagulation dysregulation and graft failure.
Transgenic expression of hTBM in donor pig is one of most important approaches to overcoming coagulopathy currently. Pig aortic endothelial cells expressing hTBM were reported to substantially suppress prothrombinase activity, delay human plasma clotting time, and exhibit less activity in inducing human platelet aggregation , In the pig-to-baboon model, hTBM expression on cardiac xenografts confers an independent protective effect for prolonging graft survival time , Another key player in the anticoagulation system is EPCR, which also mediates anti-inflammatory and cytoprotective signaling Therefore, it is speculated that overexpression of hEPCR in donor pigs is a potential solution to overcoming related barriers, providing potent local anti-inflammatory, anticoagulant, and cytoprotective cell signaling.
CD39 plays a key role in the regulation of coagulation. In addition, vWF-deficient donor pigs exhibited prolonged lung graft survival time in NHP models and caused a less substantial platelet decrease in receipts , Graft coagulation varies among different xenograft organs after transplantation, perhaps because of differences in vascular structure and protein expression pattern.
Recently, considerable progress has been made in cardiac and renal xenotransplantation. However, improvements have been limited in liver and lung xenotransplantation.
After pig liver xenotransplantation, severe thrombocytopenia can occur within minutes to hours, which exacerbates coagulation dysfunction, resulting in lethal hemorrhage PvWF is a glycoprotein that plays a key role in the pathogenesis of xenograft failure, especially in pulmonary xenotransplantation, because the lung releases more vWF than the heart or kidneys Moreover, the transcription of genes involved in coagulation, fibrinolysis, and platelet function differs in heart and kidney xenografts, which may account for the different courses of coagulation dysregulation in the recipients of these organs Pulmonary xenografts release larger quantities of vWF than do heart and kidney xenografts These data collectively suggest that successful control of coagulation dysregulation in xenotransplantation may require different genetic and pharmacological strategies for different organs.
An increasing amount of evidence suggests that inflammatory response plays a considerable role in graft failure in cases of a condition called systemic inflammatory response in xenograft recipients Therefore, the engineering of donor pigs that express one or more human anti-inflammatory or antiapoptotic genes may be an approach to xenograft protection. Transgenic pigs that express human hemeoxygenase-1 and human A20 are available , The expression of human hemeoxygenase-1 reportedly protected porcine kidneys from xenograft rejection in the case of ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells However, several human transgenes, including hHO1, hCD47, and human A20, have been introduced in pigs with multiple genetic manipulations As discussed above, numbers of genes have been found to be involved in xenograft rejection.
Because of the immune response to a pig xenograft cannot be considered in isolation, successful control of immunological rejection in xenotransplantation requires the altering of multiple genes in donor pigs. Genetically modified pigs with multiple genes, with up to seven manipulations, have been produced.
The in vivo evaluation of their individual specific benefits will be difficult, and it remains unknown whether the manipulation of so many genes in donor pigs has adverse effects. Therefore, the optimization of combinations of modified genes in donor pigs and evaluation of these xenografts in NHP models are important in further studies.
Xenotransplantation has a long history with a number of animal models, including mouse, rat, and NHP, and has been used to reveal the mechanisms of rejection responses , Old World NHPs are the preferred surrogate for humans in exploring the response to pig xenograft transplantation because of their immunological similarities to humans 6.
Today, the pig-to-NHP model is the standard model for testing the primate immune response to organs or tissue from genetically modified pigs and the effect of novel immunosuppressive regimens. It is considered the optimal testing ground for predicting human responses as the final step before a human clinical trial Two comprehensive reviews explored pig solid organ graft survival in an NHP until , More recently, several studies reported key advances in NHP models.
Paediatric xenotransplantation clinical trials and the right to withdraw. Abstract Clinical trials of xenotransplantation XTx may begin early in the next decade, with kidneys from genetically modified pigs transplanted into adult humans. Statistics from Altmetric. Competing interests None declared. Patient consent for publication Not required.
No commercial re-use. See rights and permissions. Published by BMJ. Read the full text or download the PDF:. There are now more than 26 genetically-engineered pigs for xenotransplantation research Table 2.
Cooper recently published a review on carbohydrate antigen targets on pig cells [ ]. Cowan et al also published a commentary on the importance of modifying the glycome in pigs for xenotransplantation [ ]. Timeline for application of evolving techniques for genetic engineering of pigs employed in xenotransplantation.
Table adopted from Cooper et al. With our accumulated experience [ 2 , ] and recent achievements [ 13 , 18 , 86 ] in xenotransplantation, the stage may now be set for the first-in-human exploration [ 11 ]. Although a small clinical trial of microencapsulated wild-type pig islet xenotransplantation is currently underway [ 37 , 38 ], the future is set for well-controlled trials of genetically-engineered pig islet xenotransplantation.
The xenotransplantation research community needs to decide i whether successful orthotopic heart transplantation in the pig-to-NHP model is required before proceeding to a clinical trial [ ], and ii whether the preclinical threshold for a clinical renal xenotransplantation trial can be reduced [ ].
The resurgence of xenotransplantation is now obvious [ 9 , 10 , ], with prolonged survival of cellular and solid organ xenografts Figure 2 associated with the administration of newer costimulation blockade agents [ , ] and access to genetically-engineered pigs.
Our increasing knowledge of the pig genome [ ] will almost certainly lead to further genetic manipulations. The future of xenotransplantation is vibrant. Thanks to the CRISPR technology, the production of multiple-gene pigs is easier and faster and more genetically-engineered pigs are now available for xenotransplantation research.
The International Xenotransplantation Association has recently published the first update of the consensus statement on conditions for undertaking clinical trials of porcine islet products.
Work on xenotransplantation in the Xenotransplantation Research Laboratory at Indiana University has been supported by internal funds of the Department of Surgery. Papers of particular interest, published within the annual period of review, have been highlighted as:. National Center for Biotechnology Information , U. Curr Opin Organ Transplant. Author manuscript; available in PMC Dec 1.
Cooper , MD, PhD 2. David K. Author information Copyright and License information Disclaimer. Copyright notice. The publisher's final edited version of this article is available at Curr Opin Organ Transplant. See other articles in PMC that cite the published article. Abstract Purpose of review To review the progress in the field of xenotransplantation with special attention to most recent encouraging findings which will eventually bring xenotransplantation to the clinic in the near future.
Recent findings Starting from early , with the introduction of Gal-knockout pigs, prolonged survival especially in heart and kidney xenotransplantation was recorded. Summary Clinical trials in cellular or solid organ xenotransplantation are getting closer with convincing preclinical data from many centers. Introduction Outcomes of organ and cell allotransplantation continue to improve.
Open in a separate window. Figure 1. Past The concept of xenotransplantation is not new, and there have been numerous clinical attempts during the past years or more [ 3 ]. Heart xenotransplantation Mohiuddin et al [ 13 ] demonstrated that long-term survival of genetically-engineered pig heterotopic heart grafts could be achieved in NHPs. Figure 2. Longest survival times of organ and cell xenotransplantation from pigs to nonhuman primates Microencapsulated pancreatic xeno-islets survived for days with retransplantation, but days without retransplantation.
Kidney xenotransplantation The last 2 years have shown us that we are close to clinical trials of genetically-engineered pig kidney xenotransplantation. Lung xenotransplantation Most recently, only the Maryland group has been active in exploring lung xenotransplantation. Islet xenotransplantation In , the International Xenotransplantation Association IXA published the first update on its consensus statement on conditions for undertaking clinical trials of porcine islet products in patients with type 1 diabetes [ 29 — 36 ].
Tissue cornea, heart valve, skin xenotransplantation Porcine corneal xenotransplantation shows promising application in the clinic. Cellular hepatocyte, neuronal cell xenotransplantation Machaidze et al tested porcine hepatocytes in alginate-poly-l-lysine microspheres transplanted intraperitoneally immediately after hepatectomy in a model of fulminant liver failure in baboons [ 61 ].
Inflammation and coagulation Further attention was directed to inflammation in xenotransplantation. Zoonosis The potential for the transmission of infection from animal-to-human has always been of concern. Ethics and regulatory aspects As initial clinical trials draw closer, ethics [ 79 ], acceptance of xenotransplantation by hospital personnel [ 80 ], and by the general population with different cultural and religious backgrounds [ 81 — 83 ], are topics of importance.
Genetic engineering The introduction of CRISPR clustered regularly interspaced short palindromic repeats technology in xenotransplantation has increased the speed in which genetic manipulations can be achieved in pigs. Table 1 Timeline for application of evolving techniques for genetic engineering of pigs employed in xenotransplantation.
Conclusion Future of xenotransplantation With our accumulated experience [ 2 , ] and recent achievements [ 13 , 18 , 86 ] in xenotransplantation, the stage may now be set for the first-in-human exploration [ 11 ]. Acknowledgments Work on xenotransplantation in the Xenotransplantation Research Laboratory at Indiana University has been supported by internal funds of the Department of Surgery. Footnotes Conflict of interest The authors declare no conflict of interest.
The need for xenotransplantation as a source of organs and cells for clinical transplantation. Int J Surg. Clinical xenotransplantation: the next medical revolution? A brief history of clinical xenotransplantation. Hara H, Cooper DK. The immunology of corneal xenotransplantation: a review of the literature.
Cooper DKC. Early clinical xenotransplantation experiences-An interview with Thomas E. Starzl, MD, PhD. Discordant organ xenotransplantation in primates: world experience and current status. Progress in pig-to-non-human primate transplantation models — : a comprehensive review of the literature. The role of genetically engineered pigs in xenotransplantation research. J Pathol. Perkel JM. Xenotransplantation makes a comeback.
Nat Biotechnol. Xenotransplantation 2. Schuurman HJ. Pig-to-nonhuman primate solid organ xenografting: recent achievements on the road to first-in-man explorations. Conventional FDA-approved immunosuppressive therapy is unsuccessful in preventing an adaptive immune response to pig cells, but blockade of the CDCD costimulation pathway is successful.
Survival of genetically engineered pig kidneys in immunosuppressed nonhuman primates can now be measured in months. Non-immunological aspects, for example, pig renal function, a hypovolemia syndrome, and rapid growth of the pig kidney after transplantation, are briefly discussed.
0コメント