The pathobiology of pig‐to‐primate xenotransplantation: a historical review

The immunologic barriers to successful xenotransplantation are related to the presence of natural anti‐pig antibodies in humans and non‐human primates that bind to antigens expressed on the transplanted pig organ (the most important of which is galactose‐α1,3‐galactose [Gal]), and activate the complement cascade, which results in rapid destruction of the graft, a process known as hyperacute rejection. High levels of elicited anti‐pig IgG may develop if the adaptive immune response is not prevented by adequate immunosuppressive therapy, resulting in activation and injury of the vascular endothelium. The transplantation of organs and cells from pigs that do not express the important Gal antigen (α1,3‐galactosyltransferase gene‐knockout [GTKO] pigs) and express one or more human complement‐regulatory proteins (hCRP, e.g., CD46, CD55), when combined with an effective costimulation blockade‐based immunosuppressive regimen, prevents early antibody‐mediated and cellular rejection. However, low levels of anti‐non‐Gal antibody and innate immune cells and/or platelets may initiate the development of a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. This pathogenic process is accentuated by the dysregulation of the coagulation‐anticoagulation systems between pigs and primates. The expression in GTKO/hCRP pigs of a human coagulation‐regulatory protein, for example, thrombomodulin, is increasingly being associated with prolonged pig graft survival in non‐human primates. Initial clinical trials of islet and corneal xenotransplantation are already underway, and trials of pig kidney or heart transplantation are anticipated within the next few years.     

The role of platelets in coagulation dysfunction in xenotransplantation,and therapeutic options

Xenotransplantation could resolve the increasing discrepancy between the availability of deceased human donor organs and the demand for transplantation. Most advances in this field have resulted from the introduction of genetically engineered pigs, e.g., α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs transgenic for one or more human complement‐regulatory proteins (e.g., CD55, CD46, CD59). Failure of these grafts has not been associated with the classical features of acute humoral xenograft rejection, but with the development of thrombotic microangiopathy in the graft and/or consumptive coagulopathy in the recipient. Although the precise mechanisms of coagulation dysregulation remain unclear, molecular incompatibilities between primate coagulation factors and pig natural anticoagulants exacerbate the thrombotic state within the xenograft vasculature. Platelets play a crucial role in thrombosis and contribute to the coagulation disorder in xenotransplantation. They are therefore important targets if this barrier is to be overcome. Further genetic manipulation of the organ‐source pigs, such as pigs that express one or more coagulation‐regulatory genes (e.g., thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor, CD39), is anticipated to inhibit platelet activation and the generation of thrombus. In addition, adjunctive pharmacologic anti‐platelet therapy may be required. The genetic manipulations that are currently being tested are reviewed, as are the potential pharmacologic agents that may prove beneficial.     

Justification of specific genetic modifications in pigs for clinical organ xenotransplantation

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications. We suggest that a pig with nine genetic modifications (ie, currently available) will provide organs (initially kidneys and hearts) that would function for a clinically valuable period of time, for example, >12 months, after transplantation into patients with end‐stage organ failure. The national regulatory authorities, however, will likely require evidence, based on in vitro and/or in vivo experimental data, to justify the inclusion of each individual genetic modification in the pig. We provide data both from our own experience and that of others on the advantages of pigs in which (a) all three known carbohydrate xenoantigens have been deleted (triple‐knockout pigs), (b) two human complement‐regulatory proteins (CD46, CD55) and two human coagulation‐regulatory proteins (thrombomodulin, endothelial cell protein C receptor) are expressed, (c) the anti‐apoptotic and “anti‐inflammatory” molecule, human hemeoxygenase‐1 is expressed, and (d) human CD47 is expressed to suppress elements of the macrophage and T‐cell responses. Although many alternative genetic modifications could be made to an organ‐source pig, we suggest that the genetic manipulations we identify above will all contribute to the success of the initial clinical pig kidney or heart transplants, and that the beneficial contribution of each individual manipulation is supported by considerable experimental evidence.     

Xenotransplantation in China: Present status

The main obstacle to organ transplantation is the shortage of organs from deceased individuals. Especially in China, the ratio of patients on the waiting list versus the transplant recipients is 30:1. Therefore, there is an urgent need for organ donors. Genetically modified pig organs have proved to be a new source for xenotransplantation, and Chinese scientists have made considerable progress in this area during recent years. In this paper, we review four important aspects of the xenotransplantation field in China. First, a large variety of genetically modified pigs have been generated by Chinese scientists: all these genetically modified pigs and the purpose of these modifications will be summarized. Second, the preclinical research in pig‐to‐nonhuman primate xenotransplantation is outlined. The survival time and major biochemical parameters for the xenografts are summarized. Third, regarding the bench‐to‐bed approach, more suitable organs have been developed for xenotransplantation in humans, and in particular, pig islet transplantation into diabetic patients as well as pig‐to‐human cornea and skin transplantation. Fourth, we briefly address the regulations and prospects for recruiting xenotransplantation experts in China. Based on recent progress, we anticipate that genetically modified pigs will offer suitable organs for the treatment of end‐stage organ diseases in humans in the near future. Given the recent influx of world‐renowned scientists in xenotransplantation to China, our country will definitely become one of the major centers of xenotransplantation research and development in the world.     

Progress in multiple genetically modified minipigs for xenotransplantation in China

Pig‐to‐human organ transplantation provides an alternative for critical shortage of human organs worldwide. Genetically modified pigs are promising donors for xenotransplantation as they show many anatomical and physiological similarities to humans. However, immunological rejection including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), immune cell–mediated rejection, and other barriers associated with xenotransplantation must be overcome with various strategies for the genetic modification of pigs. In this review, we summarize the outcomes of genetically modified and cloned pigs achieved by Chinese scientists to resolve the above‐mentioned problems in xenotransplantation. It is now possible to knockout several porcine genes associated with the expression of sugar residues, antigens for (naturally) existing antibodies in humans, including GGTA1, CMAH, and β4GalNT2, and thereby preventing the antigen‐antibody response. Moreover, insertion of human complement‐ and coagulation‐regulatory transgenes, such as CD46, CD55, CD59, and hTBM, can further overcome effects of the humoral immune response and coagulation dysfunction, while expression of regulatory factors of immune responses can inhibit the adaptive immune rejection. Furthermore, transgenic strategies have been developed by Chinese scientists to reduce the potential risk of infections by endogenous porcine retroviruses (PERVs). Breeding of multi‐gene low‐immunogenicity pigs in China is also presented in this review. Lastly, we will briefly mention the preclinical studies on pig‐to‐non‐human primate xenotransplantation conducted in several centers in China.     

Pig kidney graft survival in a baboon for 136 days: longest life‐supporting organ graft survival to date

The longest survival of a non‐human primate with a life‐supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α‐1,3‐galactosyltransferase gene‐knockout pig transgenic for two human complement‐regulatory proteins and three human coagulation‐regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti‐CD20mAb (induction) and anti‐CD40mAb+rapamycin+corticosteroids (maintenance). Anti‐TNF‐α and anti‐IL‐6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein‐losing nephropathy were observed. There was no evidence of an elicited anti‐pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti‐inflammatory agents prevented immune injury and a protein‐losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.     

Financial aspects of organ procurement from deceased donors in the USA—Relevance to xenotransplantation

When clinical xenotransplantation is introduced, the costs associated with acquisition of a genetically engineered pig organ are as yet unknown. How will these costs compare with those currently associated with the acquisition of deceased human organs? An understanding of the financial aspects of deceased organ and tissue procurement in the USA is therefore worthwhile. We have therefore attempted to review certain economic aspects of non‐profit and for‐profit organizations that provide cadaveric organs and/or tissues for purposes of transplantation into patients with end‐stage organ failure, cellular deficiencies, or in need of reconstructive procedures. We briefly consider the laws, organizations, and business practices that govern the acquisition, processing, and/or distribution of cadaveric organs and tissues, and the economic implications of industry practices. In particular, we explore and highlight what we perceive as a lack of transparency and oversight with regard to financial practices, and we question whether donor families would be entirely happy with the business environment that has developed from their altruistic donations. Until xenotransplantation becomes established clinically, which will negate the need for any system of organ procurement and allocation, we suggest that those involved in organ and cell transplantation, as well as those who participate in reconstructive surgery, should take responsibility to ensure that the financial practices associated with procurement are transparent, and overseen/regulated by a responsible authority. We suggest the major transplant societies should take a lead in this respect. The ability to acquire a genetically engineered pig organ whenever required through a simple commercial transaction (as in the acquisition of a life‐saving drug) will be greatly to the patient's benefit.     

Xenotransplantation: immunological barriers and strategies to overcome them

Hyperacute and acute vascular rejection of xenografts are well defined barriers to clinical pig‐to‐human xenotransplantation. Enormous progress has been made in recent years to overcome these immunological barriers. For example, transgenic expression of human complement regulatory molecules (e.g. CD46, CD55) in pigs has been shown to be an effective strategy to prevent hyperacute rejection in pre‐clinical models of xenotransplantation. Alpha1,3‐galactosyltransferase knock‐out pigs are available and provide a second possibility to avoid hyperacute rejection mediated by pre‐existing antibodies. Furthermore, transfer of protective genes (e.g. A20, HO‐1) to endothelial cells is expected to reduce their susceptibility to effector mechanisms leading to acute vascular rejection. In addition, the efficiency of strategies to avoid coagulation/thrombosis after pig‐to‐human xenotransplantation (e.g. transgenic expression of human thrombomodulin, CD39) is currently tested. Thus, for further development of clinical xenotransplantation immunological concepts are now required facilitating the control of human anti‐pig cellular immune responses. Our group focuses on the inhibition of human anti‐pig T cell responses by targeting “negative” costimulatory pathways. We tested the hypothesis that overexpression of the human negative costimulatory ligands PD‐L1 and PD‐L2 on pig antigen presenting cells will result in reduced human anti‐pig T cell responses. The data so far show that (i) human CD4⁺ T cells respond with reduced proliferation and cytokine synthesis to PD‐L1/PD‐L2 expressing pig cells, (ii) PD‐L1/PD‐L2 pig transfectants induce human regulatory T cells (T_reg) which suppress the activation of conventional T cells, and (iii) PD‐L1/PD‐L2 expressing pig cells are protected from lysis mediated by CD8⁺ human cells. Together these observations support the assumption that transgenic expression of human PD‐L1 and/or PD‐L2 in pig cells and tissues could be an approach to prevent T cell reactivity after pig‐to‐human xenotransplantation. Supported by the Deutsche Forschungsgemeinschaft (Transregio Forschergruppe “Xenotransplantation”, FOR 535).     

Systemic inflammation in xenograft recipients (SIXR)

Dysregulation of the coagulation system commonly develops in pig xenograft recipients, and remains an obstacle to successful pig organ xenotransplantation. Uncontrolled activation of coagulation leads to consumptive coagulopathy (CC) in the recipients, and thrombotic microangiopathy (TM) in the grafts. T cell‐directed immunosuppression successfully prevents the adaptive immune response to pig antigens after xenotransplantation and prolongs survival of organ xenografts. In some reports, T cell‐directed immunosuppression was able to delay (or prevent) the development of CC and/or TM. Recent reports have confirmed that inflammation can lead to activation of the coagulation system. Additionally, pro‐coagulant proteins, e.g. thrombin, are considered as pro‐inflammatory factors. In fact, an amplification loop is suggested to exist between inflammation and coagulation, leading to escalation of each other. Our in vitro data indicate that thrombin activation of pig endothelial cells is associated with upregulation of human T cell responses, suggesting that control of activation of coagulation and prevention of thrombin activation may facilitate the regulation of immune responses to xenografts in vivo. We hypothesize that a state of systemic inflammation develops after pig organ xenotransplantation, which is generated by both adaptive and innate immune responses. Even if T cell‐directed immunosuppression can control activation of coagulation induced by adaptive immune responses, pro‐inflammatory signals induced by the innate immune system can still promote activation of coagulation. We studied two models of xenotransplantation of different antigen loads; (i) pig aortic patch xenotransplantation, i.e. low antigen load, and (ii) pig organ (heart and kidney) xenotransplantation, i.e. high antigen load. We evaluated activation of coagulation, development of a T cell‐dependent immune response, and production of innate and adaptive pro‐inflammatory factors. In recipients of a low antigen load xenograft, effective prevention of the adaptive immune response by T cell‐directed immunosuppression (i.e. suppression of T cell proliferation in response to pig antigens and prevention of elicited antibody production) was associated with reduced thrombin activation. However, there was (i) upregulation of C‐reactive protein (CRP) and (ii) fibrinogen levels, (iii) increased IL‐6 production in the circulation, and (iv) an increase in the absolute number of innate immune cells (monocytes and neutrophils). Furthermore, (v) monocytes and dendritic cells showed significant upregulation of tissue factor expression, and aggregation with platelets after transplantation. In recipients with a high antigen load xenograft, short‐term organ survival was associated with high levels of CRP and IL‐6 early after transplantation. In long‐term organ survival, high levels of CRP and IL‐6 preceded the development of CC. There was intense CRP deposition in kidney xenografts (more than in heart xenografts) suggesting a stronger innate immune response. Additionally, CRP‐positive cells were detected in native lungs, suggesting an innate systemic inflammatory response. In conclusion, efficient blockade of the T cell‐dependent adaptive immune response in xenograft recipients is associated with systemic upregulation of inflammatory markers. Systemic inflammation in xenograft recipients (SIXR) is associated with upregulation of tissue factor expression on innate immune cells and their aggregation with platelets. As inflammation is known to break tolerance after transplantation, understanding the underlying mechanisms and regulation of SIXR may be necessary to achieve long‐term survival of organ xenografts (and T cell tolerance to pig antigens). Also, further genetic modifications of donor pigs to express anti‐inflammatory proteins may be essential.     

Potential pathological role of pro‐inflammatory cytokines (IL‐6, TNF‐α, and IL‐17) in xenotransplantation

The major limitation of organ transplantation is the shortage of available organs from deceased human donors which leads to the deaths of thousands of patients each year. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection and coagulation dysfunction are two major hurdles for the successful survival of pig xenografts in primate recipients. Pro‐inflammatory cytokines, such as IL‐6, TNF‐α, and IL‐17, play important roles in many diseases and in allotransplantation. However, the pathological roles of these pro‐inflammatory cytokines in xenotransplantation remain unclear. Here, we briefly review the signaling transduction and expression regulation of IL‐6, TNF‐α, and IL‐17 and evaluate their potential pathological roles in in vitro and in vivo models of xenotransplantation. We found that IL‐6, TNF‐α, and IL‐17 were induced in most in vitro or in vivo xenotransplantation model. Blockade of these cytokines using gene modification, antibody, or inhibitor had different effects in xenotransplantation. Inhibition of IL‐6 signaling with tocilizumab decreased CRP but did not increase xenograft survival. The one possible reason is that tocilizumab can not suppress IL‐6 signaling in porcine cells or organs. Other drugs which inhibit IL‐6 signaling need to be investigated in xenotransplantation model. Inhibition of TNF‐α was beneficial for the survival of xenografts in pig‐to‐mouse, rat, or NHP models. Blockade of IL‐17 using a neutralizing antibody also increased xenograft survival in several animal models. However, the role of IL‐17 in the pig‐to‐NHP xenotransplantation model remains unclear and needs to be further investigated. Moreover, blockade of TNF‐α and IL‐6 together has got a better effect in pig‐to‐baboon kidney xenotransplantation. Blockade two or even more cytokines together might get better effect in suppressing xenograft rejection. Better understanding the role of these cytokines in xenotransplantation will be beneficial for choosing better immunosuppressive strategy or producing genetic modification pig.     

40 years of heart transplantation and the DFG‐Transregio Research Group Xenotransplantation

Today, organ transplantation represents a well‐established and effective therapy of terminal organ failure revealing high actuarial survival rates. Unfortunately, the enormous potential of organ transplantation cannot be tapped due to the significant gap between organ demand and organ donation. Current statistics of the International Society of Heart and Lung Transplantation prove a continuity of depressed numbers of transplantations performed per year since the late nineties. To counteract the persisting severe shortage of human organs in Germany and worldwide suboptimal donor organs and/or organs from older donors were accepted. Both the acceptance of inferior organs and the implementation of the Transplantation Law (in Germany in 1997) could not answer this problem. Increasing the donor rates emerge difficult to achieve and will ultimately result in numbers which are not sufficient. The improvement of transplant results by e.g. a less nephrotoxic immunosuppression, or the generating of hyporeactivity or even tolerance is an additional aim important to achieve. Alternative techniques to answer the tremendous organ shortage might be the differentiation of embryonic stem cells or the reprogramming of adult stem cells as a virtually unlimited source for cell replacement to treat degenerative diseases or traumatic tissue injury. Yet, disadvantages such as ethical issues and the generation of tumorigenic cells should not be underestimated. A cellular therapy by the injection of undifferentiated bone marrow (CD133⁺ stem/progenitor) cells into the myocardium in combination with or without aortocoronary surgery for chronic ischemic heart disease as well as cells from the amniotic fluid (Wharton's jelly) might also represent possible future solutions to the organ deficit but still are far from a functional substitution of the human heart. Until now there is no in‐all implantable mechanical heart assist device which is able to completely and permanently replace the human organ and provide a quality of life comparable to that after allotransplantation. In contrast, xenotransplantation, using porcine organs for human transplantation, offers a potential solution to the world‐wide lack of donor organs. The advantages of xenotransplantation are an unlimited disposability of donor organs, an elective transplantation with a subsequent reduction of ischemic time and the possibility of a pre‐operative start of the immunosuppressive therapy of the recipient. Harmful effects of the brain death of the donor to the donor organ could be excluded. Finally, genetic modifications of compatible xenografts could be made. Substantial progress of the research in the field of xenotransplantation has been possible thanks to the introduction of organs from genetically engineered pigs transgenic for human complement regulatory proteins [e.g. human decay accelerating factor (hDAF/hCD55), human membrane cofactor (hMCP/hCD46), and human membrane inhibitor of reactive lysis (hMIRL/hCD59)]. Using an effective and persistent depletion of preformed cytotoxic anti‐Galα(1,3)Gal antibodies (IgM and IgG) by a Galα(1,3)Gal therapeutic (e.g. GAS914, TPC) in combination with these transgenic pigs hyperacute rejection can be avoided successfully. During the early phase after transplant acute vascular rejection triggered by induced anti‐Galα(1,3)Gal antibodies can be controlled. Several groups developed pigs which lack the Galα(1,3)Gal xenoantigen. Studies on xenotransplantations performed with homozygous alpha(1,3)‐galactosyltransferase gene knockout pigs demonstrated that these modified pig organs offer some progress in terms of graft survival. Thus, the major xenoantigen Galα(1,3)Gal is no longer an unsurmountable immunological barrier preventing transplantation of pig organs into humans. Acute vascular rejection, however, remains as a major hurdle to clinical application of xenotransplantation due to cytotoxic anti‐pig antibodies of other specificity than Galα(1,3)Gal. Furthermore, humoral factors are not the only players in xenograft rejection. Primate anti‐pig cellular immunity is defined by multifocal lymphocytic infiltrates, with morphologic evidence of direct tissue damage. Pre‐requisites for the clinical use of xenotransplantation are PERV‐C (porcine endogenous virus C) free animals using a PERV knock down (si‐RNA) technique. Multitransgenic αGalT‐KO [alpha(1,3)‐galactosyltransferase knockout] pigs additionally expressing human complement regulator proteins, and human anticoagulants (e.g. human thrombomodulin) are necessary to reliably prevent not only hyperacute rejection as the first immunological barrier, but also acute vascular rejection at its beginning, when serum cytotoxicity to the pig heart appears to be predominantly Galα(1,3)Gal‐specific. Further co‐stimulation blockade (e.g. PD‐1L, CTLA‐4‐Ig), HLA‐E [protection against human NK (natural killer)‐cells], or haemeoxygenase‐1 (defense against disseminated intravascular coagulation) will help to suppress acute vascular and acute cellular xenograft rejection. Special pathogen free (SPF) units and breeding conditions of pig organ donors limit the risk of microbial contamination by most pathogens liable to be transmitted from a pig graft to a human recipient. Our DFG‐(German Research Council) Transregio Research Group Xenotransplantation assembles an interdisciplinary group of leading German laboratories incl. biotechnologists, immunologists, virologists, and surgeons with vast experimental expertises in the field of experimental and clinical allotransplantation and experimental xenotransplantation. The first clinical goal of xenotransplantation is xenogeneic tissue transplantation such as the transplantation of porcine islet cells (αGalT‐KO (?), CTLA‐4‐Ig expression) in diabetic patients with hypoglycemic attacks as well as porcine cornea, porcine cardiomyocytes and porcine heart valves, possibly porcine bones and teeth (?). Thereafter, xenogeneic organ transplantation starting with the more promising use of kidneys and hearts is the definitive clinical goal. In summary, clinical heart transplantation represents an accepted method of end‐stage heart failure with an outdated “standard immunosuppression” and the need of an individualized immunosuppression adjusted to the specific needs of the individual patient. The organ shortage remains the main obstacle of the heart transplantation, and other organ transplantation, respectively. In the near future, xenotransplantation will be possible!     

Human CD59 expressed in transgenic mouse hearts inhibits the activation of complement

Porcine-to-human xenotransplantation offers a potential solution to the critical shortage of human organs. The major immunological barrier to xenotransplantation between these species is a rapid rejection process mediated by preformed natural antibodies and complement. Xenogeneic organ grafts are especially susceptible to complement mediated injury because complement regulatory proteins, which ordinarily protect cells from inadvertent injury during the activation of complement, function poorly in regulating activation of heterologous complement. Removal of xenoreactive antibodies or systemic inhibition of complement activity has been shown to prolong graft survival. As an alternative to the systemic inhibition of complement activity, we have established a model system using transgenic animals to test whether the expression of human membrane bound complement regulatory proteins on mouse endothelial cells can inhibit the activation of human complement. CD59, which acts at the terminal stage of complement activation by inhibiting the formation of the membrane attack complex, was used as a paradigm for this model. A CD59 construct containing the putative CD59 gene promoter linked to the CD59 coding region was used to demonstrate expression of the human CD59 protein in various tissues of transgenic mice, including endothelial cells in the heart. In addition, we show that the transgenic CD59 protein is biologically active as determined by the ability to inhibit the formation of membrane attack complex in transgenic mouse hearts perfused ex vivo with human plasma. These results demonstrate that expression of membrane bound complement regulatory proteins can achieve complement inhibition in a xenogeneic organ and suggest that this approach may be useful for successful xenotransplantation between discordant species.     

The complex functioning of the complement system in xenotransplantation

The role of complement in xenotransplantation is well‐known and is a topic that has been reviewed previously. However, our understanding of the immense complexity of its interaction with other constituents of the innate immune response and of the coagulation, adaptive immune, and inflammatory responses to a xenograft is steadily increasing. In addition, the complement system plays a function in metabolism and homeostasis. New reviews at intervals are therefore clearly warranted. The pathways of complement activation, the function of the complement system, and the interaction between complement and coagulation, inflammation, and the adaptive immune system in relation to xenotransplantation are reviewed. Through several different mechanisms, complement activation is a major factor in contributing to xenograft failure. In the organ‐source pig, the detrimental influence of the complement system is seen during organ harvest and preservation, for example, in ischemia‐reperfusion injury. In the recipient, the effect of complement can be seen through its interaction with the immune, coagulation, and inflammatory responses. Genetic‐engineering and other therapeutic methods by which the xenograft can be protected from the effects of complement activation are discussed. The review provides an updated source of reference to this increasingly complex subject.     

Comparison of hematologic,biochemical, and coagulation parameters in α1,3‐galactosyltransferase gene‐knockout pigs,wild‐type pigs,and four primate species

Ekser B, Bianchi J, Ball S, Iwase H, Walters A, Ezzelarab M, Veroux M, Gridelli B, Wagner R, Ayares D, Cooper DKC. Comparison of hematologic, biochemical, and coagulation parameters in α1,3‐galactosyltransferase gene‐knockout pigs, wild‐type pigs, and four primate species. Xenotransplantation 2012; 19: 342–354. © 2012 John Wiley & Sons A/S. Abstract: Background: The increasing availability of genetically engineered pigs is steadily improving the results of pig organ and cell transplantation in non‐human primates (NHPs). Current techniques offer knockout of pig genes and/or knockin of human genes. Knowledge of normal values of hematologic, biochemical, coagulation, and other parameters in healthy genetically engineered pigs and NHPs is important, particularly following pig organ transplantation in NHPs. Furthermore, information on parameters in various NHP species may prove important in selecting the optimal NHP model for specific studies. Methods: We have collected hematologic, biochemical, and coagulation data on 71 α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs, 18 GTKO pigs additionally transgenic for human CD46 (GTKO.hCD46), four GTKO.hCD46 pigs additionally transgenic for human CD55 (GTKO.hCD46.hCD55), and two GTKO.hCD46 pigs additionally transgenic for human thrombomodulin (GTKO.hCD46.hTBM). Results: We report these data and compare them with similar data from wild‐type pigs and the three major NHP species commonly used in biomedical research (baboons, cynomolgus, and rhesus monkeys) and humans, largely from previously published reports. Conclusions: Genetic modification of the pig (e.g., deletion of the Gal antigen and/or the addition of a human transgene) (i) does not result in abnormalities in hematologic, biochemical, or coagulation parameters that might impact animal welfare, (ii) seems not to alter metabolic function of vital organs, although this needs to be confirmed after their xenotransplantation, and (iii) possibly (though, by no means certainly) modifies the hematologic, biochemical, and coagulation parameters closer to human values. This study may provide a good reference for those working with genetically engineered pigs in xenotransplantation research and eventually in clinical xenotransplantation.     

The immense potential of xenotransplantation in surgery

There is a limited availability of deceased human organs and cells for the purposes of clinical transplantation. Genetically-engineered pigs may provide an alternative source. Although several immune barriers need to be overcome, considerable progress has been made in experimental models in recent years, largely through the increasing availability of pigs with new genetic modifications. Pig heterotopic heart graft survival in nonhuman primates has extended for 8 months, with orthotopic grafts supporting life for almost 2 months. Life-supporting kidney transplants have functioned for almost 3 months. The current barriers are related to coagulation dysfunction between pig and primate that results in thrombotic microangiopathy and/or a consumptive coagulopathy, which may in part be related to molecular incompatibilities in the coagulation systems of pigs and primates. Current efforts are concentrated on genetically-modifying the organ- or islet-source pigs by the introduction of 'anticoagulant' or 'anti-thrombotic' genes to provide protection from the recipient coagulation cascade and platelet activation. Progress with pig islet xenotransplantation has been particularly encouraging with complete control of glycemia in diabetic monkeys extending in one case for >12 months. Other areas where experimental data suggest the possibility of early clinical trials are corneal xenotransplantation and pig neuronal cell xenotransplantation, for example, in patients with Parkinson's disease. With the speed of advances in genetic engineering increasing steadily, it is almost certain that the remaining problems will be overcome within the foreseeable future, and clinical allotransplantation will eventually become of historical interest only.     

Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs

A combination of genetic manipulations of donor organs and target‐specific immunosuppression is instrumental in achieving long‐term cardiac xenograft survival. Recently, results from our preclinical pig‐to‐baboon heterotopic cardiac xenotransplantation model suggest that a three‐pronged approach is successful in extending xenograft survival: (a) α‐1,3‐galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal‐specific antibody‐mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co‐stimulation blockade of CD40‐CD40L pathways with anti‐CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non‐Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co‐stimulation blockade‐based immunomodulatory regimen.     

Cellular immunological barriers in xenotransplantation: current status and prospects for avoidance of graft destruction

Genetic modification of pigs (e.g. transgenic expression of human complement regulatory molecules or inactivation of α1,3galactosyltransferase) enabled the development of promising strategies to overcome hyperacute rejection after pig‐to‐primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. Cellular rejection of porcine cells in xenotransplantation models is mediated by macrophages, T cells and NK cells. Activation of human monocytes by pig cells is partly due to the incapacity of porcine ligands to bind the inhibitory receptor SIRPα (signal regulatory protein α). Thus, one approach to impair the ability of human macrophages to phagocyte porcine cells is the overexpression of the human ligand for SIRPα in porcine cells. To inhibit human NK cell reactivity after xenotranslantation transgenic expression of HLA‐E in pigs has been shown to be a promising concept. Cells from these pigs were partially protected from lysis by human NK cells. Our group focuses on manipulation of human anti‐pig T cell responses by negative costimulatory signals. Thus, we asked whether overexpression of PD‐L1 on porcine cells can (i) downregulate human anti‐pig cellular responses in vitro, and (ii) inhibit rat anti‐pig cellular immune responses in vivo. Pig cells overexpressing PD‐L1 triggered reduced proliferation and low amounts of IL‐2, IFNγ, TNF‐alpha, IL‐4, and IL‐5 in human CD4⁺ T cells compared to control pig cells. The concentration of IL‐10, however, was increased. In long‐term cultures of human CD4⁺ T cells and PD‐L1 transfectants a high frequency of CD4⁺ CD25^high FoxP3⁺ cells showed up which had the capacity to suppress the activation of conventional CD4⁺ T cells. Cytotoxic CD8⁺ T cells and NK cells lysed pig control cells very efficiently. In contrast, PD‐L1 transfected pig cells were partially protected from lysis by human effector cells. Overexpression of PD‐L1 on porcine cells was not sufficient to prevent rejection after transplantation under the rat kidney capsule. However, in rats that had been grafted with PD‐L1 expressing cells we observed reduced cellular infiltrates in the kidneys and lower antibody responses compared to rats grafted with control cells. Together these observations support the assumption that PD‐1/PD‐Ligand pathways are interesting targets to prevent cellular immune responses after xenotransplantation. PD‐L overexpression might not only impede the initiation of an anti‐pig T cell response by suppressing CD4⁺ T cells but may also protect pig cells from destruction by cytotoxic effectors. Supported by the Deutsche Forschungsgemeinschaft (Transregio Forschergruppe “Xenotransplantation”, FOR 535).     

An in vitro model of pig liver xenotransplantation--pig complement is associated with reduced lysis of wild-type and genetically modified pig cells

Hara H, Campanile N, Tai H‐C, Long C, Ekser B, Yeh P, Welchons D, Ezzelarab M, Ayares D, Cooper DKC. An in vitro model of pig liver xenotransplantation—pig complement is associated with reduced lysis of wild‐type and genetically modified pig cells. Xenotransplantation 2010; 17: 370–378. © 2010 John Wiley & Sons A/S. Abstract: Background: After pig liver transplantation in humans, the graft will produce pig complement (C). We investigated in vitro the lysis of wild‐type (WT), α1,3‐galactosyltransferase gene‐knockout (GTKO), and CD46 transgenic (CD46) pig peripheral blood mononuclear cells (PBMC) caused by human anti‐pig antibodies (Abs) + pig C. Methods: Human serum IgM/IgG binding to WT and GTKO PBMC was determined by flow cytometry, and lysis of pig PBMC by a C‐dependent cytotoxicity assay using (i) human serum (human Abs + C), (ii) GTKO pig serum (anti‐Gal Abs + pig C), (iii) heat‐inactivated human serum (human Abs) + rabbit C, or (iv) human Abs + pig C (serum). Results: Binding of human IgM and IgG to GTKO PBMC was less than to WT PBMC (P < 0.05). In the presence of human Abs, lysis of WT and GTKO PBMC by rabbit C was 87 and 13%, respectively (WT vs. GTKO, P < 0.01), but was only 37 and 0.4% in the presence of pig C (WT vs. GTKO, P < 0.05). Human/rabbit C‐induced lysis was greater than pig C‐induced lysis for both WT and GTKO PBMC. CD46 pig PBMC reduced rabbit/human C‐ and pig C‐mediated lysis (P < 0.05). Conclusions: Pig livers, particularly from GTKO and CD46 pigs, are likely to have an immunologic advantage over other organs after transplantation into humans. In the absence of pig antibodies directed to human tissues, pig complement is unlikely to cause problems after liver xenotransplantation, especially if GTKO/CD46 pigs are used as the source of the livers.     

Gal Knockout and Beyond

Recently, Galalpha1-3Galbeta1-4GlcNAc (Gal) knockout (k/o) pigs have been developed using genetic cloning technologies. This remarkable achievement has generated great enthusiasm in xenotransplantation studies. This review summarizes the current status of nonhuman primate experiments using Gal k/o pig organs. Briefly, when Gal k/o pig organs are transplanted into primates, hyperacute rejection does not occur. Although graft survival has been prolonged up to a few months in some cases, the overall results were not better than those using Gal-positive pig organs with human complement regulatory protein transgenes. Gal k/o pig kidneys rapidly developed rejection which was associated with increased anti-non-Gal antibodies. Although the precise mechanisms of Gal k/o pig organ rejection are not clear, it could result from incomplete deletion of Gal, up-regulation of new antigen (non-Gal antigen) and/or production of non-Gal antibodies. Future work in xenotransplantation should place emphasis on further modification of donors, such as combining human complement regulatory genes with Gal k/o, deleting non-Gal antigens and adding protective/surviving genes or a gene that inhibits coagulation. Induction of donor-specific T- and B-cell tolerance and promotion of accommodation are also warranted.