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Abstract

In suitable patients with end-stage organ failure, the transplantation of organs from living or deceased human donors offers a much-improved quality and length of life. However, the availability of deceased human donor organs is grossly inadequate. Gene-edited pigs might provide an alternative source of organs for clinical transplantation (xenotransplantation). However, there are major immunobiological barriers to successful pig organ transplantation in human or nonhuman primate recipients. These barriers include antibody binding, activation of complement, the innate cellular response, coagulation dysregulation between pig and primate, and a systemic inflammatory response, in addition to the T cell response. These have steadily been overcome by a combination of (i) genetic engineering of the organ-source pig (aimed mainly at the innate immune response), and (ii) the administration of novel immunosuppressive agents (directed towards the adaptive immune response). The immunological barriers that remain relate to both the innate and adaptive immune responses. Pig kidney transplants have now supported immunosuppressed (anephric) nonhuman primates for periods in excess of a year and pig heart transplants for up to 9 months, although these encouraging results cannot yet be achieved consistently.

Abbreviations

AMR: antibody-mediated rejection

GTKO: 3-galactosyltransferase gene-knockout

HLA: human leukocyte antigen

NHP: nonhuman primate

PERV: porcine endogenous retrovirus

SLA: swine leukocyte antigen

TKO: triple-knockout

INTRODUCTION

The shortage of organs from deceased human donors for transplantation into patients with end-stage organ failure is a worldwide problem. The most likely alternative source of organs is xenotransplantation (cross-species transplantation), specifically the transplantation of gene-edited pig organs into human recipients. Although patients with terminal heart failure can receive a mechanical support or replacement device, kidney failure, with the exception of dialysis, can only be treated successfully by transplantation. Given the complexity of the numerous cellular functions of the kidney, bioengineering of new kidneys will be difficult and unlikely to provide a solution within the foreseeable future 1,2. Xenotransplantation is therefore the hope for the near future.

From an immunologic perspective, nonhuman primates (NHPs) would be the preferred sources of organs for transplantation into humans, but virtually all of these species are either endangered or are too small to provide organs suitable for transplantation into large adult humans. Furthermore, concerns have been raised about the transmission of infectious agents from NHPs to humans, particularly since most NHPs are either wild-caught or have been housed under colony conditions for relatively few generations. The time and expense of breeding these animals in captivity are also prohibitive, as is a lack of experience in genetically modifying them. In addition, many members of the public would object to the use of NHPs on ethical grounds.

The pig has therefore been identified as the species most likely to be the source of organs for clinical xenotransplantation, and in recent years research efforts have been directed toward pig-to-NHP transplantation. There are several advantages for using the pig as an organ-source 3. However, a major disadvantage is that the human and NHP immune response to organs from wild-type (i.e., genetically-unmodified) pigs is rapid and intense, resulting in hyperacute rejection.

If the pig could be the organ-source, there are several potential advantages of xenotransplantation when compared to allotransplantation. The availability of an unlimited number of organs whenever required is just the most obvious. Others include the potential for infection-free organs that have not been damaged by the adverse effects of brain death or cessation of heartbeat. Xenotransplantation provides us with the first real opportunity (in > 70 years of clinical transplantation) of modifying the donor, rather than just treating the recipient. The more we can do to the donor, the less we will need to do to the recipient 4-7. This should eventually result in the need for minimal or no immunosuppressive drug therapy, leading to fewer adverse events.

IMMUNOBIOLOGICAL BARRIERS TO PIG ORGAN XENOTRANSPLANTATION

All humans and Old World NHPs have ‘natural’ antibodies to pig xenoantigens, which they develop during the first year of life (Fig. 1) as a defensive mechanism when their gastrointestinal tract is colonized by potentially pathogenic microorganisms that express some of the same antigens as pigs 8-10.

Antibody-mediated rejection (AMR) is therefore common after pig organ transplantation into Old World NHPs, even when the organ is taken from a triple-knockout (TKO) pig, i.e., a pig in which the expression of the three known pig glycan xenoantigens has been deleted (Tab. I). Whether AMR is related to the presence of natural (preformed) anti-glycan antibodies in these NHPs or to the development of elicited antibodies directed to other glycan or protein antigens expressed on the pig cells, e.g., swine leukocyte antigens (SLA), remains uncertain, but is probably associated with both. As in allotransplantation, AMR can be acute or chronic.

In our experience in xenotransplantation we have never successfully reversed acute AMR and, to our knowledge, nor has any other research group. As all NHPs have pre-existing antibodies to TKO pig organ grafts, i.e., they are sensitized 11-14, most research groups now select NHPs will low anti-pig antibody levels for their pig organ transplantation experiments. However, there is still a risk of early AMR from natural antibody or from elicited antibody (if the immunosuppressive therapy is inadequate).

Many humans, however, do not have antibodies to TKO pig xenoantigens, and in vitro studies suggest that early AMR will not occur when TKO pig organs are transplanted clinically (if the adaptive immune response is suppressed successfully) 15.

THE MECHANISM OF AMR IN XENOTRANSPLANTATION

Xenoreactive natural antibodies

Circulating natural (or preformed) antibodies are immunoglobulins found in the serum of healthy humans and NHP species without known antigenic stimulation. As part of the innate immune response, they play a key role in the recognition and neutralization of pathogens and in the stimulation of phagocytic macrophage activity. In the context of pig-to-primate organ xenotransplantation, natural antibodies to glycan xenoantigens expressed on the pig cells (Tab. I) initiate rejection through activation of the classical complement pathway 16,17.

Several studies have demonstrated that antibodies of IgM isotype are the main immunoglobulins involved in the onset of acute AMR 18, though IgG, IgA, and IgE antibodies are present19. Anti-pig aortic endothelial cell IgM antibodies are more efficient in activating the classical pathway of complement than anti-pig IgG natural antibodies 20,21. Within the pool of IgG xenoreactive antibodies, IgG1 and IgG2 subclasses are most abundant. While IgG1 and IgG3 can activate the classical pathway of complement, lgG2 can only activate the alternative complement pathway 22,23. However, in comparison to IgM, a much higher concentration of IgG antibodies is needed to achieve complement activation.

Xenoreactive natural antibodies can be directed to Gal (anti-Gal) or to nonGal (anti-nonGal) antigens. Anti-Gal antibodies account for approximately 1% of circulating immunoglobulins 24-28. Deletion of expression of Gal in the organ-source pig largely prevents hyperacute rejection 29,30, but, in the absence of effective immunosuppressive therapy, does not prevent acute AMR 31,32.

A more recent evolutionary loss of cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), an enzyme involved in sialic acid synthesis, led to the unique absence of the glycolyl form of neuraminic acid (Neu5Gc) in humans (Tab. I). All other mammals (except New World NHPs) express both the acetyl form of neuraminic acid (Neu5Ac) and the glycolyl form (Neu5Gc) at various ratios in their glycoproteins and glycolipids. In some humans, exposure to Neu5Gc expressed in food (especially milk and red meat) can induce production of anti-Neu5Gc IgG and IgM antibodies 33-36. Antibodies to Neu5Gc appear to play a greater role in the Chinese than in Western populations 37. Because all Old World NHPs express Neu5Gc, the pig-to-Old World NHP model is not suitable for investigating the effect of anti-Neu5Gc antibodies on pig xenografts 12,13,38-41.

With regard to the third known pig glycan xenoantigen, Sda (Tab. I), although commonly expressed on human gastrointestinal epithelial cells and some other tissues and on human red blood cells (RBCs), most humans produce low levels of cold-reactive anti-Sda IgM, making Sda a polyagglutinable RBC antigen. While the Sda blood group does not carry a significant transfusion risk, Sda expression on pig vascular endothelial cells may induce an antibody response in a primate host 42-44. Antibodies to Sda appear to play a greater role in the pig-to-NHP model than they will in clinical xenotransplantation 45.

The genes for the three key enzymes responsible for the production of xenoglycans in the pig have successfully been knocked out (Tab. I), producing TKO pigs. While many humans exhibit no or minimal antibody binding to cells from TKO pigs (Fig. 1) 46, complement activation and coagulation pathway dysregulation may still be observed, in part associated with molecular incompatibilities between the species 47,48. This particularly pertains to the inefficient binding of human complement and coagulation pathway proteins to pig complement-regulatory and thromboregulatory molecules, respectively.

Although clearly beneficial when a pig organ is to be transplanted into a human recipient, there are problems with TKO pig organ transplantation in Old World NHPs (Fig. 2). As all Old World NHPs express the Neu5Gc antigen, these species do not develop anti-Neu5Gc antibodies (Tab. I). Estrada et al 39 reported that, when the Neu5Gc antigen is deleted in pigs, it appears to expose another xenoantigen against which Old World NHPs, but not humans, have natural antibodies. IgM and IgG binding are higher to TKO pig cells than to GTKO cells, and serum cytotoxicity is greater than to pig cells in which Neu5Gc remains expressed (Fig. 3).

However, there are other possible contributing factors, e.g., relating to complement, that may be playing a role in the high level of NHP serum cytotoxicity to TKO pig cells (Fig. 4) 12,49. The observation that approximately half of the baboon sera tested demonstrate a high level of cytotoxicity to double-knockout pig cells (i.e., those that do not express Gal or Sda but continue to express Neu5Gc) (Fig. 3) suggests that other factors (than absence of Neu5Gc expression) are involved. Although hyperacute rejection is rare, TKO pig-to-NHP organ transplantation still results in a relatively high incidence of early graft failure from AMR (Fig. 5) 38,50, although therapy with an anti-CD154 agent appears to overcome this barrier in some cases (see below) 51.

Because all Old World NHPs have cytotoxic antibodies to TKO pig cells, the pig-to-NHP model is no longer representative of clinical pig organ xenotransplantation and has led some to advocate for the initiation of limited exploratory clinical trials 13,52.

Complement activation

The complement system is a collection of circulating and cell membrane proteins that play important roles in host defense against non-self antigens, including microbes and, unfortunately, organ grafts 17 (Fig. 6). It can be activated by these non-self antigens in the absence of antibody, as part of the innate immune response (alternative and lectin pathways) or when antibodies attach to non-self antigens (classical pathway). The complement system is important in the development of ischemia-reperfusion injury and delayed graft function, as well as in both acute and chronic AMR.

This role of complement in the humoral immune response illustrates the fundamental tenet of the two-signal hypothesis, namely that in addition to recognition of the antigen, the innate immune response to these antigens provides additional signals that are necessary for lymphocyte activation. Complement proteins bound to antigen-antibody complexes are recognized by follicular dendritic cells in germinal centers, allowing the antigen to be displayed for further B cell activation and selection of high-affinity B cells. This process is an example of an innate immune response to a non-self antigen (complement activation) enhancing an adaptive immune response to the same antigen (B cell activation and antibody production).

The binding of human serum antibodies to the xenoantigens expressed on wild-type (i.e., genetically-unmodified) pig organs results in almost immediate activation of the complement cascade, and the graft is destroyed (hyperacute rejection) 53,54. This very rarely occurs after the transplantation of TKO pig organs, but it can occur if preservation of the graft has been inadequate (Cooper DKC et al., unpublished observations) (as ischemia can result in activation of the vascular endothelial cells). Furthermore, steps have been taken to protect the pig organ from the deleterious effects of complement activation, either by drug therapy, e.g., a C1-esterase inhibitor 55 or a C5 inhibitor, or by modifying the donor pig to express one or more human complement-regulatory proteins (e.g., CD46, CD55, CD59). Pig complement-regulatory proteins expressed on pig vascular endothelial cells are effective at protecting pig cells from the effects of pig complement, but are not successful in protecting against human complement-mediated injury16,56-58.

White’s group and others demonstrated significantly prolonged survival of pig kidneys and hearts in NHPs treated with cyclosporine-based immunosuppressive therapy when human CD55 (decay-accelerating factor) was expressed on the pig vascular endothelial cells 59. The combination of GTKO and one or more human complement-regulatory proteins further prolongs graft survival 60,61.

The innate cellular response

In addition to complement activation, xenoreactive natural antibodies can lyse target cells by complement-independent pathways (i.e., by antibody-dependent cellular cytotoxicity [ADCC]). The Fab portion of xenoantibodies can bind to donor endothelial cells and the Fc receptors of innate immune cells. This triggers innate immune responses that lead to endothelial cell lysis, cytokine release, and amplification of the T cell response. Innate immune cells, e.g., macrophages, monocytes, and natural killer (NK) cells, also play significant roles 62, though these are less well-defined. The innate immune response can be inhibited by certain genetic modifications in the donor pig, e.g., expression of human CD47 and/or HLA molecules (HLA-E and/or G) that suppress macrophage and NK cell activation, respectively 63-67.

CD47’s most critical function is as a marker of self-recognition. The binding of CD47 to its ligand, signaling regulatory protein (SIRP)-, inhibits macrophage function and prevents phagocytosis of cells and platelets 66,68-70. CD47/SIRP-incompatibility, as in xenotransplantation, may also induce innate immune cell activation 71. To overcome this incompatibility, human CD47 has been transgenically expressed in the organ-source pig 72.

Coagulation dysfunction

Several early research groups provided evidence to indicate that there were several incompatibilities between the coagulation systems of pigs and primates 47,73. This was first clearly demonstrated in the pig-to-NHP model by Ierino et al. 74 and Kozlowski et al. 75. As a result of the accumulation of platelets and fibrin in the pig graft, a thrombotic microangiopathy developed, impairing the function of the graft, and leading to fatal consumptive coagulopathy 76,77.

Steps were taken to express at least one human coagulation-regulatory protein, e.g., thrombomodulin (TBM), endothelial protein C receptor [EPCR], tissue factor pathway inhibitor (TFPI), and/or CD39, on the pig vascular endothelial cells. Survival of pig kidneys and hearts transplanted into NHPs was extended to months rather than weeks 78.

Systemic inflammation

The importance of the inflammatory response to both allografts and xenografts is becoming ever more widely recognized (Fig. 7) 79. Factors that must be considered include the presence of inflammation in the recipient at the time of the transplant, e.g., associated with pre-existing comorbidities and/or chronic dialysis, and in the donor, e.g., as a result of brain death or cardiac arrest. Further inflammatory events may result from the surgical procedures. Inflammation activates recipient immune cells, e.g., neutrophils, monocytes, macrophages (which in turn produce more proinflammatory cytokines), and inflammation-mediated donor endothelial cells.

Despite the above gene-edits, evidence accumulated that suggested that, after pig organ transplantation in NHPs, systemic inflammation developed before coagulation dysfunction was obvious 80. The insertion of a human ‘apoptotic or ‘anti-inflammatory’ gene, e.g., hemeoxygenase-1 or A20, proved beneficial 72.

However, systemic anti-inflammatory drug prophylaxis in the form of an anti-interleukin-6 (IL-6) receptor mAb may also be advantageous. The anti-IL-6 receptor-blocking mAbs that have been tested, e.g., tocilizumab, block the binding of IL-6 to baboon tissue receptors, but not to pig tissues, and so their beneficial effect on the transplanted pig organ is questionable, and may even be detrimental 81. Similarly, agents that bind to soluble IL-6, e.g., siltuximab, were also found to bind only to baboon IL-6 but not to pig IL-6 81. However, the overall effect of IL-6 blockade is generally believed to be beneficial 78.

HISTOPATHOLOGICAL FEATURES OF ACUTE AMR

Immune injury after organ xenotransplantation results in an activated endothelium which leads to apoptosis and necrosis of individual endothelial cells 82. Acute AMR is characterized by endothelial injury, typically in the form of microvascular inflammation, thromboses, endothelialitis, and/or transmural vasculitis, often associated with evidence of antibody and/or complement deposition (Fig. 8). Glomerular and peritubular capillary mononuclear cells are typically present and characterize the microvascular inflammation. Subendothelial cellular infiltration or endothelial loss or detachment of the larger arteries may also be seen.

In heart xenografts, the pathologic features include interstitial edema, hemorrhage, thromboses, and myocyte necrosis 83,84, initially observed as venous thromboses, associated with capillary endothelial activation and congestion, which later can be seen as regions of interstitial hemorrhage. Myocyte coagulative necrosis can be present at a later phase 85.

THE ADAPTIVE (T AND B CELL) IMMUNE RESPONSE

When hyperacute rejection was prevented by judicious gene-editing, attention turned to the suppression of the adaptive immune response, particularly to the suppression of the T cell response. T cell-dependent elicited antibody production may be playing a major role in the development of AMR, e.g., after primate exposure to swine leukocyte antigens (SLA), which are immunogenic across species 86-89. T cell activation leads to the destruction of the graft, either by the T cells themselves or by stimulation of B cells, resulting in AMR. To overcome this barrier, immunosuppressive therapy is administered (as in allotransplantation).

Gollackner demonstrated that elicited antibodies induce endothelial cell activation and tissue factor expression far more strongly than natural antibodies and without the need for complement activation 90. Inadequate immunosuppressive therapy resulted in early AMR even when GTKO pig organs were transplanted 32. Although prevention of the initial T cell response would seem to be essential, once AMR has developed the depletion of existing T cells, e.g., by ATG, would seem unlikely to reverse the process.

In 2000, Buhler and his colleagues demonstrated that conventional (cyclosporine-based) immunosuppressive therapy did not prevent rejection to pig xenoantigens from occurring (Fig. 9) 91,92. However, this could be prevented (or at least delayed) by administration of an anti-CD154mAb to the NHP recipient. Since then, blockade of the CD40/CD154 costimulation pathway has formed the basis of all successful immunosuppressive regimens in xenotransplantation until the present day 91,93,94.

The anti-CD154mAbs available in 2000 were soon found to be thrombogenic 95-97, resulting in their withdrawal for several years until the recent introduction of Fc-modified anti-CD154 agents that are not thrombogenic. During the interim, anti-CD40mAbs, which are not thrombogenic, were administered and resulted in greatly prolonged survival of heterotopically-placed hearts (in the abdomen) in the pig-to-baboon model 98,99. (A humanized version of this agent formed the basis of the regimen used in the clinical pig heart transplant carried out at the University of Maryland at Baltimore in 2022 100.) There is increasing evidence, however, that in xenotransplantation anti-CD154 agents are preferable to anti-CD40 agents 101-103.

Although blockade of the CD40/CD154 costimulation pathway was successful, blockade of the B7/CD28 pathway, e.g., by CTLA4-Ig, was less so. Nevertheless, genetic engineering enabled CTLA4-Ig to be produced by the organ-source pig 104. The production of CTLA4-Ig was so extensive that the pigs became immunosuppressed, resulting in a high incidence of infectious complications, limiting survival. The approach of expressing an immunosuppressive agent only in the specific cells of interest (e.g., pancreatic islets) has been further explored 105 and has potential for the future.

Costimulation blockade is currently combined with a conventional agent, e.g., rapamycin or mycophenolate mofetil (MMF) 106,107 (Tab. II).

Based on (i) the innovative biotechnology for pig gene modification aimed at reducing the effect of the primate immune response to the xenograft, and (ii) the administration of novel immunosuppressive agents that block the CD40/CD154 costimulation pathway, significant progress has been made in pig-to-NHP organ xenotransplantation models 51,78,108-110. These advances have led to prolonged survival of pig kidney grafts in NHPs, and today survival is being recorded in months or years.

Potential remaining immunological challenge: control of indirect T cell recognition

In allotransplantation, the production of donor-specific HLA antibodies (DSAs), resulting from interaction between antigen-presenting cells (APCs, including B cells) and T cells through the indirect recognition pathway, hinders long-term graft survival. DSA production depends not only on the amino acid differences in the B cell epitopes recognized by the B cell receptors, but also on the type and number of T cell epitopes recognized by the T cell receptors, i.e., the donor protein (mismatched HLA)-derived unique core peptides presented by recipient HLA class II 111.

In xenotransplantation, the number of epitopes is presumably much higher. Antibody production requires strong T cell help because more peptides can be presented on xenografts than on allografts 112. Many studies have been conducted on B cell epitopes, and Gal, Neu5Gc, and Sda have attracted attention as natural anti-pig antibodies. T cell epitopes have been studied with a focus on direct recognition pathways, e.g., by the mixed lymphocyte reaction (MLR, i.e., reactions between donor antigen-presenting cells with recipient T cells).

In contrast, assays for the indirect pathway (reactions between donor-derived peptides presented by recipient APCs to recipient T cells) in xenotransplantation have not yet been fully developed and therefore have not been adequately studied 113. Naïve NHPs and humans have usually not been exposed to pig antigens, i.e., they are not sensitized. Therefore, their immune response to pig antigens might not be detected by an indirect MLR. In vivo studies, e.g., organ transplantation, will be necessary to assess the T cell response through the indirect pathway by indirect MLR 113.

Theoretically, complete blockade of the CD40/CD154 pathway should control T cell help and suppress the immune response to the donor, but it would also suppress the immune response to infection and vaccines, and so intensive immunosuppressive therapy may not be desirable.

CROSS-REACTIVITY OF ANTIBODIES BETWEEN HLA AND SLA

Although many patients with antibodies to HLA do not appear to be at any increased risk of rejection of a pig organ graft 14,44,46, cross-reactivity between anti-HLA antibodies and swine leukocyte antigens (SLA) may occur, although the incidence is low (< 5%) 14. If patients with anti-HLA antibodies that do not cross-react with SLA are identified by in vitro assays 114, then these patients should be acceptable for the initial clinical trials. For the future, methods are being developed to delete or replace specific SLA against which there might be cross-reactivity 87,115.

Of importance, if a patient receives a pig organ that is rejected with the development of elicited anti-pig antibodies, e.g., against SLA. the current (limited) evidence is that this will not preclude subsequent successful allotransplantation 14. In clinical trials, therefore, pig organ grafts could act as bridges to allotransplantation.

FUTURE DEVELOPMENTS

Organs, tissues, and cells from gene-edited pigs have great clinical therapeutic potential. Further gene-editing to protect the organ from the human adaptive immune response may include deletion of expression of SLA class 1 116, or downregulation of SLA class II 117, or expression of PD-L1 118,119. This will hopefully enable exogenous immunosuppressive therapy to be significantly reduced or, indeed, ultimately unnecessary.

However, in relation to the adaptive immune response, several questions need investigation. For example, (i) are more peptides presented when transplantation is between different species than within the same species? Whereas in allotransplantation, donor-derived peptides are limited to mismatched HLA, in xenotransplantation any pig protein (not just SLA) that differs in amino acids from human amino acids could be a target. (ii) Do these amino acids activate follicular helper T cells (effector memory T cells)? If the original pig proteins from which the peptides are derived can be identified, gene-editing may allow the pig proteins to be converted to human proteins, thus reducing the strength of the immune response.

The ultimate goal of both allotransplantation and xenotransplantation is the induction of immunologic tolerance, in which the recipient no longer attempts to reject the graft. Although efforts in this respect in xenotransplantation have to date been unsuccessful, in view of the potential offered by genetic engineering of the pig, it would seem it is more likely to be achieved in xenotransplantation than in allotransplantation.

Acknowledgements

Work on xenotransplantation in DKCC’s laboratory is supported in part by NIH NIAID U19 grant AI090959 and in part by a Kidney X Prize from the US DHHS and the American Society of Nephrology.

Conflict of interest statement

DKCC is a consultant to eGenesis Bio, Cambridge, MA, USA, but the opinions expressed in this article are those of the authors and do not necessarily represent the views of eGenesis Bio. No other author reports a conflict of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author contributions

Data were collected by LW, DKCC, KK, ZH, IR, TK, and HH; the article was prepared by DKCC, LW, KK, ZH, IR, TK, and HH, and revised and approved by all authors.

Ethical consideration

Not applicable.

Figures and tables

Figure 1. Human serum antibody binding to pig red blood cells by age. (Top): geometric mean (GM) binding and age correlation of human serum IgM (A) and IgG (B) antibodies to wild-type (WT) pig red blood cells (RBCs). There is a steady increase in IgM and IgG binding during the first year of life. (Bottom): geometric mean (GM) binding and age correlation of human serum IgM (A) and IgG (B) antibodies to TKO pig RBCs. There is virtually no increase in IgM or IgG antibodies during the first year of life, and a very low level of antibodies in adults. The dotted lines indicate no IgM or IgG binding. (Note the great difference in the scale on the Y axis between Top vs Bottom (reproduced with permission from Li Q et al. Ann Thorac Surg 2020;109:1268-1273).

Figure 2. A) Human (top) and baboon (bottom) serum antibody binding to red blood cells (RBCs) from various pigs. Human serum antibody binding to pRBCs was measured by flow cytometry using the relative geometric mean (rGM), which was calculated by dividing the GM value for each sample by the negative control. Negative controls were obtained by incubating the cells with secondary anti-human antibodies only (with no serum). (Top) Human serum (n = 14) IgM (left) and IgG (right) antibody binding to wild-type (WT), GTKO, double-knockout (i.e., deletion of expression of Gal and Sda), and triple-knockout (TKO, i.e., with additional deletion of expression of Neu5Gc) pRBCs. Human IgM and IgG binding to GTKO/4GalKO/CMAHKO (TKO) pig RBCs was almost at the level of binding to human RBCs, and there was no detectable IgM or IgG binding to TKO RBCs. Binding to TKO pig RBCs was not significantly different from human IgM and IgG binding to human RBCs of blood type O. (*p < 0.05; **p < 0.01; ns = not significant); B) Baboon (an Old World NHP, n = 14) IgM and IgG antibody binding to WT, GTKO, DKO, and TKO pig RBCs. (Note that deletion of Neu5Gc [CMAH-KO] in pig cells appears to expose a fourth xenoantigen against which baboons have natural IgM antibodies. Note also that the data support the observation that the deletion of expression of Gal has more effect in reducing the antigenicity of baboon serum (90% reduction) (A), when compared with human serum (70% reduction) (*p < 0.05, **p < 0.01; ns = not significant) (reproduced in part with permission from Cooper DKC et al. Xenotransplantation 2019;26:E12516. https://doi.org/10.1111/xen.12516).

Figure 3. Comparison of mean serum IgM (left)/IgG (middle) binding and serum complement-mediated cytotoxicity (CDC, right) of baboons (n = 72) to GTKO, GTKO/4GalNT2KO, and TKO pig peripheral blood mononuclear cells (PBMCs). On the y axis, the dotted line represents the cut-off values (IgM [rGM] 1.2, IgG [rGM] 1.1, CDC 6.4%) below which there is no binding or cytotoxicity. The red lines indicate the mean values. (*p < 0.05; **p < 0.01; ns = not significant). IgM and IgG binding and serum cytotoxicity to TKO cells were higher or comparable to binding to GTKO cells. Although mean IgM and IgG binding and mean serum cytotoxicity to DKO cells were less than to TKO cells, many baboons had a high level of cytotoxicity to DKO cells (**p < 0.01) (reproduced with permission from Yamamoto T et al. Sci Rep 2020;10:9771. https://doi.org/10.1038/s41598-020-66311-3).

Figure 4. Correlation of human (n = 9) and baboon (n = 72) serum IgM (left) and IgG (right) antibody binding with serum complement-dependent cytotoxicity (CDC, at 50% serum concentration) to TKO pPBMCs. In both humans and baboons, there was a significant increase in cytotoxicity as IgM and IgG antibody binding to TKO pPBMCs increased. In baboons, however, cytotoxicity was high whether IgM binding was high (e.g., 80% cytotoxicity at a rGM of 8), or relatively lower (e.g., 75% at a rGM of 2) (**p < 0.01) (reprinted with permission from Yamamoto T et al. Xenotransplantation 2020;27:E12596. https://doi.org/10.1111/xen.12596).

Figure 5. Rejection-free survival of GTKO pig kidneys in baboons (Group 1, in black) was significantly longer than that of TKO pig kidneys (Group 2, in red) (reproduced with permission from Iwase H, et al. Xenotransplantation 2021;28:E1700. https://doi.org/10.1111/xen.12700).

Figure 6. Schema of complement system. Classical pathway (left): activated by binding of antibodies to antigens, which triggers C1q, activates C1r, C1s, then cleaves C4 and C2 to form C4b2a (C3 convertase); Lectin pathway (middle): one of MBL, ficolin -1, -2 and -3, and collectin 10/11 and collectin-P, recognizes lipopolysaccharides, etc., and binds to one of the MASP-1, MASP-2, and MASP-3, forming C3 convertase (C4b2a) (middle). C4b2a from the classical or lectin pathway cleaves C3 into C3a and C3b. C3b binds to C4b2a to form one of the C5 convertases (C4b2a3b); Alternative pathway (right): C3 undergoes spontaneous hydrolysis to form C3(H2O), which binds to factor B, forming an unstable C3 convertase C3(H2O)Bb, generating more C3b. Activation of C3 in the presence of factor B and factor D results in the formation of C3bBb (C3 convertase) (right). Properdin stabilizes C3 and C5 convertase, and enhances the amplification loop of C3 activation, then generating C5 convertases (C3bBb3b); Activation of MAC (bottom): the C5 convertase cleaves C5 into C5a and C5b, the latter interacting with C6–C9 to form the MAC (C5b-9), which in turn results in lysis, damage, or activation of target cells (lower part). The complement system is tightly regulated by soluble inhibitors (yellow), including C1-INH, factor H (FH), factor I (FI), C4BP, anaphylatoxin inhibitor (AI) inactivating the anaphylatoxins (e.g., C5a to C5adesArg), vitronectin (VN, S-protein, Vn, and Clusterin (CL, apolipoprotein J, SP-40) maintaining continuous low-grade activation in the fluid phase in check. Host cell membranes are equipped with a number of inhibitors to protect them against attack by complement (right), including CD46, CR1, CD55, thus controlling C4 and C3 activation. CD59 protects against final assembly of the C5b-9 complex.

Figure 7. Serum C-reactive protein (C-RP) responses to gene-edited pig kidney or artery patch transplants in immunosuppressed baboons being treated with or without tocilizumab (anti-IL-6RmAb) (reproduced with permission from Li T et al. Transplantation 2017;101:2330-2339).

Figure 8. Histopathology of AMR in pig kidney and heart grafts transplanted into immunosuppressed NHPs. A) AMR in a pig kidney xenograft showing glomerular intracapillary thrombi (black arrows). Other capillaries of the glomerulus show congestion, fibrin and segmental endothelial swelling and cell loss (H&E, 400x); B) C4d deposition is present along peritubular and glomerular capillaries (C4d immunoperoxidase, 200x); C). AMR in a pig heart xenograft showing extensive interstitial edema, intracapillary mononuclear cells (arrows) and capillary thrombi (arrowheads). Interstitial hemorrhage is also evident (H&E, 400x).

Figure 9. GTKO pig kidney graft survival in baboons receiving conventional (tacrolimus-based) US FDA-approved immunosuppressive agents (Group A, in red) was much shorter than in those receiving an anti-CD40mAb-based regimen (Group B, in black) (reproduced with permission from Yamamoto T et al. Transplantation 2019;103:2090-2104).

Carbohydrate (Abbreviation) Responsible enzyme Gene-knockout pig
1.Galactose-α1,3-galactose (Gal). α1,3-galactosyltransferase GTKO
2.N-glycolylneuraminic acid (Neu5Gc). CMAH CMAH-KO
3.Sda β-1,4N-acetylgalactosaminyltransferase. β4GalNT2-KO
CMAH = Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH).
Table I. Known carbohydrate xenoantigens expressed on pig cells.
Agent Dose (duration)
Induction
Thymoglobulin (ATG) 5 mg/kg i.v. (day -3) (to reduce the CD3+T cell count to <500/mm3)
Anti-CD20mAb (rituximab) 10 mg/kg i.v. (day -2)
C1-esterase inhibitor 17.5 U/kg i.v. (days 0, 2)
Maintenance
Anti-CD154 monoclonal antibody (mAb) Dose dependent on the agent used (days 0, 2, 7, 10, 14, and weekly)
Rapamycin mg/kg i.m. daily (target trough 6-10 ng/ml), beginning on day -7.
Methylprednisolone 10 mg/kg/d on day 0, tapering to 0.25 mg/kg/d by day 7.
Anti-inflammatory
Tocilizumab 8 mg/kg i.m. on days 0, 7, 14, and then monthly
Adjunctive
Aspirin 40 mg p.o. (alternate days), from day 4.
Table II. Representative immunosuppressive regimen.

References

  1. Tsuji K, Kitamura S, Wada J. Potential strategies for kidney regeneration with stem cells: an overview. Front Cell Dev Biol 2022;10:892356. https://doi.org/10.3389/fcell.2022.892356
  2. Mou L, Chen F, Dai Y, et al. Potential alternative approaches to xenotransplantation. Int J Surg 2015;23:322-326. https://doi.org/10.1016/j.ijsu.2015.06.085
  3. Cooper DKC, Gollackner B, Sachs DH. Will the pig solve the transplantation backlog? Annu Rev Med 2002;53:133-147. https://doi.org/10.1146/annurev.med.53.082901.103900
  4. Cooper DKC, Ekser B, Ramsoondar J, et al. The role of genetically engineered pigs in xenotransplantation research. J Pathol 2016;238:288-299. https://doi.org/10.1002/path.4635
  5. Eyestone W, Adams K, Ball S, et al. Gene-edited pigs for xenotransplantation. In: Cooper DKC, Byrne G, eds. Clinical xenotransplantation: pathways and progress in the transplantation of organs and tissues between species. New York, NY: Springer International Publishing 2020, pp. 121-140.
  6. Ran FA, Hsu PD, Wright J, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013;8:2281-2308. https://doi.org/10.1038/nprot.2013.143
  7. Yue Y, Xu W, Kan Y, et al. Extensive germline genome engineering in pigs. Nat Biomed Eng 2021;5:134-143. https://doi.org/10.1038/s41551-020-00613-9
  8. Damian RT. Molecular mimicry: antigen sharing by parasite and host and its consequences. The American Naturalist 1964;98:129-149. https://doi.org/10.1086/282313
  9. Galili U, Mandrell RE, Hamadeh RM, et al. Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun 1988;56:1730-1737. https://doi.org/10.1128/iai.56.7.1730-1737.1988
  10. Li Q, Hara H, Banks CA, et al. Anti-pig antibody in infants: can a genetically engineered pig heart bridge to allotransplantation? Ann Thorac Surg 2020;109:1268-1273. https://doi.org/10.1016/j.athoracsur.2019.08.061
  11. Estrada JL, Martens G, Li P, et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/4GalNT2 genes. Xenotransplantation 2015;22:194-202. https://doi.org/10.1111/xen.12161
  12. Yamamoto T, Hara H, Iwase H, et al. The final obstacle to successful pre-clinical xenotransplantation? Xenotransplantation 2020;27:E12596. https://doi.org/10.1111/xen.12596
  13. Yamamoto T, Iwase H, Patel D, et al. Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout). Sci Rep 2020;10:9771. https://doi.org/10.1038/s41598-020-66311-3
  14. Cooper DKC, Habibabady Z, Kinoshita K, et al. The respective relevance of sensitization to alloantigens and xenoantigens in pig organ xenotransplantation. Hum Immunol 2023;84:18-26. https://doi.org/10.1016/j.humimm.2022.06.003
  15. Hara H, Yamamoto T, Wei HJ, et al. What have we learned from in vitro studies about pig-to-primate organ transplantation? Transplantation 2022;Dec 20. https://doi.org/10.1097/TP.0000000000004458 [Epub Ahead of Print]
  16. Cozzi E, White DJ. The generation of transgenic pigs as potential organ donors for humans. Nat Med 1995;1:964-966. https://doi.org/10.1038/nm0995-964
  17. Zhou H, Hara H, Cooper DKC. The complex functioning of the complement system in xenotransplantation. Xenotransplantation 2019;26:E12517. https://doi.org/10.1111/xen.12517
  18. Koren E, Neethling FA, Richards S, et al. Binding and specificity of major immunoglobulin classes of preformed human anti-pig heart antibodies. Transplant International 1993;6:351-353. https://doi.org/10.1007/BF00335975
  19. Li Q, Iwase H, Yamamoto T, et al. Anti-pig IgE and IgA antibodies in naive primates and nonhuman primates with pig xenografts. Transplantation 2021;105:318-327. https://doi.org/10.1097/TP.0000000000003408
  20. Vanhove B, Bach FH. Human xenoreactive natural antibodies – avidity and targets on porcine endothelial cells. Transplantation 1993;56:1251-1253.
  21. Bracy JL, Cretin N, Cooper DK, et al. Xenoreactive natural antibodies. Cell Mol Life Sci 1999;56:1001-1007. https://doi.org/10.1007/s000180050489
  22. Rieben R, Seebach JD. Xenograft rejection: IgG1, complement and NK cells team up to activate and destroy the endothelium. Trends Immunol 2005;26:2-5. https://doi.org/10.1016/j.it.2004.11.011
  23. Ding JW, Zhou T, Zeng H, et al. Hyperacute rejection by anti-Gal IgG1, IgG2a, and IgG2b is dependent on complement and Fc-gamma receptors. J Immunol 2008;180:261-268. https://doi.org/10.4049/jimmunol.180.1.261
  24. Galili U, Rachmilewitz EA, Peleg A, et al. A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J Exp Med 1984;160:1519-1531. https://doi.org/10.1084/jem.160.5.1519
  25. Galili U, Shohet SB, Kobrin E, et al. Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells. J Biol Chem 1988;263:17755-17762.
  26. Good AH, Cooper DK, Malcolm AJ, et al. Identification of carbohydrate structures that bind human antiporcine antibodies: implications for discordant xenografting in humans. Transplant Proc 1992;24:559-562.
  27. Cooper DK, Good AH, Koren E, et al. Identification of alpha-galactosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies: relevance to discordant xenografting in man. Transpl Immunol 1993;1:198-205. https://doi.org/10.1016/0966-3274(93)90047-c
  28. Kobayashi T, Cooper DK. Anti-Gal, alpha-Gal epitopes, and xenotransplantation. Subcell Biochem 1999;32:229-257. https://doi.org/10.1007/978-1-4615-4771-6_10
  29. Kuwaki K, Tseng YL, Dor FJMF, et al. Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med 2005;11:29-31. https://doi.org/10.1038/nm1171
  30. Yamada K, Yazawa K, Shimizu A, et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med 2005;11:32-34. https://doi.org/10.1038/nm1172
  31. Cooper DKC. Introduction: the present status of xenotransplantation research. Methods Mol Biol 2020;2110:1-25. https://doi.org/10.1007/978-1-0716-0255-3_1
  32. Chen G, Qian H, Starzl T, et al. Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys. Nat Med 2005;11:1295-1298. https://doi.org/10.1038/nm1330
  33. Leviatan Ben-Arye S, Schneider C, Yu H, et al. Differential recognition of diet-derived Neu5Gc-neoantigens on glycan microarrays by carbohydrate-specific pooled human IgG and IgA antibodies. Bioconjug Chem 2019;30:1565-1574. https://doi.org/10.1021/acs.bioconjchem.9b00273
  34. Zhu A, Hurst R. Anti-N-glycolylneuraminic acid antibodies identified in healthy human serum. Xenotransplantation 2002;9:376-381. https://doi.org/10.1034/j.1399-3089.2002.02138.x
  35. Bouhours D, Pourcel C, Bouhours JE. Simultaneous expression by porcine aorta endothelial cells of glycosphingolipids bearing the major epitope for human xenoreactive antibodies (Gal alpha 1-3Gal), blood group H determinant and N-glycolylneuraminic acid. Glycoconj J 1996;13:947-953. https://doi.org/10.1007/BF01053190
  36. Taylor RE, Gregg CJ, Padler-Karavani V, et al. Novel mechanism for the generation of human xeno-autoantibodies against the nonhuman sialic acid N-glycolylneuraminic acid. J Exp Med 2010;207:1637-1646. https://doi.org/10.1084/jem.20100575
  37. Li T, Feng H, Du J, et al. Serum antibody binding and cytotoxicity to pig cells in Chinese subjects: relevance to clinical renal xenotransplantation. Front Immunol 2022;13:844632. https://doi.org/10.3389/fimmu.2022.844632
  38. Iwase H, Jagdale A, Yamamoto T, et al. Evidence suggesting that deletion of expression of N-glycolylneuraminic acid (Neu5Gc) in the organ-source pig is associated with increased antibody-mediated rejection of kidney transplants in baboons. Xenotransplantation 2021;28:E12700. https://doi.org/10.1111/xen.12700
  39. Estrada JL, Martens G, Li P, et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/4GalNT2 genes. Xenotransplantation 2015;22:194-202. https://doi.org/10.1111/xen.12161
  40. French BM, Sendil S, Pierson RN, et al. The role of sialic acids in the immune recognition of xenografts. Xenotransplantation 2017;24. https://doi.org/10.1111/xen.12345
  41. Tector AJ, Mosser M, Tector M, et al. The possible role of anti-neu5gc as an obstacle in xenotransplantation. Front Immunol 2020;11:622. https://doi.org/10.3389/fimmu.2020.00622
  42. Zhao C, Cooper DKC, Dai Y, et al. The Sda and Cad glycan antigens and their glycosyltransferase, 1,4GalNAcT-II, in xenotransplantation. Xenotransplantation 2018;25:E12386. https://doi.org/10.1111/xen.12386
  43. Byrne GW, Du Z, Stalboerger P, et al. Cloning and expression of porcine 1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen. Xenotransplantation 2014;21:543-554. https://doi.org/10.1111/xen.12124
  44. Byrne GW. Does human leukocyte antigens sensitization matter for xenotransplantation? Xenotransplantation 2018;25:E12411. https://doi.org/10.1111/xen.12411
  45. Feng H, Li T, Du J, et al. Both Natural and induced anti-sda antibodies play important roles in GTKO pig-to-rhesus monkey xenotransplantation. Front Immunol 2022;13:849711. https://doi.org/10.3389/fimmu.2022.849711
  46. Martens GR, Reyes LM, Li P, et al. Humoral reactivity of renal transplant-waitlisted patients to cells from GGTA1/CMAH/B4GalNT2, and SLA Class I knockout pigs. Transplantation 2017;101:E86-E92. https://doi.org/10.1097/TP.0000000000001646
  47. Robson SC, Cooper DK, d’Apice AJ. Disordered regulation of coagulation and platelet activation in xenotransplantation. Xenotransplantation 2000;7:166-176. https://doi.org/10.1034/j.1399-3089.2000.00067.x
  48. Cooper DKC, Ezzelarab MB, Hara H, et al. The pathobiology of pig-to-primate xenotransplantation: a historical review. Xenotransplantation 2016;23:83-105. https://doi.org/10.1111/xen.12219
  49. Cooper DKC, Hara H, Iwase H, et al. Pig kidney xenotransplantation: progress toward clinical trials. Clin Transplant 2021;35:E14139. https://doi.org/10.1111/ctr.14139
  50. Firl DJ, Markmann JF. Measuring success in pig to non-human-primate renal xenotransplantation: systematic review and comparative outcomes analysis of 1051 life-sustaining NHP renal allo- and xeno-transplants. Am J Transplant 2022;22:1527-1536. https://doi.org/10.1111/ajt.16994
  51. Ma D, Hirose T, Lassiter G, et al. Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques. Am J Transplant 2022;22:46-57. https://doi.org/10.1111/ajt.16780
  52. Cooper DKC. The 2021 IXA Keith Reemtsma lecture: moving xenotransplantation to the clinic. Xenotransplantation 2022;29:E12723. https://doi.org/10.1111/xen.12723
  53. Cooper DK, Human PA, Lexer G, et al. Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon. J Heart Transplant 1988;7:238-246.
  54. Lexer G, Cooper DK, Rose AG, et al. Hyperacute rejection in a discordant (pig to baboon) cardiac xenograft model. J Heart Transplant 1986;5:411-418.
  55. Längin M, Mayr T, Reichart B, et al. Consistent success in life-supporting porcine cardiac xenotransplantation. Nature 2018;564:430-433. https://doi.org/10.1038/s41586-018-0765-z
  56. Zhou CY, McInnes E, Copeman L, et al. Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor. Xenotransplantation 2005;12:142-148. https://doi.org/10.1111/j.1399-3089.2005.00209.x
  57. Atkinson JP, Oglesby TJ, White D, et al. Separation of self from non-self in the complement system: a role for membrane cofactor protein and decay accelerating factor. Clin Exp Immunol 1991;86(Suppl 1):27-30. https://doi.org/10.1111/j.1365-2249.1991.tb06203.x
  58. Dalmasso AP, Vercellotti GM, Platt JL, et al. Inhibition of complement-mediated endothelial cell cytotoxicity by decay-accelerating factor. Potential for prevention of xenograft hyperacute rejection. Transplantation 1991;52:530-533. https://doi.org/10.1097/00007890-199109000-00029
  59. Schuurman HJ, Pino-Chavez G, Phillips MJ, et al. Incidence of hyperacute rejection in pig-to-primate transplantation using organs from hDAF-transgenic donors. Transplantation 2002;73:1146-1151. https://doi.org/10.1097/00007890-200204150-00024
  60. McGregor CGA, Ricci D, Miyagi N, et al. Human CD55 expression blocks hyperacute rejection and restricts complement activation in Gal knockout cardiac xenografts. Transplantation 2012;93:686-692. https://doi.org/10.1097/TP.0b013e3182472850
  61. Azimzadeh AM, Kelishadi SS, Ezzelarab MB, et al. Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein. Xenotransplantation 2015;22:310-316. https://doi.org/10.1111/xen.12176
  62. Casós K, Sommaggio R, Pérez-Cruz M, et al. Cell-based assays for modeling xenogeneic immune responses. Methods Mol Biol 2020;2110:99-113. https://doi.org/10.1007/978-1-0716-0255-3_7
  63. Miura S, Habibabady ZA, Pollok F, et al. Effects of human TFPI and CD47 expression and selectin and integrin inhibition during GalTKO.hCD46 pig lung perfusion with human blood. Xenotransplantation 2022;29:E12725. https://doi.org/10.1111/xen.12725
  64. Artrip JH, Kwiatkowski P, Michler RE, et al. Target cell susceptibility to lysis by human natural killer cells is augmented by alpha(1,3)-galactosyltransferase and reduced by alpha(1, 2)-fucosyltransferase. J Biol Chem 1999;274:10717-10722. https://doi.org/10.1074/jbc.274.16.10717
  65. Dawson JR, Vidal AC, Malyguine AM. Natural killer cell-endothelial cell interactions in xenotransplantation. Immunol Res 2000;22:165-176. https://doi.org/10.1385/IR:22:2-3:165
  66. Ide K, Wang H, Tahara H, et al. Role for CD47-SIRPalpha signaling in xenograft rejection by macrophages. Proc Natl Acad Sci U S A 2007;104:5062-5066. https://doi.org/10.1073/pnas.0609661104
  67. Laird CT, Burdorf L, French BM, et al. Transgenic expression of human leukocyte antigen-E attenuates GalKO.hCD46 porcine lung xenograft injury. Xenotransplantation 2017;24. https://doi.org/10.1111/xen.12294 [Epub Ahead of Print]
  68. Oldenborg PA, Gresham HD, Lindberg FP. CD47-signal regulatory protein alpha (SIRPalpha) regulates Fcgamma and complement receptor-mediated phagocytosis. J Exp Med 2001;193:855-862. https://doi.org/10.1084/jem.193.7.855
  69. Matozaki T, Murata Y, Okazawa H, et al. Functions and molecular mechanisms of the CD47-SIRPalpha signalling pathway. Trends Cell Biol 2009;19:72-80. https://doi.org/10.1016/j.tcb.2008.12.001
  70. Tena AA, Sachs DH, Mallard C, et al. Prolonged survival of pig skin on baboons after administration of pig cells expressing human CD47. Transplantation 2017;101:316-321. https://doi.org/10.1097/TP.0000000000001267
  71. Navarro-Alvarez N, Yang YG. Lack of CD47 on donor hepatocytes promotes innate immune cell activation and graft loss: a potential barrier to hepatocyte xenotransplantation. Cell Transplant 2014;23:345-354. https://doi.org/10.3727/096368913X663604
  72. Cooper DKC, Hara H, Iwase H, et al. Justification of specific genetic modifications in pigs for clinical organ xenotransplantation. Xenotransplantation 2019;26:E12516. https://doi.org/10.1111/xen.12516
  73. Roussel JC, Moran CJ, Salvaris EJ, et al. Pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein C and TAFI. Am J Transplant 2008;8:1101-1112. https://doi.org/10.1111/j.1600-6143.2008.02210.x
  74. Ierino FL, Kozlowski T, Siegel JB, et al. Disseminated intravascular coagulation in association with the delayed rejection of pig-to-baboon renal xenografts. Transplantation. 1998;66:1439-1450. https://doi.org/10.1097/00007890-199812150-00006
  75. Kozlowski T, Shimizu A, Lambrigts D, et al. Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation 1999;67:18-30. https://doi.org/10.1097/00007890-199901150-00004
  76. D’Apice AJ, Cowan PJ. Profound coagulopathy associated with pig-to-primate xenotransplants: how many transgenes will be required to overcome this new barrier? Transplantation 2000;70:1273-1274. https://doi.org/10.1097/00007890-200011150-00003
  77. Bühler L, Basker M, Alwayn IP, et al. Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation 2000;70:1323-1331. https://doi.org/10.1097/00007890-200011150-00010
  78. Iwase H, Hara H, Ezzelarab M, et al. Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts. Xenotransplantation 2017;24. https://doi.org/10.1111/xen.12293 [Epub Ahead of Print]
  79. Ravindranath MH, El Hilali F, Filippone EJ. The impact of inflammation on the immune responses to transplantation: tolerance or rejection? Front Immunol 2021;12:667834. [Epub https://doi.org/10.3389/fimmu.2021.667834
  80. Li J, Hara H, Wang Y, et al. Evidence for the important role of inflammation in xenotransplantation. J Inflamm (Lond) 2019;16:10. https://doi.org/10.1186/s12950-019-0213-3
  81. Zhang G, Iwase H, Wang L, et al. Is interleukin-6 receptor blockade (tocilizumab) beneficial or detrimental to pig-to-baboon organ xenotransplantation? Am J Transplant 2020;20:999-1013. https://doi.org/10.1111/ajt.15712
  82. Shimizu A, Meehan SM, Kozlowski T, et al. Acute humoral xenograft rejection: destruction of the microvascular capillary endothelium in pig-to-nonhuman primate renal grafts. Lab Invest 2000;80:815-830. https://doi.org/10.1038/labinvest.3780086
  83. Litovsky SH, Foote JB, Jagdale A, et al. Cardiac and pulmonary histopathology in baboons following genetically-engineered pig orthotopic heart transplantation. Ann Transplant 2022;27:E935338. https://doi.org/10.12659/AOT.935338
  84. Cleveland DC, Jagdale A, Carlo WF, et al. The genetically engineered heart as a bridge to allotransplantation in infants just around the corner? Ann Thorac Surg 2022;114:536-544. https://doi.org/10.1016/j.athoracsur.2021.05.025
  85. Rose AG, Cooper DK. Venular thrombosis is the key event in the pathogenesis of antibody-mediated cardiac rejection. Xenotransplantation 2000;7:31-41. https://doi.org/10.1034/j.1399-3089.2000.00042.x
  86. Martens GR, Ladowski JM, Estrada J, et al. HLA Class I-sensitized renal transplant patients have antibody binding to SLA Class I epitopes. Transplantation 2019;103:1620-1629. https://doi.org/10.1097/TP.0000000000002739
  87. Ladowski JM, Reyes LM, Martens GR, et al. Swine Leukocyte antigen Class II is a xenoantigen. Transplantation 2018;102:249-254. https://doi.org/10.1097/TP.0000000000001924
  88. Yamada K, Sachs DH, DerSimonian H. Human anti-porcine xenogeneic T cell response. Evidence for allelic specificity of mixed leukocyte reaction and for both direct and indirect pathways of recognition. J Immunol 1995;155:5249-5256.
  89. Pierson RN. Primate T-cell responses to porcine antigens: implications for clinical xenotransplantation. Xenotransplantation 2006;13:14-18. https://doi.org/10.1111/j.1399-3089.2005.00268.x
  90. Gollackner B, Goh SK, Qawi I, et al. Acute vascular rejection of xenografts: roles of natural and elicited xenoreactive antibodies in activation of vascular endothelial cells and induction of procoagulant activity. Transplantation 2004;77:1735-1741. https://doi.org/10.1097/01.tp.0000131167.21930.b8
  91. Yamamoto T, Hara H, Foote J, et al. Life-supporting kidney xenotransplantation from genetically engineered pigs in baboons: a comparison of two immunosuppressive regimens. Transplantation 2019;103:2090-2104. https://doi.org/10.1097/TP.0000000000002796
  92. Bühler L, Awwad M, Basker M, et al. High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response. Transplantation 2000;69:2296-2304. https://doi.org/10.1097/00007890-200006150-00013
  93. Samy KP, Butler JR, Li P, et al. The role of costimulation blockade in solid organ and islet xenotransplantation. J Immunol Res 2017;2017:8415205. https://doi.org/10.1155/2017/8415205
  94. Ezzelarab MB, Ekser B, Isse K, et al. Increased soluble CD154 (CD40 ligand) levels in xenograft recipients correlate with the development of de novo anti-pig IgG antibodies. Transplantation 2014;97:502-508. https://doi.org/10.1097/TP.0000000000000042
  95. Kirk AD, Knechtle SJ, Sollinger H. Preliminary results of the use of humanized anti-CD154 in human renal allotransplantation. Am J Transplant 2001;1(Suppl 1):191.
  96. Knosalla C, Gollackner B, Cooper DK. Anti-CD154 monoclonal antibody and thromboembolism revisted. Transplantation 2002;74:416-417. https://doi.org/10.1097/00007890-200208150-00024
  97. Kawai T, Andrews D, Colvin RB, et al. Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand. Nat Med 2000;6:114. https://doi.org/10.1038/72162
  98. Mohiuddin MM, Singh AK, Corcoran PC, et al. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft. Nat Commun 2016;7:11138. https://doi.org/10.1038/ncomms11138
  99. Mohiuddin MM, Singh AK, Corcoran PC, et al. Role of anti-CD40 antibody-mediated costimulation blockade on non-Gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon model. Xenotransplantation 2014;21:35-45. https://doi.org/10.1111/xen.12066
  100. Griffith BP, Goerlich CE, Singh AK, et al. Genetically modified porcine-to-human cardiac xenotransplantation. N Engl J Med 2022;387:35-44. https://doi.org/10.1056/NEJMoa2201422
  101. Perrin S, Magill M. The inhibition of CD40/CD154 costimulatory signaling in the prevention of renal transplant rejection in nonhuman primates: a systematic review and meta analysis. Front Immunol 2022;13:861471. https://doi.org/10.3389/fimmu.2022.861471
  102. Shin JS, Kim JM, Kim JS, et al. Long-term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets. Am J Transplant 2015;15:2837-2850. https://doi.org/10.1111/ajt.13345
  103. Shin JS, Kim JM, Min BH, et al. Pre-clinical results in pig-to-non-human primate islet xenotransplantation using anti-CD40 antibody (2C10R4)-based immunosuppression. Xenotransplantation 2018;25. https://doi.org/10.1111/xen.12356 [Epub Ahead of Print]
  104. Phelps CJ, Ball SF, Vaught TD, et al. Production and characterization of transgenic pigs expressing porcine CTLA4-Ig. Xenotransplantation 2009;16:477-485. https://doi.org/10.1111/j.1399-3089.2009.00533.x
  105. Klymiuk N, van Buerck L, Bähr A, et al. Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice. Diabetes 2012;61:1527-1532. https://doi.org/10.2337/db11-1325
  106. Maenaka A, Kinoshita K, Hara H, et al. The case for the therapeutic use of mechanistic/mammalian target of rapamycin (mTOR) inhibitors in xenotransplantation. Xenotransplantation. Published online March 2023.
  107. Bikhet M, Iwase H, Yamamoto T, et al. What therapeutic regimen will be optimal for initial clinical trials of pig organ transplantation? Transplantation 2021;105:1143-1155. https://doi.org/10.1097/TP.0000000000003622
  108. Adams AB, Lovasik BP, Faber DA, et al. Anti-C5 Antibody tesidolumab reduces early antibody-mediated rejection and prolongs survival in renal xenotransplantation. Ann Surg 2021;274:473-480. https://doi.org/10.1097/SLA.0000000000004996
  109. Iwase H, Liu H, Wijkstrom M, et al. Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date. Xenotransplantation 2015;22:302-309. https://doi.org/10.1111/xen.12174
  110. Kim SC, Mathews DV, Breeden CP, et al. Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on CD4 T cell depletion. Am J Transplant 2019;19:2174-2185. https://doi.org/10.1111/ajt.15329
  111. Sakamoto S, Iwasaki K, Tomosugi T, et al. Analysis of T and B Cell epitopes to predict the risk of de novo Donor-Specific Antibody (DSA) production after kidney transplantation: a two-center retrospective cohort study. Front Immunol 2020;11:2000. https://doi.org/10.3389/fimmu.2020.02000
  112. Galili U. Immune response, accommodation, and tolerance to transplantation carbohydrate antigens. Transplantation 2004;78:1093-1098. https://doi.org/10.1097/01.tp.0000142673.32394.95
  113. Kenta I, Toshihide T, Takashi S, et al. Estimation of sensitization status in renal transplant recipients by assessing indirect pathway CD4+ T cell response to donor cell-pulsed dendritic cell. Transplantation 2023:E004491. https://doi.org/10.1097/TP.0000000000004491
  114. Lucander ACK, Nguyen H, Foote JB, et al. Immunological selection and monitoring of patients undergoing pig kidney transplantation. Xenotransplantation 2021;28:E12686. https://doi.org/10.1111/xen.12686
  115. Ladowski JM, Hara H, Cooper DKC. The role of SLAs in xenotransplantation. Transplantation 2021;105:300-307. https://doi.org/10.1097/TP.0000000000003303
  116. Reyes LM, Estrada JL, Wang ZY, et al. Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease. J Immunol 2014;193.:5751-5757. https://doi.org/10.4049/jimmunol.1402059
  117. Hara H, Witt W, Crossley T, et al. Human dominant-negative class II transactivator transgenic pigs - effect on the human anti-pig T-cell immune response and immune status. Immunology 2013;140:39-46. https://doi.org/10.1111/imm.12107
  118. Buermann A, Petkov S, Petersen B, et al. Pigs expressing the human inhibitory ligand PD-L1 (CD 274) provide a new source of xenogeneic cells and tissues with low immunogenic properties. Xenotransplantation 2018;25:E12387. https://doi.org/10.1111/xen.12387
  119. Plege A, Borns K, Beer L, et al. Downregulation of cytolytic activity of human effector cells by transgenic expression of human PD-ligand-1 on porcine target cells. Transpl Int 2010;23:1293-1300. https://doi.org/10.1111/j.1432-2277.2010.01130.xacid. J Exp Med 2010;207:1637-1646. https://doi.org/10.1084/jem.20100575

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Authors

David K.C. Cooper - Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA

Liaoran Wang - The Second Affiliated Hospital of Hainan Medical University, Haikou, Hainan, China

Kohei Kinoshita - Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA

Zahra Habibabady - Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA

Ivy Rosales - Department of Pathology, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA

Takaaki Kobayashi - Aichi Medical University, Nagakute, Japan

Hidetaka Hara - College of Veterinary Medicine, Yunnan Agricultural University, Kunming, Yunnan, China

How to Cite
[1]
Cooper, D.K., Wang, L., Kinoshita, K., Habibabady, Z., Rosales, I., Kobayashi, T. and Hara, H. 2023. Immunobiological barriers to pig organ xenotransplantation. European Journal of Transplantation. 1, 3 (Oct. 2023), 167–181. DOI:https://doi.org/10.57603/EJT-266.
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