REVIEW J Am Soc Nephrol 12: 182–193, 2001

Physiologic and Immunologic Hurdles to

BENJAMIN SAMSTEIN*†‡ and JEFFREY L. PLATT* Departments of *Surgery, †, and ‡Pediatrics, Mayo Clinic, Rochester, Minnesota.

Abstract. The major problem in the field of renal transplanta- the immune response of the recipient against the ; other tion is currently the shortage of available kidneys. However, hurdles include the physiologic limitations of the transplant, the use of animals as a source of kidneys, i.e., xenotransplan- infection, and ethical considerations. This review summarizes tation, is increasingly being viewed as a potential solution to what is currently known regarding the obstacles to xenotrans- this problem. One preeminent hurdle to xenotransplantation is plantation and some potential solutions to those problems.

Organ transplantation has been one of the preeminent suc- particularly human cadavers, as sources of transplants cesses in the field of medicine. An experimental procedure 30 might avert the transmission of infectious agents such as Ep- yr ago, organ allotransplantation is now the preferred treatment stein-Barr virus and cytomegalovirus, which are major causes for failure of the kidney, heart, , and lungs and, some of morbidity after allotransplantation. would advocate, the pancreas. The 1-yr survival rate for pa- Although the potential advantages of xenotransplantation tients receiving primary kidney transplants exceeds 95% (1), generate enthusiasm, these advantages must be weighed and this survival rate seems to be significantly better than the against what may seem to be daunting hurdles to the clinical survival rate for patients treated with chronic dialysis (2). application of xenotransplantation. These hurdles include the The success of is such that the major immune response of the recipient to the transplant, the physi- problem facing the field today is neither the technical hurdles ologic limitations of the transplant, infection, and ethical con- nor the medical complications but the shortage of available cerns. This review summarizes what is currently known re- organs. The number of patients on the waiting lists for trans- garding the hurdles to xenotransplantation and some potential plantation has increased steadily, whereas the rate of organ solutions to those hurdles. donation has remained constant (1). Because of this problem, the use of animals in lieu of human subjects as a source of Early Experiences with Xenotransplantation organs, i.e., xenotransplantation, is increasingly being viewed The technical ability to perform renal transplantation was as a potential approach to organ replacement (3). achieved in the early years of the 20th century, when experi- In addition to overcoming the aggregate shortage of organs mental surgeons such as , Charles Guthrie, Em- for transplantation, xenotransplantation offers several potential rich Ulmann, and Matheiu Joubolay developed the ability to advantages compared with allotransplantation. First, an animal connect the cut ends of blood vessels, i.e., vascular anastomo- organ transplant might be, in some cases, less susceptible to the sis (4). Although vascular anastomosis made transplantation recurrence of disease or the rapid destruction caused by pre- technically feasible, it was unclear how human organs could be sensitization. Second, elimination of the donor shortage would obtained for transplantation because there was no generally allow transplantation to be considered for patients who, be- accepted definition of brain death. Thus, the initial attempts to cause of age or underlying medical diseases, would not have perform transplantation in human subjects involved xenotrans- been offered organ transplantation based on current standards plants of the kidney (4). In 1906, Joubolay performed two for listing. Third, the use of an animal source of organs would kidney xenografts (using a pig and a goat as the sources of the provide the opportunity to plan the transplant procedure; such transplants), in which the organ was connected to the arm or planning would permit approaches to immune modulation and thigh of patients with chronic renal failure. These kidneys perhaps the establishment of tolerance by pretreatment of the functioned only briefly (4). The lack of success in these efforts recipient that would not be possible with human cadaveric and a few subsequent attempts at allotransplantation discour- sources. Fourth, the use of animals instead of human subjects, aged the application of transplantation for decades. Nearly 60 yr later, the development of finally allowed kidney allotransplantation to be performed Received July 14, 2000. Accepted September 5, 2000. effectively. Questions concerning the source of organs re- Correspondence to Dr. Jeffrey L. Platt, Mayo Clinic, Medical Sciences Build- mained, because there were no generally applied laws regard- ing, Room 2-66, Transplantation Biology, 200 1st Street S.W., Rochester, MN ing brain death that would allow the ready use of cadaveric 55905. Phone: 507-538-0313; Fax: 507-284-4957; E-mail: platt.jeffrey@ mayo.edu sources. Therefore, for six patients for whom no human kid- 1046-6673/1201-0182 neys were available, Reemtsma et al. (5) transplanted kidneys Journal of the American Society of Nephrology from chimpanzees. An immunosuppression regimen consisting Copyright © 2001 by the American Society of Nephrology of , actinomycin C, steroids, and graft irradiation J Am Soc Nephrol 12: 182–193, 2001 Hurdles to Xenotransplantation 183 was used. One recipient, a 23-yr-old female schoolteacher, was for allotransplants; however, the incidence and severity are maintained for 9 mo by the xenotransplant. She died as a result much greater for xenotransplants. of an illness of undetermined cause, and the xenograft ap- peared normal at the time of her death. Although this result might have been encouraging, other attempts at using nonhu- Hyperacute Rejection man primates were less successful (6,7). Porcine organs transplanted into immunocompetent pri- mates are subject to very rapid failure because of hyperacute Donor Sources rejection (8). Hyperacute rejection is thought to occur in nearly One central question is which species would be the most every organ transplanted between widely disparate species. suitable for use as a source of organs for xenotransplantation. Combinations of donor and recipient species in which hyper- Given the results achieved by Reemtsma et al. (5), particularly acute rejection regularly occurs are called “discordant,” and the survival and function of renal xenografts for periods of combinations of donor and recipient species in which hyper- months, and given the revolutionary advances in immunosup- acute rejection rarely occurs are called “concordant” (9). Hy- pression and antimicrobial therapy in the past 37 yr, it might be peracute rejection was originally described for human kidney intuitive that primates would be the best source of kidneys for allografts transplanted into recipients with circulating antibod- xenotransplantation. However, for several reasons, investiga- ies against the donor (10). In retrospect, hyperacute rejection tors now focus on other animal sources, particularly pigs. First, was probably first observed in the early years of the 20th pigs are sufficiently plentiful to provide any conceivable num- century when experimental kidney xenografts were attempted ber of organs needed, whereas primates are available in only between disparate species. small numbers and chimpanzees are an endangered species. The clinical features of hyperacute rejection are dramatic. Although some nonhuman primates, such as baboons, might be Evidence of hyperacute rejection is usually observed upon numerous enough to meet a significant fraction of the need, reperfusion of the graft (11). The graft rapidly develops a beefy these animals tend to be significantly smaller than human red or even blue appearance and swells. An early sign of subjects, which limits their usefulness for adults. Second, pri- hyperacute rejection is a dramatic decrease in blood flow to the mates may harbor infectious agents such as herpes virus B, transplanted organ. The kidney xenograft may emit a small which can be lethal for human subjects, whereas pigs can be volume of urine, but urine output invariably slows and then bred in such a way that severe pathogens are excluded. Third, ceases in conjunction with the changes in appearance. unlike nonhuman primates, pigs can be genetically manipu- The pathology for hyperacute rejection is characterized by lated to express extrinsic genes, which may address some of formation of platelet thrombi in small blood vessels (including the biologic hurdles to xenotransplantation. The sections that glomerular capillaries), interstitial hemorrhage, and severe in- follow focus on the hurdles to transplantation of porcine kid- jury to endothelial cells (Figure 2, A and B). The immunopa- neys into human recipients. thology of hyperacute rejection is characterized by deposits of Ig and complement of the recipient along endothelial surfaces Biologic Hurdles to Xenotransplantation of graft blood vessels. A kidney transplanted from one species, such as pigs, into a The first step in the pathogenesis of hyperacute rejection is distant species, such as human patients, is subject to various the binding of xenoreactive natural antibodies, so named be- types of rejection, as listed in Figure 1. The types of rejection cause they recognize the cells of foreign species and because observed for xenotransplants are the same as those observed they are present in the circulation without a known history of exposure to the corresponding antigen (12). The natural antibodies in human subjects that bind to pig cells are predominantly directed against galactose-␣-1,3-galactose (Gal␣1,3Gal), a saccharide ex- pressed on the cells of lower mammals but not on the cells of humans, apes, and Old World monkeys (13,14). These natural antibodies are not present at birth but develop soon thereafter and are present in all humans (15). The antibodies, which recognize Gal␣1,3Gal, represent 85 to 95% of xenoreactive natural antibod- ies (16). Elimination of anti-Gal␣1,3Gal antibodies can prevent hyperacute rejection (17). Figure 1. Biologic responses to renal xenotransplantation. A porcine Binding of xenoreactive natural antibodies activates the kidney transplanted into a human patient would be subject to one or complement system, although activation of complement might, more of the immunologic reactions shown. Transplantation into a in principle, also occur via the alternative complement path- nonmanipulated recipient may give rise to hyperacute rejection. If way; however, spontaneous activation of complement does not hyperacute rejection is avoided (as, for example, by complement inhibition), then the graft may undergo accommodation, a condition in seem to occur in porcine organs transplanted into baboons and which acute vascular rejection does not occur despite the presence of Old World monkeys (and presumably humans) (18). Activated anti-donor antibodies in the circulation of the recipient. If acute complement causes endothelial cells to retract, leading to ex- vascular rejection is avoided, then the graft may undergo cellular posure of the underlying matrix. This disruption of the endo- rejection or chronic rejection. thelial monolayer results in the loss of vascular integrity and 184 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 182–193, 2001

Figure 2. (A) Hyperacute rejection in pig-to-primate renal xenotransplantation. A porcine kidney was examined 30 min after transplantation into a baboon. The tissue section was stained with hematoxylin and eosin. (B) Staining for CD41 in a tissue section from a porcine kidney undergoing hyperacute rejection, revealing platelet aggregation in a glomerulus. (C) Acute vascular rejection of a porcine kidney transplanted into a baboon. The histologic examination revealed ischemic changes. The inset demonstrates vasculitis in the same biopsy. Magnification, ϫ400.

stimulates the adhesion and activation of platelets, leading to exandre et al. (8) depleted xenoreactive antibodies and thus the typical pathology observed in hyperacute rejection. prevented hyperacute rejection of porcine kidney xenografts by One reason why xenografts may be especially susceptible to performing plasmapheresis for baboons; however, the possi- hyperacute rejection is because complement regulatory pro- bility that other elements of the blood were also depleted teins of the graft may be incompatible with the complement cannot be excluded. Recently, Lin et al. (24) and Sablinski et system of the recipient (19). Complement activation is nor- al. (25) demonstrated that specific depletion of xenoreactive mally controlled by regulatory proteins on the surfaces of cells, antibodies, using affinity columns bearing Gal␣1,3Gal, could which serve to limit the damage produced by activated com- reliably prevent hyperacute rejection in pig-to-baboon xeno- plement. These complement regulatory proteins include decay- graft models. accelerating factor, membrane cofactor protein, and CD59. The second strategy for preventing hyperacute rejection of These proteins are inefficient in regulating the activity of xenografts involves the depletion or inhibition of complement. complement of different species; therefore, porcine comple- Complement depletion has been accomplished with the admin- ment regulatory proteins do not effectively inhibit activated istration of cobra venom factor (26), which activates and thus primate complement. This lack of function between pig and consumes complement. Unfortunately, cobra venom factor en- primate proteins contributes significantly to rejection, because genders an immune response, which can limit its duration of it allows the activation of complement to proceed rapidly and action. More recently, soluble CR1, a recombinant protein that unchecked. inhibits the complement cascade at the level of C3 and C4 (27), Some recent work suggests that hyperacute rejection may has been demonstrated to effectively prevent hyperacute rejec- not occur as frequently as previously thought for some com- tion of porcine kidneys by baboons (28). One limitation of binations of donor and recipient species (20,21). However, cobra venom factor and soluble CR1 is that, by interrupting the these observations may reflect the use of very small primates complement cascade at the level of C3, these agents impair the without the high level of xenoreactive natural antibodies ob- ability of the treated individual to opsonize microorganisms served in humans (22). and they thus confer a significant risk of pyogenic infection. An approach that, in principle, confers less risk is the admin- Prevention of Hyperacute Rejection istration of anti-C5 antibodies that inhibit the cleavage of C5, Hyperacute rejection was once viewed as an absolute hurdle interrupting the complement cascade after the step at which to xenotransplantation and the major barrier to the transplan- opsonization occurs (29,30). In addition to these agents, there tation of porcine organs, such as kidneys, into human patients. is a growing list of agents that might be used to interfere with Recent years have brought significant progress in overcoming complement; readers may refer to a recent review of this this problem; therefore, hyperacute rejection is no longer con- subject (31). sidered such a daunting challenge. Indeed, three strategies have A third strategy for preventing hyperacute rejection, and the proven effective in the prevention of hyperacute rejection of strategy that has emerged as the standard, is the use of organs kidney xenografts. that express complement regulatory proteins compatible with The first strategy for preventing hyperacute rejection of the recipient. Readers may recall from the discussion above xenografts involves the depletion of xenoreactive antibodies that xenografts are especially susceptible to complement-me- from the circulation of the recipient. Depletion of xenoreactive diated injury because complement control proteins (such as antibodies was first accomplished by Perper and Najarian (23), decay-accelerating factor and CD59) in the organ, which who perfused the blood of experimental animals through the would ordinarily protect the organ from injury by complement, kidneys of potential sources of xenografts. Subsequently, Al- function poorly against complement of foreign species. To J Am Soc Nephrol 12: 182–193, 2001 Hurdles to Xenotransplantation 185 overcome this obstacle, transgenic pigs expressing human subject of debate. Various factors thought to play causative complement regulatory proteins have been generated (32). roles are listed in Table 1. Of these factors, xenoreactive Organs from pigs transgenic for human CD59 and human antibodies are now widely considered to play a critical role in decay-accelerating factor, or human decay-accelerating factor the pathogenesis of acute vascular rejection. A number of alone, did not undergo hyperacute rejection (33), even when observations led to this conclusion. The onset of acute vascular the level of expression was significantly lower than that in xenograft rejection coincides with a striking increase in the human organs (34,35). Because expression of the transgene synthesis of xenoreactive antibodies after exposure to porcine does not influence the activity of complement in the circulation tissues (44). Attempts to reduce antibody and complement of the recipient and because expression presumably continues levels have resulted in significantly improved graft survival as long as the organ remains viable, this strategy is preferred. rates (45). In fact, using transgenic pigs and anti-Ig columns to A fourth strategy for preventing hyperacute rejection has deplete xenoreactive antibodies, acute vascular rejection has been proposed but has not yet been tested. This strategy in- been prevented in a pig-to-baboon model (17). Additional volves the development of lines of pigs that express lower evidence for the role of antibodies in acute vascular rejection is levels of antigen (36,37). Efforts in this area are discussed in the finding that a similar type of rejection is observed among the next section. allograft recipients in association with the development of Using transgenic pigs and antibody depletion, hyperacute anti-donor antibodies. rejection has been overcome. Once considered the most vexing How xenoreactive antibodies might cause acute vascular problem for xenotransplantation, hyperacute rejection is now rejection is being studied in a number of laboratories. Most not a major factor preventing the use of porcine organs for investigators think that xenoreactive antibodies trigger acute human patients experiencing end-stage organ failure. vascular rejection by activating endothelial cells. Xenoreactive antibodies activate endothelial cells via three potential mech- Acute Vascular Rejection anisms. First, endothelial cell activation might be induced by When hyperacute rejection is averted, xenografts become the binding of antibodies to cell surface antigens such as subject to acute vascular rejection (38,39), so named because integrins, which can transduce signals (46). Second, xenoreac- of its similarity to acute vascular rejection of allografts (40). tive antibodies could activate complement in small amounts, Acute vascular rejection is currently the primary obstacle to the leading to activation of endothelial cells (47). Third, xenore- use of porcine organ xenografts for human subjects. Acute active antibodies could serve as targets for Fc-bearing cells, vascular rejection has been confused with hyperacute rejection; which might in turn activate endothelial cells (48). in fact, some have referred to acute vascular rejection as Some authors have suggested that macrophages, natural “delayed” xenograft rejection, but acute vascular rejection is a killer cells, and neutrophils may contribute to the pathogenesis distinct entity clinically and pathogenetically. of acute vascular rejection (42,49,50). The role of macrophages Acute vascular rejection is histologically characterized by is supported by the observation that acute vascular rejection in focal ischemia, fibrinoid necrosis, and diffuse intravascular rodents is associated with the influx of macrophages express- thrombosis, with the thrombi consisting mainly of fibrin (39– ing tissue factor (42). Natural killer cells have also been 41) (Figure 2C). Some, but not all, cases of acute vascular identified in xenografts undergoing acute vascular rejection rejection exhibit infiltrating cells. These cells may include (42). Natural killer cells are capable of binding directly to and natural killer cells and macrophages. Endothelial cell swelling inducing morphologic changes in and activation of porcine and activation are observed in acute vascular rejection (42). endothelium (51). It is thought that host natural killer cells, Xenografted kidneys undergoing acute vascular rejection may whose responses are normally limited by MHC I molecules, also demonstrate acute tubular necrosis, with degenerating may not recognize the MHC I molecules of pigs, which are proximal tubules and necrotic epithelium (21). Glomeruli ex- termed swine leukocyte antigen I molecules. Neutrophils are hibit solidification, with loss of capillary patency, as a result of occasionally observed during acute vascular rejection (38). endothelial swelling and thrombi (43). Zehr et al. (50) observed that an inhibitor of CD11b/CD18 Which factors initiate acute vascular rejection has been the expression lessened the microvascular injury in acute vascular

Table 1. Factors postulated to initiate acute vascular rejection

Component Mechanism Authors (Ref.)

Antibody Activates endothelium directly or via complement Palmetshofer et al., 1998 (109) Complement Activates endothelium, promotes inflammation Saadi et al., 1995 (110) Platelets Release thromboxane A2, induce tissue factor and Bustos and Platt, 1997 (111) E-selectin expression Monocytes Express tissue factor Blakely et al., 1994 (42) Natural killer cells Activate endothelium Malyguine et al., 1996 (51) Molecular incompatibility Decreases fibrinolytic activity Lawson et al., 1997 (54) 186 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 182–193, 2001 rejection, although only minimally. However, these cellular co-workers (60,61) proposed that accommodation is produced infiltrates are often not observed in acute vascular rejection by the expression of protective genes, including A20, bcl-2, with pig-to-primate transplants (39). Moreover, recent studies bcl-xL, and HO-1. Whether these genes play a causative role in in rodents demonstrated that the inflammatory cells accumulate accommodation has not yet been determined. after tissue damage has occurred (52). Therefore, inflammatory Another approach to the prevention of acute vascular rejec- cells may be markers of tissue injury, rather than the cause of tion is induction of tolerance, leading to decreased production acute vascular rejection. of antibodies against the graft. Tolerance could play a role in An additional factor in acute vascular rejection may be the prevention of both acute vascular rejection and cellular physiologic incompatibilities of porcine proteins, such as rejection. One method that has been proposed as a means to thrombomodulin and protein C, with human proteins (53,54). induce tolerance is to engraft marrow cells expressing Such incompatibilities may establish a thrombotic diathesis xenogeneic antigens (62,63). The enduring engraftment of and contribute to a procoagulant state involved in acute vas- donor hematopoietic cells has proven to be difficult for widely cular rejection. However, because depletion of xenoreactive disparate species because of incompatibilities of the bone mar- antibodies using absorption columns, without manipulation of row microenvironment and growth factors (64). Bracy et al. the coagulation system, can prevent acute vascular rejection, as (65) used an alternative approach, which might overcome this mentioned above, it is possible that these incompatibilities problem; they used a retroviral vector to express ␣1,3galactosyl- have limited effects on the fate of the transplant. transferase in murine bone marrow cells and then transplanted those cells into isologous glucosyltransferase knockout mice, which, like humans, produce anti-Gal␣1,3Gal antibodies. The Prevention and Treatment of Acute Vascular Rejection transplantation of ␣1,3galactosyltransferase transduced bone mar- The prevention and treatment of acute vascular rejection row led to decreased production of anti-Gal␣1,3Gal antibodies. now represent a central objective in xenotransplantation. For Acute vascular rejection might, in principle, be addressed by the most part, approaches are based on the current understand- genetic engineering of source animals to reduce the expression ing of the pathogenesis of acute vascular rejection, as summa- of antigen. The prospects for the success of this approach have rized in Table 1. been advanced by evidence that the antibodies that cause acute One approach to the prevention of acute vascular rejection vascular rejection are directed against Gal␣1,3Gal. Recent involves depletion of xenoreactive antibodies from the recipi- advances in nuclear transfer techniques have allowed the pro- ent. Depletion of xenoreactive antibodies for this purpose has duction of pigs cloned from adult somatic cells (66). In prin- been achieved using immunoabsorption columns in conjunc- ciple, the ␣1,3-galactosyltransferase gene could be targeted in tion with plasmapheresis (55,56). Effective application of im- the somatic cells by homologous recombination. Gene target- munoabsorption must be accompanied by immunosuppression, ing in sheep and the successful development to term of ovine to prevent rebound of xenoreactive antibody levels (55,56). offspring with targeted cells have been achieved, supporting The approach used in our laboratory includes splenectomy and the feasibility of such an approach (67). Targeted disruption of treatment with cyclophosphamide, which inhibits antibody the ␣1,3-galactosyltransferase gene has been achieved in pri- synthesis. These methods are clearly associated with signifi- mary porcine cells (66). These advances raise the possibility cant morbidity. that pigs lacking Gal␣1,3Gal expression might be produced. Depletion of anti-donor antibodies may allow the induction One major limitation to gene targeting is that it might of accommodation. Accommodation is a term used to describe increase the risk of infection by porcine viruses (68). Efforts to the apparent resistance of an organ graft to acute vascular decrease antigen expression have thus far used methods other rejection (19). Accommodation was first observed in the trans- than gene targeting to decrease antigen levels. Strategies have plantation of ABO-incompatible kidney allografts, where it included the expression of ␣1,2-fucosyltransferase, which cat- was produced by temporary depletion of anti-donor antibodies alyzes the formation of a nonantigen sugar (37,69) or competes (57,58). In accommodation, the return of anti-donor antibodies with ␣1,3-galactosyltransferase, and the expression of ␣galac- to the circulation does not cause graft injury, although the tosidase, which cleaves the terminal Gal residue, yielding a less corresponding antigen continues to be expressed. The mecha- immunologically reactive saccharide. In an in vitro model, cell nisms of accommodation are not certain; explanations include lines expressing ␣1,2-fucosyltransferase and ␣galactosidase decreased antibody-antigen interaction, decreased sensitivity reduced expression of the Gal␣1,3Gal epitope to negligible of the endothelium to stimulation by complement and antibod- levels and reduced reactivity with human serum to background ies, and the expression of “protective genes” in the endothe- levels (36). However, application to an in vivo model has been lium (59). In a rodent model of discordant xenotransplantation, difficult because the addition of other transgenes decreases the temporary depletion of complement and immunosuppression expression of previous transgenes. Although further improve- with cyclosporine resulted in long-term survival of heart xeno- ments will undoubtedly allow further reduction of Gal␣1,3Gal grafts in 75% of the animals, which was attributed to accom- expression, the development of xenoreactive antibodies against modation (60). Surviving hearts exhibited recipient antibodies antigens other than Gal␣1,3Gal, particularly porcine MHC and complement deposited on their endothelium. Bach and antigens, will pose a continuing challenge. J Am Soc Nephrol 12: 182–193, 2001 Hurdles to Xenotransplantation 187

Cellular Rejection in preventing cellular rejection of allografts. Whether the same If hyperacute rejection and acute vascular rejection were immunosuppressive regimens will be adequate to prevent prevented, then renal xenografts would be subject to cellular and/or treat cellular rejection of xenografts is not known. rejection. Cellular rejection is the most common form of re- One consideration in preventing or treating cellular rejection jection observed for allografts, but it has not been well char- of xenografts is whether currently available immunosuppres- acterized in xenografts, and it is not clear how similar such sive drugs would suffice. Rapamycin, which was recently rejection would be to the cellular rejection of allografts. Cel- approved for clinical use, potentiates the effects of cyclospor- lular rejection of allografts is characterized by interstitial in- ine A (77) and may enable tolerance to be induced (78,79). filtrates of T cells and macrophages (70). Infiltrates of T cells, Deoxyspergualin, which affects macrophage and T and B cell macrophages, and natural killer cells may characterize cellular function, has been demonstrated to be effective in reducing rejection of xenografts. natural antibodies in a xenograft model (80). The potent ability One aspect of the cellular immune response to xenotrans- of deoxyspergualin to suppress humoral immunity could be plants that has drawn attention in the past decade is the useful in xenotransplantation. Leflunomide inhibits prolifera- mechanism by which T cells of the recipient recognize tion of T and B cells and has been demonstrated to inhibit donor antigens. The T lymphocytes, which cause acute xenograft rejection in rodent models (81). Simultaneous block- cellular rejection of allografts, recognize predominantly ade of the costimulatory CD28 and CD40 pathways was re- MHC antigens. In doing so, alloreactive T cells recognize cently observed to prolong survival of pig-to-mouse and donor antigen by the direct pathway, with the T cell recep- rat-to-mouse cardiac xenografts (82). Indeed, blockade of both tors of the T cells of the recipient binding directly to the pathways ablated IgG responses to xenotransplantation (82). MHC molecules expressed on donor cells. It was once However, blockade of the CD28 and CD40 pathways was not thought that the direct pathway or recognition is deficient in able to induce indefinite survival of xenografts. xenogeneic responses, and there was thus some hope that In principle, another way to prevent cellular rejection of a cellular rejection of xenografts would be milder than cellular xenograft would be to induce immunologic tolerance against rejection of allografts. However, recent work indicates that human the xenograft. Some authors have argued that, given the inten- T cells recognize porcine cells by the direct pathway (71,72). sity of immune responses to xenotransplantation, tolerance Human T cells can also recognize xenografts and allografts by the induction would be necessary for the clinical application of indirect pathway, in which donor antigen is degraded to peptides xenotransplantation. However, it is not known how tolerance and presented in association with recipient MHC molecules on can be safely induced for this purpose. Several methods of recipient antigen-presenting cells. In fact, the cellular response to tolerance induction have been developed for allotransplanta- a xenograft may be markedly stronger than the cellular response tion; however, tolerance has not been induced in pig-to-primate to an allograft (73). models. The cellular immune response to a xenograft may well be Tolerance induction in primate allotransplantation has been more potent than the cellular immune response to an allograft, approached using various methods, i.e., blockade of costimu- for three reasons. First, a xenotransplant, in contrast to an lation using monoclonal antibodies against CD40L (78), use of allotransplant, may give rise to a vast array of foreign peptides, an immunotoxin that leads to T cell depletion (83), T cell stimulating a strong T cell response. Dorling et al. (73) dem- depletion combined with donor bone marrow infusion (84,85), onstrated that indirect presentation of porcine antigens led to a and establishment of mixed chimerism (43). Of those methods, detectable primary response in cell cultures, whereas such the latter two, which involve infusion of donor bone marrow or responses were not detected with allogeneic combinations. donor stem cells, seem most useful for xenotransplantation, Second, the humoral response to xenotransplants might am- because they might suppress humoral responses to Gal␣1,3Gal plify the cellular response. Kodaira et al. (74) observed that as well as cell-mediated immune responses. Most attention has complement activation and anti-endothelial cell antibodies been directed to the establishment of mixed chimerism. Mixed caused the release of heparan sulfate from the surface of chimerism aims to alter the immune repertoire of the recipient endothelial cells and heparan sulfate acted on antigen-present- by transplantation of donor bone marrow, which migrates to ing cells, resulting in increased proliferation and cytolytic T the thymus and causes the deletion of donor-reactive T cells. cell responses. Third, immunoregulatory responses that might Establishment of mixed chimerism through the transplantation depend on the compatibility of donor and recipient cytokines or of donor bone marrow is a considerable challenge, which direct recognition by suppressor leukocytes (75) might be requires “conditioning” of the recipient with whole-body irra- impaired in xenografts (76). It is possible that xenografts diation, anti-T cell antibody treatment, thymic irradiation, and would not collaborate in the immunoregulatory function of an splenectomy, with or without a short course of postoperative immune response, thus leading to more destructive outcomes. immunosuppression (86). Although allograft survival greater than 4 yr has been achieved by establishing mixed chimerism Prevention of Cellular Rejection (43), efforts to combine the induction of mixed chimerism with A critical and still unanswered question is whether the immunoabsorption of xenoreactive antibodies have thus far prevention and treatment of cellular rejection of xenografts will been unsuccessful in a pig-to-primate model, with survival require strategies different from those used, with some success, times of only 15 d (43). Although none of the regimens for allografts. Immunosuppression is currently quite effective mentioned above yielded tolerance in pig-to-primate xeno- 188 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 182–193, 2001 grafts, it is possible with further modifications that one of these erythropoietin to function in a human milieu is uncertain; regimens will be applicable to xenotransplantation in the however, there is an indication, as noted above, that porcine future. erythropoietin is insufficient to stimulate red blood cell pro- duction (21). Thus, cynomolgus monkeys with porcine kidney Physiologic Hurdles xenografts experienced severe anemia despite the production Xenografted organs must meet the physiologic demands of of porcine erythropoietin, and human erythropoietin was re- the recipient and function cooperatively with the other organs. quired to restore the hematocrit levels of the monkeys (21). Preliminary evidence suggests that a porcine kidney trans- If physiologic disturbances prove to be hurdles, it is possible planted into a primate would function adequately to maintain that genetic engineering might be used to address such prob- the water, electrolyte, and acid/base balances. Pig kidneys are lems. For example, if erythropoietin incompatibility is a seri- of very similar size and structure, compared with human kid- ous problem, then transgenic pigs that express human erythro- neys (87). The maximal concentrating ability of porcine kid- poietin might be developed. As another example, genetic neys (1080 mosmol/liter) and the GFR (126 to 175 ml/h) are manipulation might be used to generate pigs that express quite similar to those of human kidneys (87). human proteins from the coagulation cascade, to prevent There is limited information concerning the function of coagulation. porcine kidneys in primates. Sablinski et al. (25) demonstrated survival times of up to 15 d for porcine kidneys in anephric Infection cynomolgus monkeys, with creatine levels ranging from 0.8 to Infectious disease contributes significantly to the morbidity 1.3 mg/dl until the onset of rejection. and death of transplant recipients. The potential use of animals Alexandre et al. (8) transplanted porcine kidneys into ba- as a source of organs has increased the concerns regarding boons that had been subjected to bilateral nephrectomy. The infection (91). Indeed, transfer of the influenza virus from pigs recipients underwent preoperative plasmapheresis and splenec- to human subjects in 1918 is thought to have sparked a pan- tomy just before xenotransplantation. Three of the five ba- demic that led to millions of deaths. boons with porcine kidney xenografts survived more than 10 d. Issues involving the transmission of potential infectious The longest survivor (which survived for 23 d) succumbed to organisms have led to the coining of new terms to describe vascular rejection. In these xenograft recipients, the serum infections potentially transferred from animal organs into hu- creatine levels remained below 2.0 mg/dl until rejection oc- man subjects. “Zoonosis” refers to an infectious disease trans- curred. The baboons required blood transfusions to maintain mitted from an animal to a human subject under “natural” hemoglobin levels above 8 g/dl, suggesting the possibility that conditions, such as an animal bite; the terms “xenosis” and porcine erythropoietin might not be fully compatible with “xenozoonosis” were developed to describe infectious illnesses erythroid-lineage cells of baboons (8). The electrolyte levels, introduced into human subjects through medical procedures weight, and acid/base status were not reported (8). involving xenogeneic tissue (92,93). Whether distinct terms are The best evidence concerning the function of porcine kid- needed, however, is unclear. neys in primates can be found in the work of Zaidi et al. (88). Concerns regarding xenotransplantation fall into two cate- Using transgenic pigs that expressed human decay-accelerating gories. First, microbes endemic to the donor species might factor as a source of kidney xenografts, Zaidi et al. (88) infect human subjects, especially immunosuppressed trans- achieved graft survival times of up to 78 d in nephrectomized plant recipients. Second, there is the possibility that xenotrans- cynomolgus monkeys. The monkeys received immunosuppres- plantation could lead to the generation of novel pathogens sion consisting of cyclophosphamide, cyclosporine, corticoste- created by recombination of porcine retroviruses with elements roids, and splenectomy (88). When the kidneys were function- of the human genome. Both concerns focus on the potential ing, potassium, sodium, and creatine levels and the acid/base complication of infection of the recipient and the potential balance were within normal limits (88). Body weight and fluid spread of infection to others in the population. However, it is balance were apparently maintained (88). In summary, the only the second situation, i.e., the generation of novel patho- studies on xenotransplantation of porcine kidneys into primates gens, that might be unique to xenotransplantation. In any case, that have been performed to date suggest that the major factor evidence gathered to date, as summarized below, suggests that limiting kidney function is tissue injury resulting from rejec- the risks to patients and the public are low. tion and not intrinsic differences in renal function between For transplant recipients, the use of xenografts might well species. result in fewer problems attributable to infectious organisms. An area in which xenotransplants may not completely re- The urgent nature of donor-recipient matching can result in the place human organs is in the interaction of hormones or com- transplantation of organs with Epstein-Barr virus or cytomeg- plex protein cascades with the recipient. Sen et al. (89) dem- alovirus into patients. The careful breeding and housing of onstrated that heterologous renin is much less effective in animal donors could, in principle, prevent infection of the eliciting the release of angiotensin than is homologous renin. xenograft recipient by known pathogens. Therefore, xenografts We recently demonstrated that porcine thrombomodulin inter- might be “cleaner” than allografts, because the animals bred as acts poorly with human tissue factor and human protein C, a source of xenografts would be housed in pathogen-free making a porcine xenograft a potential trigger for the coagu- environments and rigorously tested for known exogenous lation cascade of the recipient (90). The ability of porcine pathogens. Xenotransplants may also be less susceptible to J Am Soc Nephrol 12: 182–193, 2001 Hurdles to Xenotransplantation 189 infection by human pathogens because animal organs fre- However, the availability of dialysis might make xenotrans- quently exhibit species-specific resistance to infection by many plantation of the kidney appealing as an experimental proce- common human pathogens. For example, neither human hep- dure because the risk to the recipient would be lower than in atitis B nor HIV-1 is capable of infecting baboons or presum- the case of other types of xenotransplants. ably pigs (94). Therefore, xenografts might be better choices in We envision that xenotransplants might be considered ini- cases where infectious diseases have produced organ failure. tially for certain small groups of patients. One group that might It is possible that some pathogenic organisms have yet to be be appropriate to consider for renal xenotransplantation con- identified. Therefore, some organisms may cause little or no sists of patients who are presensitized to multiple potential morbidity in pigs but pose a risk to human subjects. For donors and thus are unlikely to receive an allograft. The use of example, Hantaviruses, of which there are five known forms, xenotransplantation for such patients presumes that anti-HLA produce no detectable morbidity or death in rodents, which antibodies would not cross-react with porcine major histocom- constitute the natural reservoir; however, Hantaviruses can patibility antigens (swine leukocyte antigens). A second group cause disease in human subjects, with mortality rates as high as that might be appropriate to consider for renal xenotransplan- 50% (91). Because pigs and human subjects have existed in tation consists of infants with kidney failure, for whom dialysis close proximity for thousands of years, the risks of such is very difficult. Such infants are also difficult to treat with unknown pathogens seem quite small. Two exceptions, how- transplantation because of the mismatch in size between adult ever, may be a virus related to the human hepatitis E virus and kidneys and infants. A xenograft could be used in selected a new torovirus identified in pigs (95,96). cases to allow the infant to grow to a size more suitable for Of greater concern, then, is the possibility of newly emerg- allotransplantation. A third group that might be appropriate to ing pathogens developing in xenotransplant recipients and consider for renal xenotransplantation consists of patients with spreading to the general public. Concerns have focused partic- hyperoxaluria. A fourth group might be young HIV-positive ularly on endogenous retroviruses. Much significance has re- patients with HIV-associated nephropathy. A survey of 148 cently been attributed to the observation that porcine endoge- kidney transplant centers in the United States revealed that less nous retrovirus (PERV), a type C retrovirus of pigs, is capable than 10% would consider patients with HIV for allotransplan- of infecting human cells (97). Wilson et al. (98) demonstrated tation and none of the 148 centers had performed a kidney that primary porcine cells (not a cell line) are capable of transplant for a HIV-positive recipient in the past year (105). releasing PERV after activation, and the retrovirus is capable of infecting human cell lines. Porcine pancreatic islets trans- Ethical Considerations planted into immunodeficient mice were recently demonstrated The prospective removal of an organ from one individual to be capable of causing transmission of PERV to the mice and its placement in a second individual evoked tremendous (99). However, the clinical relevance of this model is unclear, debate among scientists, physicians, and ethicists when allo- because the immune system of severe combined immunodefi- transplantation was in its infancy. How organs could be ob- cient mice is much weaker than that of immunosuppressed tained for transplantation (as discussed above), how transplan- human patients and mice may be more receptive to infection tation would modify the sense of individuality of the recipient, than human subjects (68). and whether a living donor could be allowed to assume the All pigs tested to date contain multiple copies of PERV in risks involved in have stimulated debate. To- the genome, making breeding-out of the virus nearly impossi- day, however, the preeminent ethical question in the field of ble (100). However, a number of recent studies have failed to transplantation is how the very limited number of organs detect evidence that PERV can be transferred from porcine should be allocated. Clearly, the successful application of cells to human cells in vivo (101–104). For example, Paradis et xenotransplantation would solve that ethical dilemma. al. (103) studied 160 patients with exposure to living pig tissue, Xenotransplantation would raise new ethical questions. Is- including 36 patients with immunosuppression. Despite evi- sues related to xenotransplantation can be divided into three dence of porcine cells surviving in the human subjects for up categories, as follows: (1) animal welfare, (2) clinical experi- to 8.5 yr, there was no evidence of PERV infection, using PCR mentation, and (3) public health implications. Although these analysis. These studies have thus tended to diminish concerns issues are not unique to xenotransplantation, they should be regarding cross-species infection in xenotransplantation. How- considered here. ever, infectious disease will always be a concern in transplan- The question of animal welfare largely focuses on whether it tation because transplant recipients undergo immunosuppres- is justifiable to use animals as a source of organs and tissues for sion, and differences in MHC antigens may impair the host transplantation into human subjects. Arguably, because society response to infection in the transplant. allows the use of animals for food, the use of animals as a source of organs or tissues is morally acceptable. It would be Making Xenotransplantation a Clinical Reality difficult to argue against the use of pigs for xenotransplanta- Assuming that the biologic hurdles to renal xenotransplan- tion, which might be the only option for the treatment of tation can be overcome, the next critical question is which life-threatening diseases, and simultaneously countenance the patients would be most suitable as recipients of xenotrans- use of pigs for food when nonanimal sources exist. plants. This question is particularly challenging with respect to The ethical issues regarding clinical experimentation are the kidney, because dialysis is an alternative to transplantation. partly related to the issue of informed consent. The issue of 190 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 182–193, 2001 informed consent is particularly important for kidney trans- 4. Hamilton D: : A history. 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