ILAR Journal Online, Volume 37(1) 1995: Perspectives on Xenotransplantation

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ILAR Journal V37(1) 1995
Perspectives on Xenotransplantation

The Immunologic Response to Xenografts
David H. Sachs

David H. Sachs, M.D., is the Paul S. Russell/Warner-Lambert Professor of Surgery at Massachusetts General Hospital and the director of the Transplantation Biology Research Center, Harvard Medical School, Boston.

INTRODUCTION

The rejection of xenografts is clearly an immunologic phenomenon, since in the absence of an immune response, animals accept transplants even across widely disparate xenogeneic barriers. For example, nude mice, in which the absence of the thymus leads to defective T-cell immunity, have been shown to accept xenogeneic skin grafts (Manning and others 1973), even to the point of growing chicken feathers. Similarly, some of the lymphohematopoietic compartments in severe combined immune-deficient (scid) mice can be repopulated by highly disparate xenogeneic hematopoietic cells (Mosier and others 1988; McCune and others 1988). In theory, eliminating the immune response to a xenograft should be sufficient to assure its success. This premise is of more than just theoretical importance to the field of xenotransplantation, since there certainly might have been other, non-immunologic barriers that could have prevented xenotransplantation even in the absence of an immune response. For example, the cell surfaces of xenogeneic tissues might have been physiologically incompatible, or the red cells of the recipient might have been physiologically incapable of delivering oxygen to xenogeneic tissues or even unable to negotiate xenogeneic capillaries. Nevertheless, at least between mammalian species, it now seems likely that the immune response is the barrier of greatest importance.

As is the case for other immune responses, the reaction to xenografts involves both humoral and cellular immunity. Xenografts have been further categorized as concordant or discordant on the basis of phylogenetic distance and vigor of the immune response (Caine 1970). The most notable immunologic difference between concordant and discordant xenografts involves the presence in the latter of natural antibodies capable of causing hyperacute rejection of vascularized organs. As will be described in more detail below, although natural antibodies have posed a formidable barrier to discordant xenografting in the past, there are now numerous methods for eliminating these antibodies or controlling their effects, which have shown promising results in avoiding hyperacute rejection. However, both humoral and cellular immune responses to xenografts will undoubtedly be as strong as, or stronger than, responses to allografts, and will have to be overcome if xenotransplantation is to become a reality. I will review here our present understanding of these two arms of the immune response for both concordant and discordant xenografts and will try to identify strategies that may overcome the resultant barriers that each of these responses poses to successful xenotransplantation.

CONCORDANT XENOGRAFTS

Humoral responses

The most widely studied concordant xenograft systems involve closely related rodent species such as the mouse and rat. In many respects, the humoral response to transplants between such species is similar to that observed for MHC-mismatched allotransplants, and as implied by the definition of concordant species, hyperacute rejection is not observed when primarily vascularized transplants are performed. However, the absence of natural antibodies between concordant species is relative rather than absolute, and even in the rat-mouse system, natural antibodies have been detected when carefully sought (Aksentijevich and others 1991a). Thus, using flow cytometry and cytotoxicity assays, it has been determined that normal mouse serum contains natural antibodies with specific binding to, and cytotoxicity against, scid rat bone-marrow cells (Aksentijevich and others 1991a). These natural antibodies were predominantly of the lgM and IgG3 classes, and activity toward bone marrow cells was much greater than that toward spleen cells. Such antibodies probably explain the observation that much greater numbers of rat than of murine bone-marrow cells are required to achieve engraftment in mice (Ildstad and Sachs 1984). To more directly evaluate the effect of these natural antibodies on engraftment of rat bone-marrow cells in mice, adoptive transfer studies were performed using T- and B-cell-deficient scid mice as recipients (Aksentijevich and others 1991b). Because of their immunodeficiency, scid mice accepted rat bone-man'ow cells readily, with only a low dose of whole body irradiation being necessary for conditioning. Passive transfer studies showed that normal mouse serum could markedly inhibit the engraftment of rat bone-marrow cells even in this phylogenetically close species combination, consistent with the hypothesis that natural antibodies provide a barrier to engraftment of xenogeneic bone marrow. Natural antibodies are probably present in other concordant species combinations, including primates. This should hardly be surprising, given the existence of ABO antibodies even within the human species. For the most part, these antibodies are probably of sufficiently low titer that they do not lead to hyperacute rejection. Nevertheless, pretransplant cross matching will certainly be required in order to avoid those situations in which relatively high titers of such antibodies might lead to catastrophic loss of a transplanted organ. As has been demonstrated for ABO antibodies, it should be possible to eliminate hyperacute rejection due to such natural antibodies by plasmapheresis or absorption techniques (Alexandre and others 1987).

In contrast to natural antibodies, the induced humoral response across concordant species barriers is likely to pose a major barrier to xenotransplantation. In the case of rat anti-mouse responses, immunization similar to that used to produce alloantisera was effective in raising high titers of xenoantibodies very rapidly (Sachs and others 1971). Within such xenoantisera considerable titers of antibodies reactive with mouse MHC alloantigens were detected, along with other antibodies of equal or greater titer reacting with species-specific antigens (Sachs and others 1971). The initial humoral response following immunization consisted predominantly of IgM, and this shifted to IgG after 2-3 weeks, suggesting a typical T-cell-dependent response (Davie and Paul 1974).

Similarly, humoral antibodies have been a major feature of the response to concordant xenografts in several other systems. For example, the rejection of heterotopically transplanted hearts from cynomolgus monkeys to baboons was correlated with the development of cytotoxic antibodies in the recipients' sera (Sadeghi and others 1987). For this reason, splenectomy and cyclophosphamide treatment or both have been used to prolong xenograft survival in several concordant species combinations by diminishing antibody responses, presumably by removing or inhibiting antibody-producing B-cell populations (Edwards and Rose 1989; Thomas and others 1992). Antibody was likewise a prominent feature of the immune response of human-to-baboon liver transplants, which was apparently controlled by high-dose immunosuppressive medications (Starzl and others 1993b). It therefore seems clear that the measures that will have to be taken to avoid the induced antibody response to concordant xenografis will have to be as least as vigorous as they are for MHC-mismatched allografts. One fortunate note is that because the predominant problem following sensitization appears to be the T-cell-dependent IgG response, it seems possible that induction of tolerance at the T-cell level (see below) will carry with it tolerance at the B-cell level with respect to this induced humoral response.

Cellular responses

The cellular response to concordant xenografts is, in general, similar to that of allografts (Auchincloss 1988). In vitro cellular assays such as the mixed lymphocyte reaction (MLR) and the cell-mediated lympholysis (CML) assays have been shown to be qualitatively and quantitatively similar for concordant combinations of primate (Martinis and Bach 1977), canine (Hammer 1991) and rodent (Ildstad and others 1984) species to the corresponding assays within each species. Since the acute rejection of untreated vascularized allografts is predominantly a cell-mediated phenomenon, as one might expect, survival times for vascularized concordant xenografis are similar to those for MHC-mismatched allografts (Auchincloss 1988). Likewise, immunosuppressive agents such as anti-thymocyte globulin and Cyclosporin A, which are successful in controlling allogeneic cellular rejection, have also been effective in suppressing the cellular response to concordant xenografts (Russell and Monaco 1967; Sadeghi and others 1987).

Indeed, as early as 1964, Reemtsma and colleagues reported survival of a chimpanzee kidney in a patient for 9 months using azathioprine, Actinomycin C, steroids, and irradiation of the kidney transplant (all of these being the recommended treatment for allotransplants in that era) as the only immunosuppression (Reemtsma and others 1964). The long-term survival obtained suggests that for the chimpanzee-to-human combination, current drug therapy for preventing allotransplant rejection would probably suffice. However, since chimpanzees are now considered an endangered species, it is highly unlikely that these animals will see further use as clinical concordant xenograft donors. The two clinical baboon-to-human liver xenografts reported recently from Pittsburgh were likewise treated with a four-drug immunosuppressive regimen including three agents previously used in varying combinations for allotransplantation (FK506, prednisone, and prostaglandin E). Cyclophosphamide was added to the treatment for its potential effect on the humoral response. Although neither patient survived long-term (70 and 26 days, respectively) it was noteworthy that both showed little or no evidence of cellular rejection in biopsy or autopsy specimens (Starzl and others 1993a, 1994). Infectious complications were the immediate cause of death in both patients, and the investigators attributed this complication at least in part to over-immunosuppression. It therefore remains unclear whether or not levels of immunosuppression identical to those that are effective for allotransplantation would have sufficed to avoid cellular xenografi rejection for this concordant baboon-to-human xenograft combination.

DISCORDANT XENOGRAFTS

Humoral responses

As noted above, one of the most important differences between concordant and discordant xenografts is that discordant xenografts contain preformed or natural antibodies capable of causing hyperacute rejection of vascularized organs. In general, the further the phylogenetic distance between two species, the greater the detectable levels of such preformed antibodies (Hammer and others 1973), which increases the likelihood of hyperacute rejection. Although natural antibodies are defined as those that are present in the absence of immunization, it is likely that these antibodies actually represent cross-reactions between antibodies directed against bacterial cell-wall antigens and antigens on the surface of xenogeneic cells. Consistent with this hypothesis is the fact that germ-free animals have been shown to have very low levels of natural antibodies (Hammer 1987).

The majority of natural antibodies are of the IgM subclass (Hammer 1987; Latinne and others 1994). It has been clear for some time that these antibodies are cross-reactive and bind to carbohydrate determinants on glycoproteins and glycolipids (Galili and others 1984), and there is increasing evidence that the majority of natural antibodies in the human anti-pig combination are directed to the cd-3-galactose linkage (Oriol and others 1993; Sandrin and others 1993). IgM antibodies are known to be excellent activators of complement, and the effects of these antibodies on xenografts often involve activation of the complement system (Dalmasso and others 1991). For this reason, several approaches have been taken to avoid hyperacute rejection by eliminating the complement-mediated cytotoxicity pathway. These include use of complement inhibitors (Pruitt and others 1991; Miyagawa and others 1993) and potentially the production of transgenic pigs bearing species-specific complement inhibitory molecules such as decay accelerating factor (Langford and others 1994). However, natural antibodies also appear to be effective in activating endothelial cells, which may play a major role in the hyperacute rejection process (Platt and others 1991), and which may not be avoided by complement inhibition.

The fact that natural antibodies are predominantly IgM may be advantageous from the point of view of xenotransplantation, since IgM responses are generally primary and do not involve long-term immunologic memory. Memory for antibody formation is generally thought to occur at the stage of the IgM to IgG switch and to involve T-cell help (Davie and Paul 1974). Therefore, one might hope that if natural antibodies can be removed prior to xenotransplantation, and if T-cell tolerance to xenografts can then be induced, natural antibodies may not recur.

As indicated in Table 1, there are several procedures which have been suggested as potential ways to eliminate xenoreactive antibodies. Alexandre and colleagues have pioneered the use of extensive plasmapheresis as a means of removing natural antibodies (Alexandre and others 1989). In our own laboratory we have chosen an absorption technique using a pig liver to absorb antibodies in vivo prior to the xenotransplant (Latinne and others 1993; Tanaka and others in press). A one-hour perfusion was found sufficient to remove the vast majority of natural antibodies and to eliminate hyperacute rejection. More recently, we have begun to use columns in which an insoluble matrix bearing otl-3-gal epitopes is substituted for the liver in our perfusion step, and the results are very encouraging (Sablinski and others unpublished data).

The induced antibody response to discordant xenografts may pose a more difficult problem for xenotransplantation than do natural antibodies. The induced antibodies that occur in response to such transplants are predominantly IgG and result from a T-cell-dependent response. As such, they involve long-term memory, so that absorption procedures are unlikely to provide a lasting solution to their elimination. It is not yet clear how much of the induced response is directed to the same determinants that are detected by natural antibodies and how much is directed to additional antigens. Clearly, immunization across disparate species barriers leads to antibodies to many surface molecules, with antigenic determinants carried both by proteins and carbohydrates. Methods aimed at avoiding T-cell responses to xenografts should also mitigate against the generation of induced antibodies. Perhaps the most effective way to eliminate this problem will be to avoid it, by inducing tolerance at the T-cell level, as discussed below.

Cellular response

The primary cellular response to all transplants takes time, since both an afferent response (that is, sensitization and proliferation) and an efferent arm (mobilization of effector T cells) are required. Since natural antibodies cause hyperacute rejection within hours, most in vivo studies of discordant xenografts have concentrated on humoral and complement-mediated mechanisms rather than on the cellular immune response.

However, in vitro cellular responses to xenogeneic cells have been studied extensively. Early studies produced the surprising result that cellular immunity to xenogeneic antigens appeared to be weaker than the corresponding responses to allogeneic antigens (Wilson and Fox 1971; Engelhard and others 1988; Widmer and Bach 1972; Simonsen 1967). Such analyses suggested lower precursor frequencies both for proliferative and for cellular cytotoxic responses. On the basis of these lower frequencies, it was hypothesized by Jerne (1971) that the primary reactivity of T cells in the immune response was directed toward alloantigens (that is, minor variants of self-MHC antigens). However, there are numerous other reasons why cellular reactivity across discordant barriers could appear weaker than alloreactivity besides a difference in the T-cell receptor repertoire. One other potentially significant factor is the species-specificity of some accessory molecule interactions. For example, it is now clear that CD4 molecules on T-helper cells add to the affinity of the interaction of these cells with stimulator cells expressing class II antigens by interacting directly with the constant portion of class II molecules (Gay and others 1988). CD4 molecules are highly conserved within species, but divergent between species (Pames 1989), and this is also true of the constant portions of class II molecules (Klein 1986). Therefore, depending on the species combination studied, the interaction between CD4 and class II antigens could lead to diminished apparent frequencies of responder cells, because only T cells bearing receptors with high enough affinity for xenogeneic class II to react effectively even without a CD4 class II interaction would be counted as positive. Similarly, one might expect second-signal interactions (such as CD28B7), which are needed for activation (Azuma and others 1992), to depend on the effectiveness of the receptor-ligand interaction across the species difference under study. Still another possible reason for lower apparent reactivities for xenogeneic interactions may be the species-specificity of certain cytokine interactions (Benfield and others 1991).

Extensive studies by Auchincloss and colleagues using the mouse as the responding species and human, monkey and pig skin grafts as the discordant donor transplants, have demonstrated that many of these potential defects may explain the apparent decreased rejection response (Moses and others 1992). These authors have demonstrated that the failure of direct CD4 class II and CD8 class I interactions in these species combinations leads to exclusive use of the indirect pathway for sensitization to xenografts (Moses and others 1990), that is, presentation of xenogeneic class I peptides on self class 11 molecules (Sayegh and others 1994). If this were true for all xenogeneic cell-mediated responses, one might actually expect the xenograft reaction to be easier to control than an allograft reaction, which includes both direct and indirect pathways of sensitization. On the other hand, with the exception of skin grafts on mice, the cellular responses that have been encountered for discordant xenografts in vivo have been faster and stronger than those for allografts (Auchincloss 1991 ). In addition, more recent studies in several laboratories, including our own, have indicated that the human anti-pig cellular response is mediated both by direct and indirect pathways of recognition (Murray and others 1994; Kumagai-Braesch and others 1993; Yamada and others in press). Therefore, at least in this highly relevant discordant species combination, it is likely that regimens at least as potent as those required to suppress allograft rejection will undoubtedly be needed.

MIXED CHIMERISM AND TOLERANCE

Considering the nature of the discordant xenograft response, and the fact that even for allografts the titration of immunosuppressive drugs places the transplant patient on the border between rejection and infection, the amount of nonspecific immunosuppression that will be required to avoid xenograft rejection may be so great that too many patients would succumb to infectious complications. For this reason, it seems likely that the success of clinical xenografting will depend, at least in part, on finding ways of inducing tolerance across xenogeneic barriers rather than relying entirely on nonspecific immunosuppressive agents. In our laboratory, we are pursuing the use of mixed chimerism as a means of inducing tolerance across xenogeneic barriers.

The methodology we have developed is based on previous work in allogeneic and concordant xenogeneic systems in rodents (Ildstad and Sachs 1984; Sharabi and Sachs 1989; Sharabi and others 1990). The approach has also recently been extended successfully to an allogeneic system in primates (Kawai and others in press). In essence, mature T cells are depleted from the recipient animals, and sufficient ablation is administered to make room for donor bone-marrow cells to engraft. In contrast to the use of bone marrow transplantation as a treatment of leukemia, in which case complete ablation of host bone-marrow elements is required, such ablation is neither necessary nor desirable when bone marrow transplantation is used as a tolerance-inducing regimen. Instead, it is advantageous to achieve a state of mixed chimerism, in which the presence of certain donor-derived elements induce specific tolerance, while host-type antigen presenting cells maintain normal immunocompetence. Such mixed chimeras show long-term specific tolerance to transplants from the donor strain.

In our initial studies using mixed chimerism to induce allograft and concordant xenograft tolerance, we used lethal irradiation and reconstitution with mixtures of T-cell-depleted host and donor bone-marrow cells (Ildstad and Sachs 1984). These studies demonstrated that stable mixed chimerism established specific tolerance to other donor-derived tissue transplants. However, lethal irradiation is too toxic a preparative regimen to be considered for clinical transplantation. We have therefore turned more recently to a non-myeloablative regimen that produces mixed chimerism and tolerance without the use of lethal irradiation (Sharabi and Sachs 1989; Sharabi and others 1990). We have demonstrated that treatment of recipient mice with monoclonal antibodies to the two mature T-cell subsets, CD4 and CD8, followed by sublethal irradiation (300R) and a dose of irradiation to the thymus (700R thymic irradiation) permits engraftment of a subsequent injection of allogeneic bone marrow, and the production of mixed allogeneic chimeras (Sharabi and Sachs 1989). Such animals develop long-term mixed chimerism in all lymphohematopoietic compartments and are indistinguishable by two-color FACS analysis from mixed chimeras prepared by lethal irradiation and reconstitution with mixtures of T-cell depleted syngeneic plus allogeneic bone marrow. They have been shown to be stable mixed chimeras and to be specifically tolerant, as demonstrated by long-term acceptance of donor strain skin grafts and prompt rejection of third-party grafts. Using a very similar regimen, we have now demonstrated multilineage mixed chimerism and long-term tolerance to kidney allografts in a cynomolgus monkey model (Kawai and others in press).

We are now attempting to use a similar methodology to induce tolerance across the discordant xenogeneic barrier of pig-to-cynomolgus monkey. As a donor, we are using partially inbred miniature swine, which were developed in this laboratory over a 20-year period as both a large animal model for studies of transplantation biology (Sachs 1992) and a potential xenograft donor (Sachs 1994). The protocol being attempted is illustrated in Figure 1. We have recently reported the preliminary results of our first studies using this model (Latinne and others 1993; Tanaka and others in press). Recipient cynomolgus monkeys were treated with anti-thymocyte globulin to remove mature T-cell subsets and NK cells. They were treated with sublethal irradiation, similar to that used in our previous rat-mouse studies, and received bone marrow from the pig donor. At the time of operation, the recipient's blood was perfused from the aorta through a freshly isolated pig liver and back to the recipient vena cava for one hour, using Silastic catheters. Following this procedure, a pig renal xenograft was transplanted as a test organ for the induction of transplant tolerance. Our results to date have shown no sign of hyperacute rejection by the absorption , and kidney grafts have survived as long as 15 days. However, we did not achieve persistence of mixed xenogeneic chimerism in this model, nor was long-term tolerance established. Subsequent studies have administered recombinant pig cytokines (IL-3 and SCF) postoperatively in order to favor engraftment of pig bone-marrow elements. These studies remain preliminary at the time of this writing, but are encouraging, especially since the recipients have maintained normal renal function for more than 2 weeks with only a functioning pig xenograft kidney.

SUMMARY

Because xenografts are readily accepted by mutant mice that lack cellular immune function, it seems likely that successful xenografting, even across discordant species barriers, will depend predominantly on effective manipulation of immune responses. However, species-specificity of cytokine interactions and accessory molecule interactions may also have to be considered for long-term success. While immune responses to xenografts show many similarities to immune responses that have been studied extensively for allografts, they also show some differences especially for discordant species barriers. Most work to date for discordant xenografts has concentrated on the problems of humoral immunity. However, cellular responses in vivo are at least as strong or stronger than their allogeneic counterparts. For this reason, the amount of nonspecific immunosuppression required to avoid xenograft rejection is likely to lead to an unacceptably high incidence of infectious complications. It therefore seems likely that the success of clinical xenografting will depend, at least in part, on finding ways of inducing tolerance across xenogeneic barriers rather than relying entirely on nonspecific immunosuppressive agents. One such method that is being pursued in this laboratory involves the use of mixed lymphohematopoietic chimerism to establish specific transplantation tolerance, and the data so far are early but encouraging.

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TABLE 1 Strategies for eliminating natural antibodies
1. Plasmapheresis

2. Absorption

3. Anti-lg

4. Anti B kcell/Plasma-cell Rx

5. Induction of B-cell tolerance

Figure 1 Pig-to-cynomolgus monkey protocol.