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Mixed Chimerism as an Approach to Transplantation Tolerance
Clinical Immunology Vol. 95, No. 1, April, pp. S63–S68, 2000 doi:10.1006/clim.1999.4814, available online at http://www.idealibrary.com on
Mixed Chimerism as an Approach to Transplantation Tolerance David H. Sachs Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129
The induction of tolerance to transplanted organs could make transplantation safer and more uniformly successful. One of the most promising approaches currently being investigated involves the induction of deletional tolerance through the establishment of “mixed chimerism.” In this laboratory, we first studied mixed chimerism as an approach to transplantation tolerance in mice, using a nonmyeloablative preparative regimen consisting of 300 R whole-body irradiation, 700 R thymic irradiation, and treatment with monoclonal antibodies to CD4 and CD8. This approach has subsequently been extended successfully to the induction of tolerance to renal transplants in fully mismatched cynomolgus monkeys. In addition, the same approach, with minor modifications, has been found effective in producing mixed chimerism and transplantation tolerance in the concordant xenogeneic baboon to cynomolgus monkey species combination. Because pigs have many advantages as a potential xenograft donor for humans, we are also trying to extend our nonmyeloablative regimen for production of mixed chimerism to the discordant pig 3 primate combination. We have used absorption of natural antibodies to prevent hyperacute rejection and then proceeded with a mixed chimerism approach. Administration of pig hematopoietic stem cells along with pig recombinant cytokines (SCF and IL-3) to primates has enabled the pig bone marrow to survive in these xenogeneic hosts for over 6 months. This chimerism has apparently been sufficient to markedly diminish T cell immunity and the induction of new T-cell-dependent responses. However, to date we have not succeeded in preventing the return of natural antibodies, which appear to be the cause of eventual loss of organ transplants and are the subject of further intensive investigations. © 2000 Academic Press Key Words: chimerism; tolerance; transplantation; xenotransplantation.
INTRODUCTION
The fact that the major barrier to transplantation is the immune system has been made clear by an experiment of nature, the nude mouse. This animal is born without a thymus and therefore without T cell immu-
nity (it is also born without hair, which is why it is called a “nude” mouse). As part of the original description of the nude mouse, skin was grafted successfully onto these animals from donors of diverse species (1, 2). Even chicken skin was accepted and grew feathers! Thus, it is clear that without a T cell immune system, any transplant will be accepted, even from a xenogeneic source. Indeed, these studies, as well as numerous studies in other systems, indicate that the thymus plays a central role in transplantation immunity. There are three ways to overcome the immune system with respect to transplantation: matching, nonspecific immunosuppression, and the induction of transplantation tolerance. Matching is clearly the simplest path to success. Thus, if one is unfortunate enough to need a transplant, but fortunate enough to have many siblings, then one-fourth of those siblings will be matched for HLA antigens, the most important set of antigens for determining whether or not a transplant will be rejected. In these circumstances, one needs very little immunosuppression, and organ grafts are accepted with excellent long-term results. However, such matching is only applicable to organs which one can transplant without damaging the donor, such as kidney or bone marrow. Some centers are now also performing partial liver and partial lung transplantation, especially into children. However, most transplants must be performed from unmatched donors. The second method for overcoming the immune barrier to transplantation, that of nonspecific immunosuppressive drugs, has been largely responsible for the enormous success that the field of transplantation has witnessed over the past three decades. Such drugs include steroids, antimetabolites, and a variety of monoclonal antibodies. These drugs, although nonspecific, do have selectivity for the T cell compartment of the immune system and for that reason are particularly useful for suppressing the immune response to transplants. In general, such drugs are titrated, so that the patient does not reject the transplant but is not so immunosuppressed that he or she becomes overly susceptible to infections. Nevertheless, infections and malignancies are major complications of these medications. In addition, even with the best of nonspecific immunosuppressive medications, there is an inexora-
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ble loss of grafts, about 7% per year, due to chronic rejection. Therefore, while these drugs have made a major contribution to the field of transplantation, they probably do not represent the final solution for this field. Rather, the ultimate goal of transplantation immunology remains the third approach, that of inducing transplantation tolerance, the subject of the remainder of this article. THE DEFINITION OF TOLERANCE
Tolerance can be defined as the specific absence of an immune response to an antigen. By specific, we mean that the response to the transplant is absent, but immune responses to all other antigens, including bacteria and viruses, remain intact. It is now clear that tolerance is not merely the absence of a response, but may involve an active down-regulatory response and may be induced through a variety of mechanisms. Therefore, transplantation immunologists often utilize an operational definition of tolerance: The survival of a transplant without exogenous immunosuppression. We know that such tolerance occurs routinely for selfantigens, since new T cells reactive to self-antigens are produced continually in the thymus, but are eliminated by negative selection before they exit the thymus, thus avoiding autoimmunity (3). CLINICAL EXAMPLES OF TOLERANCE
We also know that tolerance can be induced for allogeneic transplants, both from a variety of experimental models and from some clinical studies in patients. Indeed, there are patients who have undergone a bone marrow transplant from an HLA identical sibling because of leukemia or another hematologic malignancy and who, years later, developed renal failure and needed a kidney transplant. Fortunately, these patients were still on sufficiently good terms with the marrow-donor sibling that the same sibling was also willing to donate a kidney. In these cases, no additional immunosuppressive drugs were required (4 – 6). The patients all accepted subsequent kidney grafts from the marrow donors long term and became specifically tolerant, showing normal responses to everything else. These cases involved HLA identical siblings; however, the same principle should apply for recipients of bone marrow transplantation (BMT) across any histocompatibility barrier. In other words, successful BMT carries with it the induction of tolerance to transplants of any other tissues from the same donor. TOLERANCE INDUCTION THROUGH MIXED CHIMERISM
BMT for treatment of hematologic malignancies involves ablation of the lymphohematopoietic system of
the host and replacement with donor bone marrow. When the primary goal of bone marrow transplantation is to induce tolerance rather than to treat a hematologic malignancy, it is neither necessary nor desirable to ablate the host’s bone marrow completely and establish a fully allogeneic chimera. This is especially true when one crosses an MHC barrier, in which case there are two major problems: (i) if mature T cells are not removed from the allogeneic bone marrow, then severe graft-versus-host disease results; and (ii) if mature T cells are removed from the allogeneic bone marrow inoculum, then the chimeras which result are relatively immunoincompetent. The probable reason for the immunoincompetence of fully allogeneic complete chimeras lies in the fact that T cells derive their specificity from education in the thymus. There they develop specificity for self-MHC plus peptides of foreign antigens (so called “Self ⫹ X”). New T cells which arise following ablative radiation and reconstitution with allogeneic bone marrow are of the allogeneic donor MHC type, but are educated in a thymus of host MHC type. These new T cells therefore acquire restriction specificities for host MHC ⫹ X. However, the antigen-presenting cells (APC), which are responsible for presenting environmental antigens to mature T cells in the periphery, are also replaced by the bone marrow transplant and are therefore of donor MHC type. The mature T cells thus encounter a different MHC ⫹ X on APC from that to which they were educated in the thymus. Some sharing of specificities is undoubtedly responsible for the weak immune responses which occur in such fully allogeneic chimeras, but the majority of MHC-restricted responses are disabled, leading to relative immunoincompetence (7–9). On the other hand, mixed chimeras have a great advantage over fully allogeneic chimeras in terms of immunocompetence. As illustrated Fig. 1, in which host MHC is indicated as “A” and donor MHC as “B,” mixed chimeras possess bone marrow precursor cells of both host and donor origin (10, 11). Both types of mature T cell populations develop in such animals and are restricted through positive selection in the thymus to the recognition of host MHC ⫹ X (i.e., A ⫹ X1, A ⫹ X2). In this case, antigen-presenting cells are present in the periphery of the host type, and immunocompetent interactions can occur. In addition, donor APC develop in such animals, and among these are dendritic cells which localize in the corticomedullary junction of the thymus. As T cells mature in the thymus, they pass through this site (Fig. 1) and are subjected to the same kind of negative selection (in this case anti-B) which occurs for self (anti-A) during T cell development (12). Thus, mixed chimeras are both immunocompetent and tolerant to self- as well as to donor MHC.
MIXED CHIMERISM AND TOLERANCE
FIG. 1. Development of T cells in the thymus of a mixed allogeneic chimera.
MIXED ALLOGENEIC CHIMERISM IN MICE
Our initial studies were carried out using lethal irradiation and reconstitution with a mixture of T-celldepleted host and donor bone marrow (13). Skin grafts placed on these animals many months later, from the fully allogeneic donor strain, survived permanently. Furthermore, the animals were fully competent to reject a third-party skin graft. However, while these studies proved the principle that mixed chimerism leads to tolerance and immunocompetence, the methodology utilized (i.e., lethal irradiation) would be too toxic to be utilized for ordinary patients in need of a transplant, who could be treated with immunosuppressive drugs. Therefore, we have subsequently concentrated on achieving mixed chimerism without the need for lethal irradiation. The methodology we have utilized most extensively is based on the nonmyeloablative protocol illustrated in Fig. 2 (14). This protocol utilizes monoclonal antibodies to the mature T cell subsets (CD4 and CD8) to eliminate the T cells which otherwise resist engraftment of allogeneic bone marrow. Such treatment permits engraftment with a very low dose (3 Gy) of whole body irradiation or even with no radiation if very large quantities of donor bone marrow are utilized (15). The methodology has also been successful in achieving mixed chimerism and tolerance in the concordant rat to mouse system (16). The nonmyeloablative preparative regimen is much less toxic than the original approach and has therefore been considered to have potential for clinical applications.
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mice, except that: (i) because there are no mAbs available to deplete all mature T cells from a primate, we have used ATG (anti-thymocyte globulin) plus 28 days of cyclosporin (Sandimmune, Novartis) to eliminate or suppress mature T cells during the period of engraftment; (ii) the animals are splenectomized at the time of transplantation, which we have found helps to prevent antibody production; and (iii) animals receive a renal transplant at the same time as they receive the donor bone marrow. In our first series of animals treated by this protocol, eight of nine animals became tolerant to fully MHC-mismatched kidneys long term. In one case, the tolerance was tested further by a skin graft from the donor almost 1 year later, and the donor skin was accepted, while third-party skin was promptly rejected. Since skin is the hardest graft to prolong (19), this finding suggests the induction of durable, systemic tolerance. Some of the animals in this series have now survived more than 5 years since transplantation, with functioning kidney grafts and with no evidence of rejection histologically. Thus, this regimen appears to avoid both acute and chronic rejection, two of the most important limitations to clinical transplantation today. We have also been successful in extending this protocol to the concordant xenograft system of baboon kidneys to cynomolgus monkey (20, 21). MIXED XENOGENEIC CHIMERISM
Finally, we have been testing the possibility of utilizing a similar tolerance-inducing regimen to induce long-term acceptance of xenografts, which, if successful would alleviate another major limitation to the field of transplantation today, namely, donor organ availability. The shortage of organs for transplantation has led many groups to search for means of permitting xenotransplantation over the past two decades (22). Many workers in this field, including the author, believe that the pig will be the donor of choice for xenotransplants. Among the advantages of this species are availability, size (especially that of minature swine (23, 24), which attain the same size as humans), and the potential for genetic engineering of the donor.
MIXED ALLOGENEIC CHIMERISM IN NON-HUMAN PRIMATES
Over the past 8 years, we have begun to apply this model to nonhuman primates, as a preclinical model (17, 18). As shown in Fig. 3, the methodology is almost identical to the nonmyeloablative protocol developed in
FIG. 2. Nonmyeloablative protocol for induction of mixed allogeneic chimerism in mice.
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FIG. 3.
Nonmyeloablative protocol for induction of mixed allogeneic chimerism in monkeys.
On the other hand, the major disadvantage of the pig is that because of its phylogenetic distance from man, there are large amounts of natural antibody in all humans and old world monkeys against all pigs (25, 26). If nothing is done to eliminate the effects of these natural antibodies (Nabs), hyperacute rejection results within minutes to hours after anastomosis of the arterial and venous supplies to a pig organ in a human or old world monkey (22). In addition, for the purposes of tolerance induction through mixed chimerism, there appear to be certain growth factors, or cytokines, that are necessary for bone marrow to survive, and some of these are species specific (27). This too would pose a problem to the extension of our tolerance-inducing regimen to this xenogeneic combination. Fortunately, there are at least two ways to interfere with hyperacute rejection: (i) by removing Nabs prior to transplantation; and (ii) by interfering with complement activation, which is the chief mechanism by
FIG. 4. Nonmyeloablative protocol for induction of mixed xenogeneic chimerism in the pig to primate combination.
which Nabs cause damage to the endothelial cells of the xenograft, resulting in hyperacute rejection. We have been utilizing an absorption procedure to remove Nabs and have thereby avoided hyperacute rejection. Since most of these antibodies are directed toward one sugar determinant, ␣-1,3-galactose, we have used plasmapheresis through columns bearing ␣-1,3-galactose (␣-1,3-Gal) residues to eliminate these antibodies. Unfortunately the Nabs appear to come back, causing a vascular form of rejection in a matter of weeks. We are currently investigating a number of ways of preventing this return of Nabs. Other groups are attempting to prevent complement activation, either by destroying the complement in vivo (28, 29) or by genetically engineering the donor pigs such that complement of the primate is inhibited from causing damage to pig endothelial cells (30, 31). The author believes that a combination of these methodologies, including tolerance induction, will be required for long-term success. The method we are using to try to induce tolerance at the T cell level in the pig to nonhuman primate combination is very similar to that described above for allogeneic models, both in mice and in primates (Fig. 3). The major differences are that (i) before the transplant we perfuse the blood of the recipient baboon through ␣-1,3-Gal columns to remove natural antibodies and thereby prevent hyperacute rejection. (ii) Posttransplant, in addition to treating with cyclosporin for 28 days, we administer one or more drugs designed to prevent Nabs from returning; several of which have helped, but none so far has been totally effective. (iii) Posttransplantation, we administer the pig recombinant growth factors IL-3 and SCF. We have found these cytokines to be effective in enabling pig bone marrow cells to survive for over 1 year in baboons,
MIXED CHIMERISM AND TOLERANCE
albeit at a very low level, detectable only by PCR (32, 33). Using this protocol, we have achieved renal xenograft survival of pig kidneys in baboons of 2–3 weeks, with normal renal function (34, 35). We have also found that the antibodies that return in these animals are the same Nabs, predominantly IgM, and directed entirely against the ␣-1,3-Gal epitope. This result is in contrast to animals exposed to pig tissues without a tolerance-inducing regimen directed toward the T cell response, in which the antibody response usually changes to predominantly IgG and is directed against other pig antigenic epitopes besides ␣-1,3-Gal (36). Nevertheless, this return of Nabs eventually causes hematuria, punctate hemorrhages in the kidney, and finally a full-blown vascular form of rejection. Much ongoing work in our laboratory is directed toward the problem of avoiding this return of Nabs. Indeed, we are hopeful that by combining an approach which can control this humoral response with the tolerance-inducing regimen we have described, we will eventually be able to achieve lasting acceptance of xenogeneic organs across the pig to primate barrier. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants 2RO1 AI31046, 2PO1 HL18646, 5RO1 AI37692, and 1RO1 HL63430. REFERENCES 1. Manning, D. D., Reed, N. D., and Shaffer, C. F., Maintenance of skin xenografts of widely divergent phylogenetic origin of congenitally athymic (nude) mice. J. Exp. Med. 138, 488 – 494, 1973. 2. Rygaard, J., Skin grafts in nude mice. 3. Fate of grafts from man and donors of other taxonomic classes. Acta Pathol. Microbiol. Scand.[A] 82, 105–112, 1974. 3. Sprent, J., Lo, D., Gao, E.-K., and Ron, Y., T cell selection in the thymus. Immunol. Rev. 101, 174 –190, 1988. 4. Helg, C., Chapuis, B., Bolle, J. F., Morel, P., Salomon, D., Roux, E., Antonioli, V., Jeannet, M., and Leski, M., Renal transplantation without immunosuppression in a host with tolerance induced by allogeneic bone marrow transplantation. Transplantation 58, 1420 –1422, 1994. 5. Jacobsen, N., Taaning, E., Ladefoged, J., Kristensen, J. K., and Pedersen, F. K., Tolerance to an HLA-B,DR disparate kidney allograft after bone-marrow transplantation from same donor. Lancet 343, 800 – 800, 1994. 6. Sayegh, M. H., Fine, N. A., Smith, J. L., Rennke, H. G., Milford, E. L., and Tilney, N. L., Immunologic tolerance to renal allografts after bone marrow transplants from the same donors. Ann. Intern. Med. 114, 954 –955, 1991. 7. Zinkernagel, R. M., Althage, A., Waterfield, E., Kindred, B., Welsh, R. M., Callahan, G., and Pincetl, P., Restriction specificities, alloreactivity, and allotolerance expressed by T cells from nude mice reconstituted with H-2-compatible or -incompatible thymus grafts. J. Exp. Med. 151, 376 –399, 1980. 8. Zinkernagel, R. M., Althage, A., Callahan, G., and Welsh, R. M., On the immunocompetence of H-2 incompatible irradiation bone marrow chimeras. J. Immunol. 124, 2356, 1980.
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9. Singer, A., Hathcock, K. S., and Hodes, R. J., Self recognition in allogeneic radiation bone marrow chimeras. J. Exp. Med. 153, 1286 –1301, 1981. 10. Ildstad, S. T., Wren, S. M., Bluestone, J. A., Barbieri, S. A., and Sachs, D. H., Characterization of mixed allogeneic chimeras: Immunocompetence, in vitro reactivity, and genetic specificity of tolerance. J. Exp. Med. 162, 231–244, 1985. 11. Sykes, M., and Sachs, D. H., Mixed allogeneic chimerism as an approach to transplantation tolerance. Immunol. Today 9, 23– 27, 1988. 12. Charlton, B., Auchincloss, H., Jr., and Fathman, C. G., Mechanisms of transplantation tolerance. Annu. Rev. Immunol. 12, 707–734, 1994. 13. Ildstad, S. T., and Sachs, D. H., Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 307(5947), 168 –170, 1984. 14. Sharabi, Y., and Sachs, D. H., Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen. J. Exp. Med. 169, 493–502, 1989. 15. Sykes, M., Szot, G. L., Swenson, K., and Pearson, D. A., Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning. Nature Med. 3, 783–787, 1997. 16. Sharabi, Y., Aksentijevich, I., Sundt, III, T. M., Sachs, D. H., and Sykes, M., Specific tolerance induction across a xenogeneic barrier: production of mixed rat/mouse lymphohematopoietic chimeras using a nonlethal preparative regimen. J. Exp. Med. 172, 195–202, 1990. 17. Kawai, T., Cosimi, A. B., Colvin, R. B., Powelson, J., Eason, J., Kozlowski, T., Sykes, M., Monroy, R., Tanaka, M., and Sachs, D. H., Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 59, 256 –262, 1995. 18. Kimikawa, M., Sachs, D. H., Colvin, R. B., Bartholomew, A., Kawai, T., and Cosimi, A. B., Modifications of the conditioning regimen for achieving mixed chimerism and donor-specific tolerance in cynomolgus monkeys. Transplantation 64, 709 –716, 1997. 19. Lin, Y., Vandeputte, M., and Waer, M., Natural killer cell- and macrophage-mediated rejection of concordant xenografts in the absence of T and B cell responses. J. Immunol. 158, 5658 –5667, 1997. 20. Ko, D. S., Bartholomew, A., Poncelet, A. J., Sachs, D. H., Huang, C., Leguern, A., Abraham, K. I., Colvin, R. B., Boskovic, S., Hong, H. Z., Wee, S. L., Winn, H. J., and Cosimi, A. B., Demonstration of multilineage chimerism in a nonhuman primate concordant xenograft model. Xenotransplantation 5, 298 –304, 1998. 21. Bartholomew, A. M., Powelson, J., Sachs, D. H., Bailin, M., Boskovic, S., Colvin, R. B., Hong, H. Z., Johnson, M., Kimikawa, M., Leguern, A., Meehan, S., Sablinski, T., Wee, S. L., and Cosimi, A. B., Tolerance in a concordant nonhuman primate xenograft model. Transplantation in press. 1998. 22. Auchincloss, H. J., and Sachs, D. H., Xenogeneic transplantation. Annu. Rev. Immunol. 16, 433– 470, 1998. 23. Sachs, D. H., MHC homozygous miniature swine. In “Swine as Models in Biomedical Research” (M. M. Swindle, D. C. Moody, and L. D. Phillips, Eds.), pp. 3–15, Iowa State Univ. Press, Ames, IA, 1992. 24. Sachs, D. H., The pig as a potential xenograft donor. Vet. Immunol. Immunopathol. 43, 185–191, 1994. 25. Oriol, R., Ye, Y., Koren, E., and Cooper, D. K., Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation. Transplantation 56, 1433–1442, 1993.
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26. Sandrin, M. S., Vaughan, H. A., Dabkowski, P. L., and McKenzie, I. F., Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1-3)Gal epitopes. Proc. Natl. Acad. Sci. USA 90, 11391–11395, 1993. 27. Yang, Y.-G., Sergio, J. J., Swenson, K., Glaser, R. M., Monroy, R., and Sykes, M., Donor-specific growth factors promote swine hematopoiesis in SCID mice. Xenotransplantation 3, 92–101, 1996. 28. Davis, E. A., Pruitt, S. K., Greene, P. S., Ibrahim, S., Lam, T. T., Levin, J. L., Baldwin, W. M., III, and Sanfilippo, F., Inhibition of complement, evoked antibody, and cellular response prevents rejection of pig-to-primate cardiac xenografts. Transplantation 62, 1018 –1023, 1996. 29. Kobayashi, T., Taniguchi, S., Neethling, F. A., Rose, A. G., Hancock, W. W., Ye, Y., Niekrasz, M., Kosanke, S., Wright, L. J., White, D. J. G., and Cooper, D. K. C., Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy. Transplantation 64, 1255–1261, 1997. 30. Wang, M. W., Wright, L. J., Sims, M. J., and White, D. J., Presence of human chromosome 1 with expression of humanX decay-accelerating factor (DAF) prevents lysis of mouse/humanX hybrid cells by human complement.X. Scand. J. Immunol. 34, 771–78X, 1991. 31. Byrne, G. W., McCurry, K. R., Kagan, D., Quinn, C., Martin, M. J., Platt, J. L., and Logan, J. S., Protection of xenogeneic cardiac endothelium from human complement by expression of Received September 1, 1999; accepted November 3, 1999
32.
33.
34. 35.
36.
CD59 or DAF in transgenic mice. Transplantation 60, 1149 – 1156, 1995. Sachs, D. H., Sykes, M., Greenstein, J. L., and Cosimi, A. B., Tolerance and xenograft survival. Nature Med. 1, 969 –969, 1995. Sablinski, T., Emery, D. W., Monroy, R., Hawley, R. J., Xu, Y., Gianello, P., Lorf, T., Kozlowski, T., Bailin, M., Cooper, D. K., Cosimi, A. B., and Sachs, D. H., Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors. Transplantation 67, 972–977, 1999. Sachs, D. H., and Sablinski, T., Tolerance across discordant xenogeneic barriers. Xenotransplantation 2, 234 –239, 1995. Kozlowski, T., Shimizu, A., Lambrigts, D., Yamada, K., Fuchimoto, Y., Glaser, R., Monroy, R., Xu, Y., Awwad, M., Colvin, R. B., Cosimi, A. B., Robson, S. C., Fishman, J., Spitzer, T. R., Cooper, D. K., and Sachs, D. H., Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation 67, 18 –30, 1999. Kozlowski, T., Monroy, R., Xu, Y., Glaser, R., Awwad, M., Cooper, D. K., and Sachs, D. H., Anti-Gal(alpha)1-3Gal antibody response to porcine bone marrow in unmodified baboons and baboons conditioned for tolerance induction. Transplantation 66, 176 –182, 1998.
Mixed Chimerism as an Approach to Transplantation Tolerance David H. Sachs Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129
The induction of tolerance to transplanted organs could make transplantation safer and more uniformly successful. One of the most promising approaches currently being investigated involves the induction of deletional tolerance through the establishment of “mixed chimerism.” In this laboratory, we first studied mixed chimerism as an approach to transplantation tolerance in mice, using a nonmyeloablative preparative regimen consisting of 300 R whole-body irradiation, 700 R thymic irradiation, and treatment with monoclonal antibodies to CD4 and CD8. This approach has subsequently been extended successfully to the induction of tolerance to renal transplants in fully mismatched cynomolgus monkeys. In addition, the same approach, with minor modifications, has been found effective in producing mixed chimerism and transplantation tolerance in the concordant xenogeneic baboon to cynomolgus monkey species combination. Because pigs have many advantages as a potential xenograft donor for humans, we are also trying to extend our nonmyeloablative regimen for production of mixed chimerism to the discordant pig 3 primate combination. We have used absorption of natural antibodies to prevent hyperacute rejection and then proceeded with a mixed chimerism approach. Administration of pig hematopoietic stem cells along with pig recombinant cytokines (SCF and IL-3) to primates has enabled the pig bone marrow to survive in these xenogeneic hosts for over 6 months. This chimerism has apparently been sufficient to markedly diminish T cell immunity and the induction of new T-cell-dependent responses. However, to date we have not succeeded in preventing the return of natural antibodies, which appear to be the cause of eventual loss of organ transplants and are the subject of further intensive investigations. © 2000 Academic Press Key Words: chimerism; tolerance; transplantation; xenotransplantation.
INTRODUCTION
The fact that the major barrier to transplantation is the immune system has been made clear by an experiment of nature, the nude mouse. This animal is born without a thymus and therefore without T cell immu-
nity (it is also born without hair, which is why it is called a “nude” mouse). As part of the original description of the nude mouse, skin was grafted successfully onto these animals from donors of diverse species (1, 2). Even chicken skin was accepted and grew feathers! Thus, it is clear that without a T cell immune system, any transplant will be accepted, even from a xenogeneic source. Indeed, these studies, as well as numerous studies in other systems, indicate that the thymus plays a central role in transplantation immunity. There are three ways to overcome the immune system with respect to transplantation: matching, nonspecific immunosuppression, and the induction of transplantation tolerance. Matching is clearly the simplest path to success. Thus, if one is unfortunate enough to need a transplant, but fortunate enough to have many siblings, then one-fourth of those siblings will be matched for HLA antigens, the most important set of antigens for determining whether or not a transplant will be rejected. In these circumstances, one needs very little immunosuppression, and organ grafts are accepted with excellent long-term results. However, such matching is only applicable to organs which one can transplant without damaging the donor, such as kidney or bone marrow. Some centers are now also performing partial liver and partial lung transplantation, especially into children. However, most transplants must be performed from unmatched donors. The second method for overcoming the immune barrier to transplantation, that of nonspecific immunosuppressive drugs, has been largely responsible for the enormous success that the field of transplantation has witnessed over the past three decades. Such drugs include steroids, antimetabolites, and a variety of monoclonal antibodies. These drugs, although nonspecific, do have selectivity for the T cell compartment of the immune system and for that reason are particularly useful for suppressing the immune response to transplants. In general, such drugs are titrated, so that the patient does not reject the transplant but is not so immunosuppressed that he or she becomes overly susceptible to infections. Nevertheless, infections and malignancies are major complications of these medications. In addition, even with the best of nonspecific immunosuppressive medications, there is an inexora-
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ble loss of grafts, about 7% per year, due to chronic rejection. Therefore, while these drugs have made a major contribution to the field of transplantation, they probably do not represent the final solution for this field. Rather, the ultimate goal of transplantation immunology remains the third approach, that of inducing transplantation tolerance, the subject of the remainder of this article. THE DEFINITION OF TOLERANCE
Tolerance can be defined as the specific absence of an immune response to an antigen. By specific, we mean that the response to the transplant is absent, but immune responses to all other antigens, including bacteria and viruses, remain intact. It is now clear that tolerance is not merely the absence of a response, but may involve an active down-regulatory response and may be induced through a variety of mechanisms. Therefore, transplantation immunologists often utilize an operational definition of tolerance: The survival of a transplant without exogenous immunosuppression. We know that such tolerance occurs routinely for selfantigens, since new T cells reactive to self-antigens are produced continually in the thymus, but are eliminated by negative selection before they exit the thymus, thus avoiding autoimmunity (3). CLINICAL EXAMPLES OF TOLERANCE
We also know that tolerance can be induced for allogeneic transplants, both from a variety of experimental models and from some clinical studies in patients. Indeed, there are patients who have undergone a bone marrow transplant from an HLA identical sibling because of leukemia or another hematologic malignancy and who, years later, developed renal failure and needed a kidney transplant. Fortunately, these patients were still on sufficiently good terms with the marrow-donor sibling that the same sibling was also willing to donate a kidney. In these cases, no additional immunosuppressive drugs were required (4 – 6). The patients all accepted subsequent kidney grafts from the marrow donors long term and became specifically tolerant, showing normal responses to everything else. These cases involved HLA identical siblings; however, the same principle should apply for recipients of bone marrow transplantation (BMT) across any histocompatibility barrier. In other words, successful BMT carries with it the induction of tolerance to transplants of any other tissues from the same donor. TOLERANCE INDUCTION THROUGH MIXED CHIMERISM
BMT for treatment of hematologic malignancies involves ablation of the lymphohematopoietic system of
the host and replacement with donor bone marrow. When the primary goal of bone marrow transplantation is to induce tolerance rather than to treat a hematologic malignancy, it is neither necessary nor desirable to ablate the host’s bone marrow completely and establish a fully allogeneic chimera. This is especially true when one crosses an MHC barrier, in which case there are two major problems: (i) if mature T cells are not removed from the allogeneic bone marrow, then severe graft-versus-host disease results; and (ii) if mature T cells are removed from the allogeneic bone marrow inoculum, then the chimeras which result are relatively immunoincompetent. The probable reason for the immunoincompetence of fully allogeneic complete chimeras lies in the fact that T cells derive their specificity from education in the thymus. There they develop specificity for self-MHC plus peptides of foreign antigens (so called “Self ⫹ X”). New T cells which arise following ablative radiation and reconstitution with allogeneic bone marrow are of the allogeneic donor MHC type, but are educated in a thymus of host MHC type. These new T cells therefore acquire restriction specificities for host MHC ⫹ X. However, the antigen-presenting cells (APC), which are responsible for presenting environmental antigens to mature T cells in the periphery, are also replaced by the bone marrow transplant and are therefore of donor MHC type. The mature T cells thus encounter a different MHC ⫹ X on APC from that to which they were educated in the thymus. Some sharing of specificities is undoubtedly responsible for the weak immune responses which occur in such fully allogeneic chimeras, but the majority of MHC-restricted responses are disabled, leading to relative immunoincompetence (7–9). On the other hand, mixed chimeras have a great advantage over fully allogeneic chimeras in terms of immunocompetence. As illustrated Fig. 1, in which host MHC is indicated as “A” and donor MHC as “B,” mixed chimeras possess bone marrow precursor cells of both host and donor origin (10, 11). Both types of mature T cell populations develop in such animals and are restricted through positive selection in the thymus to the recognition of host MHC ⫹ X (i.e., A ⫹ X1, A ⫹ X2). In this case, antigen-presenting cells are present in the periphery of the host type, and immunocompetent interactions can occur. In addition, donor APC develop in such animals, and among these are dendritic cells which localize in the corticomedullary junction of the thymus. As T cells mature in the thymus, they pass through this site (Fig. 1) and are subjected to the same kind of negative selection (in this case anti-B) which occurs for self (anti-A) during T cell development (12). Thus, mixed chimeras are both immunocompetent and tolerant to self- as well as to donor MHC.
MIXED CHIMERISM AND TOLERANCE
FIG. 1. Development of T cells in the thymus of a mixed allogeneic chimera.
MIXED ALLOGENEIC CHIMERISM IN MICE
Our initial studies were carried out using lethal irradiation and reconstitution with a mixture of T-celldepleted host and donor bone marrow (13). Skin grafts placed on these animals many months later, from the fully allogeneic donor strain, survived permanently. Furthermore, the animals were fully competent to reject a third-party skin graft. However, while these studies proved the principle that mixed chimerism leads to tolerance and immunocompetence, the methodology utilized (i.e., lethal irradiation) would be too toxic to be utilized for ordinary patients in need of a transplant, who could be treated with immunosuppressive drugs. Therefore, we have subsequently concentrated on achieving mixed chimerism without the need for lethal irradiation. The methodology we have utilized most extensively is based on the nonmyeloablative protocol illustrated in Fig. 2 (14). This protocol utilizes monoclonal antibodies to the mature T cell subsets (CD4 and CD8) to eliminate the T cells which otherwise resist engraftment of allogeneic bone marrow. Such treatment permits engraftment with a very low dose (3 Gy) of whole body irradiation or even with no radiation if very large quantities of donor bone marrow are utilized (15). The methodology has also been successful in achieving mixed chimerism and tolerance in the concordant rat to mouse system (16). The nonmyeloablative preparative regimen is much less toxic than the original approach and has therefore been considered to have potential for clinical applications.
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mice, except that: (i) because there are no mAbs available to deplete all mature T cells from a primate, we have used ATG (anti-thymocyte globulin) plus 28 days of cyclosporin (Sandimmune, Novartis) to eliminate or suppress mature T cells during the period of engraftment; (ii) the animals are splenectomized at the time of transplantation, which we have found helps to prevent antibody production; and (iii) animals receive a renal transplant at the same time as they receive the donor bone marrow. In our first series of animals treated by this protocol, eight of nine animals became tolerant to fully MHC-mismatched kidneys long term. In one case, the tolerance was tested further by a skin graft from the donor almost 1 year later, and the donor skin was accepted, while third-party skin was promptly rejected. Since skin is the hardest graft to prolong (19), this finding suggests the induction of durable, systemic tolerance. Some of the animals in this series have now survived more than 5 years since transplantation, with functioning kidney grafts and with no evidence of rejection histologically. Thus, this regimen appears to avoid both acute and chronic rejection, two of the most important limitations to clinical transplantation today. We have also been successful in extending this protocol to the concordant xenograft system of baboon kidneys to cynomolgus monkey (20, 21). MIXED XENOGENEIC CHIMERISM
Finally, we have been testing the possibility of utilizing a similar tolerance-inducing regimen to induce long-term acceptance of xenografts, which, if successful would alleviate another major limitation to the field of transplantation today, namely, donor organ availability. The shortage of organs for transplantation has led many groups to search for means of permitting xenotransplantation over the past two decades (22). Many workers in this field, including the author, believe that the pig will be the donor of choice for xenotransplants. Among the advantages of this species are availability, size (especially that of minature swine (23, 24), which attain the same size as humans), and the potential for genetic engineering of the donor.
MIXED ALLOGENEIC CHIMERISM IN NON-HUMAN PRIMATES
Over the past 8 years, we have begun to apply this model to nonhuman primates, as a preclinical model (17, 18). As shown in Fig. 3, the methodology is almost identical to the nonmyeloablative protocol developed in
FIG. 2. Nonmyeloablative protocol for induction of mixed allogeneic chimerism in mice.
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FIG. 3.
Nonmyeloablative protocol for induction of mixed allogeneic chimerism in monkeys.
On the other hand, the major disadvantage of the pig is that because of its phylogenetic distance from man, there are large amounts of natural antibody in all humans and old world monkeys against all pigs (25, 26). If nothing is done to eliminate the effects of these natural antibodies (Nabs), hyperacute rejection results within minutes to hours after anastomosis of the arterial and venous supplies to a pig organ in a human or old world monkey (22). In addition, for the purposes of tolerance induction through mixed chimerism, there appear to be certain growth factors, or cytokines, that are necessary for bone marrow to survive, and some of these are species specific (27). This too would pose a problem to the extension of our tolerance-inducing regimen to this xenogeneic combination. Fortunately, there are at least two ways to interfere with hyperacute rejection: (i) by removing Nabs prior to transplantation; and (ii) by interfering with complement activation, which is the chief mechanism by
FIG. 4. Nonmyeloablative protocol for induction of mixed xenogeneic chimerism in the pig to primate combination.
which Nabs cause damage to the endothelial cells of the xenograft, resulting in hyperacute rejection. We have been utilizing an absorption procedure to remove Nabs and have thereby avoided hyperacute rejection. Since most of these antibodies are directed toward one sugar determinant, ␣-1,3-galactose, we have used plasmapheresis through columns bearing ␣-1,3-galactose (␣-1,3-Gal) residues to eliminate these antibodies. Unfortunately the Nabs appear to come back, causing a vascular form of rejection in a matter of weeks. We are currently investigating a number of ways of preventing this return of Nabs. Other groups are attempting to prevent complement activation, either by destroying the complement in vivo (28, 29) or by genetically engineering the donor pigs such that complement of the primate is inhibited from causing damage to pig endothelial cells (30, 31). The author believes that a combination of these methodologies, including tolerance induction, will be required for long-term success. The method we are using to try to induce tolerance at the T cell level in the pig to nonhuman primate combination is very similar to that described above for allogeneic models, both in mice and in primates (Fig. 3). The major differences are that (i) before the transplant we perfuse the blood of the recipient baboon through ␣-1,3-Gal columns to remove natural antibodies and thereby prevent hyperacute rejection. (ii) Posttransplant, in addition to treating with cyclosporin for 28 days, we administer one or more drugs designed to prevent Nabs from returning; several of which have helped, but none so far has been totally effective. (iii) Posttransplantation, we administer the pig recombinant growth factors IL-3 and SCF. We have found these cytokines to be effective in enabling pig bone marrow cells to survive for over 1 year in baboons,
MIXED CHIMERISM AND TOLERANCE
albeit at a very low level, detectable only by PCR (32, 33). Using this protocol, we have achieved renal xenograft survival of pig kidneys in baboons of 2–3 weeks, with normal renal function (34, 35). We have also found that the antibodies that return in these animals are the same Nabs, predominantly IgM, and directed entirely against the ␣-1,3-Gal epitope. This result is in contrast to animals exposed to pig tissues without a tolerance-inducing regimen directed toward the T cell response, in which the antibody response usually changes to predominantly IgG and is directed against other pig antigenic epitopes besides ␣-1,3-Gal (36). Nevertheless, this return of Nabs eventually causes hematuria, punctate hemorrhages in the kidney, and finally a full-blown vascular form of rejection. Much ongoing work in our laboratory is directed toward the problem of avoiding this return of Nabs. Indeed, we are hopeful that by combining an approach which can control this humoral response with the tolerance-inducing regimen we have described, we will eventually be able to achieve lasting acceptance of xenogeneic organs across the pig to primate barrier. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants 2RO1 AI31046, 2PO1 HL18646, 5RO1 AI37692, and 1RO1 HL63430. REFERENCES 1. Manning, D. D., Reed, N. D., and Shaffer, C. F., Maintenance of skin xenografts of widely divergent phylogenetic origin of congenitally athymic (nude) mice. J. Exp. Med. 138, 488 – 494, 1973. 2. Rygaard, J., Skin grafts in nude mice. 3. Fate of grafts from man and donors of other taxonomic classes. Acta Pathol. Microbiol. Scand.[A] 82, 105–112, 1974. 3. Sprent, J., Lo, D., Gao, E.-K., and Ron, Y., T cell selection in the thymus. Immunol. Rev. 101, 174 –190, 1988. 4. Helg, C., Chapuis, B., Bolle, J. F., Morel, P., Salomon, D., Roux, E., Antonioli, V., Jeannet, M., and Leski, M., Renal transplantation without immunosuppression in a host with tolerance induced by allogeneic bone marrow transplantation. Transplantation 58, 1420 –1422, 1994. 5. Jacobsen, N., Taaning, E., Ladefoged, J., Kristensen, J. K., and Pedersen, F. K., Tolerance to an HLA-B,DR disparate kidney allograft after bone-marrow transplantation from same donor. Lancet 343, 800 – 800, 1994. 6. Sayegh, M. H., Fine, N. A., Smith, J. L., Rennke, H. G., Milford, E. L., and Tilney, N. L., Immunologic tolerance to renal allografts after bone marrow transplants from the same donors. Ann. Intern. Med. 114, 954 –955, 1991. 7. Zinkernagel, R. M., Althage, A., Waterfield, E., Kindred, B., Welsh, R. M., Callahan, G., and Pincetl, P., Restriction specificities, alloreactivity, and allotolerance expressed by T cells from nude mice reconstituted with H-2-compatible or -incompatible thymus grafts. J. Exp. Med. 151, 376 –399, 1980. 8. Zinkernagel, R. M., Althage, A., Callahan, G., and Welsh, R. M., On the immunocompetence of H-2 incompatible irradiation bone marrow chimeras. J. Immunol. 124, 2356, 1980.
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9. Singer, A., Hathcock, K. S., and Hodes, R. J., Self recognition in allogeneic radiation bone marrow chimeras. J. Exp. Med. 153, 1286 –1301, 1981. 10. Ildstad, S. T., Wren, S. M., Bluestone, J. A., Barbieri, S. A., and Sachs, D. H., Characterization of mixed allogeneic chimeras: Immunocompetence, in vitro reactivity, and genetic specificity of tolerance. J. Exp. Med. 162, 231–244, 1985. 11. Sykes, M., and Sachs, D. H., Mixed allogeneic chimerism as an approach to transplantation tolerance. Immunol. Today 9, 23– 27, 1988. 12. Charlton, B., Auchincloss, H., Jr., and Fathman, C. G., Mechanisms of transplantation tolerance. Annu. Rev. Immunol. 12, 707–734, 1994. 13. Ildstad, S. T., and Sachs, D. H., Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 307(5947), 168 –170, 1984. 14. Sharabi, Y., and Sachs, D. H., Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen. J. Exp. Med. 169, 493–502, 1989. 15. Sykes, M., Szot, G. L., Swenson, K., and Pearson, D. A., Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning. Nature Med. 3, 783–787, 1997. 16. Sharabi, Y., Aksentijevich, I., Sundt, III, T. M., Sachs, D. H., and Sykes, M., Specific tolerance induction across a xenogeneic barrier: production of mixed rat/mouse lymphohematopoietic chimeras using a nonlethal preparative regimen. J. Exp. Med. 172, 195–202, 1990. 17. Kawai, T., Cosimi, A. B., Colvin, R. B., Powelson, J., Eason, J., Kozlowski, T., Sykes, M., Monroy, R., Tanaka, M., and Sachs, D. H., Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 59, 256 –262, 1995. 18. Kimikawa, M., Sachs, D. H., Colvin, R. B., Bartholomew, A., Kawai, T., and Cosimi, A. B., Modifications of the conditioning regimen for achieving mixed chimerism and donor-specific tolerance in cynomolgus monkeys. Transplantation 64, 709 –716, 1997. 19. Lin, Y., Vandeputte, M., and Waer, M., Natural killer cell- and macrophage-mediated rejection of concordant xenografts in the absence of T and B cell responses. J. Immunol. 158, 5658 –5667, 1997. 20. Ko, D. S., Bartholomew, A., Poncelet, A. J., Sachs, D. H., Huang, C., Leguern, A., Abraham, K. I., Colvin, R. B., Boskovic, S., Hong, H. Z., Wee, S. L., Winn, H. J., and Cosimi, A. B., Demonstration of multilineage chimerism in a nonhuman primate concordant xenograft model. Xenotransplantation 5, 298 –304, 1998. 21. Bartholomew, A. M., Powelson, J., Sachs, D. H., Bailin, M., Boskovic, S., Colvin, R. B., Hong, H. Z., Johnson, M., Kimikawa, M., Leguern, A., Meehan, S., Sablinski, T., Wee, S. L., and Cosimi, A. B., Tolerance in a concordant nonhuman primate xenograft model. Transplantation in press. 1998. 22. Auchincloss, H. J., and Sachs, D. H., Xenogeneic transplantation. Annu. Rev. Immunol. 16, 433– 470, 1998. 23. Sachs, D. H., MHC homozygous miniature swine. In “Swine as Models in Biomedical Research” (M. M. Swindle, D. C. Moody, and L. D. Phillips, Eds.), pp. 3–15, Iowa State Univ. Press, Ames, IA, 1992. 24. Sachs, D. H., The pig as a potential xenograft donor. Vet. Immunol. Immunopathol. 43, 185–191, 1994. 25. Oriol, R., Ye, Y., Koren, E., and Cooper, D. K., Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation. Transplantation 56, 1433–1442, 1993.
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26. Sandrin, M. S., Vaughan, H. A., Dabkowski, P. L., and McKenzie, I. F., Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1-3)Gal epitopes. Proc. Natl. Acad. Sci. USA 90, 11391–11395, 1993. 27. Yang, Y.-G., Sergio, J. J., Swenson, K., Glaser, R. M., Monroy, R., and Sykes, M., Donor-specific growth factors promote swine hematopoiesis in SCID mice. Xenotransplantation 3, 92–101, 1996. 28. Davis, E. A., Pruitt, S. K., Greene, P. S., Ibrahim, S., Lam, T. T., Levin, J. L., Baldwin, W. M., III, and Sanfilippo, F., Inhibition of complement, evoked antibody, and cellular response prevents rejection of pig-to-primate cardiac xenografts. Transplantation 62, 1018 –1023, 1996. 29. Kobayashi, T., Taniguchi, S., Neethling, F. A., Rose, A. G., Hancock, W. W., Ye, Y., Niekrasz, M., Kosanke, S., Wright, L. J., White, D. J. G., and Cooper, D. K. C., Delayed xenograft rejection of pig-to-baboon cardiac transplants after cobra venom factor therapy. Transplantation 64, 1255–1261, 1997. 30. Wang, M. W., Wright, L. J., Sims, M. J., and White, D. J., Presence of human chromosome 1 with expression of humanX decay-accelerating factor (DAF) prevents lysis of mouse/humanX hybrid cells by human complement.X. Scand. J. Immunol. 34, 771–78X, 1991. 31. Byrne, G. W., McCurry, K. R., Kagan, D., Quinn, C., Martin, M. J., Platt, J. L., and Logan, J. S., Protection of xenogeneic cardiac endothelium from human complement by expression of Received September 1, 1999; accepted November 3, 1999
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CD59 or DAF in transgenic mice. Transplantation 60, 1149 – 1156, 1995. Sachs, D. H., Sykes, M., Greenstein, J. L., and Cosimi, A. B., Tolerance and xenograft survival. Nature Med. 1, 969 –969, 1995. Sablinski, T., Emery, D. W., Monroy, R., Hawley, R. J., Xu, Y., Gianello, P., Lorf, T., Kozlowski, T., Bailin, M., Cooper, D. K., Cosimi, A. B., and Sachs, D. H., Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors. Transplantation 67, 972–977, 1999. Sachs, D. H., and Sablinski, T., Tolerance across discordant xenogeneic barriers. Xenotransplantation 2, 234 –239, 1995. Kozlowski, T., Shimizu, A., Lambrigts, D., Yamada, K., Fuchimoto, Y., Glaser, R., Monroy, R., Xu, Y., Awwad, M., Colvin, R. B., Cosimi, A. B., Robson, S. C., Fishman, J., Spitzer, T. R., Cooper, D. K., and Sachs, D. H., Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption. Transplantation 67, 18 –30, 1999. Kozlowski, T., Monroy, R., Xu, Y., Glaser, R., Awwad, M., Cooper, D. K., and Sachs, D. H., Anti-Gal(alpha)1-3Gal antibody response to porcine bone marrow in unmodified baboons and baboons conditioned for tolerance induction. Transplantation 66, 176 –182, 1998.