- Email: [email protected]
Induction of tolerance
Volume 135 Number 4
SURGERY APRIL 2004
Surgical research review Induction of tolerance Thomas Wekerle, MD, and Megan Sykes, MD, Vienna, Austria, and Boston, Mass
From the Department of Surgery, Division of Transplantation, Vienna General Hospital/University of Vienna, Vienna, Austria; and the Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Mass
‘‘The experiments to be described in this article provide a solution—at present only a ÔlaboratoryÕ solution—of the problem of how to make tissue homografts immunologically acceptable to hosts which would normally react against them.’’1 Billingham et al During the 50 years after the publication of this paper by Billingham et al,1 the transfer of experimental tolerance protocols from the laboratory to the clinical arena has been very limited. Only when this translation is realized fully will the results of organ transplantation be optimized. We focus on recent progress in the field that we consider relevant for the development of clinically applicable tolerance strategies. Over the last 2 decades, progress in surgical technique and medical and anesthesiologic treatment has led to substantially improved short-term Supported in part by grants from the Roche Organ Transplantation Research Foundation (#110578928) and National Institutes of Health RO1 grants HL49915 and AI05558. Accepted for publication October 25, 2003. Reprint requests: Megan Sykes, MD, Transplantation Biology Research Center, Bone Marrow Transplantation Section, Massachusetts General Hospital/Harvard Medical School, MGH-East Bldg 149-5102, 13th St, Boston, MA 02129. Surgery 2004;135:359-64. 0039-6060/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.surg.2003.10.010
outcomes after organ transplantation. Long-term results, however, have improved to a lesser degree and are limited by graft loss because of chronic rejection and by the side-effects of life-long, nonspecific immunosuppressive therapy. In an attempt to reduce these effects, recent trials have been designed to minimize immunosuppression in transplant recipients. Patients were treated with conventional immunosuppressive drugs immediately after the transplant and were maintained on minimal immunosuppression.2-4 The results of such trials suggest that some organ transplant recipients tolerate very low immunosuppression and that other recipients do not tolerate reduced immunosuppression and experience acute rejection of the grafts. At present, however, we cannot predict reliably who will fall into the latter group. Longer follow-up periods and additional larger studies will reveal whether there is an overall benefit to reduced immunosuppression. These studies suggest, however, that ‘‘spontaneous’’ development of profound hyporesponsiveness or tolerance, which could allow complete drug withdrawal, is quite rare under conventional immunosuppression. Thus, the deliberate induction of robust donor-specific tolerance (ie, a state without drug therapy in which the immune system does not mount a harmful response against the graft but is left otherwise intact) is required to provide a full solution to the problems of both chronic rejection and drug toxicity. SURGERY 359
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EXPERIMENTAL TOLERANCE INDUCTION: WHICH STRATEGY COULD BECOME CLINICALLY APPLICABLE? There is an abundance of experimental tolerance protocols in rodents, and these small animal models are indispensable for the development of tolerance strategies, as evidenced by the fact that the few protocols that are successful in large animals have been based on extensive rodent studies. However, the limitations of rodent systems must be considered when the results from mouse or rat experiments are interpreted. Long-term acceptance of vascularized organs (eg, hearts) can be achieved relatively easily with numerous approaches in rodents. These successes, however, rarely are reproducible in large animal or nonhuman primate studies. To improve the chances of successful clinical development, it is therefore important to apply the strictest available tolerance tests in rodent models (often considered to be the permanent acceptance of skin from a major histocompatibility complex (MHC)–mismatched donor). Only very few strategies meet this test. Among them, the macrochimerism approach has been demonstrated most extensively by several independent groups who reproducibly induced donor-specific skin graft tolerance in various allogeneic murine models and strain combinations. Hematopoietic macrochimerism denotes the presence of substantial numbers (more than 1%) of donor hematopoietic cells in a recipient; if donor cells constitute more than 1% but less than 100%, we refer to it as mixed chimerism. The macrochimerism approach has several additional features that make it a good candidate for clinical development5: (1) Macrochimerism relies in large part on clonal deletion as a mechanism of tolerance. Because deletion leads to the physical elimination of cells with donor reactivity, it is a robust mechanism. (2) Large animal models (including nonhuman primate studies) that establish tolerance through macrochimerism have been developed successfully. (3) Macrochimerism can be measured readily and could serve as a surrogate marker of successful tolerance induction in the clinical setting. (4) Evidence that macrochimerism can also lead to tolerance in humans comes from several cases in which an organ (usually a kidney) has been transplanted into a patient who had undergone transplantation previously with the bone marrow from the same donor (for a hematologic indication) and in which the organ was accepted long-term without immunosuppression.6 (5) The first clinical tolerance studies that rely on macrochimerism have
Surgery April 2004
been initiated successfully. Given this list of attractive features, it might be surprising that macrochimerism has not made it into routine clinical practice of organ transplantation so far. Above all else, there is 1 major obstacle impeding its success: The transplantation of bone marrow (or mobilized peripheral blood leukocytes) across MHC barriers requires the conditioning of the recipient, which can be associated with considerable morbidity and death. Therefore, much effort has been focused on the development of lower-risk protocols of bone marrow transplantation (BMT) that are associated with toxicities that are acceptable for application in organ transplantation. NONTOXIC PROTOCOLS FOR THE INDUCTION OF MACROCHIMERISM AND TOLERANCE Two elements have been traditionally included in conditioning regimens in models of allogeneic macrochimerism: (1) irradiation or cytotoxic drug treatment of the recipient, primarily to promote engraftment of the infused donor hematopoietic stem cells and (2) global destruction of the host T cells (by the use of high doses of specific T cell– depleting antibodies or myeloablation) to prevent rejection of the donor cells. Both modalities have significant toxicities. The earliest macrochimerism protocols for tolerance induction included myeloablation of the recipient by lethal total-body irradiation (TBI) before the BMT.7 Subsequently, several regimens were developed that used a milder, nonmyeloablative conditioning together with nonspecific T cell depletion.8-11 However, the avoidance of myelosuppression altogether proved to be difficult because not even syngeneic hematopoietic stem cells readily engraft in unconditioned hosts when administered in conventional doses. This requirement for myelosuppression eventually was overcome in syngeneic models by the injection of very high numbers of bone marrow cells.12 When these findings were applied to the allogeneic setting, the transplantation of similarly high doses of MHCmismatched bone marrow together with T cell depletion allowed the induction of macrochimerism and tolerance without TBI or cytotoxic drug treatment, as long as a moderate dose of selective irradiation to the thymic area was given.13 However, not even the repeated injection of monoclonal cytotoxic T cell antibodies permitted the complete elimination of irradiation from this model.14 The recent introduction of costimulation-blocking reagents as components of BMT protocols has allowed the complete avoidance of non-specific T
Surgery Volume 135, Number 4
cell depletion and myelosuppression and thus has led to the hitherto mildest experimental macrochimerism protocols. The most commonly used costimulation blockers interfere with the CD28/ CTLA4–CD80/CD86 and the CD40L (CD154)– CD40 pathways. Such reagents, when used alone (without BMT), have profound immunosuppressive properties in various transplantation models.15-17 Costimulation blockade alone, however, usually does not induce tolerance robust enough to meet the most stringent test, namely permanent skin graft acceptance across MHC barriers in euthymic recipients (except in a susceptible strain when combined with the use of rapamycin).18,19 Certain mouse strains are more susceptible to the tolerogenic effects of costimulation blockade than others,20 and the most stringent tests of tolerance should include the ‘‘difficult’’ strains. Costimulation blockade with CTLA4Ig, anti-CD80/86, antiCD40, anti-CD154 monoclonal antibodies, and various combinations thereof has also been effective as immunosuppressive treatment in non-human primate kidney transplantation models, but again did not induce tolerance.16,21,22 These antibodies have a unique effect, however, when used together with the transplantation of bone marrow. The use of an anti-CD154 monoclonal antibody, with or without CTLA4Ig, allowed the induction of high levels of lasting, multilineage macrochimerism in several nonmyeloablative murine BMT models without cytotoxic T cell monoclonal antibodies, even in strains that were quite resistant to the effects of these monoclonal antibodies in the absence of BMT.23-27 The progressive extrathymic deletion of donor-reactive T cells immediately after BMT and anti-CD154 treatment seems to be a critical mechanism by which these protocols achieve macrochimerism in the presence of an essentially intact peripheral and intrathymic host T cell repertoire.23 However, tolerance occurs in these models before deletion is complete,28 which points to an important role for nondeletional mechanisms during the induction phase of tolerance immediately after BMT. As in previous macrochimerism models, tolerance seems to be maintained long-term predominantly through intrathymic clonal deletion. A course of conventional immunosuppression can be added to a protocol of BMT plus costimulation blockade without interference with tolerance induction and even allows reduction in the dose of irradiation that is required to allow the establishment of lasting macrochimerism with the use of standard doses of bone marrow,29 which further increases the clinical feasibility of this approach.
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By the use of costimulation blockade (again either anti-CD154 alone or combined with CTLA4Ig) together with very high doses of bone marrow (approximately 12-fold the usual dose), lasting allogeneic macrochimerism and robust tolerance were induced without any cytoreductive host conditioning (no irradiation, cytotoxic drugs, or monoclonal antibodies).30,31 Although such high doses of bone marrow would not be available routinely in the clinical setting, these models are proof that completely nonmyelosuppressive BMT regimens are possible. Protocols that achieve macrochimerism and tolerance with the use of clinically feasible numbers of bone marrow without cytoreduction, however, still must be developed. PRECLINICAL LARGE ANIMAL PROTOCOLS FOR THE INDUCTION OF MACROCHIMERISM AND TOLERANCE Macrochimerism and tolerance are induced with greater difficulty in large animals than in rodents. Although substantial progress has been achieved in large animals, the latest, least toxic rodent regimens still must be adapted for large animals. One reason for the increased resistance to macrochimerism and tolerance may be that outbred large animals live in a less sheltered environment than inbred mice or rats and are exposed to a much wider range of antigens. They consequently have a higher frequency of primed T cells, which cross-react with transplantation antigens. Because primed T cells are more difficult to make tolerant,32 intensified conditioning may be required in large animals to obtain lasting chimerism and tolerance. Furthermore, a cohort of outbred animals demonstrates a substantially higher variability in their immune responses than a particular inbred strain of rodents. A further potential reason for the differences in results between studies of tolerance in small and large animals is the lack of equally effective reagents for use in large animals, as are available for rodents. As a case in point, when an effective T cell–depleting antibody-toxin conjugate became available for swine,33 stable mixed chimerism was achieved with nonmyeloablative conditioning in pigs for the first time.34 In this MHC-matched model, long-term macrochimerism and donor-specific skin graft tolerance were induced after either bone marrow or cytokinemobilized peripheral blood stem-cell35 transplantation in hosts that had been conditioned with an immunotoxin-conjugated anti-CD3 monoclonal antibody and nonmyeloablative conditioning with TBI (3 Gy) and thymic irradiation (7 Gy). Durable macrochimerism and donor-specific tolerance have
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been achieved across MHC barriers for the first time with nonmyeloablative conditioning in a large animal model by the extension of this protocol to a full haplotype, MHC-mismatched, donor-recipient combination.36,37 In these studies, high doses of mobilized donor peripheral blood progenitor cells were given to animals that received the antiCD3 immunotoxin without TBI. These results demonstrate, in a large animal model, that TBI can be avoided by the use of very high doses of stem cells. Macrochimerism was also induced successfully in MHC-identical dogs with a nonmyeloablative conditioning regimen that consists of 2-Gy TBI and treatment with cyclosporine and mycophenolate mofetil for the first 4 weeks after BMT.38 The dose of irradiation could be further reduced to 1 Gy in this model through pretreatment with CTLA4Ig and peripheral blood mononuclear cells.39 The risk of graft-versus-host disease, however, has still posed a substantial impediment when the induction of macrochimerism has been attempted across MHC barriers in dogs.40 A macrochimerism protocol for the induction of tolerance has been developed for nonhuman primates. In this model, cynomolgus monkeys are conditioned with high doses of anti-thymocyte globulin, fractionated TBI (3 Gy), local thymic irradiation (7 Gy), and splenectomy before transplantation of unseparated bone marrow and a kidney from the same donor.41 Cyclosporine is given for 4 weeks after BMT,42 with no immunosuppression thereafter. Multilineage macrochimerism is transient with this regimen (detectable in peripheral blood lymphocytes for only up to 68 days after BMT), but long-term stable graft function can be achieved (for more than 10 years [T. Kawai, written communication, December 5, 2003]). The use of a short course of anti-CD154 monoclonal antibody treatment avoids the need for splenectomy from this protocol and leads to improved chimerism levels.43 Control animals that received the conditioning without bone marrow infusion consistently rejected the grafts,42 which indicates that BMT was necessary for tolerance to occur in this model. Because tolerance persisted long after macrochimerism disappeared, however, the renal graft itself also probably participated in the long-term maintenance of tolerance. CLINICAL PILOT STUDIES FOR TOLERANCE INDUCTION AFTER ORGAN TRANSPLANATION On the basis of the progress that has been achieved in the development of progressively less toxic rodent and non-human primate protocols, as
Surgery April 2004
well as initial evaluations in patients with hematologic malignancies, the first clinical trials for the deliberate induction of hematopoietic macrochimerism and tolerance after renal transplantation were initiated. In a small series, patients with refractory multiple myeloma and end-stage renal disease have been treated with simultaneous bone marrow and renal transplants that were obtained from the same HLA-identical living-related donor.44 The conditioning consisted of cyclophosphamide, antithymocyte globulin and thymic irradiation (7 Gy). Cyclosporine was given for approximately 2 months after transplantation and was tapered thereafter. Most patients also received 1 or several donor lymphocyte infusions after the transplantation, in an attempt to augment antitumor effects. Multilineage hematopoietic macrochimerism was induced reliably with this regimen but was transient in some patients. Despite the loss of chimerism, the renal grafts retained normal function and remained free of histologic signs of acute or chronic rejection without any long-term immunosuppression; the current follow-up period has been up to 5 years.44 This BMT protocol has been well tolerated in all patients. A similar protocol was initiated recently that used a more powerful T cell–depleting agent, MEDI-507, for haploidentic transplants in renal transplantation candidates with no concomitant malignancy. In another ongoing pilot study, transient multilineage macrochimerism was achieved in 3 of 4 kidney transplant recipients who were co-transplanted with selected CD34+ mobilized hematopoietic cells from the same HLA-mismatched living donor.45 Hosts were conditioned with total lymphoid irradiation and antithymocyte globulin and received cyclosporine and prednisone after the transplantation. Although the protocol was well tolerated without signs of graft-versus-host disease, episodes of acute rejection have been encountered, and tolerance was not achieved. SUMMARY The induction of donor hematopoietic macrochimerism is a promising approach for the induction of tolerance in the clinical setting of organ transplantation. Recent progress has resulted in substantially less toxic murine tolerance models that are based on macrochimerism. Currently, these regimens are being adapted to large animal models, in which macrochimerism and tolerance have already been achieved by more intense conditioning protocols. Results from the first clinical macrochimerism trial in renal transplant recipients demonstrate that transplantation
Surgery Volume 135, Number 4
tolerance can be induced deliberately in the clinical setting. Taken together, these advances fuel the hope that the 5 decades of tolerance research will lead finally to clinical success. We thank Ms Robin Laber for assistance with the manuscript.
REFERENCES 1. Billingham RE, Brent L, Medawar PB. ‘‘Actively acquired tolerance’’ of foreign cells. Nature 1953;172:603-6. 2. Swanson SJ, Hale DA, Mannon RB, Kleiner DE, Cendales LC, Chamberlain CE, et al. Kidney transplantation with rabbit antithymocyte globulin induction and sirolimus monotherapy. Lancet 2002;360:1662-4. 3. Starzl TE, Murase N, Abu-Elmagd K, Gray EA, Shapiro R, Eghtesad B, et al. Tolerogenic immunosuppression for organ transplantation. Lancet 2003;361:1502-10. 4. Calne R, Friend P, Moffatt S, Bradley A, Hale G, Firth J, et al. Proper tolerance, perioperative campath 1H, and low-dose cyclosporine monotherapy in renal allograft recipients. Lancet 1998;351:1701-2. 5. Wekerle T, Sykes M. Mixed chimerism and transplantation tolerance. Annu Rev Med 2001;52:353-70. 6. Dey B, Sykes M, Spitzer TR. Outcomes of recipients of both bone marrow and solid organ transplants: a review. Medicine 1998;77:355-69. 7. Ildstad ST, Sachs DH. Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 1984;307:168-70. 8. Sharabi Y, Sachs DH. Mixed chimerism and permanent specific transplantation tolerance induced by a non-lethal preparative regimen. J Exp Med 1989;169:493-502. 9. Colson YL, Li H, Boggs SS, Patrene KD, Johnson PC, Ildstad ST. Durable mixed allogeneic chimerism and tolerance by a nonlethal radiation-based cytoreductive approach. J Immunol 1996;157:2820-9. 10. De Vries-van der Zwan A, Besseling AC, De Waal LP, Boog CJP. Specific tolerance induction and transplantation: a single-day protocol. Blood 1997;89:2596-601. 11. Mayumi H, Good RA. Long-lasting skin allograft tolerance in adult mice induced across fully allogeneic (multimajor H-2 plus multiminor histocompatibility) antigen barriers by a tolerance-inducing method using cyclophosphamide. J Exp Med 1989;169:213-38. 12. Stewart FM, Crittenden RB, Lowry PA, Pearson-White S, Quesenberry PJ. Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice. Blood 1993;81:2566-71. 13. Sykes M, Szot GL, Swenson K, Pearson DA. Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning. Nature Med 1997;3:783-7. 14. Wekerle T, Nikolic B, Pearson DA, Swenson KG, Sykes M. Minimal conditioning required in a murine model of T cell depletion, thymic irradiation and high-dose bone marrow transplantation for the induction of mixed chimerism and tolerance. Transplant Int 2002;15:248-53. 15. Larsen CP, Elwood ET, Alexander DZ, Ritchie SC, Hendrix R, Tucker-Burden C, et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 1996;381:434-8.
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16. Kirk A, Burkly L, Batty D, Baumgartner R, Berning J, Buchanan K, et al. Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nature Med 1999;5: 686-93. 17. Sayegh MH, Turka LA. The role of T cell costimulatory activation pathways in transplant rejection. N Engl J Med 1998;338:1813-21. 18. Li Y, Li XC, Zheng XX, Wells AD, Turka LA, Strom TB. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nature Med 1999;5:1298302. 19. Markees TG, Phillips NE, Gordon EJ, Noelle RJ, Shultz LD, Mordes JP, et al. Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4+ T cells, interferon-c, and CTLA4. J Clin Invest 1998;101:2446-55. 20. Williams MA, Trambley J, Ha J, Adams AB, Durham MM, Rees P, et al. Genetic characterization of strain differences in the ability to mediate CD40/CD28-independent rejection of skin allografts. J Immunol 2000;165:6849-57. 21. Ossevoort MA, Ringers J, Kuhn EM, Boon L, Lorre K, van den Hout Y, et al. Prevention of renal allograft rejection in primates by blocking the B7/CD28 pathway. Transplantation 1999;68:1010-8. 22. Pearson TC, Trambley J, Odom K, Anderson DC, Cowan S, Bray R, et al. Anti-CD40 therapy extends renal allograft survival in rhesus macaques. Transplantation 2002;74: 933-40. 23. Wekerle T, Sayegh MH, Hill J, Zhao Y, Chandraker A, Swenson KG, et al. Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance. J Exp Med 1998;187:2037-44. 24. Adams AB, Durham MM, Kean L, Shirasugi N, Ha J, Williams MA, et al. Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression. J Immunol 2001; 167:1103-11. 25. Quesenberry PJ, Zhong S, Wang H, Stewart M. Allogeneic chimerism with low-dose irradiation, antigen presensitization, and costimulator blockade in H-2 mismatched mice. Blood 2001;97:557-64. 26. Seung E, Iwakoshi N, Woda BA, Markees TG, Mordes JP, Rossini AA, et al. Allogeneic hematopoietic chimerism in mice treated with sublethal myeloablation and anti-CD154 antibody: absence of graft-versus-host disease, induction of skin allograft tolerance, and prevention of recurrent autoimmunity in islet-allografted NOD/Lt mice. Blood 2000;95: 2175-82. 27. Taylor PA, Lees CJ, Waldmann H, Noelle RJ, Blazar BR. Requirements for the promotion of allogeneic engraftment by anti-CD154 (anti-CD40L) monoclonal antibody under nonmyeloablative conditions. Blood 2001;98:467-74. 28. Kurtz J, Ito H, Wekerle T, Shaffer J, Sykes M. Mechanisms involved in the establishment of tolerance through costimulatory blockade and BMT: Lack of requirement for CD40L-mediated signaling for tolerance or deletion of donor-reactive CD4+ cells. Am J Transplant 2001;1:339-49. 29. Blaha P, Bigenzahn S, Koporc Z, Schmid M, Langer F, Selzer E, et al. The influence of immunosuppressive drugs on tolerance induction through bone marrow transplantation with costimulation blockade. Blood 2003;101:2886-93.
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30. Wekerle T, Kurtz J, Ito H, Ronquillo JV, Dong V, Zhao G, et al. Allogeneic bone marrow transplantation with costimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nature Med 2000;6:464-9. 31. Durham MM, Bingaman AW, Adams AB, Ha J, Waitze S-Y, Pearson TC, et al. Administration of anti-CD40 ligand and donor bone marrow leads to hematopoietic chimerism and donor-specific tolerance without cytoreductive conditioning. J Immunol 2000;165:1-4. 32. Valujskikh A, Pantenburg B, Heeger PS. Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice. Am J Transplant 2002;2:501-9. 33. Huang CA, Yamada K, Murphy MC, Shimizu A, Colvin RB, Neville DM Jr, et al. In vivo T cell depletion in miniature swine using the swine CD3 immunotoxin, pCD3-CRM9. Transplantation 1999;68:855-60. 34. Huang CA, Fuchimoto Y, Scheier-Dolberg R, Murphy MC, Neville DM Jr, Sachs DH Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model. J Clin Invest 2000;105:173-81. 35. Colby C, Chang Q, Fuchimoto Y, Ferrara V, Murphy M, Sackstein R, et al. Cytokine-mobilized peripheral blood progenitor cells for allogeneic reconstitution of miniature swine. Transplantation 2000;69:135-40. 36. Fuchimoto Y, Huang CA, Shimizu A, Kitamura H, Colvin RB, Ferrara V, et al. Mixed chimerism without whole body irradiation in a large animal model. J Clin Invest 2000; 105:1779-89. 37. Gleit ZL, Fuchimoto Y, Yamada K, Melendy E, ScheierDolberg R, Manajati L, et al. Variable relationship between chimerism and tolerance after hematopoietic cell transplantation without myelosuppressive conditioning. Transplantation 2002;74:1535-44. 38. Storb R, Yu C, Wagner JL, Deeg HJ, Nash RA, Leisenring W, et al. Stable mixed hematopoietic chimerism in DLA-
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39.
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44.
45.
identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood 1997;89:3048-54. Storb R, Yu C, Zaucha JM, Deeg HJ, Georges G, Kiem HP, et al. Stable mixed chimerism in dogs given donor antigen, CTLA4Ig, and 100 cGy total body irradiation before and pharmacologic immunosuppression after marrow transplant. Blood 1999;94:2523-9. Yu C, Linsley P, Seidel K, Sale G, Sale G, Deeg HJ, Nash RA, et al. Cytotoxic T lymphocyte antigen 4-immunoglobulin fusion protein combined with methotrexate/cyclosporine as graft-versus-host disease prevention in a canine dog leukocyte antigen-nonidentical marrow transplant model. Transplantation 2000;69:450-4. Kawai T, Cosimi AB, Colvin RB, Powelson J, Eason J, Kozlowski T, et al. Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 1995;59:256-62. Kimikawa M, Sachs DH, Colvin RB, Bartholomew A, Kawai T, Cosimi AB. Modifications of the conditioning regimen for achieving mixed chimerism and donor-specific tolerance in cynomolgus monkeys. Transplantation 1997;64:70916. Kawai T, Abrahamian G, Sogawa H, Wee S, Boskovic S, Andrew D, et al. Costimulatory blockade for induction of mixed chimerism and renal allograft tolerance in nonhuman primates. Transplant Proc 2001;33:221-2. Bu¨hler LH, Spitzer TR, Sykes M, Sachs DH, Delmonico FL, Tolkoff-Rubin N, et al. Induction of kidney allograft tolerance after transient lymphohematopoietic chimerism in patients with multiple myeloma and end-stage renal disease. Transplantation 2002;74:1405-9. Millan MT, Shizuru JA, Hoffmann P, Dejbakhsh-Jones S, Scandling JD, Grumet FC, et al. Mixed chimerism and immunosuppressive drug withdrawal after HLA- mismatched kidney and hematopoietic progenitor transplantation. Transplantation 2002;73:1386-91.
SURGERY APRIL 2004
Surgical research review Induction of tolerance Thomas Wekerle, MD, and Megan Sykes, MD, Vienna, Austria, and Boston, Mass
From the Department of Surgery, Division of Transplantation, Vienna General Hospital/University of Vienna, Vienna, Austria; and the Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Mass
‘‘The experiments to be described in this article provide a solution—at present only a ÔlaboratoryÕ solution—of the problem of how to make tissue homografts immunologically acceptable to hosts which would normally react against them.’’1 Billingham et al During the 50 years after the publication of this paper by Billingham et al,1 the transfer of experimental tolerance protocols from the laboratory to the clinical arena has been very limited. Only when this translation is realized fully will the results of organ transplantation be optimized. We focus on recent progress in the field that we consider relevant for the development of clinically applicable tolerance strategies. Over the last 2 decades, progress in surgical technique and medical and anesthesiologic treatment has led to substantially improved short-term Supported in part by grants from the Roche Organ Transplantation Research Foundation (#110578928) and National Institutes of Health RO1 grants HL49915 and AI05558. Accepted for publication October 25, 2003. Reprint requests: Megan Sykes, MD, Transplantation Biology Research Center, Bone Marrow Transplantation Section, Massachusetts General Hospital/Harvard Medical School, MGH-East Bldg 149-5102, 13th St, Boston, MA 02129. Surgery 2004;135:359-64. 0039-6060/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.surg.2003.10.010
outcomes after organ transplantation. Long-term results, however, have improved to a lesser degree and are limited by graft loss because of chronic rejection and by the side-effects of life-long, nonspecific immunosuppressive therapy. In an attempt to reduce these effects, recent trials have been designed to minimize immunosuppression in transplant recipients. Patients were treated with conventional immunosuppressive drugs immediately after the transplant and were maintained on minimal immunosuppression.2-4 The results of such trials suggest that some organ transplant recipients tolerate very low immunosuppression and that other recipients do not tolerate reduced immunosuppression and experience acute rejection of the grafts. At present, however, we cannot predict reliably who will fall into the latter group. Longer follow-up periods and additional larger studies will reveal whether there is an overall benefit to reduced immunosuppression. These studies suggest, however, that ‘‘spontaneous’’ development of profound hyporesponsiveness or tolerance, which could allow complete drug withdrawal, is quite rare under conventional immunosuppression. Thus, the deliberate induction of robust donor-specific tolerance (ie, a state without drug therapy in which the immune system does not mount a harmful response against the graft but is left otherwise intact) is required to provide a full solution to the problems of both chronic rejection and drug toxicity. SURGERY 359
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EXPERIMENTAL TOLERANCE INDUCTION: WHICH STRATEGY COULD BECOME CLINICALLY APPLICABLE? There is an abundance of experimental tolerance protocols in rodents, and these small animal models are indispensable for the development of tolerance strategies, as evidenced by the fact that the few protocols that are successful in large animals have been based on extensive rodent studies. However, the limitations of rodent systems must be considered when the results from mouse or rat experiments are interpreted. Long-term acceptance of vascularized organs (eg, hearts) can be achieved relatively easily with numerous approaches in rodents. These successes, however, rarely are reproducible in large animal or nonhuman primate studies. To improve the chances of successful clinical development, it is therefore important to apply the strictest available tolerance tests in rodent models (often considered to be the permanent acceptance of skin from a major histocompatibility complex (MHC)–mismatched donor). Only very few strategies meet this test. Among them, the macrochimerism approach has been demonstrated most extensively by several independent groups who reproducibly induced donor-specific skin graft tolerance in various allogeneic murine models and strain combinations. Hematopoietic macrochimerism denotes the presence of substantial numbers (more than 1%) of donor hematopoietic cells in a recipient; if donor cells constitute more than 1% but less than 100%, we refer to it as mixed chimerism. The macrochimerism approach has several additional features that make it a good candidate for clinical development5: (1) Macrochimerism relies in large part on clonal deletion as a mechanism of tolerance. Because deletion leads to the physical elimination of cells with donor reactivity, it is a robust mechanism. (2) Large animal models (including nonhuman primate studies) that establish tolerance through macrochimerism have been developed successfully. (3) Macrochimerism can be measured readily and could serve as a surrogate marker of successful tolerance induction in the clinical setting. (4) Evidence that macrochimerism can also lead to tolerance in humans comes from several cases in which an organ (usually a kidney) has been transplanted into a patient who had undergone transplantation previously with the bone marrow from the same donor (for a hematologic indication) and in which the organ was accepted long-term without immunosuppression.6 (5) The first clinical tolerance studies that rely on macrochimerism have
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been initiated successfully. Given this list of attractive features, it might be surprising that macrochimerism has not made it into routine clinical practice of organ transplantation so far. Above all else, there is 1 major obstacle impeding its success: The transplantation of bone marrow (or mobilized peripheral blood leukocytes) across MHC barriers requires the conditioning of the recipient, which can be associated with considerable morbidity and death. Therefore, much effort has been focused on the development of lower-risk protocols of bone marrow transplantation (BMT) that are associated with toxicities that are acceptable for application in organ transplantation. NONTOXIC PROTOCOLS FOR THE INDUCTION OF MACROCHIMERISM AND TOLERANCE Two elements have been traditionally included in conditioning regimens in models of allogeneic macrochimerism: (1) irradiation or cytotoxic drug treatment of the recipient, primarily to promote engraftment of the infused donor hematopoietic stem cells and (2) global destruction of the host T cells (by the use of high doses of specific T cell– depleting antibodies or myeloablation) to prevent rejection of the donor cells. Both modalities have significant toxicities. The earliest macrochimerism protocols for tolerance induction included myeloablation of the recipient by lethal total-body irradiation (TBI) before the BMT.7 Subsequently, several regimens were developed that used a milder, nonmyeloablative conditioning together with nonspecific T cell depletion.8-11 However, the avoidance of myelosuppression altogether proved to be difficult because not even syngeneic hematopoietic stem cells readily engraft in unconditioned hosts when administered in conventional doses. This requirement for myelosuppression eventually was overcome in syngeneic models by the injection of very high numbers of bone marrow cells.12 When these findings were applied to the allogeneic setting, the transplantation of similarly high doses of MHCmismatched bone marrow together with T cell depletion allowed the induction of macrochimerism and tolerance without TBI or cytotoxic drug treatment, as long as a moderate dose of selective irradiation to the thymic area was given.13 However, not even the repeated injection of monoclonal cytotoxic T cell antibodies permitted the complete elimination of irradiation from this model.14 The recent introduction of costimulation-blocking reagents as components of BMT protocols has allowed the complete avoidance of non-specific T
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cell depletion and myelosuppression and thus has led to the hitherto mildest experimental macrochimerism protocols. The most commonly used costimulation blockers interfere with the CD28/ CTLA4–CD80/CD86 and the CD40L (CD154)– CD40 pathways. Such reagents, when used alone (without BMT), have profound immunosuppressive properties in various transplantation models.15-17 Costimulation blockade alone, however, usually does not induce tolerance robust enough to meet the most stringent test, namely permanent skin graft acceptance across MHC barriers in euthymic recipients (except in a susceptible strain when combined with the use of rapamycin).18,19 Certain mouse strains are more susceptible to the tolerogenic effects of costimulation blockade than others,20 and the most stringent tests of tolerance should include the ‘‘difficult’’ strains. Costimulation blockade with CTLA4Ig, anti-CD80/86, antiCD40, anti-CD154 monoclonal antibodies, and various combinations thereof has also been effective as immunosuppressive treatment in non-human primate kidney transplantation models, but again did not induce tolerance.16,21,22 These antibodies have a unique effect, however, when used together with the transplantation of bone marrow. The use of an anti-CD154 monoclonal antibody, with or without CTLA4Ig, allowed the induction of high levels of lasting, multilineage macrochimerism in several nonmyeloablative murine BMT models without cytotoxic T cell monoclonal antibodies, even in strains that were quite resistant to the effects of these monoclonal antibodies in the absence of BMT.23-27 The progressive extrathymic deletion of donor-reactive T cells immediately after BMT and anti-CD154 treatment seems to be a critical mechanism by which these protocols achieve macrochimerism in the presence of an essentially intact peripheral and intrathymic host T cell repertoire.23 However, tolerance occurs in these models before deletion is complete,28 which points to an important role for nondeletional mechanisms during the induction phase of tolerance immediately after BMT. As in previous macrochimerism models, tolerance seems to be maintained long-term predominantly through intrathymic clonal deletion. A course of conventional immunosuppression can be added to a protocol of BMT plus costimulation blockade without interference with tolerance induction and even allows reduction in the dose of irradiation that is required to allow the establishment of lasting macrochimerism with the use of standard doses of bone marrow,29 which further increases the clinical feasibility of this approach.
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By the use of costimulation blockade (again either anti-CD154 alone or combined with CTLA4Ig) together with very high doses of bone marrow (approximately 12-fold the usual dose), lasting allogeneic macrochimerism and robust tolerance were induced without any cytoreductive host conditioning (no irradiation, cytotoxic drugs, or monoclonal antibodies).30,31 Although such high doses of bone marrow would not be available routinely in the clinical setting, these models are proof that completely nonmyelosuppressive BMT regimens are possible. Protocols that achieve macrochimerism and tolerance with the use of clinically feasible numbers of bone marrow without cytoreduction, however, still must be developed. PRECLINICAL LARGE ANIMAL PROTOCOLS FOR THE INDUCTION OF MACROCHIMERISM AND TOLERANCE Macrochimerism and tolerance are induced with greater difficulty in large animals than in rodents. Although substantial progress has been achieved in large animals, the latest, least toxic rodent regimens still must be adapted for large animals. One reason for the increased resistance to macrochimerism and tolerance may be that outbred large animals live in a less sheltered environment than inbred mice or rats and are exposed to a much wider range of antigens. They consequently have a higher frequency of primed T cells, which cross-react with transplantation antigens. Because primed T cells are more difficult to make tolerant,32 intensified conditioning may be required in large animals to obtain lasting chimerism and tolerance. Furthermore, a cohort of outbred animals demonstrates a substantially higher variability in their immune responses than a particular inbred strain of rodents. A further potential reason for the differences in results between studies of tolerance in small and large animals is the lack of equally effective reagents for use in large animals, as are available for rodents. As a case in point, when an effective T cell–depleting antibody-toxin conjugate became available for swine,33 stable mixed chimerism was achieved with nonmyeloablative conditioning in pigs for the first time.34 In this MHC-matched model, long-term macrochimerism and donor-specific skin graft tolerance were induced after either bone marrow or cytokinemobilized peripheral blood stem-cell35 transplantation in hosts that had been conditioned with an immunotoxin-conjugated anti-CD3 monoclonal antibody and nonmyeloablative conditioning with TBI (3 Gy) and thymic irradiation (7 Gy). Durable macrochimerism and donor-specific tolerance have
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been achieved across MHC barriers for the first time with nonmyeloablative conditioning in a large animal model by the extension of this protocol to a full haplotype, MHC-mismatched, donor-recipient combination.36,37 In these studies, high doses of mobilized donor peripheral blood progenitor cells were given to animals that received the antiCD3 immunotoxin without TBI. These results demonstrate, in a large animal model, that TBI can be avoided by the use of very high doses of stem cells. Macrochimerism was also induced successfully in MHC-identical dogs with a nonmyeloablative conditioning regimen that consists of 2-Gy TBI and treatment with cyclosporine and mycophenolate mofetil for the first 4 weeks after BMT.38 The dose of irradiation could be further reduced to 1 Gy in this model through pretreatment with CTLA4Ig and peripheral blood mononuclear cells.39 The risk of graft-versus-host disease, however, has still posed a substantial impediment when the induction of macrochimerism has been attempted across MHC barriers in dogs.40 A macrochimerism protocol for the induction of tolerance has been developed for nonhuman primates. In this model, cynomolgus monkeys are conditioned with high doses of anti-thymocyte globulin, fractionated TBI (3 Gy), local thymic irradiation (7 Gy), and splenectomy before transplantation of unseparated bone marrow and a kidney from the same donor.41 Cyclosporine is given for 4 weeks after BMT,42 with no immunosuppression thereafter. Multilineage macrochimerism is transient with this regimen (detectable in peripheral blood lymphocytes for only up to 68 days after BMT), but long-term stable graft function can be achieved (for more than 10 years [T. Kawai, written communication, December 5, 2003]). The use of a short course of anti-CD154 monoclonal antibody treatment avoids the need for splenectomy from this protocol and leads to improved chimerism levels.43 Control animals that received the conditioning without bone marrow infusion consistently rejected the grafts,42 which indicates that BMT was necessary for tolerance to occur in this model. Because tolerance persisted long after macrochimerism disappeared, however, the renal graft itself also probably participated in the long-term maintenance of tolerance. CLINICAL PILOT STUDIES FOR TOLERANCE INDUCTION AFTER ORGAN TRANSPLANATION On the basis of the progress that has been achieved in the development of progressively less toxic rodent and non-human primate protocols, as
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well as initial evaluations in patients with hematologic malignancies, the first clinical trials for the deliberate induction of hematopoietic macrochimerism and tolerance after renal transplantation were initiated. In a small series, patients with refractory multiple myeloma and end-stage renal disease have been treated with simultaneous bone marrow and renal transplants that were obtained from the same HLA-identical living-related donor.44 The conditioning consisted of cyclophosphamide, antithymocyte globulin and thymic irradiation (7 Gy). Cyclosporine was given for approximately 2 months after transplantation and was tapered thereafter. Most patients also received 1 or several donor lymphocyte infusions after the transplantation, in an attempt to augment antitumor effects. Multilineage hematopoietic macrochimerism was induced reliably with this regimen but was transient in some patients. Despite the loss of chimerism, the renal grafts retained normal function and remained free of histologic signs of acute or chronic rejection without any long-term immunosuppression; the current follow-up period has been up to 5 years.44 This BMT protocol has been well tolerated in all patients. A similar protocol was initiated recently that used a more powerful T cell–depleting agent, MEDI-507, for haploidentic transplants in renal transplantation candidates with no concomitant malignancy. In another ongoing pilot study, transient multilineage macrochimerism was achieved in 3 of 4 kidney transplant recipients who were co-transplanted with selected CD34+ mobilized hematopoietic cells from the same HLA-mismatched living donor.45 Hosts were conditioned with total lymphoid irradiation and antithymocyte globulin and received cyclosporine and prednisone after the transplantation. Although the protocol was well tolerated without signs of graft-versus-host disease, episodes of acute rejection have been encountered, and tolerance was not achieved. SUMMARY The induction of donor hematopoietic macrochimerism is a promising approach for the induction of tolerance in the clinical setting of organ transplantation. Recent progress has resulted in substantially less toxic murine tolerance models that are based on macrochimerism. Currently, these regimens are being adapted to large animal models, in which macrochimerism and tolerance have already been achieved by more intense conditioning protocols. Results from the first clinical macrochimerism trial in renal transplant recipients demonstrate that transplantation
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tolerance can be induced deliberately in the clinical setting. Taken together, these advances fuel the hope that the 5 decades of tolerance research will lead finally to clinical success. We thank Ms Robin Laber for assistance with the manuscript.
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