- Email: [email protected]
Clinical Transplantation Tolerance: A Myth No More, But …
MASSRY LECTURE Clinical Transplantation Tolerance: A Myth No More, But . . . Jamil Azzi, MD, and Mohamed H. Sayegh, MD
The Shaul G. Massry Distinguished Lecture is delivered at the National Kidney Foundation’s annual Spring Clinical Meetings. This lectureship was established to honor Dr Massry for his scientific achievements and his contributions to the kidney health care community and the National Kidney Foundation. The 2009 lecturer was Mohamed H. Sayegh, MD, and his remarks form the basis for this article.
M
ore than 55 years ago, a report published in Nature by Billingham et al1 widened the horizons of modern medicine by describing the phenomenon of acquired immunologic tolerance to transplant antigens. Shortly thereafter, one of the first kidney transplants was performed at the Peter Bent Brigham Hospital in Boston, MA, by a team headed by Joseph Murray, a plastic surgeon, that included John Merrill, a nephrologist.2 These 2 great milestones laid the foundation for tremendous advances in the field of transplantation immunology during the past 5 decades.3 Together, they launched a worldwide search for the holy grail of transplant tolerance, which is defined as the absence of pathologic immune response against a graft in an immunocompetent host not using maintenance immunosuppressive drugs.4 In this review, we highlight recent advances in the field of immunologic tolerance in organ transplantation that have made that
More than 55 years after the description of the phenomenon of acquired immunologic tolerance to transplant antigens by Medawar and colleagues, the holy grail of transplantation is a myth no more. Establishment of the Immune Tolerance Network in 1999 changed the approach used to achieve clinical tolerance by translating advances in the field of immunology in general and in tolerance in particular from experimental animal strategies into human clinical trials, and initial results have been promising. Despite these advances, scientific and operational challenges still face the transplantation community and affect progress. Overcoming those challenges is a shared responsibility of scientists, transplant professionals, and general nephrologists alike. Achieving tolerance could not only revolutionize the field of transplantation through avoidance of toxicity associated with immunosuppressive agents, but also influence the treatment of autoimmune diseases, in which the immune system attacks self, as well as cancer, in which tolerance of the immune system toward malignant cells may permit disease development and progression. Am J Kidney Dis 54:1005-1011. © 2009 by the National Kidney Foundation, Inc.
holy grail a myth no more. For the sake of clarity, terminology used in this article is defined in the glossary (Box 1).
THE RISE OF A NEW ERA IN TRANSPLANTATION Early Efforts to Achieve Transplant Tolerance After the pioneering era of Peter Medawar, John Merrill, Joseph Murray, and others, a second era can be described in which the major advances in immunology focused on the development of strategies to induce tolerance in experimental animal models. In addition, although several reports described the phenomenon of immunologic tolerance in some humans, these interventions were not rigorously attempted in controlled trials.5 During the pre-1999 era, tolerance was reported first by a group from Stanford in 3 patients who underwent total-body lymphoid irradiation before receiving a cadaveric kidney transplant.6 Soon thereafter, we reported 2 leukemic patients
complicated by end-stage renal disease (ESRD) who received a bone marrow transplant. Each of these patients subsequently received a successful kidney transplant several years later from their bone marrow donor without using immunosuppressive drugs, except for a low dose of prednisone for other indications.7 Similarly, a team at the Massachusetts General Hospital treated a patient with multiple myeloma complicated by ESRD
From the Transplantation Research Center, Renal Division, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, MA. Originally published online as doi: 10.1053/j.ajkd.2009.08.005 on October 12, 2009. Address correspondence to Mohamed H. Sayegh, MD, Transplant Research Center, Brigham and Women’s Hospital, Children’s Hospital Boston, 221 Longwood Ave, Boston MA 02115. Email: [email protected] © 2009 by the National Kidney Foundation, Inc. 0272-6386/09/5406-0006$36.00/0 doi:10.1053/j.ajkd.2009.08.005
American Journal of Kidney Diseases, Vol 54, No 6 (December), 2009: pp 1005-1011
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Azzi and Sayegh Box 1. Glossary
Immunologic tolerance: a state of immunologic unresponsiveness toward a given set of antigens in an immunocompetent individual Transplantation tolerance: the absence of pathologic immune response against a graft in an immunocompetent host not using maintenance immunosuppressive drugs Mixed lymphohematopoietic chimerism: the state after bone marrow transplant in which hematopoietic cells of donor and recipient origin coexist Nonmyeloablative bone marrow transplant: allogeneic bone marrow transplant that uses lower doses of chemotherapy and/or radiation with the goal to make room for the donor cells and prevent their rejection without destroying the recipient’s bone marrow Regulatory T cell (Treg): T lymphocyte that turns off specific immune responses Mixed lymphocyte reaction (MLR): the proliferative response that occurs when lymphocytes from 2 individuals are cultured together, as the result of reactions of T cells of 1 individual to MHC antigens on the other individual’s cells Cytotoxic T lymphocyte (CTL): a T lymphocyte (which usually expresses CD8) that kills its target cell on recognizing complexes of peptides and MHC molecules on the target-cell membrane Central deletion: a mechanism of immune tolerance using lymphoablative treatments to create a mixed hematopoietic chimeric state. In this instance, donor cells seed the thymus, and maturing donorreactive T-cell clones are deleted through intrathymic apoptosis Peripheral deletion: another mechanism of immune tolerance by which donor-reactive cells can undergo apoptosis outside the thymus, in either the allograft itself or in draining lymph nodes Abbreviation: MHC, major histocompatibility complex.
with both a bone marrow and kidney transplant from the same donor, inducing mixed lympho-
hematopoietic chimerism (defined as the persistence of donor hematopoietic cells in the recipient). This has been suggested to modulate immune responsiveness to donor alloantigens as a mechanism of transplant tolerance.8 Finally, some transplant recipients would play Russian roulette by discontinuing their immunosuppressive medications, with a lucky few serendipitously winning tolerance.9 Successes in Liver Transplant Tolerance The liver is considered an immune-privileged organ characterized by an anatomic structure believed to facilitate mixed chimerism through bidirectional interaction of donor and recipient leukocytes, resulting in elimination of alloreactive T cells to donor and recipient cells consecutively.10 These observations encouraged prospective drug withdrawal programs in ⬎ 90 liver transplant recipients between 1992 and 1996, with very promising results for the transplant community at the time.11 After this initial trial, a smaller cohort of 18 patients had immunosuppression therapy withdrawn at King’s College, London, with 28% of patients reported completely free of medications at 3 years.12 Operational liver tolerance trials have continued to emerge in the last few years. Takatsuki et al13 in Japan successfully achieved complete withdrawal of tacrolimus therapy in 38% of their 63 liver transplant recipients, and a Kyoto group reported the withdrawal of immunosuppressants in 87 of 659 (15%) children who underwent living donor liver transplant be-
tween 1990 and May 2005.14 Results from a Pittsburgh group have been particularly notable, with a mean time off immunosuppression therapy ⬎ 10 years in a cohort of 48 liver transplant recipients. Return to immunosuppression therapy has been rare (1 of 48 patients).15 The Era of the Immune Tolerance Network A new era emerged with the rise of the new millennium after establishment of the ITN (Immune Tolerance Network: www.immunetolerance.org), a clinical research consortium founded by the US National Institutes of Health in 1999 for the purpose of focusing efforts on the goal of achieving immune tolerance therapies. The formation of this consortium has resulted in many clinical trials, not only in islet and solid-organ transplant, but also in autoimmune disease and allergy/asthma (Table 1). One of the first fruits born of the ITN was the publication from the Massachusetts General Hospital group of a case series composed of 6 patients with multiple myeloma and ESRD who received simultaneous kidney and bone marrow transplants from HLAidentical sibling donors after a nonmyeloablative regimen. Immunosuppressive drugs were completely withdrawn within 2 months after transplant. Chimerism was transient in 4 patients. One patient experienced rejection. This was treated successfully, and immunosuppressive drugs were withdrawn successfully in that patient 1 year later. Two other patients developed graft-versus-host disease,
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1007 Table 1. Recent and Ongoing Clinical Trials of Immune Tolerance Therapies
Procedure
Islet transplant
Tolerance Protocol
Edmonton protocol of steroid-free immunosuppression
Alemtuzumab,a tacrolimusb for 60 d, sirolimus (rapamycin) for 1 y Kidney transplant Daclizumab, LEA29Y (belatacept), MMF, sirolimus for 1 y Kidney transplant Alemtuzumab, donor stem-cell infusion, tacrolimus/sirolimus and MMF for 1 y Kidney & bone marrow Cyclophosphamide, MEDI-507 (siplizumab), 7 Gy thymic transplant irradiation, donor bone marrow, cyclosporine for 60 d Kidney & bone marrow Cyclophosphamide, ATG, 7 Gy thymic irradiation, donor transplant for bone marrow, cyclosporine for 36-60 d myeloma patients Liver transplant Alemtuzumab, tacrolimus slowly withdrawn Liver transplant Gradual withdrawal of maintenance immunosuppression in patients who underwent living donor liver transplants as children Liver transplant Alemtuzumab vs 3 mo of steroids followed by tacrolimus slowly withdrawn Kidney transplant
Study Status
Completed; 44% (16/36) insulin free at 1 y; 14% (5/36) insulin free at 2 y16 Active Active Active Completed; 4/5 patients withdrawn from all immunosuppressants18 Completed; all 6 patients withdrawn from all immunosuppressants17 Active Active
Active
Abbreviations: ATG, antithymocyte globulin; MMF, mycophenolate mofetil. a Humanized anti-CD52 antibody (Campath-1H; Berlex Laboratories, www.berlex.com). b Prograf (Astellas Pharma US Inc, www.astellas.us).
and immunosuppressive medications were resumed.17 A recent New England Journal of Medicine report published by the same Massachusetts General Hospital group reported on 5 patients with ESRD with combined bone marrow and kidney transplants from HLA single-haplotype– mismatched living related donors.18 They used a nonmyeloablative preparative regimen with cyclophosphamide at days ⫺5 and ⫺4 with respect to transplant; humanized antiCD2 monoclonal antibody on days ⫺1, 0, and 1; and intravenous cyclosporine and thymic irradiation on day ⫺1. They intravenously infused donor bone marrow after the kidney transplant, trying to induce mixed chimerism. One patient developed humoral rejection that required modifying the protocol for future patients to
induce B-cell depletion with monoclonal antibody against CD20 (rituximab) on days ⫺7 and ⫺2. The other 4 recipients discontinued immunosuppressive therapy at 9-14 months after transplant with stable renal function, now going on ⬎ 6 years for patients 1 and 2 and ⬎ 4 and 3 years for patients 4 and 5, respectively (B. Cosimi, personal communication, July 2009). Interestingly, although the chimerism induced by infusion of the bone marrow transplant was transient, tolerance persisted. Hypothesized mechanisms include elimination of cells reactive to donor antigens in the thymus (called central deletion) or generation of regulatory T cells with high expression levels of FOXP3 (a marker of regulatory T cells) in the renal allograft protocol biopsy specimens.18 Close follow-up of these patients is necessary to under-
stand the impact of losing the lymphohematopoietic mixed chimerism and its effect on tolerance, the risk of graft-versushost disease, and the emergence of other types of rejection, for example, humoral rejection. In the same New England Journal of Medicine issue, a Stanford group described a recipient of a combined kidney and hematopoietic cell transplant from an HLA-matched donor followed by total lymphoid irradiation and antithymocyte globulin with ⬎ 2 years of follow-up with no episodes of rejection or graft-versushost disease.19
CHALLENGES IN TRANSLATING TOLERANCE STRATEGIES INTO THE CLINIC The lack of reliable assays to assess immune function is the most significant challenge
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to develop clinical tolerance strategies.20 Given that acquired tolerance is weaker than natural tolerance and also may be transient, long-term monitoring is vital.21 Many immune function assays have been developed during the last decade, but none can accurately predict the development of tolerance.22 Accordingly, we still rely on measuring drug levels of immunosuppressive agents (which affect individuals’ immune systems differently), and we evaluate graft function (usually a late marker for events). Currently available assays can be antigen specific, meant to evaluate donor-specific T-cell responses against recipient antigens by measuring either: (1) proliferation of donor T cells in a mixed lymphocyte reaction, (2) lysis of recipient T cells in a cytotoxic T-lymphocyte assay,9 or (3) production of interleukins with either an enzyme-linked immunosorbent assay or enzyme-linked immunospot assay.23 The transvivo delayed-type hypersensitivity assay uses an immune-deficient mouse as an “in vivo milieu” for the interaction between donor T cells and recipient antigens. T Cells and antigens are injected into the footpads of the mice, causing swelling that is quantified relative to the level of sensitivity that can be measured using a caliper.24 Tetramer technology also may be used to detect the T-cell receptors that are specific for the recipient peptide– major histocompatibility complex molecule complex.25 The classic pretransplant cross-match is still used to detect donorspecific antibodies.
Azzi and Sayegh Box 2. Types of Immune Function Assays Antigen-specific assays T-Cell alloreactivity: -Proliferation of donor T cells in MLR -Lysis of recipient T cell in CTL assay -Production of cytokines in ELISA or Luminex or ELISpot assays -Transvivo delayed-type hypersensitivity Humoral immune responses: -HLA and non-HLA alloantibodies Non–antigen-specific assays Expression profiling of cellular surface markers: -Flow cytometry for regulatory T cells -Flow cytometry for plasmacytoid dendritic cells Measuring T-cell response to nonspecific polyclonal stimulus: -Intracellular ATP production RNA analysis (eg, microarrays) and proteomics: -Identify signature of tolerance Note: Luminex (www.luminexcorp.com) is a proprietary fluorescent bead immunoassay. Abbreviations: ATP, adenosine triphosphate; MLR, mixed lymphocyte reaction; CTL, cytotoxic T lymphocyte; ELISA, enzyme-linked immunosorbent assay; ELISpot, enzyme-linked immunosorbent spot.
Assays also may be non– antigen specific, measuring regulatory cells, such as the CD4⫹CD25high T cells known as Tregs,26 plasmacytoid dendritic cells that were reported to promote a type 2 T-helper cell– type response that is more tolerogenic.27 An assay commercialized as ImmunoKnow (Cylex Inc, www.cylex.net) measures T-cell response to a nonspecific polyclonal stimulus by quantifying intracellular adenosine triphosphate production as a marker of the level of cell immune function.28 Another revolutionary measure is a gene signature or gene expression profiling, recently explored by identifying a number of genes differentiating a tolerant from a nontolerant recipient with high accuracy.29,30 Finally, proteomics, which studies protein expression, can be a necessary complement to gene expression
because levels of protein synthesis may not correspond well to measured amounts of the respective messenger RNA transcripts31 (Box 2). Another major obstacle in developing clinical tolerance is the lack of the ideal animal model that will translate accurately in humans. For example, strategies in rodents have been unsuccessful in nonhuman primates32-34 and, in turn, may not be applicable in humans.35,36 Those significant immunologic differences mainly manifest in the proportion of memory and immunologically naive T cells.32 Rodents, for example, which are considered immunologically naive with a lower proportion of memory cells than primates or humans, respond successfully to many costimulatory blockade strategies that fail when used in humans.34 However, memory
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cells are very resistant to depletion therapy, which may deplete regulatory cells, promoting expansion of the memory T-cell compartment and making tolerance induction even more difficult.37 These unsuccessful outcomes in humans complicate selection of the best strategy to induce tolerance. Is central or peripheral deletion in association with drug minimization the answer, or does a completely novel therapy need to be developed?21 Evolving data incriminate infectious agents through stimulation of Toll-like receptors in preventing and reversing tolerance.38-40 Toll-like receptors, expressed on a variety of cell types, such as endothelial cells, epithelial cells, macrophages, neutrophils, and antigen-presenting cells, have a major role in innate immunity by recognizing different pathogens and differentiating self from nonself.41 In antigenpresenting cells exposed to an infection, Toll-like receptors stimulate cell maturation by increasing expression of CD80, CD86, and major histocompatibility complex class II antigens, which enhances T-cell responses, directly inducing survival and proliferation of CD4 cells42 and allogeneic CD8 T cells.40 This may explain the differences in resistance to tolerance protocols among transplanted organs because organs that are most exposed to infectious agents tend to be the most recalcitrant. Another scientific nonimmunologic challenge facing the transplantation community is the best end points to use in a clinical trial. Coupling observa-
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tion of short-term outcomes (1year survival) with longer term results when infectious and malignant complications are most likely to occur (2-3 years or beyond) is necessary to ensure safety. Furthermore, other operational challenges can be difficult to overcome. We need to reach consensus on when to move from a pilot study to a larger trial without exposing patients to intensive protocols if long-term success is unlikely.21 Similarly, it is extremely important to decide on the best management strategy for patients in tolerance trials who experience organ rejection: whether to withdraw them from the trial and return them to conventional therapy or modify therapeutic strategies according to a specific protocol. The most challenging issue facing modern medicine today, including our quest for tolerance strategies, is ethics. One major issue is eligibility criteria. This is particularly notable for age, given the recognition that children receiving organ transplants may derive the greatest benefit from immunosuppression withdrawal because of their potential longer exposure to immunosuppression, but may derive the greatest risk because a failed organ in a child carries a worse long-term prognosis than in an adult because of sensitization and compromise of a future transplant. A second issue is choosing a suitable organ for these clinical trials given that the consequences of failed liver, heart, and lung transplants are acutely life threatening. Should we compromise a
living donor organ or would a cadaveric organ for our initial trials be more appropriate? These issues require collaboration and consensus-building among all members of the transplantation community. ITN began this initiative by presenting a platform on which to share ideas. Diverging interests between pharmaceutical companies and the transplantation community regarding tolerogenic strategies may impair progress because the pharmaceutical industry may be perceived as being interested in keeping patients on maintenance immunosuppressive medication therapy. Therefore, cooperation between pharmaceutical companies that develop and market agents to induce tolerance and the transplantation community is essential for success in achieving the goal of tolerance. Facilitating future success in refining tolerance-inducing strategies is a responsibility shared by scientists and transplant and general nephrologists alike. Resistance to minimizing or halting immunosuppression is an issue for both patients and their physicians, especially when patients currently are stable with maintenance or low-dose immunosuppression. Rightfully, patients and physicians both ask: Is low-dose immunosuppression really an alternative to complete withdrawal from drug therapy? Do low-dose safe levels exist?
CONCLUSION Improving our communication and collaboration is integral to achieving tolerance, which will have a direct impact on the morbidity and mor-
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tality of transplant recipients, mainly through avoidance of toxicity associated with immunosuppressive agents. Nonspecific immunosuppression increases the risk of malignancies, infections, and metabolic diseases, such as diabetes and dyslipidemia, thus increasing the rate of cardiovascular mortality.43 Furthermore, calcineurin inhibitors have been incriminated in shortening graft survival, increasing the demand for retransplant, and worsening the organ shortage.44 Beyond transplantation, better understanding the mechanics of tolerance could profoundly influence the treatment of autoimmune diseases, such as type 1 diabetes and lupus, in which the immune system attacks self. In addition, therapeutic approaches to cancer might evolve by better understanding the tolerance of the immune system toward malignant cells. Implementation of a successful safe tolerance regimen also has the potential to create a framework for new treatments of genetic diseases through stem cell therapy, reestablishing islet cell transplant in patients with type 1 diabetes, and launching regenerative medicine by allogeneic stem cell transplant, for which the achievement of tolerance is not a mere option, but an absolute necessity.
ACKNOWLEDGEMENTS Financial Disclosure: Dr Sayegh is a consultant to Genzyme, but has no financial interest related to the subject matter of this article. Dr Azzi has no relevant financial interests.
Azzi and Sayegh
REFERENCES 1. Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature. 1953; 172(4379):603-606. 2. Merrill JP, Murray JE, Harrison JH, Guild WR. Successful homotransplantation of the human kidney between identical twins. J Am Med Assoc. 1956;160(4):277-282. 3. Sayegh MH, Carpenter CB. Transplantation 50 years later—progress, challenges, and promises. N Engl J Med. 2004;351(26):2761-2766. 4. Suthanthiran M. Transplantation tolerance: fooling Mother Nature. Proc Natl Acad Sci U S A. 1996;93(22): 12072-12075. 5. Ansari MJ, Sayegh MH. The arduous road to achieving an immunosuppression-free state in kidney transplant recipients. Nat Clin Pract Nephrol. 2007; 3(9):464-465. 6. Strober S, Dhillon M, Schubert M, et al. Acquired immune tolerance to cadaveric renal allografts. A study of three patients treated with total lymphoid irradiation. N Engl J Med. 1989; 321(1):28-33. 7. Sayegh MH, Fine NA, Smith JL, Rennke HG, Milford EL, Tilney NL. Immunologic tolerance to renal allografts after bone marrow transplants from the same donors. Ann Intern Med. 1991;114(11):954-955. 8. Spitzer TR, Delmonico F, TolkoffRubin N, et al. Combined histocompatibility leukocyte antigen-matched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease: the induction of allograft tolerance through mixed lymphohematopoietic chimerism. Transplantation. 1999;68(4):480-484. 9. Burlingham WJ, Grailer AP, Fechner JH, Jr, et al. Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant recipient. Transplantation. 1995;59(8): 1147-1155. 10. Sanchez-Fueyo A, Strom TB. Immunological tolerance and liver transplantation. J Hepatol. 2004;41(5): 698-705. 11. Mazariegos GV, Reyes J, Marino IR, et al. Weaning of immunosuppression in liver transplant recipients. Transplantation. 1997;63(2):243-249.
12. Devlin J, Doherty D, Thomson L, et al. Defining the outcome of immunosuppression withdrawal after liver transplantation. Hepatology. 1998;27(4): 926-933. 13. Takatsuki M, Uemoto S, Inomata Y, et al. Weaning of immunosuppression in living donor liver transplant recipients. Transplantation. 2001;72(3): 449-454. 14. Koshiba T, Li Y, Takemura M, et al. Clinical, immunological, and pathological aspects of operational tolerance after pediatric living-donor liver transplantation. Transpl Immunol. 2007; 17(2):94-97. 15. Mazariegos GV, Sindhi R, Thomson AW, Marcos A. Clinical tolerance following liver transplantation: long term results and future prospects. Transpl Immunol. 2007;17(2):114119. 16. Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med. 2006;355(13): 1318-1330. 17. Fudaba Y, Spitzer TR, Shaffer J, et al. Myeloma responses and tolerance following combined kidney and nonmyeloablative marrow transplantation: in vivo and in vitro analyses. Am J Transplant. 2006;6(9):21212133. 18. Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med. 2008;358(4):353-361. 19. Scandling JD, Busque S, Dejbakhsh-Jones S, et al. Tolerance and chimerism after renal and hematopoietic-cell transplantation. N Engl J Med. 2008;358(4):362-368. 20. Salama AD, Remuzzi G, Harmon WE, Sayegh MH. Challenges to achieving clinical transplantation tolerance. J Clin Invest. 2001;108(7):943948. 21. Salama AD, Womer KL, Sayegh MH. Clinical transplantation tolerance: many rivers to cross. J Immunol. 2007; 178(9):5419-5423. 22. Najafian N, Albin MJ, Newell KA. How can we measure immunologic tolerance in humans? J Am Soc Nephrol. 2006;17(10):2652-2663. 23. Salama AD, Najafian N, Clarkson MR, Harmon WE, Sayegh MH. Regulatory CD25⫹ T cells in human
Massry Lecture kidney transplant recipients. J Am Soc Nephrol. 2003;14(6):1643-1651. 24. VanBuskirk AM, Burlingham WJ, Jankowska-Gan E, et al. Human allograft acceptance is associated with immune regulation. J Clin Invest. 2000;106(1):145-155. 25. Cai J, Lee J, Jankowska-Gan E, et al. Minor H antigen HA-1-specific regulator and effector CD8⫹ T cells, and HA-1 microchimerism, in allograft tolerance. J Exp Med. 2004; 199(7):1017-1023. 26. Li Y, Koshiba T, Yoshizawa A, et al. Analyses of peripheral blood mononuclear cells in operational tolerance after pediatric living donor liver transplantation. Am J Transplant. 2004;4(12):2118-2125. 27. Mazariegos GV, Zahorchak AF, Reyes J, et al. Dendritic cell subset ratio in peripheral blood correlates with successful withdrawal of immunosuppression in liver transplant patients. Am J Transplant. 2003;3(6):689-696. 28. Kowalski R, Post D, Schneider MC, et al. Immune cell function testing: an adjunct to therapeutic drug monitoring in transplant patient management. Clin Transplant. 2003;17(2): 77-88. 29. Brouard S, Mansfield E, Braud C, et al. Identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance. Proc Natl Acad Sci U S A. 2007;104(39):15448-15453.
1011 30. Martinez-Llordella M, Lozano JJ, Puig-Pey I, et al. Using transcriptional profiling to develop a diagnostic test of operational tolerance in liver transplant recipients. J Clin Invest. 2008;118(8):2845-2857. 31. Fathman CG, Soares L, Chan SM, Utz PJ. An array of possibilities for the study of autoimmunity. Nature. 2005;435(7042):605-611. 32. Lakkis FG, Sayegh MH. Memory T cells: a hurdle to immunologic tolerance. J Am Soc Nephrol. 2003;14(9): 2402-2410. 33. Koehn B, Gangappa S, Miller JD, Ahmed R, Larsen CP. Patients, pathogens, and protective immunity: the relevance of virus-induced alloreactivity in transplantation. J Immunol. 2006;176(5):2691-2696. 34. 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(6):501-509. 35. Kirk AD, Hale DA, Mannon RB, et al. Results from a human renal allograft tolerance trial evaluating the humanized CD52-specific monoclonal antibody alemtuzumab (Campath1H). Transplantation. 2003;76(1):120129. 36. Wadman M. London’s disastrous drug trial has serious side effects for research. Nature. 2006;440(7083): 388-389. 37. Neujahr DC, Chen C, Huang X, et al. Accelerated memory cell ho-
meostasis during T cell depletion and approaches to overcome it. J Immunol. 2006;176(8):4632-4639. 38. Turgeon NA, Iwakoshi NN, Phillips NE, et al. Viral infection abrogates CD8(⫹) T-cell deletion induced by costimulation blockade. J Surg Res. 2000;93(1):63-69. 39. Chen L, Wang T, Zhou P, et al. TLR engagement prevents transplantation tolerance. Am J Transplant. 2006;6(10):2282-2291. 40. Thornley TB, Brehm MA, Markees TG, et al. TLR agonists abrogate costimulation blockade-induced prolongation of skin allografts. J Immunol. 2006;176(3):1561-1570. 41. Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol. 2009;21(4): 317-337. 42. Gelman AE, LaRosa DF, Zhang J, et al. The adaptor molecule MyD88 activates PI-3 kinase signaling in CD4⫹ T cells and enables CpG oligodeoxynucleotide-mediated costimulation. Immunity. 2006;25(5):783793. 43. Denton MD, Magee CC, Sayegh MH. Immunosuppressive strategies in transplantation. Lancet. 1999;353(9158): 1083-1091. 44. Sho M, Sandner SE, Najafian N, et al. New insights into the interactions between T-cell costimulatory blockade and conventional immunosuppressive drugs. Ann Surg. 2002; 236(5):667-675.
The Shaul G. Massry Distinguished Lecture is delivered at the National Kidney Foundation’s annual Spring Clinical Meetings. This lectureship was established to honor Dr Massry for his scientific achievements and his contributions to the kidney health care community and the National Kidney Foundation. The 2009 lecturer was Mohamed H. Sayegh, MD, and his remarks form the basis for this article.
M
ore than 55 years ago, a report published in Nature by Billingham et al1 widened the horizons of modern medicine by describing the phenomenon of acquired immunologic tolerance to transplant antigens. Shortly thereafter, one of the first kidney transplants was performed at the Peter Bent Brigham Hospital in Boston, MA, by a team headed by Joseph Murray, a plastic surgeon, that included John Merrill, a nephrologist.2 These 2 great milestones laid the foundation for tremendous advances in the field of transplantation immunology during the past 5 decades.3 Together, they launched a worldwide search for the holy grail of transplant tolerance, which is defined as the absence of pathologic immune response against a graft in an immunocompetent host not using maintenance immunosuppressive drugs.4 In this review, we highlight recent advances in the field of immunologic tolerance in organ transplantation that have made that
More than 55 years after the description of the phenomenon of acquired immunologic tolerance to transplant antigens by Medawar and colleagues, the holy grail of transplantation is a myth no more. Establishment of the Immune Tolerance Network in 1999 changed the approach used to achieve clinical tolerance by translating advances in the field of immunology in general and in tolerance in particular from experimental animal strategies into human clinical trials, and initial results have been promising. Despite these advances, scientific and operational challenges still face the transplantation community and affect progress. Overcoming those challenges is a shared responsibility of scientists, transplant professionals, and general nephrologists alike. Achieving tolerance could not only revolutionize the field of transplantation through avoidance of toxicity associated with immunosuppressive agents, but also influence the treatment of autoimmune diseases, in which the immune system attacks self, as well as cancer, in which tolerance of the immune system toward malignant cells may permit disease development and progression. Am J Kidney Dis 54:1005-1011. © 2009 by the National Kidney Foundation, Inc.
holy grail a myth no more. For the sake of clarity, terminology used in this article is defined in the glossary (Box 1).
THE RISE OF A NEW ERA IN TRANSPLANTATION Early Efforts to Achieve Transplant Tolerance After the pioneering era of Peter Medawar, John Merrill, Joseph Murray, and others, a second era can be described in which the major advances in immunology focused on the development of strategies to induce tolerance in experimental animal models. In addition, although several reports described the phenomenon of immunologic tolerance in some humans, these interventions were not rigorously attempted in controlled trials.5 During the pre-1999 era, tolerance was reported first by a group from Stanford in 3 patients who underwent total-body lymphoid irradiation before receiving a cadaveric kidney transplant.6 Soon thereafter, we reported 2 leukemic patients
complicated by end-stage renal disease (ESRD) who received a bone marrow transplant. Each of these patients subsequently received a successful kidney transplant several years later from their bone marrow donor without using immunosuppressive drugs, except for a low dose of prednisone for other indications.7 Similarly, a team at the Massachusetts General Hospital treated a patient with multiple myeloma complicated by ESRD
From the Transplantation Research Center, Renal Division, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, MA. Originally published online as doi: 10.1053/j.ajkd.2009.08.005 on October 12, 2009. Address correspondence to Mohamed H. Sayegh, MD, Transplant Research Center, Brigham and Women’s Hospital, Children’s Hospital Boston, 221 Longwood Ave, Boston MA 02115. Email: [email protected] © 2009 by the National Kidney Foundation, Inc. 0272-6386/09/5406-0006$36.00/0 doi:10.1053/j.ajkd.2009.08.005
American Journal of Kidney Diseases, Vol 54, No 6 (December), 2009: pp 1005-1011
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Azzi and Sayegh Box 1. Glossary
Immunologic tolerance: a state of immunologic unresponsiveness toward a given set of antigens in an immunocompetent individual Transplantation tolerance: the absence of pathologic immune response against a graft in an immunocompetent host not using maintenance immunosuppressive drugs Mixed lymphohematopoietic chimerism: the state after bone marrow transplant in which hematopoietic cells of donor and recipient origin coexist Nonmyeloablative bone marrow transplant: allogeneic bone marrow transplant that uses lower doses of chemotherapy and/or radiation with the goal to make room for the donor cells and prevent their rejection without destroying the recipient’s bone marrow Regulatory T cell (Treg): T lymphocyte that turns off specific immune responses Mixed lymphocyte reaction (MLR): the proliferative response that occurs when lymphocytes from 2 individuals are cultured together, as the result of reactions of T cells of 1 individual to MHC antigens on the other individual’s cells Cytotoxic T lymphocyte (CTL): a T lymphocyte (which usually expresses CD8) that kills its target cell on recognizing complexes of peptides and MHC molecules on the target-cell membrane Central deletion: a mechanism of immune tolerance using lymphoablative treatments to create a mixed hematopoietic chimeric state. In this instance, donor cells seed the thymus, and maturing donorreactive T-cell clones are deleted through intrathymic apoptosis Peripheral deletion: another mechanism of immune tolerance by which donor-reactive cells can undergo apoptosis outside the thymus, in either the allograft itself or in draining lymph nodes Abbreviation: MHC, major histocompatibility complex.
with both a bone marrow and kidney transplant from the same donor, inducing mixed lympho-
hematopoietic chimerism (defined as the persistence of donor hematopoietic cells in the recipient). This has been suggested to modulate immune responsiveness to donor alloantigens as a mechanism of transplant tolerance.8 Finally, some transplant recipients would play Russian roulette by discontinuing their immunosuppressive medications, with a lucky few serendipitously winning tolerance.9 Successes in Liver Transplant Tolerance The liver is considered an immune-privileged organ characterized by an anatomic structure believed to facilitate mixed chimerism through bidirectional interaction of donor and recipient leukocytes, resulting in elimination of alloreactive T cells to donor and recipient cells consecutively.10 These observations encouraged prospective drug withdrawal programs in ⬎ 90 liver transplant recipients between 1992 and 1996, with very promising results for the transplant community at the time.11 After this initial trial, a smaller cohort of 18 patients had immunosuppression therapy withdrawn at King’s College, London, with 28% of patients reported completely free of medications at 3 years.12 Operational liver tolerance trials have continued to emerge in the last few years. Takatsuki et al13 in Japan successfully achieved complete withdrawal of tacrolimus therapy in 38% of their 63 liver transplant recipients, and a Kyoto group reported the withdrawal of immunosuppressants in 87 of 659 (15%) children who underwent living donor liver transplant be-
tween 1990 and May 2005.14 Results from a Pittsburgh group have been particularly notable, with a mean time off immunosuppression therapy ⬎ 10 years in a cohort of 48 liver transplant recipients. Return to immunosuppression therapy has been rare (1 of 48 patients).15 The Era of the Immune Tolerance Network A new era emerged with the rise of the new millennium after establishment of the ITN (Immune Tolerance Network: www.immunetolerance.org), a clinical research consortium founded by the US National Institutes of Health in 1999 for the purpose of focusing efforts on the goal of achieving immune tolerance therapies. The formation of this consortium has resulted in many clinical trials, not only in islet and solid-organ transplant, but also in autoimmune disease and allergy/asthma (Table 1). One of the first fruits born of the ITN was the publication from the Massachusetts General Hospital group of a case series composed of 6 patients with multiple myeloma and ESRD who received simultaneous kidney and bone marrow transplants from HLAidentical sibling donors after a nonmyeloablative regimen. Immunosuppressive drugs were completely withdrawn within 2 months after transplant. Chimerism was transient in 4 patients. One patient experienced rejection. This was treated successfully, and immunosuppressive drugs were withdrawn successfully in that patient 1 year later. Two other patients developed graft-versus-host disease,
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1007 Table 1. Recent and Ongoing Clinical Trials of Immune Tolerance Therapies
Procedure
Islet transplant
Tolerance Protocol
Edmonton protocol of steroid-free immunosuppression
Alemtuzumab,a tacrolimusb for 60 d, sirolimus (rapamycin) for 1 y Kidney transplant Daclizumab, LEA29Y (belatacept), MMF, sirolimus for 1 y Kidney transplant Alemtuzumab, donor stem-cell infusion, tacrolimus/sirolimus and MMF for 1 y Kidney & bone marrow Cyclophosphamide, MEDI-507 (siplizumab), 7 Gy thymic transplant irradiation, donor bone marrow, cyclosporine for 60 d Kidney & bone marrow Cyclophosphamide, ATG, 7 Gy thymic irradiation, donor transplant for bone marrow, cyclosporine for 36-60 d myeloma patients Liver transplant Alemtuzumab, tacrolimus slowly withdrawn Liver transplant Gradual withdrawal of maintenance immunosuppression in patients who underwent living donor liver transplants as children Liver transplant Alemtuzumab vs 3 mo of steroids followed by tacrolimus slowly withdrawn Kidney transplant
Study Status
Completed; 44% (16/36) insulin free at 1 y; 14% (5/36) insulin free at 2 y16 Active Active Active Completed; 4/5 patients withdrawn from all immunosuppressants18 Completed; all 6 patients withdrawn from all immunosuppressants17 Active Active
Active
Abbreviations: ATG, antithymocyte globulin; MMF, mycophenolate mofetil. a Humanized anti-CD52 antibody (Campath-1H; Berlex Laboratories, www.berlex.com). b Prograf (Astellas Pharma US Inc, www.astellas.us).
and immunosuppressive medications were resumed.17 A recent New England Journal of Medicine report published by the same Massachusetts General Hospital group reported on 5 patients with ESRD with combined bone marrow and kidney transplants from HLA single-haplotype– mismatched living related donors.18 They used a nonmyeloablative preparative regimen with cyclophosphamide at days ⫺5 and ⫺4 with respect to transplant; humanized antiCD2 monoclonal antibody on days ⫺1, 0, and 1; and intravenous cyclosporine and thymic irradiation on day ⫺1. They intravenously infused donor bone marrow after the kidney transplant, trying to induce mixed chimerism. One patient developed humoral rejection that required modifying the protocol for future patients to
induce B-cell depletion with monoclonal antibody against CD20 (rituximab) on days ⫺7 and ⫺2. The other 4 recipients discontinued immunosuppressive therapy at 9-14 months after transplant with stable renal function, now going on ⬎ 6 years for patients 1 and 2 and ⬎ 4 and 3 years for patients 4 and 5, respectively (B. Cosimi, personal communication, July 2009). Interestingly, although the chimerism induced by infusion of the bone marrow transplant was transient, tolerance persisted. Hypothesized mechanisms include elimination of cells reactive to donor antigens in the thymus (called central deletion) or generation of regulatory T cells with high expression levels of FOXP3 (a marker of regulatory T cells) in the renal allograft protocol biopsy specimens.18 Close follow-up of these patients is necessary to under-
stand the impact of losing the lymphohematopoietic mixed chimerism and its effect on tolerance, the risk of graft-versushost disease, and the emergence of other types of rejection, for example, humoral rejection. In the same New England Journal of Medicine issue, a Stanford group described a recipient of a combined kidney and hematopoietic cell transplant from an HLA-matched donor followed by total lymphoid irradiation and antithymocyte globulin with ⬎ 2 years of follow-up with no episodes of rejection or graft-versushost disease.19
CHALLENGES IN TRANSLATING TOLERANCE STRATEGIES INTO THE CLINIC The lack of reliable assays to assess immune function is the most significant challenge
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to develop clinical tolerance strategies.20 Given that acquired tolerance is weaker than natural tolerance and also may be transient, long-term monitoring is vital.21 Many immune function assays have been developed during the last decade, but none can accurately predict the development of tolerance.22 Accordingly, we still rely on measuring drug levels of immunosuppressive agents (which affect individuals’ immune systems differently), and we evaluate graft function (usually a late marker for events). Currently available assays can be antigen specific, meant to evaluate donor-specific T-cell responses against recipient antigens by measuring either: (1) proliferation of donor T cells in a mixed lymphocyte reaction, (2) lysis of recipient T cells in a cytotoxic T-lymphocyte assay,9 or (3) production of interleukins with either an enzyme-linked immunosorbent assay or enzyme-linked immunospot assay.23 The transvivo delayed-type hypersensitivity assay uses an immune-deficient mouse as an “in vivo milieu” for the interaction between donor T cells and recipient antigens. T Cells and antigens are injected into the footpads of the mice, causing swelling that is quantified relative to the level of sensitivity that can be measured using a caliper.24 Tetramer technology also may be used to detect the T-cell receptors that are specific for the recipient peptide– major histocompatibility complex molecule complex.25 The classic pretransplant cross-match is still used to detect donorspecific antibodies.
Azzi and Sayegh Box 2. Types of Immune Function Assays Antigen-specific assays T-Cell alloreactivity: -Proliferation of donor T cells in MLR -Lysis of recipient T cell in CTL assay -Production of cytokines in ELISA or Luminex or ELISpot assays -Transvivo delayed-type hypersensitivity Humoral immune responses: -HLA and non-HLA alloantibodies Non–antigen-specific assays Expression profiling of cellular surface markers: -Flow cytometry for regulatory T cells -Flow cytometry for plasmacytoid dendritic cells Measuring T-cell response to nonspecific polyclonal stimulus: -Intracellular ATP production RNA analysis (eg, microarrays) and proteomics: -Identify signature of tolerance Note: Luminex (www.luminexcorp.com) is a proprietary fluorescent bead immunoassay. Abbreviations: ATP, adenosine triphosphate; MLR, mixed lymphocyte reaction; CTL, cytotoxic T lymphocyte; ELISA, enzyme-linked immunosorbent assay; ELISpot, enzyme-linked immunosorbent spot.
Assays also may be non– antigen specific, measuring regulatory cells, such as the CD4⫹CD25high T cells known as Tregs,26 plasmacytoid dendritic cells that were reported to promote a type 2 T-helper cell– type response that is more tolerogenic.27 An assay commercialized as ImmunoKnow (Cylex Inc, www.cylex.net) measures T-cell response to a nonspecific polyclonal stimulus by quantifying intracellular adenosine triphosphate production as a marker of the level of cell immune function.28 Another revolutionary measure is a gene signature or gene expression profiling, recently explored by identifying a number of genes differentiating a tolerant from a nontolerant recipient with high accuracy.29,30 Finally, proteomics, which studies protein expression, can be a necessary complement to gene expression
because levels of protein synthesis may not correspond well to measured amounts of the respective messenger RNA transcripts31 (Box 2). Another major obstacle in developing clinical tolerance is the lack of the ideal animal model that will translate accurately in humans. For example, strategies in rodents have been unsuccessful in nonhuman primates32-34 and, in turn, may not be applicable in humans.35,36 Those significant immunologic differences mainly manifest in the proportion of memory and immunologically naive T cells.32 Rodents, for example, which are considered immunologically naive with a lower proportion of memory cells than primates or humans, respond successfully to many costimulatory blockade strategies that fail when used in humans.34 However, memory
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cells are very resistant to depletion therapy, which may deplete regulatory cells, promoting expansion of the memory T-cell compartment and making tolerance induction even more difficult.37 These unsuccessful outcomes in humans complicate selection of the best strategy to induce tolerance. Is central or peripheral deletion in association with drug minimization the answer, or does a completely novel therapy need to be developed?21 Evolving data incriminate infectious agents through stimulation of Toll-like receptors in preventing and reversing tolerance.38-40 Toll-like receptors, expressed on a variety of cell types, such as endothelial cells, epithelial cells, macrophages, neutrophils, and antigen-presenting cells, have a major role in innate immunity by recognizing different pathogens and differentiating self from nonself.41 In antigenpresenting cells exposed to an infection, Toll-like receptors stimulate cell maturation by increasing expression of CD80, CD86, and major histocompatibility complex class II antigens, which enhances T-cell responses, directly inducing survival and proliferation of CD4 cells42 and allogeneic CD8 T cells.40 This may explain the differences in resistance to tolerance protocols among transplanted organs because organs that are most exposed to infectious agents tend to be the most recalcitrant. Another scientific nonimmunologic challenge facing the transplantation community is the best end points to use in a clinical trial. Coupling observa-
1009
tion of short-term outcomes (1year survival) with longer term results when infectious and malignant complications are most likely to occur (2-3 years or beyond) is necessary to ensure safety. Furthermore, other operational challenges can be difficult to overcome. We need to reach consensus on when to move from a pilot study to a larger trial without exposing patients to intensive protocols if long-term success is unlikely.21 Similarly, it is extremely important to decide on the best management strategy for patients in tolerance trials who experience organ rejection: whether to withdraw them from the trial and return them to conventional therapy or modify therapeutic strategies according to a specific protocol. The most challenging issue facing modern medicine today, including our quest for tolerance strategies, is ethics. One major issue is eligibility criteria. This is particularly notable for age, given the recognition that children receiving organ transplants may derive the greatest benefit from immunosuppression withdrawal because of their potential longer exposure to immunosuppression, but may derive the greatest risk because a failed organ in a child carries a worse long-term prognosis than in an adult because of sensitization and compromise of a future transplant. A second issue is choosing a suitable organ for these clinical trials given that the consequences of failed liver, heart, and lung transplants are acutely life threatening. Should we compromise a
living donor organ or would a cadaveric organ for our initial trials be more appropriate? These issues require collaboration and consensus-building among all members of the transplantation community. ITN began this initiative by presenting a platform on which to share ideas. Diverging interests between pharmaceutical companies and the transplantation community regarding tolerogenic strategies may impair progress because the pharmaceutical industry may be perceived as being interested in keeping patients on maintenance immunosuppressive medication therapy. Therefore, cooperation between pharmaceutical companies that develop and market agents to induce tolerance and the transplantation community is essential for success in achieving the goal of tolerance. Facilitating future success in refining tolerance-inducing strategies is a responsibility shared by scientists and transplant and general nephrologists alike. Resistance to minimizing or halting immunosuppression is an issue for both patients and their physicians, especially when patients currently are stable with maintenance or low-dose immunosuppression. Rightfully, patients and physicians both ask: Is low-dose immunosuppression really an alternative to complete withdrawal from drug therapy? Do low-dose safe levels exist?
CONCLUSION Improving our communication and collaboration is integral to achieving tolerance, which will have a direct impact on the morbidity and mor-
1010
tality of transplant recipients, mainly through avoidance of toxicity associated with immunosuppressive agents. Nonspecific immunosuppression increases the risk of malignancies, infections, and metabolic diseases, such as diabetes and dyslipidemia, thus increasing the rate of cardiovascular mortality.43 Furthermore, calcineurin inhibitors have been incriminated in shortening graft survival, increasing the demand for retransplant, and worsening the organ shortage.44 Beyond transplantation, better understanding the mechanics of tolerance could profoundly influence the treatment of autoimmune diseases, such as type 1 diabetes and lupus, in which the immune system attacks self. In addition, therapeutic approaches to cancer might evolve by better understanding the tolerance of the immune system toward malignant cells. Implementation of a successful safe tolerance regimen also has the potential to create a framework for new treatments of genetic diseases through stem cell therapy, reestablishing islet cell transplant in patients with type 1 diabetes, and launching regenerative medicine by allogeneic stem cell transplant, for which the achievement of tolerance is not a mere option, but an absolute necessity.
ACKNOWLEDGEMENTS Financial Disclosure: Dr Sayegh is a consultant to Genzyme, but has no financial interest related to the subject matter of this article. Dr Azzi has no relevant financial interests.
Azzi and Sayegh
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