On the Horizon of Immunosuppression

TRANSPLANTATION REVIEWS V O L 14, N O 4

OCTOBER 2000

On the Horizon of Immunosuppression j. HaroldHelderman The history of immunosuppression in renal transplantation and trends that might inform its future are discussed. A philosophical paradigm shift away from additive to reductive immunosuppressive protocols is announced. Newer immunosuppressive strategies that use the deeper understanding of the immunobiology of organ transplantation is detailed, with particular reference to inhibition of costimulatory pathways and blockade of antigen-specific signaling, as well as to the potential for tolerance development. Agents that might use novel immunosuppressive pathways are discussed. The future of transplantation immunosuppression is bounded only by our imagination and our understanding of the basic science of the field. Copyright © 2000 by W.B. Saunders Company

linically successful renal transplantation began nearly 50 years ago as a cooperative venture between interested scientists and clinicians. Few could predict that the investigative problems of immunosuppression, surgical technique, and tissue matching would advance to the point that renal replacement by transplantation has become the mode of choice. As the history of renal transplantation is examined, the discovery of various immunosuppressive strategies have been the milestones. Specific historic eras have been defined by immunosuppression. The first historic era was a period of little clinical success and a great deal of basic and clinical research. This era permitted the problems of surgical technique to be explored while successfully engrafting identical twins who posed r~o immunologic barriers to transplantation success. The second historic era, the azathioprine period in human transplantation, began in the early 1960s as

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From theDepartmentofMedicine,DivisionofNephrology,Vanderbilt TransplantCenter,VanderbiltUniversityMedicalCenter,Nashville, TN. Address reprint requeststo J. Harold Helderman, MD, Vanderbilt University Medical Center, Division of Nephrology, S-3305 Medical CenterNorth,Nashville, TN37232-2372. Copyright© 2000by W.B. SaundersCompany 0955-470X/00/1404-0003$10.00/0 doi.'lO.lO53/trre.2000.16514

Sir Roy Calne studied the immunosuppressant synthesized by Hitchings and Elion. The advent of azathioprine permitted the extension of renal transplantation to live nontwin donor transplantation and cadaveric transplantation with modest success. This period was characterized by a great deal of clinical rejection episodes, with cadaveric graft survival rates of approximately 50% at 1 year and decreasing mortality as experience with the use of the new agent grew. The discovery of the calcineurin' inhibitor drugs, beginning with cyclosporine A, launched the third historic era. Acute rejection rates were halved, and markedly improved early graft survival was the rule. Soon after Borel, Calne, and White discovered cyclosporine A as an important clinical immunosuppressant, the monoclonal antibody directed to the CD3 constellation of proteins became available for the successful treatment of acute rejection episodes. Recently, new immunosuppressive strategies have rapidly been found, holding the promise of the near elimination of clinically apparent acute rejection of the kidney transplant. Not only have these newer immunosuppressive strategies, coupled to an immunosuppressant backbone of calcineurn inhibition, improved early graft survival, but there has been a steady and even more rapid increase in long-term graft outcomes.

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As recently shown by Hariharan et al in a seminal article in transplant outcomes research using the graft half-life metaphor, there has been steady improvement in long-term outcomes to match the 1-year graft survival rate. j Analyzing the United Network for Organ Sharing database of almost 94,000 renal transplantations from 1988 to 1996, rejection rates were reduced from 46% to 24%, graft half-lives for recipients of cadaveric allografts have increased from 7.9 to 13.8 years, and graft half-lives of living donor grafts have increased from 12.7 to 21.6 years. Moreover, Hariharan et al clearly showed the advantages of any strategy to reduce acute and clinically apparent renal transplant rejection on favorable long-term renal transplant outcomes. Careful analysis of the tempo of the half-life improvements suggests that the continued discovery of such new agents as mycophenolate mofetil and the monoclonal anti-CD25, interleukin-2-receptor antibodies, will continue to contribute to improved long-term outcomes.

As one looks to the future ofimmunosuppression, questions are raised about the need for continued research into newer immunosuppressive strategies. Of course, continued long-term patient and graft survival improvements will always be a stimulus for new research. An equally persuasive argument for continued research into new immunosuppressive strategies is to discover agents that balance efficacy and safety. The discovery of immunosuppressive strategies that can provide equally superb results to those presently obtained with markedly reduced toxicities will always be an important stimulus for new investigation. Translating these outcome results to high-risk patients who have not shared equally in the improved survival rates is another goad to new drug development. Recently, the pioneering and even heroic studies of Rush et al from the University of Winnipeg that uncovered the importance of subclinical rejection and its relation to chronic allograft nephropathy and loss provide an additional rationale for continued new immunosuppression studies; the severing of the link between subclinical rejection and its long-term consequences.2 Two additional intrinsic goals stimulate a great deal of research in immunosuppression: overcoming the xenograft immunologic barriers and developing true immunologic tolerance. Both these topics deserve an extensive and individual treatise beyond the scope of this article.

ParadigmS h i f t The future of immunosuppression will begin with a philosophic paradigm shift in the practice of renal transplant medicine. In the first 3 historic eras of renal transplantation, the dominant philosophy of immunosuppression was one that I call an additive approach to immunosuppression, in which each new agent was discovered and shown to be clinically useful which was added to those already used or substituted for 1 of the elements in a multidrug regimen. The corollary to this philosophic approach has been the move away from fixed standard immunosuppressive regimens that governed practice throughout the transplant world to a wide variety of approaches that vary among institutions because the transplant team can choose from up to 20 different options for immunosuppressive protocols. One of the most important changes in immunosuppression evolving or already in use is a change in philosophy to a more reductive approach, in which the fewest agents that provide the maximal protection from acute rejection episodes with the least side effects will be used, with careful individualized patient selection further destro)4ng the notion of a standard immunosuppressive regimen for most renal transplant recipients. The search for this deductive approach with the avoidance or minimization of important elements of immunosuppression that have defined our clinical practice in the last 2 decades is underway with a vengeance. At a recent national meeting of the American Society of Transplant Surgeons, more than 35 separate communications discussed calcineurninhibitor minimization, and an additional 25 submissions discussed steroid withdrawal. The steroid withdrawal story is as old as clinical transplantation. Each time a new immunosuppressive agent has been brought to the clinic, an attempt to minimize or withdraw steroids has been undertaken. The past universal outcomes from steroid withdrawal have been improvements in the steroidrelated side-effect profile, with a small percentage of patients experiencing an acute rejection episode temporally related to steroid withdrawal. In addition, there has even been evidence for reduced long-term graft survival in those patients with steroid therapy, successfully withdrawn with respect to acute rejection prophylaxis, which appears 3 to 5 years after withdrawal has been accomplished. A recent attempt under the auspices of the National Institutes of Health to use mycophenolate mofetil in a multidrug regimen to withdraw steroid therapy in patients

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experiencing no early acute rejection episodes was met with an unacceptable acute rejection rate in the steroid-withdrawn group, leading to the discontinuance of new enrollment on the recommendation of the safety monitoring board. More recently, total steroid avoidance or rapid early withdrawal has been tested with better early success, but long-term outcomes in this setting remain to be determined. Some might argue that the future of immunosuppression includes a new era of steroid freedom. Despite the observation that early and late graft outcomes have benefited immensely from the use of calcineurin drugs as the backbone ofimmunosuppressire regimens, series of arguments can be constructed for attempting to match present graft outcome success with regimens that minimize or avoid the use of this class of agents. Some begin this argument by repeating the false mantra that longterm graft survival has not kept pace with short-term results under the aegis of these agents. Clearly, the 2 candidate drugs in this class, cyclosporine A and tacrolimus, have a substantial toxicity profile, including nephrotoxicity, cosmetic derangements, diabetogenesis, hyperlipidemia, hyperkalemia, hyperuricemia, and neurotoxicity, which might be avoided or reduced in minimization regimens. Total acute rejection prophylaxis has been achieved in animal studies with these agents at doses prohibitive on the grounds of toxicity. Some argue that the positive effects that flow from acute rejection episode reduction may be balanced by nephrotoxicity and the propensity of both members of the calcineurin inhibitor class to activate profibrotic growth factor genes that contribute to the histologic derangement called chronic allograft nephropathy. Three specific strategies have been attempted in cyclosporine-sparing regimens: (1) cyclosporine withdrawal from the initial regimen alone or after the addition of other immunosuppression, such as azathioprine or mycophenolate mofetil; (2) the use of lowdose cyclosporine in triple-therapy regimens that include mycophenolate mofetil; and (3) the introduction of such newer agents as sirolimus while reducing cyclosporin A or tacrolimus. In 2 small series in which clinically suspected or biopsy-proven chronic allograft nephropathy was present, Weir et al and Huasso discontinued cyciosporine therapy and added mycophenolate mofetil.3 In both cases, no acute rejection episodes were encountered, renal function improved, serum cholesterol levels and blood pressure improved, and in the latter case, transforming growth factor-J3 plasma levels

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decreased. Grinyo studied the reduction in cyclospofine dose from a triple-therapy regimen including mycophenolate mofetil and prednisone in stable renal transplant recipients without acute rejection episodes, and reported improved renal function, blood pressure, and transforming growth factor-J3 serum levels. Kasiske used the statistical approach of metaanalysis for a multiplicity of cyclosporine-withdrawal trials involving more than 1,000 patients in the premycophenolate era and concluded that a small fraction of patients would experience rejection temporally related to cyclosporine withdrawal without detriment to short-term graft survival rates. 4 He cautioned that the impact on long-term outcomes consequent to this increased acute early rejection rate was not discernible from the studies analyzed. The advent of such newer agents as" mycophenolate mofetil and sirolimus have permitted continued testing of the hypothesis that calcineurin inhibitors may be avoided. Vincente et al attempted to use newer agents to avoid the use of calcineurin inhibitor. 5 In a multicenter, randomized, prospective protocol in which anti-CD25 antibody was used for induction, followed by mycophenolate mofetil and prednisone maintenance, calcineurin inhibitors were avoided in 98 transplant recipients followed up for I year. Such a strategy may not be acceptable because the rejection rate in this cohort was 50%, 5-fold greater than that experienced by many centers with multidrug regimens using newer immunosuppression with calcineurin inhibitors. However, the combined use of several new agents without calcineurin inhibitors continues to hold promise. Recently Kahan studied the use of sirolimus, mycophenolate mofetil, and prednisone to avoid the use of a calcineufin inhibitor early in the face of delayed graft function, with no acute rejection episodes in a small cohort as proof of the principle that calcineurin inhibitors can successfully be avoided. This served as the scientific underpinning for a multicenter trial of the combination of sirolimus and mycophenolate mofetil with prednisone after antibody induction without cyclosporine or tacrolimus. 6

Immunosuppressionby Interfering With Lymphocyte Activation The immunosuppressive milestones that defined the history of renal transplantation have grown out of a research paradigm that takes a molecule shown to be an effective inhibitor of a number of in vitro assays of alloimmune responses into animal models, with a final test in the clinic. As our understanding of the

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molecular pathways of lymphocyte activation and response expands, a second paradigm shift for immunosuppressive drug development will define the transplant immunosuppression future. A plethora of developing strategies for immunosuppression use our newer understanding of the immunobiologic characteristics of alloresponse as the target for discrete pathway inhibition. The 3-signal metaphor for alloimmune T-lymphocyte activation popularized by Halloran is a useful starting point to explicate this new paradigm and discuss strategies for clinical immunosuppression that might be used in the near future) In this formulation, the first activation signal flows from the apprehension of the alloantigen by the naive T lymphocyte using the T-cell-antigen receptor and the signal transduction pathway afforded by the CD3 constellation of proteins. The recognition step for signal 1 involves the provision of the signal, along with major histocompatibility complex (MCH) elements directly on donor-specific professional antigenpresenting cells (APCs) or indirectly by recipient MHC on recipient APCs. Essential for the initial events of T-cell activation is a second signal, an antigen-independent interaction between the T cell and the APC through a series ofligand-receptor pairs called costimulatory molecules. The provision of signal 1 without signal 2, either by presenting alloantigen on nonprofessional APCS without the costimulatory pathways or by blocking these pathways, leads to a state ofimmunosuppression called T-cell anergy, in which the cells are still present but neither enter the activation-directed cell cycle nor are capable of further activation by antigen for a finite period of time. Consequent to the provision of signals 1 and 2, the T cell enters G1A and G1B of the cell cycle with the synthesis of an array of new molecules, which together provide the machinery necessary for the creation of a third signal of lymphocyte activation, which is absolutely essential to the clonal expansion of the activated T-cell effector cells ultimately responsible for allograft rejection. Many of these new molecules are elements of high-affinity growth factor receptors not constitutively expressed on quiescent T cells; others are the growth factors themselves. Signal 3 flows from the interaction of specific growth factors and cytokines, with their cell-surface receptors stimulating the activation of regulatory molecules called cyclins, permitting the T cell to double its DNA, array the chromosomes in a metaphase configuration, and divide. Recently, the laboratories of Turka and Strom have shown that a balancing

signal for lymphocyte inhibition through an apoptotic mechanism can also result from signal 3, which has important consequences for the design of potential future immunosuppressive strategies.

Inhibition of the Costimulatory Pathways The basic immunobiologic process supporting several strategies to block the costimulatory pathway, signal 2 of the metaphoric description of T-cell activation, with the goal of establislfing anergy of antigen-specific T-cell clones, has led to clinical strategies of immunosuppression presently under study that might become part of the new treatment landscape. The first strategy extends the fertile use of monoclonal antibody development in transplant immunosuppression to antibodies that might block either partner of the costimulatory ligand-receptor pair. Recently, an antibody directed against the CD40-CD40 ligand system, anti-CD154, prolonged ailograft survival in primate models. The initial human trial of the first-generation anti-CD154 was started but was halted secondary to unanticipated thrombogenic complications. Based on the sound immunologic theory and superb primate results of such newer antibodies, perhaps using recently developed genomically engineered molecules to fully humanize the antibody, this strategy is predicted to be examined in human studies to determine whether the xenogeneic component of the original monoclonal antibody was the thrombogenic culprit. A second strategy of inhibition of costimulatory molecular pairs is equally attractive. This second strategy targets the B7-CD28 pair of costimulatory molecules using a natural ligand for the B7 molecule, CTLA4, made into a fusion protein called CTLA4IG. This approach has had substantial success in animal models of organ transplantation in the laboratories of Turka and Sayegh. These reagents will soon undergo human trials in the United States. The ferment to use the knowledge of the importance of the signals derived from the costimulatory pathways and their inhibition ~11 certainly drive continued clinical research. It is highly likely that reagents targeting those systems will become a routine component ofimmunosuppression in the future.

Blockade of Antigen-Specific Signaling The steps that initiate T-cell activation at the inception of donor-recipient interaction that provides sig-

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nal 1 are vulnerable to abrogation. Such a strategy for immunosuppression flows from the observations of Krensky et al that specific synthetic peptides derived from the sequences of informative regions of the MHC corresponding to unique extracellular domains of both class I and class II molecules may be immunoregulatory. The use of a small sequence of peptides derived from class I MI-IC shown to inhibit T-cell activation has lead to the development of a clinical reagent called Allotrap (SangStat Medical, Fremont, CA), which has been effective in models of primate heart transplantation. The laboratory of Krensky et al has an array of such immunoregulatory peptides, some that might be more attractive than the Allotrap. Regardless of the specific reagent currently available, it is likely that an immunosuppressant strategy using such synthetic peptides will form the near future of the transplant discipline.

Tolerance Development Since the infancy of transplantation, the dream of the scientist and the clinician has been to create true immunologic tolerance in the clinic, defined as graft retention without immunosuppression. The National Institutes of Health has taken ultimate this goal, an allegorical search for the Holy Grail of transplantation, into the development of the multicenter Tolerance Network to foster clinical investigation into promising strategies that might culminate in the discovery of this mythical goal. In the development of adult acquired tolerance, almost all experimental strategies have involved the initial manipulation of the immune system, some form of immune-conditioning regimen, after which tolerance may ensue. This approach of immune conditioning provides a newer rationale for the development of novel immunosuppressant regimens that might alter how immunosuppression is viewed and performed in the future. The 3 signal model of lymphocyte activation is an apposite means to understand at least 1 approach of tolerance development using immunosuppression as effective conditioning regimens in the future. Recall that signal 3, generated from growth factor signal transduction, provides not only a positive signal for lymphocyte activation, but a signal for potential tolerance development through apoptosis. In an interesting series of animal experiments from the laboratory of Turka and Strom, prolonged graft survival after just such conditioning immunosuppression was a consequence of the apprehension of the alloantigen with intact signal 1 and signal 2 pathways. Thus, some

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growth factor elaboration was present, followed by a reduction in the magnitude of this cell cycle signal with antibody to CD25 added to the blockade of the cell cycle stimulatory pathway by sirolimus. This approach activated the apoptosis pathway, culminating in tolerance. This strategy totally avoids cyclosporine and uses newer agents in this novel conditioning to achieve the long-sought transplant outcome.

Novel Immunosuppressive Pathways The future of immunosuppression will not exclusively be consequent to abrogation of the T-cell activation pathways. Immunosuppressive reagents are under development that have revolutionary and novel mechanisms of action unrelated to signals for lymphocyte activation. Immunosuppressive molecules that use such novel pathways have already been developed and are in clinical testing. One such novel approach involves alteration of lymphocyte homing or trafficking pathways. The agent, F'FY720, a synthetic myriocin analogue isolated from the fungus, Ischaria sinclairii, is an effective immunosuppressive molecule in animal models and reduces peripheral lymphocyte counts by approximately 50% and the movement of lymphocytes into inflammatory tissues and grafts while increasing the component of lymphocytes in gut collections and Peyer's patches in the intestine. Single- and multiple-dose, phase 1, dose-ranging studies of humans have been recently concluded, enhancing optimism that this revolutionary approach to immunosuppression will be available for clinical trials in the near future and.will be part of a group of new medicines and strategies available to the clinician.

Conclusion The era of immunosuppression in which only a few reagents were available to achieve modest success, leading to a 1-size-fits-all approach to therapy, is past. A philosophic revolution in immunosuppression is before us, and an entirely individualized approach to immunosuppression is at hand. The explosion in research into the immunobiologic state of organ transplantation is the intellectual driver in this philosophic revolution. Although we can see the pattern of the fabric ofimmunosuppression for organ transplantation at the horizon, the future is only limited by our understanding of immunology and our imagination.

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References 1. Hariharan S,Johnson CP, Bresnahan BA, et al: Improved graft survival after transplantation in the United States, 1988 to 1996. N EnglJ Med 342:605-612, 2000 2. Rush D, Nickerson P, Cough J, et al: Beneficial effects of treatment of early subclinical rejection: A randomized stud),. J Am Soc Nephrol 9:2129-2134, 1998 3. Weir M:R,FinkJC, Hanes DS, et ah Chronic allograft nephropathy: Effect ofcyclosporin in reduction and addition of mycophenolate mofetil on progression of renal disease. Transplant Proc 31:1286-1287, 1999 4. Kasiske BL, Heim-Duthoy K, Ma JZ: Elective cyclosporine withdrawal after renal transplantation. JAMA 269:396-400, 1993

5. Vincente F: Sparing of calcineurin antagonists: The use of interleukin-2 monoclonal antobodies in conjunction with mycophenolate mofetil. Transplant Immunol Lett 16:4-6, 2000 6. Gauthier P, HeldermanJH: Cyclosporine avoidance.J Am Soc Nephrol 2000 (in press) 7. Halloran PF: T-cell activation pathways: A transplantation perspective. Transplant Proc 31:769-771, 1999 8. Li XC, Dodge I, et ah Induction of allograft tolerance in the absence of Fas-mediated apoptosis. J Immunol 163:2500-2507, 1999 9. Issazadeh S, Abdallah K, Chitnis T, et al: Role of passive T-cell death in chronic experimental autoimmune encephalomyelitis. J Clin Invest 105:1109-1116,2000