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Transplantation Tolerance: From Bench to Bedside Dela Golshayan and Manuel Pascual 3 Early or Late Calcineurin Inhibitor Withdrawal and Mycophenolate Mofetil-Based Immunosuppression to Prevent Graft Loss in Patients with Suboptimal Kidney Transplant Function Ravinder K. Wali and Matthew R. Weir 13 Long-Term Immunosuppression in Pediatric Liver Transplantation Paloma Jara and Loreto Hierro 28

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Trends in Transplant. 2009;1:3-12 Dela Golshayan, Manuel Pascual: Transplantation Tolerance: From Bench to Bedside

Transplantation Tolerance: From Bench to Bedside Dela Golshayan and Manuel Pascual Transplantation Centre and Transplantation Immunopathology Laboratory, Centre Hospitalier Universitaire Vaudois (CHUV), University of Lausanne, Lausanne, Switzerland

Abstract During the past twenty years, solid organ transplantation has become the therapy of choice for many end-stage organ diseases. While short-term graft and patient outcome has greatly improved with the development of new immunosuppressive protocols, long-term results remain relatively disappointing, mainly because of the side effects associated with chronic drug exposure. Thus, the induction of donor-specific immune tolerance remains an important goal in transplantation. Here, we first describe the pathways underlying allograft rejection and the potential targets for immune intervention. We then review the strategies that could lead to the induction of transplantation tolerance based on experimental models and discuss the protocols that are currently evaluated in clinical transplantation. (Trends in Transplant. 2009;1:3-12) Corresponding author: Dela Golshayan, [email protected]

Key words Allorecognition. Transplantation tolerance. T-cells. Regulatory T-cells.

Introduction Within the past twenty years, the successes achieved in clinical solid organ transplantation have been tightly linked to the use of powerful immunosuppressive drugs that can control the rejection process. Based on extensive data obtained in rodents and large-animal experimental transplantation models, newer immunosuppressive strategies have progressively been developed and transposed to routine clinical practice1,2. Shortterm outcomes such as patient and allograft survival, as well as rates of acute allograft rejection episodes in the first year after transplantation, have

Correspondence to: Dela Golshayan Division of Nephrology and Transplantation Centre, BU 17, CHUV, 1011 Lausanne, Switzerland E-mail: [email protected]

steadily improved. However, the full potential of organ transplantation has not yet been realized because of the unwanted side effects and increased patient morbidity and mortality associated with chronic drug exposure3-5. Besides the complications related to nonspecific suppression of the immune response, there is an inexorable loss of transplanted organs due to chronic allograft dysfunction, a process involving immunologic (inadequate immunosuppression) and non-immunologic factors (drug-related toxicities)6,7. Because of the ever increasing number of potential transplant candidates and the shortage of donor organs8, the long-term outcome of clinical transplantation must be optimized. The ultimate goal in clinical transplantation remains, therefore, to safely achieve long-term allograft acceptance in the recipient, which implies sustained donor-specific T- and B-cell non-responsiveness with preserved allograft function, in the absence of (operational tolerance) or minimal (near-tolerance) chronic immunosuppression. 3

Trends in Transplantation 2009;1

Over the past 50 years, experimental models have become an invaluable tool for elucidating the mechanisms underlying the induction and maintenance of tolerance to alloantigens9. If these experimental protocols can be successfully transposed to clinical transplantation, the induction of donor-specific transplantation tolerance could result in long-term allograft survival while preserving the host from drug-related adverse effects.

Mechanisms of allograft rejection Allorecognition Adaptive immune responses to grafted tissues are the major obstacle to successful transplantation. The term “allorecognition” refers to T-cell recognition of genetically encoded polymorphisms between members of the same species, mainly major histocompatibility complex (MHC) molecules which are expressed on donor cells. Although MHC incompatibility can provoke a strong immune response, graft rejection can also occur in MHC-matched donor-torecipient combinations due to the recognition of minor histocompatibility antigens10. These antigens are peptides derived from polymorphic proteins other than MHC molecules presented in the context of MHC class I and II molecules. Tissues that are mismatched for MHC antigens can provoke antidonor immunity and allograft rejection via three distinct pathways, mediated primarily by T-cells: the direct, indirect, and semidirect pathways11. T-cells with direct allospecificity are activated after recognition of intact MHC alloantigens displayed at the surface of donor professional antigen-presenting cells (APC). T-cells with indirect allospecificity recognize donor alloantigens as processed peptides associated with recipient MHC molecules in a self-restricted manner. Both pathways were shown to help cytotoxic CD8+ T-cells, induce macrophage responses and allospecific antibody production by B-cells. The high frequency of T-cells with direct allospecificity and the relative low frequency of T-cells with indirect allospecificity in the T-cell repertoire has led to the concept that the direct alloresponse dominates the early posttransplantation period and is mainly involved in acute transplant rejection, while the indirect pathway plays a major role in later forms of alloresponses and in chronic transplant rejection11-15. However, animal models also support a role for the indirect pathway in acute rejection as 4

this pathway has been shown to be sufficient to elicit allograft destruction in the absence of direct allorecognition. Thus, current data suggest that the indirect pathway might be the one to target to achieve long-term graft survival, particularly since experimental models have shown the importance of indirect allorecognition in the induction of transplantation tolerance11-17. A third mechanism of allorecognition has recently been described (the semi-direct pathway), where trafficking recipient dendritic cells (DC) could acquire intact donor MHC:peptide complexes from donor DC or endothelial cells and induce proliferation of antigen-specific T-cells18,19.

T-cell activation The recognition by recipient T-cells of MHC-mismatched antigens is the primary event that ultimately leads to graft rejection. In the early stages after transplantation, proinflammatory signals produced as a result of initial ischemia/reperfusion tissue injury promote the maturation of donor DC and their migration out of the allograft towards secondary lymphoid organs to encounter recipient alloreactive T-cells20,21. Once activated by donor DC (direct pathway), alloantigen-specific T-cells will home to the allograft and initiate the acute rejection process. At later stages, with the decline of donor-derived DC, the immune response against an allograft is maintained by recipient APC (DC and B-cells) that process and present allogeneic MHC molecules shed from the graft (indirect and semidirect pathways). In addition to antigen recognition, full T-cell activation requires a second distinct co-stimulatory signal. The first signal (signal 1) is delivered through the T-cell receptor (TCR) by recognition of peptide antigen presented in the context of MHC molecules on the APC. Co-stimulatory signals (signal 2) are delivered via constitutive or inducible receptors on the responding T-cell surface interacting with their ligands constitutively expressed or upregulated on the activated APC. A growing number of co-stimulatory receptor/ligand molecules have been identified. The CD28/ B7.1(CD80)-B7.2 (CD86), CD40L(CD154)/CD40, ICOS/ICOSL positive activating co-stimulatory pathways have been best characterized so far in in vivo experimental transplantation models. These signals are balanced by inhibitory inducible signals such as CD152(CTLA-4)/B7 and PD-1/PD-L,

Dela Golshayan, Manuel Pascual: Transplantation Tolerance: From Bench to Bedside

allowing a regulation of the T-cell response22-24. If partial activation occurs, as for instance in the absence of co-stimulation, T-cells become hyporesponsive to subsequent antigen-specific TCR signals (donor-specific anergy) or die by apoptosis25,26. The engagement of the TCR/CD3 complex (signal 1) in the presence of signal 2 activates the intracellular calcium-calcineurin signaling pathway and the induction of transcription factors, leading to the expression of new surface molecules, such as inducible co-stimulatory molecules and cytokine receptors, that deliver growth and proliferation signals (signal 3) via the downstream phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathways.

T-cell effector function The encounter of naive T-cells with DC modulates their differentiation into polarized T helper (Th) cells and thus is a major component in the regulation of T-cell responsiveness (Fig. 1)21,27-29. Depending on the type of DC, the type and dose of antigens, and the local cytokine microenvironment, T-cells can differentiate into various subsets of effector T-cells, determining the outcome of the immune response towards immunity or tolerance. Mature DC and the presence of interleukin (IL)-12 promote the development of Th1 cells that secrete cytokines usually associated with inflammation (interferon-g, IL-2 and tumor necrosis factor-a) and induce cell-mediated immune responses. Interleukin-4 skews towards Th2 cells, which are mainly involved in humoral immunity and allergic responses30. More recently, a subset of IL-17-producing cells (Th17) has been identified in infectious and autoimmune disease models. In vitro, this subset of cells develops in the presence of transforming growth factor (TGF)-b and the proinflammatory cytokine IL-631-33. These pathogenic effector T-cells are counterbalanced by a subset of naturally occurring regulatory T-cells, the CD4+CD25+ Foxp3+ T regulatory (Treg) cells. This small population of CD4+ T-cells is selected in the thymus, and constitutively co-expresses the IL-2 receptor α-chain (CD25) and the forkhead box protein 3 transcription factor (Foxp3). These cells were shown to be crucial for the control of autoreactive T-cells in autoimmune-disease models, as well as being potent suppressors of the effector function of alloreactive T-cells in transplantation models in vivo34-36. There is increasing evidence that in the

peripheral immune system and under specific conditions, uncommitted naive T-cells can be skewed toward T-cells with regulatory potentials, also referred to as induced regulatory T-cells (iTreg). The presence of “regulatory” cytokines such as TGFb or IL-10 and antigen presentation by immature DC have been shown to favor the generation of antigen-specific T-cells with regulatory properties in vitro and in vivo27,28. Recent data suggest that naive T-cells could be induced to differentiate in the periphery into Th17 or iTreg cells in a mutually exclusive manner33. Thus, skewing of the immune response away from Th17 or Th1 proinflammatory cells (for example by blocking critical cytokines such as IL-6) and towards Treg cells may prevent transplant rejection.

Immune tolerance in transplantation Immune tolerance is defined as the ability of the immune system to distinguish between self and nonself harmful antigens, leading to a specific, protective, cell-mediated and humoral response. Experimental models have shown that the immunologic mechanisms that normally maintain immune homeostasis and tolerance to selfantigens were basically the same as those involved in the induction of tolerance to alloantigens. T-cell tolerance to self-antigens can be established centrally during lymphocyte development in the thymus, or in the periphery at sites of antigen recognition and processing. Self-tolerance is partly achieved by intra-thymic deletion of selfreactive lymphocytes from the immune repertoire (clonal selection). As not all self-antigens are expressed in the thymus, other mechanisms exist in the peripheral immune system to maintain a safe T-cell repertoire. Peripheral tolerance can be established and maintained by various mechanisms, including deletion of activated/effector Tcells, anergy induction, and active regulation of effector T-cells (Fig. 2)26,37.

Central tolerance Many experimental data support the role of the thymus in the induction of sustained robust tolerance to alloantigens38,39. During the normal maturation process in the thymus, T-cells with high avidity for thymic-expressed self-antigens are deleted (negative selection) so that potentially deleterious antigen-reactive T-cells will not reach the periphery. This physiologic process 5

Trends in Transplantation 2009;1

THYMUS

CD4+CD25+Foxp3+

CD4+CD25–Foxp3– PERIPHERY DC

Pathogenic T-cells

Th0

Foxp3–

Regulatory T-cells TGFβ

IL-12 Th1

TGF β+IL-6

IL-4

T-bet

Th2

GATA-3 Th17

Foxp3

Foxp3+

Naturally occurring + induced Treg

ROR-γt

Figure 1. T-cell activation and differentiation into effector T-cell. The encounter of naive T-cells with antigen-presenting DC modulates their differentiation into polarized Th cells and thus is a major component in the regulation of T-cell responsiveness. The committed T-cells are characterized by their cytokine profile and the expression of specific transcription factors: T-bet for Th1, GATA-3 for Th2, FoxP3 for Treg cells and ROR-γt for Th17 cells. To maintain immune homeostasis, the pathogenic Th1, Th2 and Th17 effector T-cells are counterbalanced by a subset of naturally occurring or induced Treg cells. DC: dendritic cells; Th: T helper; IL: interleukin; TGF: transforming growth factor; Treg: regulatory T-cell.

could be exploited experimentally to induce specific tolerance to the allograft by the delivery of donor alloantigens to the thymus prior to organ transplantation. Experimentally, donor-derived allopeptides can be directly injected into the thymus40. Another possibility, which is more appropriate for clinical application, is the induction of a state of hematopoietic mixed chimerism in the recipient’s repertoire, that is to say the presence of cells from both recipient and donor origin. This implies the transfusion of donor whole bone marrow (BM) cells into a cell-depleted recipient. Experimental studies have demonstrated that after the transfer of donor hematopoietic cells, donorderived APC migrate to the recipient’s thymus and induce clonal deletion of donor-reactive T-cells9,38,41-43. Thus, hematopoietic chimerism results in education of the immune system to recognize the donor as self. It was initially thought that to establish central tolerance in solid organ transplantation, total ablation of the preexisting, cross-reactive, peripheral T-cells and a state of hematopoietic donor-type full chimerism would be necessary. This implied myeloablation with total body irradiation or highdose chemotherapeutic agents, similar to protocols used in clinical BM transplantation for malig6

nant disorders. It was reported that such recipients who achieved full chimerism could subsequently accept a kidney allograft from the original BM donor without immunosuppression44. However, because of its toxicity, this approach could not be justified to achieve tolerance in solid organ transplant recipients without malignant diseases. Extensive work performed in small and large animal models have since demonstrated that sustained donor-specific transplantation tolerance can be induced through the generation of a state of hematopoietic mixed chimerism (reconstitution of the recipient with donor and recipient hematopoietic cells). Thus, high-intensity myeloablative preconditioning protocols could be successfully replaced by other less-toxic approaches (highdose BM combined to co-stimulatory blockade and thymic irradiation)45-48.

Peripheral tolerance Circulating alloreactive T-cells are crucial in the initiation and coordination of the immune response to an allograft, and, to promote tolerance, the alloreactive effector T-cell pool must be minimized while enhancing regulatory mech-

Dela Golshayan, Manuel Pascual: Transplantation Tolerance: From Bench to Bedside

anisms37. In addition, in the peripheral immune system, T-cells respond to alloantigens if they are presented in the context of appropriate costimulatory signals and within specialized structures of secondary or tertiary lymphoid organs49-51. Experimental transplantation models have explored the following strategies to achieve peripheral transplantation tolerance: - deletion of peripheral effector T-cells (lymphocyte-depleting protocols); - inhibition of T-cell activation by blocking or modifying co-stimulatory signals (co-stimulatory blockade, manipulation of DC); - interference with the effector function and homing of activated T-cells (anti-chemokines); - active regulation of effector T-cells by antigen-specific Treg cells. After extensive studies in rodent and largeanimal models, some of these approaches are now being progressively transposed to the clinic.

Translating tolerance into the clinic Spontaneously tolerant transplant recipients A number of clinical reports of occasional “tolerant” transplant recipients, characterized by prolonged allograft survival with minimal or no immunosuppression, suggest that immunologic tolerance may indeed be achievable in some patients52. Spontaneous operational tolerance is rare in kidney transplant recipients, but has been more frequently reported in liver transplant recipients, suggesting that various mechanisms may be involved such as the induction of mixed chi­ merism38,53 or the promotion of peripheral regulatory mechanisms54-56. Collaborative international studies are currently underway to pool these tolerant transplant recipients in order to determine more precisely how this state arises and to develop reliable tests to predict tolerance52,57,58.

Mixed chimerism The proof-of-concept of the clinical applicability of hematopoietic mixed-chimerism approaches to deliberately induce transplantation

tolerance was first established in a small number of patients with hematopoietic malignant disorders (e.g. end-stage renal failure secondary to refractory multiple myeloma). These highly selected recipients underwent HLA-matched BM and kidney transplantation from the same living donor, and had long-term acceptance of their renal allograft in the absence of ongoing immunosuppression44,59-62. More recently, these protocols have been adapted to patients without concomitant malignancy. Following on their previous protocols developed in animal models and in small clinical trials63, the Stanford group recently started a study on patients with end-stage renal disease without malignancy, undergoing combined HLA-matched kidney and hematopoietic cell transplantation, using a low-intensity conditioning regimen (total lymphoid irradiation and antithymocyte globulin)64. Their first transplant recipient in this protocol had persistent mixed chimerism and normal kidney allograft function more than two years after discontinuation of all immunosuppressive drugs. Of interest, a few years ago the same group had attempted a similar protocol for the induction of allograft tolerance in HLAmismatched recipients63, but rejection generally developed following immunosuppression withdrawal. However, the recent report of the Massachusetts General Hospital group brings some hope of the possibility to extend these clinical protocols to HLA-mismatched patients65. In their study, five patients with end-stage renal disease and no malignancy received combined BM and one-haplotype HLA-mismatched living-donor kidney transplantation, with the use of a nonmyeloablative preconditioning regimen. Except for one patient who developed non-reversible humoral rejection, immunosuppression was withdrawn in the remaining patients at 9-14 months after transplantation, with stable kidney function and donor-specific T-cell unresponsiveness. Interestingly, all the recipients displayed only transient chimerism posttransplantation, suggesting that, while the induction of tolerance is dependent on central deletion of donor-reactive T-cells, peripheral mechanisms may be involved in the long-term maintenance of a tolerant state. Clearly, because these trials have involved a relatively low number of patients, there is a need to confirm these results in larger groups in order to fully assess the clinical potential of the “mixed-chimerism strategy”. It also would be desirable to try to simplify this type of clinical protocol as, at least for now, these ap7

Trends in Transplantation 2009;1

THYMUS

PERIPHERY

Treg

Negative selection APC

MHC:peptide Positive selection

Co-stimulation

TCR/CD3 Th

Th0 T-cell activation Thymic selection Cytokines

Effector function

Figure 2. Mechanisms of T-cell activation and targets for the induction of transplantation tolerance. (A) Central tolerance. During T-cell maturation, T-cells with high or no affinity for thymically expressed antigens undergo intra-thymic deletion. As a result, only T-cells with moderate affinity to host MHC are selected and released to the periphery. In hematopoietic mixed chimerism, both host and donor type APC can migrate to the thymus, and dictate thymic selection. (B) Peripheral tolerance. Normal T-cell activation requires the interaction of MHC:peptide on the APC with the TCR, together with a co-stimulatory signal. The following peripheral mechanisms control effective T-cell activation, and could be exploited to induce immune tolerance: i) Co-stimulatory blockade, manipulation of dendritic cells. Antigen-recognition in the absence of an appropriate signal 2 leads to antigen-specific anergy. ii) T-cell depleting antibodies. All T-cells are depleted from the periphery, irrelevant of their specificity or activation state. iii) Anti-cytokines, anti-chemokines. Alloreactive T-cells are activated, but they cannot home to the allograft and exert their effector function. iv) T-cell regulation. T-cells are present, but their activation and effector function is controlled by Treg cells. Treg: regulatory T-cell; APC: antigen-presenting cells; MHC: major histocompatibility complex; TCR: T-cell receptor; Th: T helper.

proaches have required a high degree of technical expertise and medical “know how”, so that patients had to be referred to specific transplantation centers to be enrolled in such protocols.

T-cell depletion The primary mediators of the immune response are T and B lymphocytes that have antigen-specific receptors, recognizing alloantigens and inducing donor-specific cellular and humoral responses. With the development of polyclonal and monoclonal antibodies (MAb), newer therapeutic strategies have been developed based on powerful cell-depletion at the time of transplantation (induction therapy) when immune activation is most intense. In various experimental rodent and nonhuman primate (NHP) models, anti-T-cell antibodies have been used either alone or in combination with other strategies that aim to limit the clonal expansion of effector T-cells such as co-stimulatory blockade or transfusion of donorderived peptides66. T-cell depletion strategies have been extensively studied in NHP transplantation models, and encouraging results have led 8

to clinical trials using polyclonal antithymocyte globulins, a humanized anti-CD52 MAb (alemtuzumab, Campath-1H), or anti-CD3 MAb. Polyclonal antithymocyte globulins and Campath-1H induce profound and durable reduction of circulating leucocytes capable of mounting an alloresponse at a time when the allograft is already susceptible to inflammatory damage following the peri-transplantation ischemia/reperfusion injury. Lymphocytes will gradually repopulate the host weeks to months later when the innate immune response has resumed and the allograft is more quiescent. Calne, et al. first published interesting clinical data in deceased-donor kidney transplantation, using lymphocyte depletion with Campath-1H, which provided long-term, rejection-free allograft survival with minimal maintenance therapy (low-dose cyclosporine) in most patients. Because these recipients did not exhibit true operational tolerance, the authors coined the term “prope or near-tolerance” to describe their immunologic state67. Other groups have extended this approach of drug minimization by combining polyclonal rabbit ATG or Campath-1H together with 15-deox-

Dela Golshayan, Manuel Pascual: Transplantation Tolerance: From Bench to Bedside

yspergualin (DSG, a monocyte inhibitor), rapamycin, or tacrolimus. These subsequent clinical studies confirmed that, despite profound peri-transplant T-cell depletion, consistent transplantation tolerance could not be achieved when anti-T-cell Ab were used in monotherapy, and immunosuppressive therapy could rarely be completely withdrawn68-74. Of note, the use of Campath-1H alone or in combination with sirolimus or DSG was associated with an unusually high rate of early cellular rejection episodes, characterized by predominant monocytic infiltrates, as well as antibody mediated rejection episodes, so that minimal immunosuppression had to be maintained in later studies68,71-74. Non-activating humanized Fc-receptornon-binding anti-CD3 MAb (teplizumab, ChAglyCD3, visilizumab) are currently being tested in phase I and II clinical trials in autoimmune diseases settings (type 1 diabetes, Crohn’s disease, arthritis) as well as in renal and pancreatic islet transplantation75. Whether these antibodies will find a place in the transplant drug armamentarium still needs to be determined.

Co-stimulatory blockade Current data suggest that the interruption of T-cell signaling pathways at specific steps prevents the differentiation of alloreactive T-cells into proliferating effector T-cells and may promote immune tolerance in some circumstances. By inhibiting T-cell activation rather than eliminating all T-cells, as in depleting protocols, the strategy of co-stimulatory blockade might more selectively target effector T-cells and spare beneficial Treg cells. In many experimental rodent and NHP transplantation models, dual blockade of the CD154(CD40L):CD40 and the CD28:B7 pathways was shown to act synergistically to promote tolerance76. Of the various co-stimulatory molecules that have been targeted in animal models, the most successful results were obtained using an anti-CD40L MAb77. However, the administration of humanized anti-CD40L MAb in transplant recipients resulted in unexpected thromboembolic complications, possibly due to the expression of CD40L on platelets, and all clinical trials with this agent were therefore terminated78. CD28 is constitutively expressed on CD4+ T-cells and up to 50% of CD8+ T-cells, and its ligation by B7 molecules (CD80, CD86) expressed on

DC synergizes with TCR signaling to lower the activation threshold of T-cells. CD28 may also deliver a reverse signal to DC, inducing the production of IL-679,80. Cytotoxic T-lymphocyte-associated antigen (CTLA)-4 Ig, a fusion protein combining the extracellular binding domain of CTLA-4 with the Fc portion of IgG1 and with specificity for CD80/86 expressed on APC, was first used in experimental models with excellent outcomes. In NHP models, CTLA-4 Ig prolonged pancreatic islet survival and, in combination with anti-CD40L, induced indefinite acceptance of renal and heart allografts, while allowing prolonged skin-graft survival81-83. Following these encouraging preclinical results, clinical trials were initiated with LEA29Y (belatacept), a high-affinity variant of CTLA-4 Ig84. In a recent phase II renal transplantation clinical trial, after induction with anti-IL-2 receptor MAb (basiliximab), belatacept was administered every four weeks together with mycophenolate mofetil and steroids. The reported short-term results were promising in terms of safety and rates of biopsyproven acute rejection within the first year, when compared to a “classical” cyclosporine-based maintenance regimen85. But, co-stimulatory blockade has been so far used in the clinic more as an immunosuppressive agent and an alternative to calcineurin inhibitors. Longer follow-up data will have to establish if this drug can prevent chronic allograft dysfunction and/or favor the induction of transplantation tolerance, as was suggested in animal models.

Regulatory T-cells The use of powerful induction therapies may promote regulatory mechanisms and the induction of peripheral transplantation tolerance by depleting or interfering with the activation and/or effector function of alloreactive T-cells at the time of transplantation. However, unlike in animal models, only a small proportion of patients can completely discontinue therapy (operational tolerance), or be kept on minimal immunosuppression (near-tolerance) safely, that is in the absence of donor-specific alloresponses and with long-term normal graft function. In the absence of the induction of robust, donor-specific tolerogenic mechanisms (as may occur following a state of donor-type mixed chimerism), some degree of immunosuppression must be maintained as long as the allograft is in place. Thus, transplant immunobiologists are now exploring newer individualized therapies based on 9

Trends in Transplantation 2009;1

manipulated donor or recipient cells that specifically target donor alloantigens27,86. There is increasing evidence that in many experimental protocols where robust peripheral transplantation tolerance is achie­ved, immunoregulatory mechanisms dependent on donor-specific Treg cells are critical in the induction and maintenance of the tolerant state35,87. Based on these observations, strategies exploiting antigenspecific Treg cells in the induction of transplantation tolerance are currently being explored. A first strategy to promote peripheral tolerance would be to manipulate host immune responses in order to delete peripheral, effector, alloreactive T-cells, and at the same time favor the development and expansion of donor-specific Treg cells37,88,89. In accordance with previous experimental results, induction regimens based on T-cell-depleting antibodies90-91 or co-stimulatory blockade, as well as the use of mTOR inhibitors, appear to be promising protocols in clinical transplantation. In experimental transplantation models, mTOR inhibitors were shown to facilitate peripheral deletion of effector T-cells by promoting activation-induced cell death, leaving a small pool of residual alloreactive T-cells which could be regulated by Treg cells88. Furthermore, recent data have demonstrated that the mTOR inhibitor rapamycin could selectively expand Treg cells in vitro and in vivo, while calcineurin inhibitors appear to have a deleterious effect92-94. Another strategy that is currently under investigation would be to achieve regulation of in vivo alloresponses by the transfer to the recipient of customized donor-specific Treg cells, selected and expanded ex vivo86,95,96.

Conclusion and perspectives Encouraging results from experimental transplantation models have led to preliminary clinical trials with immunomodulatory therapies aiming at promoting donor-specific hyporesponsiveness and the induction of operational tolerance. However, the translation of these successful experimental tolerogenic protocols across species and into the clinic in human transplant recipients remains a major challenge. One of the major hurdle is the human T-cell repertoire that, compared to laboratory animals that have not been exposed to as many environmental antigens, contains a larger proportion of cross-reactive memory T-cells97,98. Memory T-cells differ from naive T-cells by their activation requirements and 10

homing properties, and they have been shown to undergo homeostatic proliferation in T-cell-depleted hosts, thus rendering them more resistant to co-stimulatory blockade and anti-T-cell antibodies99,100. Newer strategies are currently being investigated in experimental transplantation models to more efficiently target this population101. In addition, detailed characterization of rare, spontaneously “tolerant” transplant recipients, that are drug-free or only minimally immunosuppressed, may help in defining the mechanisms involved in clinical transplantation tolerance and designing new tolerogenic protocols. A new era, therefore, has been opened in the field of immunomodulation in clinical transplantation. Ultimately, tolerance-inducing strategies will need to be compared to modern immunosuppressive drug regimens in order to define the optimal antirejection management for transplant recipients.

Acknowledgments D. Golshayan is supported by a SCORE grant (N° 3232BO-111370/1) from the Swiss National Science Foundation and a grant from the Faculty of Biology and Medicine (FBM), University of Lausanne, Switzerland.

References 1. Halloran PF. Immunosuppressive drugs for kidney transplantation. N Engl J Med. 2004;351:2715-29. 2. Sayegh MH, Carpenter CB. Transplantation 50 years later - progress, challenges, and promises. N Engl J Med. 2004;351:2761-6. *A historical perspective of solid organ transplantation, describing past successes, highlighting progresses as well as current limitations and future challenges in the field. 3. Magee CC, Pascual M. Update in renal transplantation. Arch Intern Med. 2004;164:1373-88. 4. Pascual M, Theruvath T, Kawai T, Tolkoff-Rubin N, Cosimi AB. Strategies to improve long-term outcomes after renal transplantation. N Engl J Med. 2002;346:580-90. **A detailed description of the mechanisms leading to late allograft loss and of current strategies that may help to prevent or minimize this growing problem. 5. Meier-Kriesche HU, Schold JD, Srinivas TR, Kaplan B. Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era. Am J Transplant. 2004;4:378-83. *A large study analyzing the outcome of kidney transplantation over time in the USA, focusing on the evolution of the rates of acute rejection and its impact on graft survival in the recent era of immunosuppression. 6. Hernandez-Fuentes MP, Lechler RI. Chronic graft loss. Immunologic and non-immunologic factors. Contrib Nephrol. 2005;146:54-64. 7. Nankivell BJ, Borrows RJ, Fung CL, O’Connell PJ. The natural history of CAN. N Engl J Med. 2003;349:2326-33. *The best prospective study describing time course development and evolution of chronic allograft nephropathy up to 10 years after transplantation. 8. Evans RW, Orians CE, Ascher NL. Need, demand, and supply in kidney transplantation: a review of the data, an examination of the issues, and projections through the year 2000. Semin Nephrol. 1992;12:234-55. 9. Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature. 1953;172:603-6.

Dela Golshayan, Manuel Pascual: Transplantation Tolerance: From Bench to Bedside 10. Simpson E, Scott D, James E, et al. Minor H antigens: genes and peptides. Transpl Immunol. 2002;10:115-23. 11. Jiang S, Herrera O, Lechler RI. New spectrum of allorecognition pathways: implications for graft rejection and transplantation tolerance. Curr Opin Immunol. 2004;16:550-7. 12. Benichou G, Valujskikh A, Heeger PS. Contributions of direct and indirect T-cell alloreactivity during allograft rejection in mice. J Immunol. 1999;162:352-8. *A detailed analysis in a murine model of the frequency and cytokine phenotype of T-cells responding via direct and indirect pathways to alloantigens, at various time-points after transplantation. 13. Vella JP, Spadafora-Ferreira M, Murphy B, et al. Indirect allorecognition of MHC allopeptides in human renal transplant recipients with chronic graft dysfunction. Transplantation. 1997;64:795-800. 14. Hornick PI, Mason PD, Baker RJ, et al. Significant frequencies of T-cells with indirect anti-donor specificity in heart graft recipients with chronic rejection. Circulation. 2000;101:2405-10. 15. Baker RJ, Hernandez-Fuentes MP, Brookes PA, Chaudhry AN, Cook HT, Lechler RI. Loss of direct and maintenance of indirect alloresponses in renal allograft recipients: implications for the pathogenesis of chronic allograft nephropathy. J Immunol. 2001;167:7199-206. 16. Wise MP, Bemelman F, Cobbold SP, Waldmann H. Linked suppression of skin graft rejection can operate through indirect recognition. J Immunol. 1998;161:5813-16. 17. Yamada A, Chandraker A, Laufer TM, Gerth AJ, Sayegh MH, Auchincloss H. Recipient MHC class II expression is required to achieve long-term survival of murine cardiac allografts after co-stimulatory blockade. J Immunol. 2001;167:5522-6. 18. Smyth LA, Herrera OB, Golshayan D, Lombardi G, Lechler RI. A novel pathway of antigen presentation by dendritic and endothelial cells: Implications for allorecognition and infectious diseases. Transplantation. 2006;82:S15-18. 19. Herrera OB, Golshayan D, Tibbott R, et al. A novel pathway of alloantigen presentation by dendritic cells. J Immunol. 2004;173:4828-37. 20. Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: endogenous activators of dendritic cells. Nat Med. 1999;11:1249-55. 21. Lanzavecchia A, Sallusto F. Regulation of T-cell immunity by dendritic cells. Cell. 2001;106:263-6. 22. Sayegh M, Turka L. The role of T cell co-stimulatory activation pathways in transplant rejection. N Eng J Med. 1998;338:1813-21. 23. Salomon B, Bluestone JA. Complexities of CD28/B7: CTLA4 co-stimulatory pathways in autoimmunity and transplantation. Annu Rev Immunol. 2001;19:225-52. 24. Khoury SJ, Sayegh MH. The roles of the new negative T-cell co-stimulatory pathways in regulating autoimmunity. Immunity. 2004;20:529-38. 25. Harding FA, McArthur JG, Gross JA, Raulet DH, Allison JP. CD28-mediated signaling co-stimulates murine T-cells and prevents induction of anergy in T-cell clones. Nature. 1992;356:607-9. 26. Lechler RI, Chai JG, Marelli-Berg F, Lombardi G. The contributions of T-cell anergy to peripheral T-cell tolerance. Immunology. 2001;103:262-9. 27. Morelli AE, Thomson AW. Tolerogenic dendritic cells and the quest for transplant tolerance. Nature Rev Immunol. 2007;7:610-21. **An excellent review on the role of DC as regulators of the immune response and their therapeutic potential in the induction of transplantation tolerance. 28. Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH. Induction of IL 10-producing, non-proliferating CD4+ T-cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med. 2000;192:1213-22. 29. Lechler R, Ng WF, Steinman RM. Dendritic cells in transplantation: Friend or foe? Immunity. 2001;14:357-68. 30. Constant SL, Bottomly K. Induction of Th1 and Th2 CD4+ T-cell responses: the alternative approaches. Annu Rev Immunol. 1997;15:297-322. 31. Mangan PR, Harrington LE, O’Quinn DB, et al. Transforming growth factor-beta induces development of the Th17 lineage. Nature. 2006;441:231-4. 32. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T-cells. Immunity. 2006;24:179-89. 33. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector Th17 and regulatory T-cells. Nature. 2006;441:235-8. 34. Sakaguchi S. Naturally arising CD4+ regulatory T-cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol. 2004;22:531-62. **A thorough review of current knowledge on CD4+CD25+ regulatory T-cells























by the scientist who first described this subpopulation of CD4+ T-cells and launched this field of research. 35. Wood KJ, Sakaguchi S. Regulatory T-cells in transplantation tolerance. Nature Rev Immunol. 2003;3:199-210. 36. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T-cells. Nature Immunol. 2003;4:330-6. 37. Lechler RI, Garden OA, Turka LA. The complementary roles of deletion and regulation in transplantation tolerance. Nat Rev Immunol. 2003;3:147-58. 38. Starzl TE, Demetris AJ, Murase N, Ildstad S, Ricordi C, Trucco M. Cell migration, chimerism, and graft acceptance. Lancet. 1992;339:1579-82. 39. Remuzzi G. Cellular basis of long-term transplant acceptance: pivotal role of intra-thymic clonal deletion and thymic dependence of bone marrow microchimerism-associated tolerance. Am J Kidney Dis. 1998;31:197-212. 40. Spriewald BM, Ensminger SM, Jenkins S, Morris PJ, Wood KJ. Intra-thymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival. Transpl Int. 2004;17:458-62. 41. 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. 42. Sykes M. Mixed chimerism and transplant tolerance. Immunity. 2001;14:417-24. 43. Khan A, Tomita Y, Sykes M. Thymic dependence of loss of tolerance in mixed allogeneic bone marrow chimeras after depletion of donor antigen. Peripheral mechanisms do not contribute to maintenance of tolerance. Transplantation. 1996;62:380-7. 44. 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;114:954-5. 45. Sharabi Y, Sachs DH. Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen. J Exp Med. 1989;169:493-502. 46. Sykes M, Szot GL, Swenson KA, Pearson DA. Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning. Nat Med. 1997;3:783-7. 47. Wekerle T, Kurtz J, Ito H, et al. Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat Med. 2000;6:464-9. 48. Fuchimoto Y, Huang CA, Yamada K, et al. Mixed chimerism and tolerance without whole body irradiation in a large animal model. J Clin Invest. 2000;105:1779-89. 49. Golshayan D, Lechler R. Priming of alloreactive T cells where does it happen? Eur J Immunol. 2004;34:3301-4. 50. Lakkis FG, Arakelov A, Konieczny BT, Inoue Y. Immunologic ‘ignorance’ of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat Med. 2000;6:686-8. *The authors have used an elegant experimental model to demonstrate that secondary lymphoid organs provide the proper environment for the priming of naive T- and B-cells, and, that the alloimmune response to a vascularized allograft cannot be initiated in the graft itself. 51. Nasr IW, Reel M, Oberbarnscheidt MH, et al. Tertiary lymphoid tissues generate effector and memory T-cells that lead to allograft rejection. Am J Transplant. 2007;7:1071-9. 52. Ashton-Chess J, Giral M, Brouard S, Soulillou JP. Spontaneous operational tolerance after immunosuppressive drug withdrawal in clinical renal allotransplantation. Transplantation. 2007;84:1215-9. 53. Starzl TE. Immunosuppressive therapy and tolerance of organ allografts. N Engl J Med. 2008;358:407-11. 54. VanBuskirk AM, Burlingham WJ, Jankowska-Gan E, et al. Human allograft acceptance is associated with immune regulation. J Clin Invest. 2000;106:145-55. 55. Baeten D, Louis S, Braud C, et al. Phenotypically and functionally distinct CD8+ lymphocyte populations in long-term drug-free tolerance and chronic rejection in human kidney graft recipients. J Am Soc Nephrol. 2006;17:294-304. 56. Louis S, Braudeau C, Giral M, et al. Contrasting CD25hiCD4+ T-cells/FOXP3 patterns in chronic rejection and operational drug-free tolerance. Transplantation 2006;81:398-407. *An extensive phenotypic analysis of the peripheral blood of operationally tolerant kidney transplant patients compared to a large number of control groups. 57. Bluestone JA, Matthews JB, Krensky AM. The Immune Tolerance Network: The “Holy Grail” comes to the clinic. J Am Soc Nephrol. 2000;11:2141-6. **This report summarizes the rationale for emphasizing clinical research on immune tolerance and highlights the progresses of the Immune Tolerance Network

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(ITN), an international consortium of basic and clinical immunologists dedicated to the evaluation of novel tolerance-inducing therapies in autoimmune diseases and transplantation. 58. Goldman M, Wood K. European research on cell and organ transplantation: towards novel opportunities? Transplant Int. 2007;20:1016-19. **A description of the ongoing collaborative effort across the European Union in basic and translational immunology, including the development of novel immunosuppressive agents, new diagnostic tools and validation of biomarkers for the prediction of rejection as well as the induction of tolerance. 59. Spitzer TR, Delmonico F, Tolkoff-Rubin 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;68:480-4. 60. Buhler LH, Spitzer TR, Sykes M, 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. 61. Strober S, Benike C, Krishnaswamy S, Engleman EG, Grumet FC. Clinical transplantation tolerance 12 years after prospective withdrawal of immunosuppressive drugs: studies of chimerism and antidonor reactivity. Transplantation. 2000;69:1549-54. 62. Fudaba Y, Spitzer TR, Shaffer J, et al. Myeloma responses and tolerance following combined kidney and non-myeloablative marrow transplantation: in vivo and in vitro analyses. Am J Transplant. 2006;6:2121-33. 63. Millan MT, Shizuru JA, Hoffmann P, et al. Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation. Transplantation. 2002;73:1386-91. 64. Scandling JD, Busque S, Dejbakhsh-Jones S, et al. Tolerance and chimerism after renal and hematopoietic-cell transplantation. N Engl J Med. 2008;358:362-8. 65. Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med. 2008;358:353-61. *A description of the hematopoietic mixed chimerism approach to induce tolerance in clinical kidney transplantation, in recipients without concomitant malignancy. 66. Elster EA, Hale DA, Mannon RB, Cendales LC, Swanson SJ, Kirk AD. The road to tolerance: renal transplant tolerance induction in nonhuman primate studies and clinical trials. Transplant Immunol. 2004;13:87-99. 67. Calne R, Friend P, Moffatt S, et al. Prope tolerance, perioperative Campath 1H, and low-dose cyclosporin monotherapy in renal allograft recipients. Lancet. 1998;351:1701-2. 68. Kirk AD, Hale DA, Mannon RB, et al. Results from a human renal allograft tolerance trial evaluating the humanized CD52-specific MAb alemtuzumab (Campath-1H). Transplantation. 2003;76:120-9. 69. Shapiro R, Jordan ML, Basu A, et al. Kidney transplantation under a tolerogenic regimen of recipient pretreatment and low-dose postoperative immunosuppression with subsequent weaning. Ann Surg. 2003;238:520-5. 70. Swanson SJ, Hale DA, Mannon RB, et al. Kidney transplantation with rabbit antithymocyte globulin induction and sirolimus monotherapy. Lancet. 2002;360:1662-4. 71. Kirk AD, Mannon RB, Kleiner DE, et al. Results from a human renal allograft tolerance trial evaluating T-cell depletion with alemtuzumab combined with deoxyspergualin. Transplantation. 2005;80:1051-9. 72. Barth RN, Janus CA, Lillesand CA, et al. Outcomes at three years of a prospective pilot study of Campath-1H and sirolimus immunosuppression for renal transplantation. Transplant Int. 2006;19:885-92. 73. Tan HP, Kaczorowski DJ, Basu A, et al. Living donor renal transplantation using alemtuzumab induction and tacrolimus monotherapy. Am J Transplant. 2006;6:2409-17. 74. Golshayan D, Pascual M. Drug-minimization or tolerance-promoting strategies in human kidney transplantation: is Campath1H the way to follow? Transplant Int. 2006;19: 881-4. 75. Chatenoud L, Bluestone JA. CD3-specific antibodies: a portal to the treatment of autoimmunity. Nature Rev Immunol. 2007;7:622-32. 76. Larsen CP, Elwood ET, Alexander DZ, et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature. 1996;381:434-8. 77. Larsen CP, Pearson TC. The CD40 pathway in allograft rejection, acceptance, and tolerance. Curr Opin Immunol. 1997;9:641-7. 78. Kawai T, Andews D, Colvin RB, Sachs DH, Cosimi AB. Thromboembolic complications after treatment with MAb against CD40 ligand. Nat Med. 2000;6:114.

79. Alegre ML, Frauwirth KA, Thompson CB. T-cell regulation by CD28 and CTLA-4. Nat Rev Immunol. 2001;1:220-8. 80. Orabona C, Grohmann U, Belladonna ML, et al. CD28 induces immunostimulatory signals in dendritic cells via CD80 and CD86. Nat Immunol. 2004;5:1134-42. 81. Adams AB, Shirasugi N, Jones TR, et al. Development of a chimeric anti-CD40 MAb that synergizes with LEA29Y to prolong islet allograft survival. J Immunol. 2005;174:542-50. 82. Kirk AD Harlan DM, Armstrong NN, et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci USA. 1997;94:8789-94. 83. Adams AB, Shirasugi N, Durham MM, et al. Calcineurin inhibitor-free CD28 blockade-based protocol protects allogeneic islets in nonhuman primates. Diabetes. 2002;51:265-70. 84. Larsen CP, Pearson TC, Adams AB, et al. Rational development of LEA29Y (belatacept), a high-affinity variant of CTLA4-Ig with potent immunosuppressive properties. Am J Transplant. 2005:5:443-53. 85. Vincenti F, Larsen C, Durrbach A, et al. Belatacept Study Group. Co-stimulation blockade with belatacept in renal transplantation. N Engl J Med. 2005;353:770-81. *The first report of the use of co-stimulatory blockade with CTLA-4 Ig (belatacept) in a clinical kidney transplantation trial, as compared to cyclosporine-based immunosuppression. 86. Bluestone JA, Thomson AW, Shevach EM, Weiner HL. What does the future hold for cell-based tolerogenic therapy? Nature Rev Immunol. 2007;7:650-4. **A discussion of the potential of cell-based tolerogenic therapies in transplantation and autoimmune diseases, highlighting some important issues that must be considered before this form of immunotherapy can become a reality. 87. Graca L, Cobbold SP, Waldmann H. Identification of regulatory T-cells in tolerated allografts. J Exp Med. 2002;195: 1641-6. 88. Zheng XX, Sanchez-Fueyo A, Sho M, Domenig C, Sayegh MH, Strom TB. Favorably tipping the balance between cytopathic and regulatory T-cells to create transplantation tolerance. Immunity. 2003;19:503-14. **The proof of concept in an experimental model of the efficacy of an immunotherapeutic strategy to induce peripheral transplantation tolerance by preferentially targeting activated pathogenic donor-reactive T-cells and sparing Treg cells. 89. Wells AD, Li XC, Li Y, et al. Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat Med. 1999;5:1303-7. 90. Lopez M, Clarkson MR, Albin M, Sayegh MH, Najafian N. A novel mechanism of action for antithymocyte globulin: induction of CD4+CD25+Foxp3+ regulatory T-cells. J Am Soc Nephrol. 2006;17:2844-53. 91. Bloom DD, Chang Z, Fechner JH, et al. CD4+CD25+FOXP3+ regulatory T-cells increase de novo in kidney transplant patients after immunodepletion with Campath-1H. Am J Transplant. 2008;8:793-802. 92. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 2005;105(12):4743-8. 93. Zeiser R, Nguyen VH, Beilhack A, et al. Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent IL-2 production. Blood. 2006;108:390-9. 94. Segundo DS, Ruiz JC, Izquierdo M, et al. Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+FOXP3+ regulatory T-cells in renal transplant recipients. Transplantation. 2006;82:550-7. 95. Golshayan D, Jiang S, Tsang J, Garin MI, Mottet C, Lechler RI. In vitro-expanded donor alloantigen-specific CD4+CD25+ regulatory T-cells promote experimental transplantation tolerance. Blood. 2007;109:827-35. 96. Joffre O, Santolaria T, Calise D, et al. Prevention of acute and chronic allograft rejection with CD4+CD25+Foxp3+ regulatory T lymphocytes. Nat Med. 2008;14:88-92. 97. Lakkis FG, Sayegh MH. Memory T-cells: a hurdle to immunologic tolerance. J Am Soc Nephrol. 2003;14:2402-10. 98. Adams AB, Williams MA, Jones TR, et al. Heterologous immunity provides a potent barrier to transplantation tolerance. J Clin Invest. 2003;111:1887-95. 99. Pearl JP, Parris J, Hale DA, et al. Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody-mediated T-cell depletion. Am J Transplant. 2005;5:465-74. 100. Wu Z, Bensinger SJ, Zhang J, et al. Homeostatic proliferation is a barrier to transplantation tolerance. Nat Med. 2004;10:8792. 101. Koyama I, Nadazdin O, Boskovic S, et al. Depletion of CD8 memory T-cells for induction of tolerance of a previously transplanted kidney allograft. Am J Transplant. 2007;7:1055-61.

Trends in Transplant. Ravinder 2009;1:13-27 K. Wali, Matthew R. Weir: CNI Withdrawal and mTOR Based Immunosuppression

Early or Late Calcineurin Inhibitor Withdrawal and Mycophenolate Mofetil-Based Immunosuppression to Prevent Graft Loss in Patients with Suboptimal Kidney Transplant Function Ravinder K. Wali and Matthew R. Weir University of Maryland School of Medicine, Department of Medicine, Division of Nephrology, Baltimore, MD, USA

Abstract The rate of graft loss in kidney transplant recipients after the first year of transplantation has remained unchanged despite the short-term gains achieved with the recent advances in immunosuppression therapy. Progressive allograft dysfunction after transplantation is an important risk factor for future graft failure. The most common histologic finding in graft biopsies of patients with progressive loss of graft function in the absence of known causes is the development of interstitial fibrosis and tubular atrophy, not otherwise specified, defined in the past as chronic allograft nephropathy. Nearly one-third of recipients of kidney transplants demonstrate de novo interstitial fibrosis and tubular atrophy not otherwise specified by three months after transplantation in the absence of changes in serum creatinine values. The pathogenesis of interstitial fibrosis and tubular atrophy following kidney transplantation is complex. One of the critical mechanisms for the onset and progression of interstitial injury is the result of epithelial-mesenchymal transition due to time-dependent immunologic and nonimmunologic graft injury. Markers of epithelial-mesenchymal transition can be found in more than 40% of allograft biopsies by the third month after transplantation. Calcineurin inhibitors have the potential of inducing epithelial-mesenchymal transition, and their toxicity could perpetuate tubular injury and interstitial inflammation, leading to interstitial fibrosis and tubular atrophy. These interstitial changes progress despite reductions in calcineurin inhibitor dose. Early detection of interstitial fibrosis and tubular atrophy not otherwise specified may indicate the need for calcineurin inhibitor withdrawal. The calcineurin inhibitor can be replaced with anti-metabolites such as mycophenolate mofetil in those who are on azathioprine-based maintenance therapy. If patients are already on mycophenolate mofetil-based therapy, conversion from calcineurin inhibitor- to sirolimus-based therapy may be another option to prevent further deterioration of allograft function. Conversion therapy can improve graft function and may prolong graft survival.

Correspondence to: Ravinder K Wali University of Maryland School of Medicine Department of Medicine, Division of Nephrology 22 South Greene Street Baltimore, MD 21201, USA E-mail: [email protected]

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To prevent progressive nephron loss, every effort should be made to detect interstitial fibrosis and tubular atrophy by allograft biopsy before measurable changes in serum creatinine level. Once the serum creatinine has increased, often the histologic damage is irreversible. As a result, conversion to calcineurin inhibitor-sparing regimens in patients with advanced graft dysfunction with estimated glomerular filtration rate of less than 30 ml/min/1.73 m2 may not be as beneficial as earlier conversion therapy. (Trends in Transplant. 2009;1:13-27) Corresponding author: Ravinder K. Wali, [email protected]

Key words Chronic allograft nephropathy. Interstitial fibrosis and tubular atrophy of not otherwise specified. Calcineurin inhibitors. Mammalian target of rapamycin inhibitors. Mycophenolate mofetil. Epithelial to mesenchymal transition. Graft failure. Acute rejection.

Introduction During the first year after kidney transplantation, the primary goal is to use maintenance immunosuppression to prevent acute cellular and antibody mediated rejection. In fact, during the past decade, due to significant advances in immunosuppression therapy, the rate of acute cellular rejection during the first year after transplantation has decreased to less than 20%, a significant achievement in short-term gains. These gains have not so far translated into long-term gains since the absolute rate of graft loss either due to patient death (death with a functioning graft) or censored after patient death has not improved significantly despite these advances. In addition, long-term deterioration in allograft function with a consequent increase in cardiovascular disease remains a daunting challenge. Worsening allograft function that develops in the absence of histologic features of other well-defined causes is usually defined as chronic allograft nephropathy (CAN). It was also referred to as chronic rejection by the Banff 1997 classification. Often the histologic features of CAN are nonspecific1 and could be due to a combination of factors such as donor-related nephron loss, posttransplant hypertension, and diabetes, or as a consequence of ischemia-reperfusion injury, drug toxicity, and viral infections such as reactivation of polyomavirus or cytomegalovirus. However, the most prominent histologic 14

features of CAN are interstitial fibrosis and tubular damage. The terminology of CAN was redefined by the recent Banff meeting, and has been labeled as chronic interstitial fibrosis and tubular atrophy, not otherwise specified (IF/TA NOS)2.

Prevalence of interstitial fibrosis and tubular atrophy, not otherwise specified Analysis of the data from the centers that have performed protocol biopsies during the past ten years would suggest that IF/TA NOS is an early event following successful transplantation regardless of the origin of the kidney, i.e. deceased donor or living donor kidney transplants3. Protocol biopsies have demonstrated that IF/TA NOS can be detected in 25% of allografts at three months4, 40% of grafts at two years5 and almost 99% of grafts at 10 years6. Similarly, histologic features at three months have also been associated with long-term graft survival. Serón, et al. demonstrated that protocol biopsies at three months in patients with stable serum creatinine could predict the 10-year graft survival. The CAN score and not acute rejection correlated with long-term prognosis; patients with no CAN had a 10-year graft survival of 95.4% compared with 82.3% in patients with interstitial fibrosis, and 41.3% in patients with combination of both interstitial and tubular lesions7.

Ravinder K. Wali, Matthew R. Weir: CNI Withdrawal and mTOR Based Immunosuppression

50

A

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Figure 1. Projected graft half-life in recipients of deceased and living donor kidney transplants based on serum creatinine values at 12 months posttransplant. Projected median kidney graft half-life in years, for living donor (A), and deceased donor (B) kidney grafts based on the serum creatinine values (mg/dl) at one year posttransplant without and with censoring for death with functioning graft (DWFG). Modified and reproduced with permission from Siddiqi, et al.12

Similarly, early tubulointerstitial damage at three months profoundly influenced graft survival beyond 10 years8, and changes in IF/TA over time from the baseline biopsies at three months could also predict the graft survival9. Freeze, et al. described that IF/TA are more prominent features of chronic graft damage than vascular changes. When the biopsies were scored according to Banff 97 criteria, 48% of biopsies had CAN grade II, and 7.5% had CAN grade III. Arterial wall thickening was present in 66% of the late biopsies. The Banff CAN

score and serum creatinine levels were independent predictors of future graft survival. Intriguingly, the presence or absence of arterial wall thickening had no prognostic impact10. In addition to histologic features, surrogate markers of graft function during the first six months after transplantation also predicts graft as well as patient survival. In the United Network for Organ Sharing (UNOS) data set of recipients of kidney transplants with a functioning graft at one year (n = 85,135), nearly half of the recipients (n = 41,299; 48.5%) had serum creatinine 15

Trends in Transplantation 2009;1

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Figure 2. Prevalence of calcineurin inhibitor toxicity in recipients of kidney and kidney-pancreas transplants. Point prevalence of histologically defined calcineurin-inhibitor nephrotoxicity during the follow-up period on yearly biopsies after first year of transplantation. Modified and reproduced with permission from Nankivell, et al.6

> 1.5 mg/dl. Among these recipients, the graft half-life was reduced by more than 50% compared to those with serum creatinine ≤ 1.5 mg/ dl at one year posttransplantation. This accelerated graft failure was similar in the recipients of either living or deceased donor kidneys (Fig. 1). Furthermor these differences in graft survival persisted even after censoring for death with functioning graft11,12 (Fig. 2). Therefore, the cumulative evidence would suggest that the presence of IF/TA of any degree during the first year posttransplant, even in the absence of detectable changes in the graft function as measured by either serum creatinine or estimated/measured glomerular filtration rate (GFR), are independently associated with immediate and long-term graft survival13-15.

Novel insights into the molecular mechanisms of interstitial fibrosis and tubular atrophy, not otherwise specified Tubulointerstitial fibrosis is an important hallmark of future deterioration of renal function in any type of kidney disease16. Epithelial-to16

mesenchymal transition (EMT) is the result of injury to epithelial cells, which is responsible for the initiation and progression of interstitial fibrosis in different types of kidney disease. An interesting and novel study by Strutz, et al.17 showed that as a result of injury to tubular epithelial cells, these cells have the potential to express fibroblast markers, which is the harbinger of EMT. The majority of renal tubules in adult kidney, other than the collecting duct, are developmentally derived from the metanephric mesenchyme through mesenchymal to epithelial trans-differentiation18. The healthy kidney either lacks or has a sparse population of fibroblasts compared to other organs, with a change in this pattern during different pathologic conditions. The active process of EMT in the kidney correlates with the expression of a novel cytokine, S100A4. It is the human homolog of mouse fibroblast-specific protein-1+, a mesenchymal marker19. Increased expression of fibroblast-specific protein-1+ is associated with changes in the epithelial phenotype to mesenchymal phenotype. Fibroblast-specific protein-1+ expression is induced early by transforming growth factor-β1 (TGF-β1) and epithelial growth factor. These two cytokines are considered as

Ravinder K. Wali, Matthew R. Weir: CNI Withdrawal and mTOR Based Immunosuppression

potent stimuli for inducing EMT in the allograft20. Following tubular injury, fibroblast-specific protein-1+ epithelial cells cross the damaged tubular basement membrane and accumulate in the interstitium of the kidney21. While in the interstitium, these damaged epithelial cells lose their epithelial markers and, under the influence of different cytokines, they change their phenotype to fibroblasts21. During the process of renal fibro­genesis, one-third of new fibroblasts are derived from local EMT. Twenty percent are deri­ ved from the bone marrow stem cells, and the remaining numbers develop from the local proliferation of EMT-derived fibroblasts22. These findings reinforce the notion that fibrogenesis is a local epithelial event. Among the network of cytokines, profibrotic TGF-β1 is the main driving force to maintain the phenomenon of EMT. The TGF-β1 initiates as well as propagates the pathway of EMT process in allograft fibrosis23. Long-term calcineurin inhibitor (CNI), either cyclosporine (CsA) or tacrolimus, use is considered to be tubulotoxic through renal vasoconstriction effects and the ability of the CNI to induce EMT. Experimental evidence has substantiated the longstanding notion that CNI use is associated with IF/TA. In vitro culture studies of human renal tubular epithelial cells in the presence of CsA have consistently demonstrated that CsA has the potential of inducing EMT24,25. Recently, it was demonstrated that biopsies from kidney transplants with allograft nephropathy showed a significant increase in S100A4, a marker of EMT, along with an increase in CD8 lymphocytes. Comparison of implantation biopsies with the biopsies obtained after the onset of allograft nephropathy with IF/TA demonstrated increased expression of S100A4 and other markers of EMT. EMT markers in biopsies with IF/TA was markedly increased compared to protocol biopsies in patients with stable graft function or minor degrees of interstitial fibrosis20. One of the interesting features of EMT is its reversibility. The reversibility is partly determined by the surviving cells that are able to repopulate the injured tubules with new functional epithelia. The plasticity of tubular epithelial cells is considered to be a unique phenomenon that is regulated by several growth factors. Major regulators of renal epithelial cell plasticity are two multifunctional growth factors, bone morphogenetic protein-7 (BMP-7) and TGF-β1.

While TGF-β1 promotes EMT, BMP-7 reverses EMT by directly counteracting TGF-β1. The antagonistic actions of these two cytokines are important regulators of the repair process in injured kidneys. Due to plasticity of epithelial cell, it could be modulated to reverse the process of IF/TA26,27. Preclinical studies of novel therapeutic strategies such as hepatocyte growth factor26 or BMP-727 for blocking TGF-β1 and other signaling involved in the regulation of EMT, could lead to clinical applications to slow or arrest nephron loss due to IF/TA NOS. As we wait for these preclinical experimental studies to reach the clinical arena, strategies to slow or arrest the progression of IF/TA and block the EMT pathway could include careful minimization of CNI, or replacement of CNI with other immunosuppressants such as mammalian target of rapamycin (mTOR) inhibitors and potent anti-metabolites such as mycophenolate mofetil (MMF). The following two studies demonstrate that at present, the use of sirolimus can block the EMT pathways in recipients of kidney transplants. Stallone, et al. evaluated the histologic and clinical effects of sirolimus on biopsy proven CAN. They used confocal microscopy to estimate the magnitude of alpha-smooth muscle actin protein expression as a marker of fibroblast activation in the biopsies of kidney transplant recipients with CAN at baseline and again at 24 months. These patients were randomized to 40% CNI reduction plus MMF (group I; 50 patients) or immediate CNI withdrawal using sirolimus with MMF (group II; 34 patients). At the end of 24 months, graft survival was significantly better in the group II patients. There was a significant decrease in the level of alpha-smooth muscle actin expression in the allograft biopsies of group II patients. On the contrary, CAN grading worsened significantly along with an increase in alpha-smooth muscle actin expression at the interstitial and vascular level in the biopsies of group 1 patients, who were maintained on minimized doses of CNI. Hence, this study, albeit small, indicated that the progression in IF/TA can be modified with the use of sirolimus therapy and discontinuation of CNI28 (Figs. 3 and 4). Pontrelli, et.al.29 performed morphometric analysis of kidney biopsies in patients with CAN 17

Trends in Transplantation 2009;1

80

3 2.5

60

proteinuria (gm/24 h)

creatinine clearance (mg/dl)

70

50 40 30 20

2 1.5

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1 0.5

10

0

0

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Randomization

2 years later

Figure 3. Changes in creatinine clearance (mg/ml/24 hours) and 24-hour proteinuria in patients with chronic allograft nephropathy while maintained on calcineurin inhibitor therapy and after conversion to sirolimus-based therapy. (A) Creatinine clearance in ml/min (CrCl by Nankivell formula): Group I: (50.8±22.9 vs. 47.8±17.6) vs. Group II: (50.1±19.3 vs. 53.1±21.5). (B) Proteinuria (g/24 hours) at baseline and at two years: Group I (0.75±0.43 vs. 0.92±0.52) vs. Group II (0.83±0.19 vs. 1.2±0.69). Modified and reproduced with permission from Stallone, et al.28

45 Quantitative a-SMA expression in pixels

40 35 30

Pre Post

25 20 15 10 5 0 Group I

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Figure 4. Markers of epithelial-mesenchymal transition in graft biopsies while on calcineurin inhibitor (CNI) therapy and after conversion to sirolimus-based therapy. Quantification of alpha-smooth muscle actin (α-SMA) expression in the interstitial areas in the allograft biopsies in Group 1 (maintained on CNI-based therapy) and Group II (converted from CNI- to sirolimus-based therapy) at the time of enrollment and after two years of follow-up. Alpha-SMA expression is an indirect marker of epithelial-mesenchymal transition. Modified and reproduced with permission from Stallone, et al.28

18

Ravinder K. Wali, Matthew R. Weir: CNI Withdrawal and mTOR Based Immunosuppression

before and after conversion to sirolimus therapy. Six patients remained on CNI therapy, and in other 12 patients CNI was converted to sirolimus therapy. Morphometric analysis and gene expression of plasminogen activator inhibitor-1 was performed at baseline and again at the end of 24 months in both groups of patients. The conversion from CNI to sirolimus was associated with a significant regression of glomerulosclerosis and a significant reduction in the rate of progression in fibrogenesis. The degree of interstitial fibrosis increased only 26% from baseline in the conversion group, compared to an increase of 112% in those patients who remained on CNI therapy. Sirolimus use was associated with a significant decrease in glomerular and tubulointerstitial plasminogen activator inhibitor-1 expression in the kidney biopsy tissues in the conversion group. In addition, in vitro study showed that plasminogen activator inhibitor-1 gene expression in the cultured renal proximal tubular cells decreased on exposure to sirolimus. These observations indicate that sirolimus has the potential of decreasing plasminogen activator inhibitor-1 expression, which in turn prevents extracellular matrix deposition that can prevent progression of interstitial fibrosis. Another therapeutic intervention that has been demonstrated to reduce fibrogenesis in the kidney is the use of MMF-based therapy. Besides the clinical studies that demonstrate that MMF can prevent acute rejection in the recipients of kidney allografts, MMF also has the potential of preventing progression of interstitial fibrosis and proteinuria in a model of chronic scarring30-32.

Strategies to prevent the progression of graft dysfunction due to interstitial fibrosis and tubular atrophy (CAN) The use of CNI is considered critical during the first year of transplantation, when the primary clinical goal is to prevent acute cellular rejection and graft failure. During the subsequent years, however, the major goal of maintenance immunosuppression therapy should be to prevent the insidious onset of progressive allograft dysfunction. During the past several years it became apparent that long-term exposure of the allograft to CNI can result in cumulative damage to the interstitium and tubules. Bi-

opsy studies of the natural history of CAN demonstrate that by five years posttransplantation, 93.5% of patients have evidence of CsA nephrotoxicity and nearly 70% have evidence of CAN Banff grade II or III6,33. A meta-analysis has shown that withdrawal of CsA from conventional triple therapy of CsA, azathioprine, and prednisone is associated with an increased risk of acute rejection without affecting the rate of graft failure34. Treatment strategies that can replace the long-term use of CNI must prevent the development of acute rejection, including subclinical rejection, and yet at the same time prevent the progression of graft dysfunction due to IF/TA NOS. These strategies could include either replacement of CNI with MMF in patients who are not on such therapy. Or replacement of CNI with Mamalian Target of rapamycin inhibitors (mTOR-I).

Replacement of calcineurin inhibitors or azathioprine with mycophenolate mofetil in patients on triple therapy with cyclosporine, azathioprine and/or steroids Dudley, et al. randomized 144 patients with negative slopes of 1/serum creatinine for at least three months while on the combination of CsA and azathioprine. These patients were randomized to either weaning of CsA over six weeks and replacement with MMF 2-3 g/day (n = 74), or continuation of the baseline CsA and azathioprine dose (n = 70). Both groups were similar in their demographics, and baseline creatinine clearance was < 38 ml/min. At the end of one year of follow-up, the CNI withdrawal group had positive slopes of 1/serum creatinine in 49% of patients compared to 26% of those who remained on CsA-based therapy. Allograft function assessed by serum creatinine or calculated creatinine clearance improved significantly in the CsA withdrawal group, whereas progressive deterioration of allograft function occurred in the group who continued on CsA. There were no acute rejection episodes. However, six patients developed graft loss, and four of these graft losses developed in the CsA continuation group35. Weir, et al. studied 118 kidney transplant recipients with CAN and deteriorating graft func19

Trends in Transplantation 2009;1

tion; MMF therapy was initiated to either eliminate CNI (n = 18) or a 50% reduction in CNI dose (n = 100). During a mean follow-up period of less than two years, more than 50% of patients in the CNI minimization group showed a positive slope (improvement) graft function36. Ojo, et al. analyzed the renal transplant scientific registry data with 66,000 patient observations and showed statistically significant improvement in graft survival in those patients whose immunosuppressive regimens contained MMF compared to those who did not receive MMF. In addition, the use of MMF resulted in 27% reduction in the relative risk of allograft failure37. Data analysis of 49,666 primary renal allograft recipients reported between October 31, 1988 and June 30, 1998 from the UNOS database demonstrated that continuous use of MMF compared to azathioprine was associated with improved allograft function after one year of continuous therapy, and this protective effect persisted after two years of treatment38. These prospective studies and registry data analysis support the concept that the use of MMF is associated with preservation of kidney allograft function.

Withdrawal of calcineurin inhibitors and their replacement with mammalian target of rapamycin inhibitors Sirolimus and everolimus are mTOR inhibitors. Sirolimus is a microcyclic lactone antibiotic produced from Streptomyces hygroscopicus. Everolimus is a derivative of sirolimus. Both bind to the intracellular immunophilin (FK506-binding protein 12), but without inhibiting calcineurin. The mTOR inhibitors inhibit the progression from G0 to G1 cell phase, and interleukins IL-2 and IL-4 dependent proliferation of T as well as B lymphocytes by suppressing the ribosomal protein synthesis. They also block the maturation of the G1-S phase of the cell cycle39 and inhibit growth factors such as fibroblast growth factor, platelet derived factor, vascular endothelial growth factor and, more importantly, TGF-β1. These pleiotropic effects of mTOR inhibitors have the potential of preventing the proliferation of both hematopoietic and non-hematopoietic cells. These antiproliferative activities also mediate the potential side effects 20

that can be encountered in patients using sirolimus or everolimus40. Furthermore, sirolimus inhibits metastatic tumor growth and tumor angiogenesis in in vivo mouse models, and therapeutic doses also inhibit the growth of established tumors by decreasing the production of vascular endothelial growth factor. These effects also limit endothelial cell proliferation. The use of sirolimus compared to CNI may reduce the chance of recurrent as well as de novo cancers in high cancer-risk transplant patients41. Both preclinical and phase II clinical studies demonstrated that addition of sirolimus to the regimen of CsA and azathioprine, with or without prior induction therapy, results in a significant reduction in the rate of acute rejection. However, contrary to these studies, a paradox was noted that combined use of sirolimus and CsA resulted in graft dysfunction. It became apparent that the use of sirolimus with CsA was associated with increased nephrotoxicity. Subsequent studies of CsA withdrawal in such patients resulted in improvement of renal function, and decreased the burden of other CNI-associated side effects. Data analysis of the Rapamune Maintenance Regimen Study Group42-45 established that withdrawal of CsA in de novo renal transplant recipients who had been on the combination of sirolimus and CsA therapy, resulted in significant improvement in graft function at one year and at four years, along with an improvement in CAN histology scores, albeit at the cost of an increased incidence of acute rejection. Despite an increased rate of acu­te rejection, graft function at one to four years was significantly superior in the CsA withdrawal group versus the group that remained on a combination of CsA and sirolimus therapy. These observations have been confirmed by others as well, such as the Sirolimus Renal Function Study Group46 and the UK sirolimus study47. The results of these studies showing that CNI withdrawal in kidney transplant recipients on CsA- and sirolimus-based therapies was associated with increased graft and patient survival despite an increase in the rate of acute rejection, led to a wave of single-center studies to explore the impact of CNI elimination in patients with rising creatinine (creatinine creep) in the absence of other reversible factors.

Ravinder K. Wali, Matthew R. Weir: CNI Withdrawal and mTOR Based Immunosuppression

Cohort studies of conversion from calcineurin inhibitors to sirolimus in patients with interstitial fibrosis and tubular atrophy, not otherwise specified (CAN)

In other small sample studies of late conversion, Kruger, et al. (n = 12)54, Renders, et al. (n = 13)55, and Wu, et al. (n = 16)56 reported somewhat similar results as described by Diekmann, et al.53.

Early experience with de novo substitution of sirolimus for CNI in kidney recipients with worsening graft function was largely based on small cohort studies from different centers. Each cohort was less than 15 patients. The results were inconsistent on graft function and graft survival due to the heterogeneity of the patient population in these different cohorts48-50. However, these results did not deter other investigators from exploring the benefit of converting from CNI- to sirolimus-based therapy in patients with different degrees of graft dysfunction. Invariably, all of these studies were performed in kidney recipients who were on combination of CNI, MMF and low-dose corticosteroid therapy.

Another late conversion study included 60 renal transplant recipients with median serum creatinine of 1.9 mg/dl and median GFR 51 ml/ min. During a follow-up of 12 months, less than 4% developed acute rejection. The patient and graft survival rates were 96.7 and 95%, respectively. These patients had several different types of adverse events, including hypercholesterolemia, diarrhea, peripheral edema, rash, and anemia57.

Citterlo, et al. used sirolimus with immediate withdrawal of CsA in 19 patients with CAN and worsening graft function. Renal function improved in 36%, remained stable in 21%, and continued to deteriorate in another 42% of patients. Patients with advanced graft dysfunction with mean serum creatinine of more than 2.9±0.9 mg/dl continued to have progressive deterioration of graft function despite withdrawal of CNI therapy51. Diekmann, et al.52 used a strategy of slow withdrawal (1-2 months) of CNI with sirolimus substitution in 59 renal transplant patients with CAN. During the first year of follow-up, improvement in graft function was noted in 54% of the cohort, and graft function continued to deteriorate in another 46%. After controlling for baseline creatinine, the histologic grade of CAN, prior episodes of acute rejection, and baseline proteinuria were the major determinants for progression in CAN despite discontinuation of CNI. As in other studies, all patients were on MMF and steroid therapy. In continued follow-up for five years following sirolimus conversion in this cohort, the patient and graft survivals were 88 and 38%, respectively. These patients had stable creatinine clearance (33.7±14 ml/min), and with progression in proteinuria (826 ± 860 mg/day) after 3.2 to 6.8 years of follow-up. Baseline proteinuria < 800 mg/ day was associated with better graft survival, with a positive predictive value of 90% to predict positive response with sirolimus-based therapy53.

In a large single center study, recipients of living and deceased kidney transplants (n = 159; two thirds of these patients were high-risk African American) with progressive deterioration in graft function due to biopsy proven CAN were converted from tacrolimus- to sirolimus -based therapy in combination with MMF. An intent-to-treat analysis was performed after excluding the patients (n = 23) in whom sirolimus was discontinued due to different side effects before completing the first three months of therapy while using the loading dose of sirolimus. We demonstrated that 74% of patients had an improvement of graft function. Those who continued to deteriorate following con­ version therapy had a mean baseline serum creatinine of 3.8 mg/dl and estimated GFR of < 19 ml/min/1.7 m2. In addition, time to conversion therapy was more than 34 months is those who continued to deteriorate, compared to 17 months in those with marked improvement in allograft function. The changes in biopsy findings were interesting. A comparison of the allograft biopsies before and at 12 months after conversion demonstrated significant improvements in tubular degeneration, tubular atrophy, and arteriolar hyalinosis, with some progression in interstitial fibrosis58. These cohort studies51,53,55,57-59 demonstrated that: (i) late conversion is not an effective strategy for the prevention of further deterioration in graft function, (ii) advanced allograft dysfunction at the time of conversion therapy is a predictor of continuing deterioration in graft function, and (iii) proteinuria > 800 mg/24 hours is a bad prognostic factor and these patients do not benefit from such conversion therapy. 21

Trends in Transplantation 2009;1

Randomized controlled studies of conversion from calcieurin inhibitors to sirolimus in patients with interstitial fibrosis and tubular atrophy, not otherwise specified (CAN) The first randomized study that explored withdrawal of CsA in stable renal transplant patients was reported by Abramowicz, et al.60. The recipients of kidney transplants (n = 187) with stable graft function were randomized to continue a baseline triple-drug immunosuppressive regimen of MMF, CsA (Neoral®), and steroids (n = 85), or CsA was converted to sirolimus (n = 85). During nine months of follow-up, biopsy proven acute rejection developed in 11% on sirolimus therapy and < 3% who remained on CsA-based therapy. Conversion was associated with a significant improvement in graft function only after excluding patients who developed acute rejection. In 2005, Watson, et al.61 reported the first randomized study (n = 40) in patients with biopsy proven CAN. Discontinuation of CsA with sirolimus substitution resulted in a mean improvement in GFR of 8.5 ml/min from the baseline. This increase was apparent within the first three months after conversion therapy. The patients who remained on CsA continued to lose graft function with a mean loss of GFR of 4.3 ml/ min from baseline. Baseline GFR was an important predictor of improvement in graft function following discontinuation of CsA therapy. Early conversion was important to preserve the graft function. More importantly, however, no significant change in proteinuria or rate of acute rejection was noted following conversion to sirolimus therapy in this small group of patients. Convert Study: This multicenter study enrolled patients with established diagnosis of CAN within 6-60 months posttransplant (n = 830). These patients were randomized either to remain on the CNI-based therapy (n = 275), or CNI was withdrawn and replaced with sirolimusbased therapy (n = 555). At the end of two years, data analysis was stratified according to GFR* groups. All patients remained on centerspecific choice of MMF or azathioprine and steroid doses.

*GFR was calculated by Nankivell formula.

22

For patients with baseline estimated GFR > 40 ml/min at the time of enrollment, on-therapy analysis revealed an overall improvement in GFR in patients randomized to conversion to sirolimus versus maintenance CsA therapy (62.6 ml/min, n = 370, versus 59.9 ml/min, n = 201; p = 0.009) at the end of two years of follow-up. However, nearly 20% of patients randomized to sirolimus had to discontinue the assigned therapy because of adverse events; the most common adverse events included hyperlipidemia, diarrhea, anemia, and edema. The incidence of biopsy proven acute rejection was nearly similar in both groups62. At the end of the first two years of the enrollment process, it was apparent that those with baseline GFR < 40 ml/min developed worsening of GFR after conversion and had more adverse events. Subsequent enrollment of patients with GFR < 40 ml/min was terminated at the recommendation of the Data Safety Monitoring Board of the study63. An important finding of the CONVERT trial was that improvement in GFR after conversion to sirolimus was inversely related to the degree of urine protein excretion at baseline. The tendency for worsening proteinuria was related to baseline histopathology. A higher Banff total sum score for CAN with a higher percentage of sclerotic glomeruli at baseline was significantly correlated with worsening proteinuria after conversion to sirolimus therapy63. Spare the Nephron (STN) study: This was an open-label, prospective, multicenter study that randomized kidney transplant recipients within 30-180 days posttransplant (n = 305) either to continue the current center-specific CNI therapy (CsA or tacrolimus), or CNI was converted to sirolimus-based therapy. All patients received MMF and center-specific doses of steroids. The data analysis of the first 249 patients who completed 12 months of follow-up (CNI/MMF, n = 126 and SRL/MMF, n = 123) was presented during the 2007 American Transplant Congress meeting. Conversion to sirolimus-based therapy was associated with a gain of nearly 6 ml/min iothalamate-based GFR at 12 months (primary endpoint of the study). Biopsy proven acute rejection, graft loss, and patient death were similar in both groups. However, during the first year after randomization nearly 19% of patients in the sirolimus/MMF group had to discontinue the assigned therapy due to adverse events64.

Ravinder K. Wali, Matthew R. Weir: CNI Withdrawal and mTOR Based Immunosuppression

A recent meta-analysis by Mulay, et al. of conversion studies (CNI to sirolimus-based therapy) with MMF and steroids included five randomized studies (n = 1,040) and 25 cohort studies with different inclusion/exclusion criteria (n = 977) with different degrees of allograft dysfunction and different time intervals after transplantation. Withdrawal of CNI was associated with > 6 ml/min improvement in creatinine clearance65. These randomized controlled studies and smaller cohort studies illustrate the risk factors associated with lack of benefit of conversion therapy: (i) advanced graft dysfunction defined either by baseline serum creatinine > 3.0 mg/dl or estimated GFR < 30 ml/min/1.73 m2, and (ii) proteinuria > 800 mg/24 hours are associated with poor graft outcome following sirolimusbased therapy51,57,59,66.

Everolimus use in interstitial fibrosis and tubular atrophy, not otherwise specified, (CAN) To date, the role of everolimus in patients with established diagnosis of CAN has been reported in small cohort studies. A single-center study of 20 kidney transplant patients reported an acute rejection rate of 15% following conversion from CsA to everolimus at seven weeks after transplantation. During a short follow-up period of six months, a significant increase in calculated GFR was described67. In a small Chilean study, everolimus was substituted for CsA in patients with Banff grade I and II CAN. Forty-two percent of the cohort had improvement in graft function and none of these patients developed proteinuria68.

Controversies regarding the conversion from calcineurin inhibitor- to sirolimus-based therapy in patients with interstitial fibrosis and tubular atrophy, not otherwise specified (CAN) Time to conversion therapy The optimal timing of conversion in recipients of kidney transplants is unknown, but likely should occur in low-risk patients before significant graft damage occurs. Since it is dif-

ficult to routinely perform measured GFR in transplant recipients, often conversion occurs late, which may limit the benefits. Hopefully, newer creatinine or non-creatinine based formulae that will allow precise evaluation of graft function and clinical availability of transcriptome analysis will help to identify at-risk patients for graft damage at an early stage. However, protocol biopsies at different time intervals in the posttransplant period will help to identify recipients with subclinical rejection and the onset of IF/TA NOS at an early stage, before the onset of graft dysfunction. A change in estimated GFR of > 10% is also considered to be a better predictor of graft loss69. While most centers perform biopsies based on the changes in the graft function, this deprives us from detecting the early changes as seen in protocol biopsies at one month, three months, and one year posttransplant70. Therefore, it is important to consider intervention before changes in surrogate markers of graft function (serum creatinine or estimated GFR) become apparent. This is also conceptually supported by the fact that EMT can be reversed before the onset of advanced fibrogenesis71.

De novo onset or worsening of existing proteinuria The development and progression of proteinuria in patients with different degrees of allograft dysfunction is multifactorial and rather complex due to the existence of different degrees of glomerular, endothelial, epithelial, and tubular damage in patients with IF/TA NOS72. Several studies have evaluated the clinical implications of pro­teinuria on the success of CNI conversion to sirolimus-based therapy. Ruiz, et al.73,74 evaluated the impact of CNI to sirolimus conversion on progression of proteinuria. They assessed proteinuria at baseline and again at six months in 149 patients. These patients were categorized in three groups, based on mean proteinuria at baseline (before conversion): Group 1: ≤ 300 mg/day, n = 64; Group II: > 300-3,500 mg/day, n = 79; Group III: > 3.5 g/ day, n = 6 (Fig. 5). Three important findings were established by this study: (i) patients with CAN have varying degrees of proteinuria at baseline, and increasing degree of proteinuria at baseline was associated with increased levels of creatinine; (ii) during a follow-up period of six months after conversion, proteinuria increased in all 23

Trends in Transplantation 2009;1

5 4.5

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Figure 5. Serum creatinine values based on the level of proteinuria before and after conversion to sirolimus-based therapy. Mean serum creatinine values of patients with an increase of proteinuria 500 mg/day after conversion are shown with white bars, patients with an increase of > 500 mg/day with black bars. Modified and reproduced with permission from Ruiz, et al.74

three groups, but more significantly in group I and II, and, (iii) graft function as assessed by serum creatinine improved in group I patients, remained unchanged in group II, and worsened in group III patients. Hence, baseline proteinuria should be considered before conversion. Diekman, et al.52,59,75 suggested that patients with proteinuria > 800 mg/24 hours do not benefit from sirolimus-based therapy. Recent evidence would suggest that worsening of proteinuria after initiation of sirolimus-based therapy could be due to reduced tubular reabsorption of protein76. Given these challenges, more work needs to be done to understand the pathophysiology of proteinuria in patients with IF/TA NOS as well as with sirolimus-based therapy.

Minimizing the side effects associated with combination of sirolimus therapy with either mycophenolate mofetil or azathioprine Most of the cohort and randomized studies have demonstrated that nearly 15-20% of 24

patients may need to discontinue sirolimus therapy due to different types of side effects. These side effects can develop due to bone marrow suppression leading to neutropenia, thrombocytopenia, and anemia, and suppression of cell proliferation in the colon leading to diarrhea. Both hematologic and gastrointes­ti­nal side effects are amenable to modification of the MMF dose, or tapering the sirolimus dose to reduce the sirolimus trough levels too58. We usually achieve a target sirolimus trough level of 8-10 ng/ml during the first year, 6-8 ng/ml during the second year, and 4-6 ng/ml during the subsequent years after transplantation. Dyslipidemia manifests itself mostly in the form of increased LDL-cholesterol and triglyceride levels. Most of these lipid abnormalities plateau at three to six months after conversion therapy. In our center, we start pa­tients on statins on the day of sirolimus therapy and monitor serum lipid profile every three months during the first year and subsequently every six months afterwards. Increases in serum lipids may require increasing the dose of statins to the maximum recommended dose58,77. Other side effects

Ravinder K. Wali, Matthew R. Weir: CNI Withdrawal and mTOR Based Immunosuppression

associated with sirolimus therapy include acne, mouth ulcers, lymphedema, or peripheral edema that can at times be asymmetric. Therefore, it is important that patients should be counseled regarding the nature and severity of these side effects. On the contrary, sirolimus use has other long-term benefits such as antifibrotic effects28,29 that can prevent the progression in interstitial and vascular fibrosis, and antineoplastic properties78 that may benefit transplant recipients who are at risk for malignancy.

Conversion procedure The safe procedure of stopping the CNI with initiation of sirolimus therapy is not well de­ fined. In our center, we usually stop the CNI after achieving the therapeutic trough level of si­rolimus. Since we started this technique of con­ version and without using the loading dose of sirolimus, the rate of acute side effects of sirolimus as well as early acute rejection following conversion therapy were minimized58.

Conclusion Treatment of progressive graft dysfunction is a challenge for the future, but it is a daunting task. Due to the lack of robust randomized studies and the lack of generalizability of the currently existing randomized as well as cohort studies, it is difficult to develop a uniform line of action. However, the use of revised Banff criteria that will allow early identification of histologic changes and graft dysfunction may help to optimize the treatment strategies to prevent progressive graft dysfunction. Late CNI withdrawal has achieved variable results, possibly because withdrawal was attempted after the kidney damage was irreversible. Early CNI withdrawal, prior to significant graft damage, has generally improved the graft function, including biomarkers of ongoing fibrosis, and decreased CNI-associated tubular toxicity. Successful withdrawal of CNI appears to be more effective than CNI minimization. However, CNI withdrawal and conversion to sirolimus-based therapy in combination with MMF and with or without corticosteroids improves graft function in patients with different degrees of allograft dysfunction and effectively protects from acute cellular re-

jection. However, many questions remain, and the tolerability of therapy remains an important concern.

References

1. Halloran PF, Langone AJ, Helderman JH, Kaplan B. Assessing long-term nephron loss: is it time to kick the CAN grading system? Am J Transplant. 2004;4:1729-30. 2. Mengel M, Sis B, Halloran PF. SWOT analysis of Banff: strengths, weaknesses, opportunities and threats of the international Banff consensus process and classification system for renal allograft pathology. Am J Transplant. 2007;7:2221-6. **The revisions in the Banff 97 criteria for the classification and identification of chronic allograft nephropathy and the caveats of using the term chronic allograft nephropathy. 3. Cosio FG, Grande JP, Larson TS, et al. Kidney allograft fibrosis and atrophy early after living donor transplantation. Am J Transplant. 2005;5:1130-6. 4. Legendre C, Thervet E, Skhiri H, et al. Histologic features of CAN revealed by protocol biopsies in kidney transplant recipients. Transplantation. 1998;65:1506-9. 5. Isoniemi H, Taskinen E, Hayry P. Histologic chronic allograft damage index accurately predicts chronic renal allograft rejection. Transplantation. 1994;58:1195-8. 6. Nankivell BJ, Borrows RJ, Fung CL, et al. The natural history of CAN. N Engl J Med. 2003;349:2326-33. **This study has given us the insights regarding the development and progression of chronic allograft damage that develops over a long period of time. The time course of different histologic events in the posttransplant period. 7. Seron D, Moreso F, Bover J, et al. Early protocol renal allograft biopsies and graft outcome. Kidney Int. 1997;51:310-16. 8. Nankivell BJ, Fenton-Lee CA, Kuypers DR, et al. Effect of histologic damage on long-term kidney transplant outcome. Transplantation. 2001;71:515-23. 9. Nankivell BJ, Borrows RJ, Fung CL, et al. Delta analysis of posttransplantation tubulointerstitial damage. Transplantation. 2004;78:434-1. 10. Freese P, Svalander CT, Molne J, et al. Chronic allograft nephropathy–biopsy findings and outcome. Nephrol Dial Transplant. 2001;16:2401-6. 11. Hariharan S, McBride MA, Cherikh WS, et al. Posttransplant renal function in the first year predicts long-term kidney transplant survival. Kidney Int. 2002;62:311-18. 12. Siddiqi N, McBride MA, Hariharan S. Similar risk profiles for posttransplant renal dysfunction and long-term graft failure: UNOS/OPTN database analysis. Kidney Int. 2004;65:190613. Impact of serum creatinine at one year posttransplant on the graft life of deceased as well as living donors. 13. Benigni A, Bruzzi I, Mister M, et al. Nature and mediators of renal lesions in kidney transplant patients given cyclosporine for more than one year. Kidney Int. 1999;55:674-85. 14. Kuypers DR, Chapman JR, O’Connell PJ, et al. Predictors of renal transplant histology at three months. Transplantation. 1999;67:1222-30. 15. Yilmaz S, Tomlanovich S, Mathew T, et al. Protocol core needle biopsy and histologic CADI as surrogate endpoint for long-term graft survival in multicenter studies. J Am Soc Nephrol. 2003;14:773-9. 16. Liu Y. EMT in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol. 2004;15:1-12. 17. Strutz F, Okada H, Lo CW, et al. Identification and characterization of a fibroblast marker: FSP1. J Cell Biol. 1995;130: 393-405. 18. Horster MF, Braun GS, Huber SM. Embryonic renal epithelia: induction, nephrogenesis, and cell differentiation. Physiol Rev. 1999;79:1157-91. 19. Masszi A, Di Ciano C, Sirokmany G, et al. Central role for Rho in TGFβ1-induced α-SMA expression during EMT. Am J Physiol Renal Physiol. 2003;284:F911-24. 20. Vongwiwatana A, Tasanarong A, Rayner DC, et al. EMT during late deterioration of human kidney transplants: the role of tubular cells in fibrogenesis. Am J Transplant. 2005;5:1367-74. **The role of EMT in the pathogenesis of interstitial fibrosis.

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Trends in Transplantation 2009;1 21. Okada H, Ban S, Nagao S, et al. Progressive renal fibrosis in murine polycystic kidney disease: an immunohistochemical observation. Kidney Int. 2000;58:587-97. 22. Iwano M, Plieth D, Danoff TM, et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 2002;110:341-50. 23. Rastaldi MP, Ferrario F, Giardino L, et al. EMT of tubular epithelial cells in human renal biopsies. Kidney Int. 2002;62:137-46. 24. McMorrow T, Gaffney MM, Slattery C, et al. CsA-induced EMT in human renal proximal tubular epithelial cells. Nephrol Dial Transplant. 2005;20:2215-25. 25. Slattery C, Campbell E, McMorrow T, Ryan MP. CsA-induced renal fibrosis: a role for EMT. Am J Pathol. 2005;167:395-407. 26. Yang J, Liu Y. Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis. J Am Soc Nephrol. 2002;13:96-107. 27. Zeisberg M, Hanai J, Sugimoto H, et al. BMP-7 counteracts TGFβ-1-induced EMT and reverses chronic renal injury. Nat Med. 2003;9:964-8. 28. Stallone G, Infante B, Schena A, et al. Rapamycin for treatment of CAN in renal transplant patients. J Am Soc Nephrol. 2005;16:3755-62. ** Sirolimus use is associated with decreased levels of markers of EMT, which in turn could reduce the rate of progression in interstitial fibrosis. 29. Pontrelli P, Rossini M, Infante B, et al. Rapamycin inhibits PAI-1 expression and reduces interstitial fibrosis and glomerulosclerosis in CAN. Transplantation. 2008;85:125-34. *The use of sirolimus can result in decreased expression of cytokines that prevent progression in interstitial fibrosis in recipients of kidney transplants. 30. Azuma H, Nadeau KC, Ishibashi M, Tilney NL. Prevention of functional, structural, and molecular changes of chronic rejection of rat renal allografts by a specific macrophage inhibitor. Transplantation. 1995;60:1577-82. 31. Remuzzi G, Zoja C, Gagliardini, E et al. Combining an antiproteinuric approach with MMF fully suppresses progressive nephropathy of experimental animals. J Am Soc Nephrol. 1999;10:1542-9. 32. Romero F, Rodriguez-Iturbe B, Parra G, et al. MMF prevents the progressive renal failure induced by 5/6 renal ablation in rats. Kidney Int. 1999;55:945-55. 33. Colvin RB. Chronic allograft nephropathy. N Engl J Med. 2003;349:2288-90. 34. Kasiske BL, Chakkera HA, Louis TA, Ma JZ. A meta-analysis of immunosuppression withdrawal trials in renal transplantation. J Am Soc Nephrol. 2000;11:1910-17. 35. Dudley C, Pohanka E, Riad H, et al. MMF substitution for CsA in renal transplant recipients with chronic progressive allograft dysfunction: the “creeping creatinine” study. Transplantation. 2005;79:466-75. 36. Weir MR, Blahut S, Drachenburg C et al. Late CNI withdrawal as a strategy to prevent graft loss in patients with suboptimal kidney transplant function. Am J Nephrol. 2004; 24:379-86. 37. Ojo AO, Hanson JA, Wolfe RA, et al. Long-term survival in renal transplant recipients with graft function. Kidney Int. 2000;57:307-13. 38. Meier-Kriesche HU, Steffen BJ, Hochberg AM, et al. MMF versus azathioprine therapy is associated with a significant protection against long-term renal allograft function deterioration. Transplantation. 2003;75:1341-6. 39. Halloran PF. Immunosuppression in the post-adaptation period. Transplantation. 2000;70:3-5. 40. Murgia MG, Jordan S, Kahan BD. The side effect profile of sirolimus: a phase I study in quiescent cyclosporine-prednisonetreated renal transplant patients. Kidney Int. 1996;49:209-16. 41. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of VEGF. Nat Med. 2002;8:128-35. 42. Johnson RW, Kreis H, Oberbauer R, et al. Sirolimus allows early cyclosporine withdrawal in renal transplantation resulting in improved renal function and lower blood pressure. Transplantation. 2001;72:777-86. 43. Kreis H, Oberbauer R, Campistol JM, et al. Long-term benefits with sirolimus-based therapy after early cyclosporine withdrawal. J Am Soc Nephrol. 2004;15:809-17. 44. Oberbauer R, Kreis H, Johnson RW, et al. Long-term improvement in renal function with sirolimus after early cy-

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closporine withdrawal in renal transplant recipients: 2-year results of the Rapamune Maintenance Regimen Study. Transplantation. 2003;76:364-70. 45. Oberbauer R, Segoloni G, Campistol JM, et al. Early cyclosporine withdrawal from a sirolimus-based regimen results in better renal allograft survival and renal function at 48 months after transplantation. Transpl Int. 2005;18:22-8. 46. Gonwa TA, Hricik DE, Brinker K, et al. Improved renal function in sirolimus-treated renal transplant patients after early cyclosporine elimination. Transplantation. 2002;74:1560-7. 47. Baboolal K: A phase III prospective, randomized study to evaluate concentration-controlled sirolimus (Rapamune) with cyclosporine dose minimization or elimination at six months in de novo renal allograft recipients. Transplantation. 2003; 75:1404-8. 48. Dominguez J, Mahalati K, Kiberd B, et al. Conversion to rapamycin immunosuppression in renal transplant recipients: report of an initial experience. Transplantation. 2000;70:1244-7. 49. Sundberg AK, Rohr MS, Hartmann EL, et al. Conversion to sirolimus-based maintenance immunosuppression using daclizumab bridge therapy in renal transplant recipients. Clin Transplant. 2004;18(Suppl 12):61-6. 50. Wyzgal J, Paczek L, Senatorski G, et al. Sirolimus rescue treatment in CNI nephrotoxicity after kidney transplantation. Transplant Proc. 2002;34:3185-7. 51. Citterlo F, Scata MC, Violi P, et al. Rapid conversion to sirolimus for chronic progressive deterioration of the renal function in kidney allograft recipients. Transplant Proc. 2003;35:1292-4. 52. Diekmann F, Budde K, Oppenheimer F, et al. Predictors of success in conversion from CNI to sirolimus in chronic allograft dysfunction. Am J Transplant. 2004;4:1869-75. 53. Diekmann F, Budde K, Slowinski T, et al. Conversion to sirolimus for chronic allograft dysfunction: long-term results confirm predictive value of proteinuria. Transpl Int. 2008;21:152-5. 54. Kruger B, Fischereder M, Jauch KW, et al. Five-year followup after late conversion from CNI to sirolimus in patients with chronic renal allograft dysfunction. Transplant Proc. 2007; 39:518-21. 55. Renders L, Steinbach R, Valerius T, et al. Low-dose sirolimus in combination with MMF improves kidney graft function late after renal transplantation and suggests pharmacokinetic interaction of both immunosuppressive drugs. Kidney Blood Press Res. 2004;27:181-5. 56. Wu MS, Chang CT, Hung CC. Rapamycin in patients with chronic renal allograft dysfunction. Clin Transplant. 2005;19:236-42. 57. Peddi VR, Jensik S, Pescovitz M, et al. An open-label, pilot study evaluating the safety and efficacy of converting from CNI to sirolimus in established renal allograft recipients with moderate renal insufficiency. Clin Transplant. 2005;19:130-6. 58. Wali RK, Mohanlal V, Ramos E, et al. Early withdrawal of CNI and rescue immunosuppression with sirolimus-based therapy in renal transplant recipients with moderate to severe renal dysfunction. Am J Transplant. 2007;7:1572-83. **A large but uncontrolled single center study that demonstrated the advantages of substitution of tacrolimus with sirolimusbased therapy in patients with advanced graft dysfunction. 59. Diekmann F, Campistol JM: Conversion from CNI to sirolimus in CAN: benefits and risks. Nephrol Dial Transplant. 2006;21: 562-8. 60. Abramowicz D, Manas D, Lao M, et al. Cyclosporine withdrawal from a MMF-containing immunosuppressive regimen in stable kidney transplant recipients: a randomized, controlled study. Transplantation. 2002;74:1725-34. 61. Watson CJ, Firth J, Williams PF, et al. A randomized controlled trial of late conversion from CNI-based to sirolimusbased immunosuppression following renal transplantation. Am J Transplant. 2005;5:2496-503. 62. Oberbauer R, Schena FP, Wali R, et al. Conversion from CNI to sirolimus compared with continued use of CNI in renal allograft recipients: 24-months safety and efficacy results from the CONVERT trial. J Am Soc Nephrol. 2006;17 [abstract]. 63. Univariate and multivariate analyses of factors affecting renal allograft function after conversion from CNI to sirolimus based immunosuppression: preliminary results of CONVERT study. Transplantation. 2006;82:412. 64. Pearson T, Patel A, Scandling J, et al. Final Results of the Spare-the-Nephron (STN) Trial: A CNI withdrawal trial in renal transplant recipients. Transplantation. 2008 [abstract].

Ravinder K. Wali, Matthew R. Weir: CNI Withdrawal and mTOR Based Immunosuppression 65. Mulay AV, Cockfield S, Stryker R, et al. Conversion from CNI to sirolimus for chronic renal allograft dysfunction: a systematic review of the evidence. Transplantation. 2006;82:115362. 66. Abramowicz D, Hadaya K, Hazzan M, et al. Conversion to sirolimus for chronic renal allograft dysfunction: risk factors for graft loss and severe side effects. Nephrol Dial Transplant. 2008 [Epub ahead of print]. 67. Holdaas H, Bentdal O, Pfeffer P, et al. Early, abrupt conversion of de novo renal transplant patients from cyclosporine to everolimus: results of a pilot study. Clin Transplant. 2008; 22:366-71. 68. Morales J, Fierro A, Benavente D, et al. Conversion from a CNI-based immunosuppressive regimen to everolimus in renal transplant recipients: effect on renal function and proteinuria. Transplant Proc. 2007;39:591-3. 69. Marques GG, Goenaga PE, Royo FJ, et al. Evolution of the renal function is a better predictor of long-term survival than serum creatinine. Transplant Proc. 2005;37:3701-4. 70. Nankivell BJ, Chapman JR. The significance of subclinical rejection and the value of protocol biopsies. Am J Transplant. 2006;6:2006-12. 71. Sugimoto H, Grahovac G, Zeisberg M, Kalluri R. Renal fibrosis and glomerulosclerosis in a new mouse model of diabetic nephropathy and its regression by bone morphogenic protein-7 and advanced glycation end product inhibitors. Diabetes. 2007;56:1825-33. **The progression in EMT can be arrested that can prevent the progression in interstitial fibrosis. 72. Wavamunno MD, O’Connell PJ, Vitalone M, et al. Transplant glomerulopathy: ultrastructural abnormalities occur early in longitudinal analysis of protocol biopsies. Am J Transplant. 2007;7:2757-68. **Importance of serial longitudinal biopsies











to detect the histologic changes and the evolution of these changes with time. 73. Ruiz JC, Diekmann F, Campistol JM, et al. Evolution of proteinuria after conversion from CNI to SRL in renal transplant patients: a multicenter study. Transplant Proc. 2005;37:3833-5. 74. Ruiz JC, Campistol JM, Sanchez-Fructuoso A, et al. Increase of proteinuria after conversion from CNI to sirolimus-based treatment in kidney transplant patients with chronic allograft dysfunction. Nephrol Dial Transplant. 2006;21:3252-7. *A systemic study that evaluated the development and progression of proteinuria while on CNI- compared to sirolimusbased therapy. 75. Diekmann F, Rovira J, Carreras J, et al. Mammalian target of rapamycin inhibition halts the progression of proteinuria in a rat model of reduced renal mass. J Am Soc Nephrol. 2007;18:2653-60. 76. Straathof-Galema L, Wetzels JF, Dijkman HB, et al. Sirolimus-associated heavy proteinuria in a renal transplant recipient: evidence for a tubular mechanism. Am J Transplant. 2006;6:429-33. 77. Kasiske BL, de Mattos A, Flechner SM, et al. Mammalian target of rapamycin inhibitor dyslipidemia in kidney transplant recipients. Am J Transplant. 2008;8:1384-92. **A detailed review of the types of different lipid abnormalities that may develop in the posttransplant period in patients treated with sirolimus-based therapy. 78. Campistol JM, Eris J, Oberbauer R, et al. Sirolimus therapy after early cyclosporine withdrawal reduces the risk for cancer in adult renal transplantation. J Am Soc Nephrol. 2006;17:581-9. **Important role of sirolimus in solid organ transplant recipients at risk to develop different types of malignancy in the posttransplant period.

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Trends in Transplant. Transplantation 2009;1:28-34 2009;1

Long-Term Immunosuppression in Pediatric Liver Transplantation Paloma Jara and Loreto Hierro Hospital Infantil Universitario “La Paz”, Madrid

Abstract Pediatric liver transplantation is a successful treatment with prolonged survival in 80-90% of patients. Immunosuppression has a crucial role in allowing graft survival. The importance of an appropriate balance between protection from rejection and avoidance of the side effects of immunosuppressive drugs is universally recognized. Most trials have been focused on minimizing acute rejection in the early postoperative period. The management of immunosuppression in the long term has been a subject of general description with few detailed studies or trials. Long-term immunosuppression in pediatric liver transplant patients consists of a calcineurin inhibitor at lower blood levels compared to the target in the early posttransplant period, to which low-dose steroids or mycophenolate may be added. Nearly 30% of patients exhibit graft dysfunction of various causes. Once a biliary problem is ruled out, the reasons for dysfunction are rejection, autoimmune hepatitis, or idiopathic problems in which an immunologic basis is highly suspected. Most of these cases are detected in the subclinical stages, and are managed with increased immunosuppression. Noncompliance complicates the evolution of the graft in adolescents and young adults. Altogether, very few patients lose the graft in the long term; however, protocol biopsies indicate a high rate of abnormal histology in contrast to normal biochemistry. Published information on immunosuppression in the long term, the choice of drugs, drug monitoring and methods to evaluate compliance, immune-related causes of graft dysfunction, and attitudes to renal function sparing have been reviewed. Tacrolimus and cyclosporine are both safe options for primary immunosuppression, but tacrolimus is preferred by most centers because of the reduced risk of refractory rejection and cosmetic benefits. Eighty percent of the patients do not need steroids, but no trial has adequately compared the benefit/risk ratio of steroid maintenance at low doses, still used in many centers. Mycophenolate allows decreasing the calcineurin inhibitor level and is applied for renal sparing in selected children, showing a decrease in glomerular filtration rate, which can be detected early by measuring cystatin C levels. Blood levels are the current method for drug monitoring. Some biomarkers of immune cell function are starting clinical

Correspondence to: Paloma Jara Servicio de Hepatología y Trasplante Hospital Infantil Universitario La Paz Paseo Castellana 261 28046 Madrid, Spain E-mail: [email protected]

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Paloma Jara and Loreto Hierro: Long-Term Immunosuppression in Pediatric Liver Transplantation

use. An excessive variation of blood levels is the best marker of noncompliance, which affects up to 40% of patients in adolescence. The incidence of late acute rejection is 10%. It is a difficult diagnosis in the long term even after a panel of experts’ revision of criteria. No gross differences in incidence or outcome are seen in centers with different immunosuppression protocols. Chronic rejection and autoimmune hepatitis occur in 5-10% overall. Long-term liver pathology indicates lesions resembling chronic hepatitis or early stages of chronic rejection that are observed in many cases in contrast to minor biochemical abnormalities or normal liver function. Current immunosuppression practices achieve equivalent numbers of patients with relevant conditions related to under- or over-immunosuppression, but should be the subject of more investigations for the correct management of late graft dysfunction and biochemically silent graft damage. (Trends in Transplant. 2009;1:28-34) Corresponding author: Paloma Jara, [email protected]

Key words Liver transplantation. Children. Long-term follow-up. Graft function. Immunosuppression.

Introduction Liver transplantation has been applied for children for more than two decades, with increasing patient and graft survival rates. The number of children undergoing liver transplantation in Europe in the period 1988-2006 was 6,089 (European Liver Transplant Registry, September 2007); a total of 5,675 children, approximately 600 per year, underwent liver transplantation in the USA and Canada from 1996 to 20051. Current patient survival approximates 90% at year 10 in the main centers of Europe, Japan, and the USA. The USA database from 1994 to 2006 shows that actuarial graft survival was 84.0% at one year and 77.3% at four years; patient survival at four years was 85.5%2. The improved patient and graft survival is attributed to advances in surgery and improved immunosuppression regimens. Immunosuppression has evolved over time subject to two caveats: the availability of cyclosporine (cyclosporin A, CsA) and of tacrolimus (TAC). Additional drugs designed for intense acute immunosuppression (rabbit antithymocyte globulin, anti-CD25, anti-CD52) or for chronic use (mycophenolate, mTOR inhibitors) have become available as well. Different combinations are possible, with a calcineurin inhibitor (CNI) as main therapy.

The best immunosuppression regimen is the one allowing a balanced risk of rejection and adverse effects. Early and late postoperative periods have special features, and priorities change from avoiding rejection in the first period to maintaining the patient with minimal toxicities and infection in the long term.

Primary immunosuppression Calcineurin inhibitors Only one trial is available to compare tacrolimus and cyclosporin microemulsion as primary immunosuppression in children3. The study was multicentre: 181 patients were recruited, 91 children received TAC (target level 10-15 ng/ml) and 90 received cyclosporine (CsA), combined with steroids and, in the cyclosporine arm only, associated to azathioprine. The 12-month results were published in 20043. Patient and graft survival were equally high in both groups (patient: 93.4% TAC vs. 92.2% CsA; graft: 92.3% TAC vs. 85.4% CsA). Tacrolimus allowed higher rejection-free survival, with a significantly lower risk of steroid-resistant rejection (5.5% TAC vs. 26.7% CsA; p = 0.0001). Bacterial, fungal, and cytomegalovirus infections affected nearly the same number of 29

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children in both arms; no differences were noticed in the average decrease of glomerular filtration rate (GFR). A long-term analysis was done with 146 patients (74 TAC, 72 CsA) participating in the above study4. Two to five years after liver transplantation, there were 97 patients still receiving the drug to which they were originally randomized (59 TAC, 38 CsA), and 49 patients had been withdrawn from receiving it for various reasons (15 TAC, 34 CsA). A lower risk of chronic rejection was observed in the TAC group (completers: 1/59 TAC vs. 6/38 CsA; withdrawn: 0/15 TAC vs. 2/34 CsA). The incidence of posttransplant lymphoproliferative disease was 3/74 TAC and 5/72 CsA. Five years after randomization, 78% of children in the TAC arm and only 33% in the CsA arm maintained the original CNI. Patient survival up to nine years after transplantation remained similar according to the initial CNI randomization. The conclusion of the study indicates that TAC is more effective in preventing acute and chronic rejection with fewer adverse effects. Both CsA and TAC offer equivalent rates of patient survival and are safe alternatives for children undergoing liver transplantation. However, the higher efficacy of TAC explains the change over the past 10 years towards a TAC-based primary immunosuppression in most centers. Data from the USA show that in the last five years, around 90% of pediatric liver transplant recipients received a TAC-based maintenance immunosuppression therapy; CsA use has decreased from 22% of patients to only 4% in 20051. The relative risk of rejection is 1.49 in children on primary immunosuppression with CsA compared to those on TAC, but episodes of rejection in the first six months are not predictors of graft failure2. Chronic rejection justifies only 14% of the retransplantation procedures in children5. These facts are arguments for avoiding aggressive primary immunosuppression in the early posttransplant period, as infection causes more morbidity than rejection and is the main cause of mortality. Chronic rejection is rare, both in children on TAC and in patients who receive CsA but are changed to TAC in the case of steroid-resistant rejection. A German series observed that 19% of the CsA-treated patients needed conversion to TAC6. 30

The reasons for TAC preference in the long-term follow-up are the exceptionality of chronic rejection, unchanged physical appearance, and the avoidance of side effects related to CsA (hirsutism, gum hyperplasia). The main reason for conversion from TAC to CsA is the appearance of food allergy, a problem presented in 10% of children who underwent liver transplantation at a very young age; food allergy usually disappears on CsA treatment7.

Steroids Steroid-free protocols are frequently discussed; however, 84% of pediatric liver transplant recipients in the USA were discharged on maintenance corticosteroids in 20052. Most European centers apply steroids in the early period after liver transplantation. Nearly 50% of European and USA centers do seek steroid-free regimens, starting between 3-12 months posttransplantation. In the experience of Pittsburgh, 98.5% of children on TAC could be weaned off steroids; only 22% needed reinstitution of steroids for rejection or renal dysfunction8. Using primary immunosuppression with CsA, most children at Hamburg were off steroids in the follow-up6. Maintenance steroids at low doses (usually on alternate-day basis) do not apparently make differences in growth compared to steroidfree regimens. The anti-inflammatory properties of steroids could be of value in preventing graft damage in the long term.

Monitoring immunosuppressive treatment Blood levels of CsA or TAC are determined to assess immunosuppression. Levels are checked every 2-3 months in stable patients. Maintenance at a level of TAC 4-6 ng/ml or trough CsA 80-120 ng/ml is common beyond one year. Trough CsA blood level has been substituted by two hours post-dose (C2) levels in many centers because of the better correlation to drug exposure. Trough mycophenolic acid levels ranging from 1.5-3 mg/l are adequate for liver transplanted children receiving mycophenolate mofetil (MMF) in association to a CNI9. Mycophenolic acid blood levels do not correlate to the area under the curve and

Paloma Jara and Loreto Hierro: Long-Term Immunosuppression in Pediatric Liver Transplantation

novel methods to guide MMF dosing are proposed with the measurement of inosine monophosphate dehydrogenase activity10. The usual practice of assessing drug trough levels may not reflect overall immune suppression. Some assays have been developed to estimate immune response such as the measurement of soluble CD30, nuclear factor of activated T-cellregulated gene expression, profiles of circulating cytokines, and circulating regulatory T-cells, but these have not translated into clinical value11,12. The immune cell function assay (ImmuKnow®, ViraCor Laboratories, USA) for assessment of cell-mediated immunity in an immunosuppressed population is designed to measure increases in intracellular adenosine triphosphate (ATP) of CD4 T-cells following activation by the mitogen PHA (phytohemagglutinin). The ImmuKnow assay is an additional tool in transplant patient management, but probably it evaluates the effect of steroids and CNI, and not that derived from mammalian target of rapamycin (mTOR) inhibitors or MMF. In adults, the degree of immune function as assessed by the ImmuKnow assay helps to predict patients at risk for infection or rejection. Trials have compared immune responses in healthy adults and stable transplant recipients so that three zones of immune response were established: strong (≥ 525 ng/ml ATP), moderate (226-524 ng/ml ATP) and low (≤ 225 ng/ml ATP). In adult patients, low ATP values (< 25 ng/ml) predispose to infection (12-fold) and high ATP (> 700) increased the odds of rejection 30-fold13. The study in children revealed that healthy children (< 12 years) had statistically significantly lower immune function values than healthy adults, and that pediatric renal transplant recipients were more immunosuppressed than adult transplant recipients. The adjusted zones for children under 12 years are: strong ≥ 395, moderate 176-394, and low ≤ 175 ng/ml ATP14. The ImmuKnow assay has been applied in the evaluation of liver transplanted children with Epstein-Barr virus (EBV) infection. Patients with low EBV loads had a significantly (p < 0.04) stronger immune response to PHA than patients with EBV load > 1,000 copies/μg DNA. All patients with ATP < 125 ng/ml showed a high EBV load (> 4,000 copies/μg DNA). When immunosuppression was reduced, an increase of the ATP release was observed that correlated with a decrease of the EBV viral load15.

Nonadherence to the immunosuppressive regimen In the pediatric transplant setting, it is common to encounter adolescent patients who take their medications with admitted accidentally omitted doses, but the extent of missed doses is usually difficult to assess. Nonadherence to medication is significantly associated to late acute rejection. In a series of 111 patients 12-21 years old, 45% were identified as nonadherent, defined by at least one episode of admission by the patient of not taking immunosuppressive medications or not attending any clinical visit in a retrospective review of a one-year period. Among 30 cases with sporadic or complete discontinuation of drugs, late acute rejection occurred in 33%, compared to 9.3% in adherent patients16. In adolescents and young adults, measuring adherence is crucial to maintain graft function, allowing earlier psychosocial and behavioral interventions. Several studies concluded that the degree of fluctuation of levels of TAC over successive outpatient visits was the best measure of adherence. A standard deviation (SD) higher than 2, 2.5, or 3 ng/ml is an indicator of noncompliance. Dose modifications made by the physician may also contribute to variations of TAC levels. A study was taken to minimize that confounder by standardizing physician practice patterns in adjusting TAC dosing, and by recognizing many explainable reasons for variations of levels so that dose was not modified routinely. Over the period of the study, an increasing proportion of patients (initial 55%, evolutive 75-85%) had their TAC levels within the therapeutic range, which was interpreted as the effect of improved compliance by patients who had been informed about the program of vigilance of levels in order to assess adherence to treatment. Eleven episodes of late acute rejection occurred during the study period of one year in 101 patients; 10 of the 11 episodes occurred in patients who had TAC level SD > 2. The incidence of rejection was 1% in patients with SD < 2, while 28% of patients with TAC SD > 2, and 67% of those with SD > 3 developed acute rejection. Good compliance and not a higher mean TAC level influenced the risk of rejection, as only 2% of 50 children with mean TAC blood levels < 5.27 ng/ ml had rejection, compared to 18% in the remaining 51 patients with mean values > 5.27 ng/ml17. Other investigators found the cutoff value of SD ≥ 2.5 in TAC values was useful to guide clini31

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cians, as patients had about eight-times higher odds to develop rejection, and it provided a satisfactory balance to differentiate true nonadherence and the variations of levels attributable to changes in absorption, body mass, intervening illness, and drug interactions18. Children with chronic disease often experience a delay in personal maturation and independence19. Chronologic age alone is not an adequate guide to initiate the full responsibility in taking immunosuppressive drugs. The essence of timing of all aspects of transition is that of flexibility, timing of events in the transitional process of a young patient taking a primary role in the medical consultation until transition to adult services must be individualized and planned years beforehand20. Difficulties of adherence in late adolescence can deteriorate further with the transition from pediatric care to adult services. A short series of 14 patients showed median TAC SD was 3.2 before transition, but 4.08 and 5.09 in the first and second years, respectively, under follow-up in adult clinics21. Adherence was significantly poorer for transitioned patients than for a cohort of adolescent patients still handled by pediatricians. Proposals to manage the problem include “teen clinics”, or a delay of transition to adult services until the age of risk is overcome.

Rejection and other immunemediated causes of graft damage in the long term Most late causes of liver allograft injury are first detected because of abnormalities in routinely monitored liver tests; clinical signs and symptoms are much less common. The problem, usual in adult patients, of differentiating recurrence of pretransplantation disease is limited to very few children who needed transplantation for viral or autoimmune hepatitis. Most commonly, biliary disease due to anastomotic strictures, or intrahepatic strictures secondary to previous ischemic damage constitute the main conditions to be ruled out along with late rejection. Posttransplant de novo autoimmune hepatitis should be included in the differential diagnosis of pediatric liver transplant patients without previous autoimmune liver disease who develop late graft dysfunction. 32

Rejection The definite diagnosis of rejection can be difficult when facing liver allograft dysfunction occurring more than one year after transplantation. Histopathologic features of late rejection are somewhat different from acute rejection occurring early after transplantation. The Banff Working Group on Liver Allograft Pathology described late acute rejection as having fewer blastic lymphocytes, slightly greater interface activity, less venous subendothelial inflammation, and slightly more lobular activity. It can also present as isolated perivenular inflammation and hepatocyte dropout (so-called “central perivenulitis”) and evolve into typical chronic rejection with ductopenia. Subendothelial inflammation of portal or central veins is not a required finding in such cases. Late acute rejection, however, is still most commonly characterized by predominantly mononuclear portal inflammation containing lymphocytes, neutrophils and eosinophils, venous subendothelial inflammation of portal or central veins or perivenular inflammation, and inflammatory bile duct damage22. Children followed in the long term develop a 10% annual rate of biopsy proven rejection or a liver dysfunction with nonspecific histologic changes that ultimately receives treatment as a rejection episode17,23-25. A cumulative rate of chronic rejection occurs in 6% of children followed in the long term 26. Most cases are thought to be related to nonadherence to immunosuppressive treatment.

Autoimmune hepatitis Posttransplant de novo autoimmune hepatitis (d-AIH) is increasingly described as a long-term complication after pediatric liver transplantation. It is characterized by graft dysfunction, the development of autoimmune antibodies, and histologic evidence of hepatitis in liver transplant recipients without a previous history of autoimmune liver disease. This disorder affects 2.1-5.2% of pediatric liver transplanted patients. It remains unclear whether d-AIH represents an autoimmune condition or a form of chronic rejection. Forty-one of 619 patients in the UCLA series were ultimately identified as having hepatitis-AIH (incidence, 6.6%).The median duration be-

Paloma Jara and Loreto Hierro: Long-Term Immunosuppression in Pediatric Liver Transplantation

tween transplantation and the development of dAIH was 6.7 years. Specific differences at the time of diagnosis revealed that prior to the diagnosis of d-AIH, patients had more episodes of rejection, an increased dependence on steroids, and overall greater immunosuppression requirements than their matched controls. Fewer of the cases (46%) versus controls (81%) were off prednisone, fewer of the cases (29%) versus controls (63%) were on monotherapy CsA or TAC, and a trend was noted that most of the cases (39%) versus controls (24%) required MMF or azathioprine as maintenance medications. Additionally, there was a statistically significant difference in the mean TAC levels at diagnosis of the d-AIH patients (7.1 ± 1.2 ng/ml) relative to the controls (5.3 ± 1.0 ng/ml)27.

Idiopathic graft damage A substantial proportion of children show inflammatory liver lesions that usually go unrecognized by a normal biochemistry. Protocol biopsies were performed in a series of 158 children28. Normal or near-normal histology was reported in 77 of 113 (68%), 61 of 135 (45%), and 20 of 64 (31%) at one, five, and ten years, respectively. The commonest histologic abnormality was chronic hepatitis, the incidence of which increased with time: 22, 43, and 64% at one, five, and ten years, respectively. The incidence of fibrosis associated with chronic hepatitis increased with time: 52, 81, and 91% at one, five, and ten years, respectively. Aspartate aminotransferase (AST) levels were slightly elevated in children with chronic hepatitis (median levels 52, 63, and 48 IU/l at one, five, and ten years, respectively), but this did not reach statistical significance compared with those with normal histology. The most important factor associated with chronic hepatitis was the presence of autoantibodies, noted in 72 and 80% of cases at five and ten years, respectively, compared with 13 and 10% of cases with normal or near-normal histology. However, only four children with chronic hepatitis and autoantibodies had other features supporting a diagnosis of de novo AIH. Chronic hepatitis may represent a form of chronic rejection related to under-immunosuppression. Most of the children in that study received CsA as monotherapy from one year posttransplantation, and steroids were usually withdrawn at three months28. As pointed out in that study, the prevalence of autoantibodies in pediatric liver recipients is

high in the long term. In the series of Hamburg, positive markers were detected in 74%, while liver dysfunction was observed in 46% of these children (none had AIH) and in 35% of the children seronegative for autoantibodies29. In an Italian series, 24% of patients had positive autoantibodies, of whom 37% suffered graft disease with either early chronic rejection (perivenular drop-out and venulitis without ductopenia) or autoimmune hepatitis. Autoimmune hepatitis improves with conventional steroid plus azathioprine treatment26.

Renal-sparing immunosuppression regimens The monitoring of renal function and blood pressure is a key part of post-liver transplant care. Calcineurin inhibitor-induced acute and chronic arterial vasoconstriction mediates nephrotoxicity leading to a decrease in GFR and tubular damage. Severe renal insufficiency develops in 5% of liver transplant patients in the long term30. The proportion of children with significant renal dysfunction rises to 30%. A median 30% fall in GFR is observed in the long term compared to pretransplant values in children on TAC or CsA31 Creatinine-based estimates are not very sensitive to moderate decreases of GFR, and early recognition of CNI-induced nephropathy is important since safe, alternative immunosuppressive regimens can be applied. Cystatin C > 1.06 mg/l is an easy and reliable index of 51Chromium-ethylenediamine tetraacetic acid GFR < 80 ml/min, a reasonable threshold for actions towards prevention of further deterioration32. The method for sparing renal function has consisted of CNI reduction (usually plus azathioprine/MMF)33. Reduced doses of CNI associated to MMF led to improvement in renal function parameters in 82% of cases with renal dysfunction secondary to the prolonged use of CsA or TAC, and none experienced rejection34. The dosage of MMF required is lower in children on TAC compared to those on CsA35. In a short series of children who had received a liver transplant more than six months prior the study, a 12-hour pharmacokinetic profile showed, when used in combination with CsA, a MMF dose of 740 mg/m2 twice-daily would be recommended in pediatric liver transplant recipients to achieve mycophenolic acid exposures similar to those observed in adult liver transplant recipients36 . 33

Trends in Transplantation 2009;1

A CNI-devoid regimen (transfer to MMF or sirolimus) has been experienced in selected patients. Transfer to MMF (20-40 mg/kg/day) in 48 children (median 4 years posttransplantation) significantly improved the calculated GFR from 54 to 77 ml/min/1.73 m2 (median at baseline and second month, respectively). Beneficial effect with recovery was seen in a group, while patients with endstage renal failure at baseline just sustained calculated GFR values. Since 14% of patients experienced liver function test abnormalities, association of steroids for an initial three-month period was recommended37.

References

1. Horslen S, Barr ML, Christensen LL, Ettenger R, Magee JC. Pediatric transplantation in the USA, 1996-2005. Am J Transplant. 2007;7:1339-58. *Trends in early immunosuppression in the USA. 2. Shepherd RW, Turmelle Y, Nadler M, et al. SPLIT Research Group. Risk factors for rejection and infection in pediatric liver transplantation. Am J Transplant. 2008;8:396-403. *Comparative importance of infection and rejection causing morbidity and mortality. 3. Kelly D, Jara P, Rodeck B, et al. Tacrolimus and steroids versus cyclosporine microemulsion, steroids, and azathioprine in children undergoing liver transplantation: randomized European multicentre trial. Lancet. 2004;364:1054-61. *Trial comparing cyclosporine and tacrolimus primary immunosuppression. 4. LLoyd C, Jara P, de Ville de Goyet J, et al. Long term follow of children receiving tacrolimus or cyclosporin A-microemulsion post liver transplantation. Hepatology. 2005;42(Suppl 1):323A. 5. Ng V, Anand R, Martz K, Fecteau A. liver retransplantation in children: a SPLIT database analysis of outcome and predictive factors for survival. Am J Transplant. 2008;8:386-95. *Relative importance of rejection as a cause of retransplantation in children. 6. Broering DC, Kim JS, Mueller T, et al. One hundred thirty-two consecutive pediatric liver transplants without hospital mortality: lessons learned and outlook for the future. Ann Surg. 2004;240:1002-12. 7. Lykavieris P, Frauger E, Habes D, Bernard O, Debray D. Angioedema in pediatric liver transplant recipients under tacrolimus immunosuppression. Transplantation. 2003;75:152-5. 8. Jain A, Mazariegos G, Kashyap R, et al. Reasons why some children receiving tacrolimus therapy require steroids more than 5 years post liver transplantation. Pediatr Transplant. 2001:5:93-8. 9. Tredger JM, Brown NW, Adams J, et al. Monitoring mycophenolate in liver transplant recipients: toward a therapeutic range. Liver Transpl. 2004;10:492-502. 10. Weimert NA, Derotte M, Alloway RR, Woodle ES, Vinks AA. Monitoring of inosine monophosphate dehydrogenase activity as a biomarker for mycophenolic acid effect: potential clinical implications. Ther Drug Monit. 2007;29:141-9. 11. Truong DQ, Darwish AA, Gras J, et al. Immunological monitoring after organ transplantation: potential role of soluble CD30 blood level measurement. Transpl Immunol. 2007;17:283-7. 12. Reding R, Gras J, Truong DQ, Wieërs G, Latinne D. The immunological monitoring of alloreactive responses in liver transplant recipients: a review. Liver Transpl. 2006;12:373-83. 13. Kowalski RJ, Post DR, Mannon RB, et al. Assessing relative risks of infection and rejection: a meta-analysis using an immune function assay. Transplantation. 2006;82:663-8. 14. Hooper E, Hawkins DM, Kowalski RJ, et al. Establishing pediatric immune response zones using the Cylex® ImmuKnow™ assay. Clin Transplant. 2005:19:834-9. *Reference values of ImmuKnow™ assay in children. 15. Lee TC, Goss JA, Rooney CM, et al. Quantification of a low cellular immune response to aid in identification of pediatric liver transplant recipients at high-risk for EBV infection. Clin Transplant. 2006:20:689-94. *Management of patients with high risk for developing PTLD.

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16. Berquist RK, Berquist WE, Esquivel CO, Cox KL, Wayman KI, Litt IF. Non-adherence to posttransplant care: Prevalence, risk factors and outcomes in adolescent liver transplant recipients. Pediatr Transplant. 2008:12:194-200. 17. Venkat VL, Nick TG, Wang Y, Bucuvalas JC. An objective measure to identify pediatric liver transplant recipients at risk for late allograft rejection related to non-adherence. Pediatr Transplant. 2008:12:67-72. 18. Stuber ML, Shemesh E, Seacord D, Washington J III, Hellemann G, McDiarmid S. Evaluating non-adherence to immunosuppressant medications in pediatric liver transplant recipients. Pediatr Transplant. 2008 [in press]. 19. Nevins TE. Non-compliance and its management in teenagers. Pediatr Transplant. 2002:6:475-9. *A review of non compliance and approaches to management. 20. McDonagh JE. Growing up and moving on: Transition from pediatric to adult care. Pediatr Transplant. 2005:9:364-72. 21. Annunziato RA, Emre S, Shneider BL, Barton C, Dugan CA, Shemesh E. Adherence and medical outcomes in pediatric liver transplant recipients who transition to adult services. Pediatr Transplant. 2007:11:608-614. 22. Banff Working Group. Liver biopsy interpretation for causes of late liver allograft dysfunction. Hepatology. 2006;44:489501. *Histological criteria of late rejection. 23. Avitzur Y, De Luca E, Cantos M, et al. Health status ten years after pediatric liver transplantation-looking beyond the graft. Transplantation. 2004;78:566-73. 24. Sundaram SS, Melin-Aldana H, Neighbors K, Alonso EM. Histologic characteristics of late cellular rejection, significance of centrilobular injury, and long-term outcome in pediatric liver transplant recipients. Liver Transpl. 2006:12:58-64. 25. D’Antiga L, Dhawan A, Portmann B, et al. Late cellular rejection in pediatric liver transplantation: etiology and outcome. Transplantation. 2002;73:80-84. 26. Riva S, Sonzogni A, Bravi M, et al. Late graft dysfunction and autoantibodies after liver transplantation in children: preliminary results of an Italian experience. Liver Transpl. 2006;12:573-7. *Comparative features of late rejection and autoimmune hepatitis. 27. Venick RS, McDiarmid SV, Farmer DG, et al. Rejection and steroid dependence: unique risk factors in the development of pediatric posttransplant de novo autoimmune hepatitis. Am J Transplant. 2007;7:955-63. **Risk factors for de novo autoimmune hepatitis. 28. Evans HM, Kelly DA, McKiernan PJ, Hubscher SG. Progressive histologic damage in liver allografts following pediatric liver transplantation. Hepatology. 2006;43:1109-17. **Data of protocol liver biopsies in the long term. 29. Richter A, Grabhorn E, Helmke K, Manns MP, Ganschow R, Burdelski M. Clinical relevance of autoantibodies after pediatric liver transplantation. Clin Transplant. 2007:21:427-32. 30. Kelly DA. Current issues in pediatric transplantation. Pediatr Transplant. 2006;10:712-20. **A review of long-term problems in pediatric liver transplantation. 31. Arora-Gupta N, Davies P, McKiernan P, Kelly DA. The effect of long-term calcineurin inhibitor therapy on renal function in children after liver transplantation. Pediatr Transplant. 2004;8:145-50. 32. Samyn M, Cheeseman P, Bevis L, et al. Cystatin C, an easy and reliable marker for assessment of renal dysfunction in children with liver disease and after liver transplantation. Liver Transpl. 2005;11:344-9. 33. Tonshoff B, Hocker B. Treatment strategies in pediatric solid organ transplant recipients with calcineurin inhibitor-induced nephrotoxicity. Pediatr Transplant. 2006:10:721-9. 34. Tannuri U, Gibelli NE, Maksoud-Filho JG, et al. Mycophenolate mofetil promotes prolonged improvement of renal dysfunction after pediatric liver transplantation: experience of a single center. Pediatr Transplant. 2007;11:82-6. 35. Brown NW, Aw MM, Mieli-Vergani G, Dhawan A, Tredger JM. Mycophenolic acid and mycophenolic acid glucuronide pharmacokinetics in pediatric liver transplant recipients: effect of cyclosporine and tacrolimus co-medication. Ther Drug Monit. 2002;24:598-606. 36. Lobritto SJ, Rosenthal P, Bouw R, Leung M, Snell P, Mamelok RD. Pharmacokinetics of mycophenolate mofetil in stable pediatric liver transplant recipients receiving mycophenolate mofetil and cyclosporine. Liver Transpl. 2007;13:1570-5 37. Evans HM, McKiernan PJ, Kelly DA. Mycophenolate mofetil for renal dysfunction after pediatric liver transplantation. Transplantation. 2005;79:1575-80. **Experience on MMF therapy and calcineurin inhibitor withdrawal in children with renal dysfunction.

Trends María G. in Transplant. Crespo-Leiro, 2009;1:35-40 et al.: Molecular Testing for Monitoring of Rejection in Heart Transplant Recipients

Molecular Testing for Early Detection and Monitoring of Graft Rejection by Heart Transplant Recipients María G. Crespo-Leiro1, María J. Paniagua-Martín1 and Manuel Hermida-Prieto2 1 2

Unit for Cardiac Insufficiency and Heart Transplantation, Hospital Universitario A Coruña, La Coruña, Spain; Health Sciences Institute, Universidad de A Coruña, La Coruña, Spain

Abstract Heart transplantation is a life-prolonging procedure for many patients with stage D heart failure and other forms of advanced heart disease. However, even with the latest advances in immunosuppression, graft rejection is a major cause of death among heart transplant patients. It would be desirable to be able to detect rejection early enough and specifically enough to prevent allograft dysfunction without unnecessary over-immunosuppression. Hitherto, the main technique employed to monitor rejection status has been endomyocardial biopsy, which is invasive, prone to tissue sampling error, and placed in question by interobserver variability, but is unmatched by any non-gene-based noninvasive technique. Currently, a multi-parametric approach is employed that comprises clinical examination for signs or symptoms of heart failure, endomyocardial biopsies, drug level monitoring, allograft function tests (mainly echocardiographic studies), and screening for allograft vasculopathy. Gene expression profiling can now be used in the USA to screen heart transplant patients for risk of current rejection, thereby sparing the majority from endomyocardial biopsy, and the possibility of its application in Europe is currently being studied, as is its performance in comparison to endomyocardial biopsy in regard to relevant clinical outcomes, quality of life, and resource utilization. In future it may also be useful for classification of patients as regards risk of future rejection, for monitoring weaning from steroids, and for detection of allograft vasculopathy and antibody mediated rejection. (Trends in Transplant. 2009;1:35-40) Corresponding author: Maria G. Crespo-Leiro, [email protected]

Key words Heart transplantation. Cardiac rejection. Gene expression profiling.

Correspondence to: María G. Crespo-Leiro Unidad de Insuficiencia Cardiaca y Trasplante Cardiaco Hospital Universitario A Coruña Xubias 84 15006 La Coruña E-mail: [email protected]

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Trends in Transplantation 2009;1

Introduction Heart transplantation is a life-prolonging procedure for many patients with stage D heart failure and other forms of advanced heart disease1. However, even with the latest advances in immunosuppression, graft rejection is a major cause of death among heart transplant patients2. According to the registry of the International Society for Heart and Lung Transplantation (ISHLT), rejection causes 12% of deaths occurring between one and 12 months after heart transplantation3, and 20-50% of heart transplant patients suffer at least one rejection episode during the first year following transplantation4. It would be desirable to be able to detect rejection early enough and specifically enough to prevent allograft dysfunction without unnecessary overimmunosuppression. Gene expression profiling seems to be a promising tool for this purpose5.

Screening for and monitoring cardiac rejection: clinical methods Hitherto, the main technique employed in monitoring the rejection status of a transplanted heart has been endomyocardial biopsy (EMB), which allows rejection to be screened for and monitored on the basis of the extent and distribution of lymphocytic infiltrates and associated myocardial damage6. The goal of periodic EMB is to detect acute rejection before allograft dysfunction occurs. The latest version of the ISHLT EMB grading scheme7 establishes four categories: 0R (absence of rejection), 1R (mild rejection, defined as the presence of an interstitial and/or perivascular infiltrate, with or without a focus of myocyte damage, 2R (moderate rejection, the presence of two or more infiltrate foci with associated myocyte damage, and 3R (severe rejection, the presence of a diffuse infiltrate with multifocal myocyte damage and or edema, and/or vasculitis and/or hemorrhage). The letter “R” denotes “Revised Classification” to avoid confusion with the previous scheme, the 1990 working formulation8. Endomyocardial biopsy has significant limitations. It is invasive, it is expensive9, its sensitivity is limited by sampling efficacy, it suffers from considerable interobserver variability, and it is difficult to interpret nodular endomyocardial infiltrates (so-called “Quilty lesions”)10. Also, al36

though the incidence of complications is very low when EMB is performed by experienced staff, severe complications can arise, including pneumothorax, bleeding, pericardial tamponade secondary to perforation of the right ventricle, arrhythmias, fistulae between a coronary artery and the right ventricle, tricuspid regurgitation, damage to the carotid or femoral artery, and arterial-venous fistulae11. There is no consensus among heart transplant centers or countries regarding the frequency with which EMB should be performed, or for how long they should continue to be performed, but the current trend is to reduce their number; most U.S. centers limit periodic EMB to the first five years posttransplantation12, and most Spanish centers to the first year13. The role of EMB for rejection screening continues to be debated14,15. Although many noninvasive techniques have been investigated as regards their capacity for early detection of rejection, including echocardiography, radionuclide imaging, magnetic resonance imaging, intramyocardial electrogram, immune system monitoring and biochemical parameters16-18, none has so far proved able to match the performance of EMB. Currently, a multi-parametric approach is employed that comprises clinical examination for signs or symptoms of heart failure, EMB, drug level monitoring, allograft function tests (mainly echocardiographic studies), and screening for allograft vasculopathy. At the same time it is mandatory to be on the look-out for side effects or complications of immunosuppressive therapy, particularly nephrotoxicity, infection, and cancer. Table 1 lists the most important procedures, including some that are currently still being validated for clinical use.

Gene-based methods in heart transplantation The traditional genetic approaches of the pre-genomic era were designed to identify single loci or genes responsible for Mendelian disorders such as familial hypercholesterolemia. In these conditions, alteration of a single DNA codon results in pathological changes in protein abundance or function, and persons with the clinical signs of the pathology in question invariably exhibit the genetic alteration. However, pathologies of this kind are usually rare. The genetic component of more common disorders

María G. Crespo-Leiro, et al.: Molecular Testing for Monitoring of Rejection in Heart Transplant Recipients

Table 1. Immune and functional monitoring of heart transplant recipients Monitoring tool

Type

Value

Endomyocardial biopsy

Histology and immunohistochemistry or immunofluorescence

Drug monitoring and pharmacogenomics

Drug level or AUC

Gold standard for the diagnosis of rejection. Disadvantage of being invasive and susceptible to sampling errors and variability in interpretation. Trough levels are usually monitored for practical reasons, although peak levels usually correlate better with AUC; gene polymorphisms of CYP3A5 and MDR1 correlate with calcineurin inhibitor levels.

Functional monitoring

Diastolic parameters

Moderate correlation with significant rejection.

Tissue Doppler

Δ tissue Doppler systolic velocities vare sensitive although less specific for the diagnosis of significant rejection.

B-type natriuretic peptide

Correlates with significant rejection; no specific threshold has good discrimination capacity.

Genomic markers of rejection

AlloMap® Mollecular Expression Test (GEP)

Sensitive marker for cellular rejection although lower specificity; not validated for antibody mediated rejection.

T-cell function assays

ImmuKnow®

Marker of T-cell activation, currently under validation in heart transplantation.

ELISpot

Marker of cytokine-producing T-cells; currently under validation.

Donor-specific antibodies

The presence of DSA has been associated with an increased risk of rejection and allograft vasculopathy.

Antibody monitoring

AUC: area under curve; GEP: gene expression profile: DSA: donor-specific antibodies. Modified from Hunt, et al.12

generally involves variant alleles of multiple genes that interact to modulate the individual’s response to non-genetic risk factors19. The problem for the clinical geneticist is to identify pathological patterns of variation. Human populations exhibit about three million single nucleotide polymorphisms (SNP), i.e. about 0.1% of the three  billion base pairs in the human genome are polymorphic5. In the field of transplantation, several SNP have been associated with heart transplant outcome, which correlates with the possession of variant alleles by donor or recipient. These SNP are clustered in genes involved in alloimmune or pharmacogenetic interactions, the renin-angiotensin-aldosterone system, proclivity to renal dysfunction, and transforming growth factorbeta (TGFβ) signaling5,20-24. However, studies seeking to establish the relevance of a candidate polymorphism are often hampered by their observational character and small sample size, while meta-analyses are faced with selection bias (the non-publication of studies with negative results) and study weaknesses such as lack of clarity and uniformity regarding outcome measures, or poor evaluation of the ethnic characteristics of populations5. As a result, although SNP have thrown light on heart transplant out-

comes, the possibility of using them to predict propensity for rejection remains uncertain.

Gene expression profiling An approach that currently promises to be of much more immediate utility than SNP analysis is based on correlations between clinical states and the expression of certain genes. Although DNA defines a person’s biological potential, it is the active transcription of DNA to RNA, followed by translation to protein, that realizes this potential in accordance with his or her history, environmental context, and clinical situation. If a gene expression profile can be identified that is sufficiently characteristic of a given physiological state, this profile can then be used to test whether the individual patient exhibits the state in question. If the gene expression profile becomes manifest before clinical, biochemical, or histologic signs, this allows earlier detection of disease states, and if it appears in tissue that can be obtained noninvasively, it may allow noninvasive diagnosis of conditions that were previously best diagnosed invasively. In certain cases, a gene expression profile may indicate not a current or imminent disorder, but a physiological state that correlates with the future development of disease. 37

Trends in Transplantation 2009;1

Genes that are upregulated during acute cellular rejection after heart transplantation are involved in a wide range of functions, including T-cell activation and migration, natural killer cell activation, stem cell mobilization, hematopoiesis, platelet function, alloimmune recognition, and steroid responsiveness. Gene expression profiles have been obtained both from heart tissue25 and from peripheral blood mononuclear cells (PBMC)26,27. For screening and monitoring purposes, the latter source has the great advantage over EMB of being noninvasive, which not only eliminates the risk of EMB-related complications but also allows more frequent testing. Schoels, et al.26 took 58 blood samples from 44 heart transplant patients at the time of EMB, and used real-time quantitative PCR to study the expression of 39 genes, including genes for cytokines and chemokines, in PBMC. The PBMC from patients with ISHLT EMB grades ≥ 2 (according to the 1990 classification8) differed significantly from those of patients graded