Challenges in therapeutic strategies for transplantation: Where now from here?

Transplant Immunology 15 (2005) 149 – 155 www.elsevier.com/locate/trim

Challenges in therapeutic strategies for transplantation: Where now from here? David A. Bruno a,b, Kiran K. Dhanireddy a,b, Allan D. Kirk a,* a

Transplantation Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Building 10, Room 5-5750, Bethesda, MD 20892, United States b Georgetown University Hospital, Department of Surgery, Washington, District of Columbia, United States Received 23 June 2005; accepted 12 July 2005

Abstract The current standard of care in transplantation reliably achieves acceptable graft and patient survival but still depends on life long immunosuppression in most patients. Current strategies employ medications that, in general, inhibit distal events mediating rejection, namely T cell activation and cytotoxicity. They do not typically interfere with initial allorecognition or the factors that influence the direction of an immune response (towards cytotoxicity as opposed to anergy or regulation). Given the exponential amplification of immune responses, these proximal targets may be more efficient in preventing rejection. Recent laboratory investigations have identified several approaches, e.g., costimulation blockade, depletion, and hematopoietic chimerism, that influence the initial stages of the alloimmune response, or establish self-perpetuating means of eliminating rejection without chronic immunosuppression. This manuscript reviews methods of immune manipulation that the authors view as promising for future exploitation and transfer to the clinic. These therapies are similar in that they are viewed as attempts to influence the ability of the body to mount an immune response and its subsequent direction, as opposed to supplying late effector phase inhibition. While it is recognized as unlikely that any one therapy will universally lead to tolerance, the authors propose that these concepts will make immunosuppressive drug minimization more readily successful. D 2005 Elsevier B.V. All rights reserved. Keywords: Transplantation; T cell activation; Cytotoxicity

Transplantation has been on the cutting edge of medical technology since the middle of the last century. Its current status as an accepted therapy for end stage organ failure is no doubt the result of its use of new, increasingly potent, immunosuppressive agents. Indeed, clinicians now have an unprecedented array of drugs available to prevent rejection; so much so that there is no regimen defined as ‘‘the’’ standard of care. Instead, many combinations of induction and maintenance medications have been adopted as standard based on their ability to deliver comparably high patient and graft survival. Most of these approaches have in common a strategy of preventing late effector phase T cell function, rather than influencing the character of early allorecogni-

* Corresponding author. Tel.: +1 301 496 3047; fax: +1 301 451 6989. E-mail address: [email protected] (A.D. Kirk). 0966-3274/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2005.07.001

tion. This requires multi-drug regimens to limit the exponential expansion characteristic of an immune response. We suggest that drug use influencing events more proximal, such as antigen presentation efficiency and T cell precursor frequency, might allow the field to progress from an era of drug sufficiency, to one of drug efficiency. Drug efficiency is optimized by rational application of the available drugs in a manner that complements the physiological mechanisms of self-tolerance and perhaps eschews the general concept of immune suppression in favor of immune redirection. To accomplish this, we must move away from treatments aimed at the distal effector mechanisms directly mediating rejection (namely T cells), and toward early manipulation of the proximal factors that increase the probability of an alloimmune response [1]. Similarly, we should recognize rejection and tolerance for what they are, aggregate and opposite effects of factors that

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influence the probability of an effector immune response, namely allospecific T cell precursor frequency, effective antigen presentation and costimulation (and the required access to the secondary lymphoid tissue), innate responses to injury that spur antigen presentation and lower the activation threshold of effector cells, and effective chemotaxis directing the effector response to the target organ. The aforementioned factors, when unchecked, make alloimmunity a probable event and autoimmunity is an improbable event. However, neither of these immune responses are inevitable nor impossible, and both can be manipulated by similar means, with allograft tolerance achieved when the aggregate effects of tolerizing processes overcome the aggregate effects of pro-immune processes. Currently, multiple therapies that influence the direction of an immune response are being pursued, specifically, costimulation blockade, depletional induction and mixed hematopoietic chimerism. Use of these approaches and application of the lessons they have spawned to date can improve the efficiency with which we treat patients and are leading to successful drug minimization regimens.

1. Costimulation blockade—context based therapy The two-signal theory of lymphocyte activation of Bretscher and Cohn, and Cunningham and Lafferty [2,3] has evolved considerably over the past 2 decades. Though first a proposed requirement for two signals for efficient lymphocyte activation (signal one, antigen recognition by the TCR, and a second co-stimulatory signal indicating that a response is appropriate), costimulation is now a more complex recognition that immune responses are governed by many ligands with the potential for enhancing the efficiency of lymphocyte activation or conversely interrupting a response through anergy or apoptosis. Accordingly, manipulation of costimulation should be seen as a means of changing the efficiency with which immune activation occurs and hence the probability of a biologically relevant response, not simply establishing whether cells are activated in a binary on/off manner. The best characterized activating costimulatory signals include CD28 and CD154 on T cells and their ligands on APCs, B7-1/B7-2 (CD80/CD86) and CD40 respectively. Appropriate costimulatory input allows for exponential T cell proliferation and lowers the threshold for activation [4]. The counter-balance to activating costimulation is mediated through molecules like CD152 (CTLA-4) that compete with CD28 for ligation of CD80/ CD86. CTLA-4 signaling is believed to be involved in the processes that restrain an aggressive immune response by attenuating the activation and proliferation of T cells [5– 7]. There are now many constitutive and inducible costimulatory molecules, but their aggregate effect is that they mediate changes in both the activating potential of T cells and the efficiency of a T cell mediated response [8]. Changes in activation efficiency can result in augmented or

stalled immune responses—immune responses that never reach the threshold for effective immunity (Fig. 1). Thus, changes in the magnitude and/or the type of costimulation can change a vigorous response with certain in vivo relevance, to an inefficient response that, although still present, fails to make a meaningful change in graft function. By influencing the response early, small changes can have a major influence on the magnitude of the subsequent response. Interestingly, many of the factors that are known to influence the incidence of rejection, ischemia, trauma, immunosuppression, mediate their effects at least in part through changes in the availability and type of costimulation molecules [9,10]. Many laboratories including our own have explored the power of costimulation blockade in non-human primates [6,11 –16]. Interruption on B7 ligation using either CD152 fusion proteins (CTLA4-Ig and LEA29Y) and blockade of CD40-CD154 interactions with monoclonal antibodies specific for CD154 (hu5c8 and IDEC-131) have led to prolongation of graft survival to months and years respectively and occasionally resulted in long-term allograft survival even after withdrawal of therapy. The B7 strategies are now entering pivotal clinical trials with the fusion protein LEA29Y and are poised to be an important addition to our armamentarium [17]. Unfortunately, phase 2 clinical trial to test CD154 based therapies have stalled over concerns for thrombotic complications [18]. An important concept emerging from the growing experience with costimulation is that TCR mediated antigen recognition is required for many of its salutary effects. As such, indiscriminate use of TCR inhibiting immunosuppressants and limitations in the availability of donor antigen have both been shown to limit the effect. Specifically, studies in mice have shown that calcineurin inhibition prevents costimulatory blockade from favoring tolerance presumably by preventing the activation induced apoptosis of alloreactive cells [19,20]. Drawing on these concepts, we have recently investigated a strategy that both preserves TCR signal transduction and encourages engagement with alloantigen to decrease the length of anti-CD154 therapy and perhaps avoid thrombotic complications. We have used the CD154specific antibody IDEC-131 combined with sirolimus and donor-specific transfusion in non-human primates [13]. This therapy has provided consistent prevention of rejection during therapy both in skin and renal transplantation, similar to that of monotherapy hu5c8. Furthermore, 3 of 5 renal allograft recipients treated with this triple therapy have demonstrated operational tolerance manifested by long-term renal allograft survival and subsequent donor specific skin graft acceptance off therapy. Importantly, using a clinically relevant prophylaxis regimen with perioperative heparin and aspirin, no animals treated with IDEC-131 developed thrombotic complications [21]. To date, the mechanisms by which anti-CD154 therapy prolongs allograft survival are not fully elucidated, but the drug likely exerts a number of

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Fig. 1. In order for an adaptive immune response to occur, T cells (for example) must reach a critical number (the number required to evoke a noticeable phenotype of immunity, or the threshold for immunity) without exceeding their physiological homeostatic parameters (an excessive number of cells, e.g., a lymphoproliferative disorder or point of futility). Thus, an optimal immune response (perfect cell doubling beginning from a precursor frequency of 1, black solid line) reaches this point (in this example 200,000 cells) after a considerable number of cell divisions (in this case ¨23, represented by the arrow) and enters a zone of anticipated regulation (a cell number or activity for which the body has regulatory control, grey box). Thus, under physiological conditions, an immune response will proceed at an anticipatable rate and can be controlled by means that cover an anticipatable cell density. To prevent an immune response, the efficiency of cell division need only be reduced to maintain a cell number below the threshold for effective immunity. Thus, by reducing the division efficiency (e.g., removing costimulation inducing a 6% division efficiency, dotted black line), even though some immune activity has occurred it fails to reach a point where it can effect the body or register as an immune phenotype. When the precursor frequency is raised above that which is typical (in this example from 1 to 100, solid grey line), an immune response proceeds at a rate far faster than would be anticipated under physiological conditions. A phenotype of immunity occurs outside the bounds of physiological regulation. More importantly, when means of attenuating an immune response are applied to a non-physiological precursor frequency (e.g., removing costimulation and applying a 6% division efficiency, dotted grey line), the resulting response still exceeds the rate and progression of an optimal immune response under physiological conditions. An inefficient immune response with a high precursor frequency can exceed a perfectly efficient immune response with a low precursor frequency in such a way that the typical regulatory means by which autoimmunity are prevented fail to prevent the response.

effects on both the innate and acquired immune system. Among these are down regulation of CD80/CD86, direct blockade of CD154 on activated lymphocytes, and interference with APC and endothelial cell antigen presentation [22]. All of the aforementioned mechanisms likely contribute to modest changes in individual lymphocyte activation potential and the number of cells recruited into a response, thus preventing the threshold for immunity from being exceeded. In addition to these effects, we increasingly believe that a substantial effect of CD154 based therapies is mediated by its effect on interactions between platelets and antigen presenting cells [23,24]. CTLA4-Ig is a fusion protein designed to block the interactions of CD80 and CD86 with their activating ligand on T cells, CD28. In rodent models, CTLA4-Ig has been shown to have tolerizing effects [25 – 27], but when used in non-human primates, the results are less dramatic [11,12]. Recently, Larsen et al., in collaboration with Bristol MyersSquibb, have developed a high affinity variant of CTLA4-Ig named LEA29Y (Belatacept) [17]. They paired LEA29Y with conventional immunosuppressants and found prolonged renal allograft survival in non-human primates. Specifically, the combination of LEA29Y and basiliximab resulted in 5 of 6 animals with greater than 100 days of

survival. LEA29Y administration was well tolerated and also blocked the development of anti-donor antibody. Although the use of LEA29Y did not result in tolerance, it has shown promise as a maintenance immunosuppressant. Therefore, human trials are currently investigating this possibility. Although not a tolerance trial per se, the mechanism of action of Belatacept will open up several new avenues of clinical investigation that will have bearing on the development of pro-tolerant immune therapies. As discoveries continue to be made regarding costimulation biology, the limitations of therapies targeting CD40CD154 and CD28-CD80/CD86 interactions are becoming apparent. Namely, costimulation blockade is most relevant for naı¨ve immune responses. One relevant difference between adolescent non-human primates and adult human transplant recipients is their level of antigen experience. As individuals age, they are likely to have developed memory responses to multiple environmental exposures and infections. This is evidenced by an ever enlarging peripheral pool of memory T cells [28,29]. Recently, virally induced memory T cells with heterologous cross-reactivity to alloantigens have been shown to mediate allograft rejection in mice [30]. Furthermore, immunization with viral antigens has been shown to counter otherwise successful tolerance

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induction schemes. This heterologous immunity is a potent barrier to tolerance induction that likely has significant influence in adult humans. A second unique aspect of memory cells is their more efficient response to antigenic challenge. Lakkis et al., have shown that memory cells, in contrast to naı¨ve cells, can mount a vigorous immune response which leads to allograft rejection in the absence of secondary lymphoid structures [31]. Taken together, these observations help explain a critical limitation of costimulation blockade, particularly in light of the high memory cell presence in adult humans [32]. Another potential obstacle to tolerance induction by costimulation blockade is the act of transplantation itself. The danger model of immunity suggests that APCs can provide costimulation for T cells upon detection of adjacent cell stress or tissue damage—for example, ischemia and reperfusion during a transplant operation [9,10]. In response to tissue damage, the first mediators of costimulation to arrive at the site of injury are perhaps not APC or T cells, but rather platelets. Recent evidence suggests that plateletderived CD154 may be a key link between the innate and acquired immune systems. Czapiga et al. demonstrated that platelet-derived CD154 was able to cause dendritic cell maturation, secretion of IL-12 and upregulation of costimulatory molecules and can in fact chemotax toward a site of inflammation via functional formyl peptide receptors [23,24]. Clearly an area of study that is only now developing is examining the modes of communication between the innate and acquired immune systems. Apparently, costimulation may play a crucial role in this process. In spite of previous disappointments in translating preclinical success into human trials, costimulation pathways remain compelling targets for immune manipulation. With greater understanding of the strengths and limitations of these therapies, costimulation blockade may be paired with other therapies to produce as yet unseen synergy.

2. Depletional therapy The current enthusiasm for depletional induction therapy can be traced back to the independent work of Knechtle and Thomas during the 1990s. Both investigators were able to show long-term renal allograft survival in non-human primates treated with FN18-CRM9 immunotoxin induction therapy [33,34]. Immunotoxin is an anti-CD3 monoclonal antibody conjugated to a mutated diphtheria toxin developed by Neville et al. that mediates potent peripheral and nodal T cell depletion [35]. By non-specifically reducing the precursor frequency of alloreactive T cells, they were able to induce tolerance. One of the first clinical trials to demonstrate the potential of depletional induction to dramatically reduce maintenance immunosuppression in humans was performed by Calne et al. [36]. He postulated that alemtuzumab (Campath-1H, an anti-CD52 humanized monoclonal antibody) could serve as

a surrogate for immunotoxin, and in a limited trial, was able to demonstrate a reduced requirement for maintenance immunosuppression after depletion, which he termed ‘‘prope,’’ or almost, tolerance. Since that time, multiple studies have been conducted using depletional induction with maintenance immunosuppression [36 –38]. Our laboratory has looked at this phenomenon to ask whether prope tolerance really tolerance or merely manageable immunosuppression? In two trials, one using depletion with alemtuzumab alone and another investigating high dose rabbit anti-thymocyte globulin, we have begun to examine the effects of depletional induction with limited or no maintenance immunosuppression [38,39]. Both therapies result in profound and persistent T cell depletion. Monocytes were more resilient to depletion but were greatly reduced nonetheless. In both studies, a requirement for some maintenance immunosuppression was established and rejections, when they occurred, were lymphopenic. The rejections were coincident with a rebound in the peripheral monocyte count and were characterized by macrophage infiltration and increased TNFa transcription on biopsy specimens. Despite the clear evidence that depletion of this magnitude did not induce tolerance, all of the patients were maintained even after rejection on low dose monotherapy immunosuppression. Although tolerance was not induced with depletional induction alone, many insights were gained through these limited trials. Most notably, there exists an immunocompetent population of T cells that are resistant to antibodymediated depletion capable of causing rejection even in very small numbers [32]. Pearl et al. showed that post-depletional T cells were of a singular effector memory phenotype (CD3 + CD4 + CD45 CD62L CCR7 ). These cells are the first to begin repopulating at the 1-month time point and have a skewed Vh repertoire indicating oligoclonality. Although functional, their limited number substantially limits the allospecific precursor frequency available for an immune response. As such, rejections are manageable. Furthermore, we have shown that these cells are very responsive to calcineurin inhibition but not other maintenance immunosuppressants. As with costimulation blockade, heterologous memory remains a significant barrier to tolerance. Simultaneously, Turka et al. have shown in rodents that homeostatic proliferation, the reactive expansion of mature peripheral lymphocytes in a lymphopenic environment, induces naı¨ve cells to become memory-like in that they are have a lower threshold for activation and are less dependent on costimulation than naı¨ve cells [40,41]. They demonstrated that following depletion, the resultant pool of repopulated lymphocytes is generally resistant to tolerance induction [42]. Thus, T cells with either a true memory or pseudo memory phenotype remain barriers to tolerance induction. This fits well with the general theme of probability related immunity. A high precursor frequency with low activation potential is as potent as a low precursor

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frequency with high activation potential. Both conditions reach an effective threshold for immunity. As with costimulation blockade, our understanding of the biology of lymphocyte depletion is incomplete. Depletional induction, through its induction of homeostatic activation, may have inadvertently created a circumstance where tolerance induction is highly unlikely. This does not mean that that depletion should be abandoned, but rather, that its effects will need to be tempered by some other adjuvant therapy. Even without tolerance, however, depletion will result in reduced immunosuppression requirements.

3. Mixed hematopoietic chimerism A third prominent strategy aimed at transplantation tolerance is hematopoietic mixed chimerism—the coexistence of hematopoietic cells from both the donor and recipient within the donor [43]. This has been demonstrated to be a robust means of achieving tolerance, predominantly through the thymic clonal deletion of donor-specific cells. Its association with tolerance is well established, but it is a state that is increasingly difficult to achieve as the recipient ascends the phylogenetic tree. Rodents can be induced into a chimeric state relatively easily and tolerance invariably follows. Conversely, primates typically exhibit only transient chimerism even after vigorous induction regimens. Interestingly, even a brief period of chimerism appears necessary to facilitate prolonged rejection-free survival [44,45]. Chimerism approaches are also moving into the clinic. Millan et al. reported a trial utilizing total lymphoid irradiation, donor CD34+ cells, and anti-rabbit thymocyte globulin followed by kidney transplantation [44]. Of the four patients, three were demonstrated to have macrochimerism, and one was taken off all immunosuppression. In another more recent clinical study, two patients with multiple myeloma had cyclophosphamide bone marrow conditioning, thymic radiation, and rabbit anti-thymocyte globulin followed by bone marrow and renal transplantation. Although chimerism was achieved in both patients, it was fleeting. Of note, these patients had no episodes of rejection after medications were withdrawn despite the disappearance of microchimerism [46]. The initial rejectionfree period in these patients is likely due to the intense immunosuppression associated with the induction. However, thereafter chimeric cells may influence thymic selection and lead to a sustainable method by which donor specific precursor frequency can be controlled without ongoing immunosuppression. The role of the thymus, particularly in adult humans, is an important area of investigation at present. Although these studies show promise for tolerance, some themes emerge from both preclinical and clinical attempts at chimerism and graft acceptance. First, these regimens are rigorous and the concern for potential morbidity is not insignificant at present. Second, these studies result in

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tolerance of some but not all patients who undergo these therapies. A correlation between graft tolerance and lesser degrees of donor/recipient HLA mismatch is implied. Thirdly, rejection in these groups can occur after relatively long periods without warning.

4. The future In the last 50 years, the treatment of end stage organ disease has gone from the extraordinary to the ordinary. Through a variety of advances in progressively more potent immunosuppression, transplantation has become a routinely effective treatment for organ failure. With the best intentions, the science of transplantation has focused on the terminal events of graft loss—namely T cell mediated rejection. However, given the exponential nature of immune amplification, intervention late in the immune cascade necessitates broader and more aggressive intervention. Conversely, by looking toward early events, we may be capable of achieving a similar effect with more subtle manipulations. The aforementioned strategies have all proven themselves in rigorous preclinical biological models, yet there appropriate use in humans remains to be defined. Much work remains to be done. A more thorough understanding of the interaction between all the actors of the immune response is necessary. Platelets, B-cells, and graft endothelium most certainly play a role in the eventual immune response that results in organ rejection—yet they have thus far been under-appreciated in attempts to achieve durable tolerance. If immunity is considered in terms a probability, we must accept that it is unlikely that we will find a single, universal therapy that shifts probability towards allograft acceptance. Instead we must strive for a thorough understanding of the mechanisms that lead to immunity or anergy, and then be able to determine which of a variety of therapies that exert fine control over these processes will lead to tolerance in a given individual. It is likely that therapy will need to be applied at a number of different times to steer immunity towards the tolerant state. It is also likely that curative therapy applied to one patient will be ineffective in another. Greater understanding and mastery of the immune response will be achieved only with rigorous basic science research applying current and future technology. As we work to advance the science of transplantation in an effort to increase the probability of tolerance, we should recognize that even now with our limited successes, most people can be transplanted with less immunosuppression than is currently considered standard of care.

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