The Target: Transplantation Tolerance
The ability to consistently induce robust, sustained, donor-specific tolerance would offer many benefits to transplant recipients. However, although the theoretical basis for acquired tolerance was established in the mid-20th century, clinical success has been rare and unpredictable. Advances in our understanding of the mechanisms that mediate rejection and tolerance increasingly allow the induction of tolerance in many experimental models. This, together with a rapidly expanding armamentarium of more selective biologic immunosuppressants, has rekindled interest in transitioning tolerance to the clinic.
Balancing this enthusiasm is the realization that achieving tolerance has proven to be significantly more difficult in humans than in animals. Together with the improving outcome of clinical transplantation, this has caused some to question the need for transplantation tolerance. Nevertheless, chronic immunosuppression remains associated with expense, risks of infections and malignancies, and drug-specific toxicities including hypertension, glucose intolerance, and hypercholesterolemia. These unwanted consequences of chronic immunosuppression result in a 5–10 fold increase in the all-cause mortality of transplant recipients relative to the general population independent of the effects of rejection (1). The nephrotoxicity of calcineurin inhibitors also affects recipients of renal and nonrenal allografts and contributes to the increasingly common development of chronic allograft nephropathy and end-stage renal disease in recipients of extrarenal organs (2). Lastly, recent data suggest that, unlike immediate outcomes, long-term outcomes of transplantation may not be improving (3). Viewed as a whole, these problems continue to fuel enthusiasm for tolerance.
How is the Target Moving?
Barriers to Attaining Transplantation Tolerance
Several features of the immune system conspire against the development of transplantation tolerance. First, the innate immune system is designed to detect threats to homeostasis. The act of transplantation itself causes injury which in turn triggers responses by multiple components of the innate immune system. Two models have been proposed to describe how innate immune responses are initiated. The danger model hypothesizes that cellular components of the innate immune system respond to tissue injury by producing soluble mediators that perpetuate the inflammatory state and promote the maturation of adaptive immune responses (4). The pattern recognition receptor model postulates that highly conserved molecules expressed on the surface of injured cells engage receptors expressed by cells comprising the innate immune system (e.g., toll-like receptors) thereby triggering innate immunity (5).
A second factor that likely poses a barrier to tolerance is the unusually large proportion of the T-cell repertoire capable of recognizing alloantigens. Although it has been estimated that fewer than one in 100,000 T cells recognize a given nominal antigen, it has been reported that 7% of the T cell repertoire undergoes proliferation in response to alloantigens (6). This large clone size may mediate an early, aggressive immune response that irreversibly damages a transplanted organ before potentially protective responses can develop. Recognition of alloantigens can occur via two distinct pathways. The first and dominant process begins upon organ reperfusion when passenger leukocytes or APCs migrate to recipient secondary lymphoid organs where direct donor antigen recognition occurs (7, 8). It is now clear that naïve responses are critically dependent on secondary lymphoid organs and rejection is greatly attenuated in their absence (9, 10). As donor APCs are unlikely to be replenished, the direct response would be predicted to dissipate over time. At later time points, the recognition of alloantigens is believed to be primarily via the indirect pathway of antigen presentation (11). Indirect presentation refers to the recognition of donor peptides presented by recipient MHC molecules and APCs. The magnitude of the indirect response is significantly less than that of the direct response, possibly reflecting the lower clone size of T cells capable of recognizing indirectly presented antigens. It has been suggested that the indirect pathway plays a particularly important role in chronic rejection (12). Distinguishing between these two methods of alloantigen recognition may have therapeutic implications. For example, costimulation blockade has been reported to inhibit T cell priming via the indirect, but not direct, pathway (13).
The development of immunologic memory poses a third potential impediment to the development of transplantation tolerance. Memory cells differ from naïve cells in that they have higher functional avidity, lower activation thresholds, the ability to rapidly engage effector functions, and the potential to circulate widely through peripheral tissues. By virtue of these properties, allospecific memory cells represent a clear threat to transplanted organs. Until recently, many have viewed one’s initial encounter with an alloantigen as a naïve response. However, the growing recognition that alloimmunity is strongly influenced by heterologous responses to previously encountered antigens suggests that even first exposures to alloantigen provoke responses by crossreactive memory cells (14, 15). Thus, memory cells may be important mediators of allograft damage even in “naïve” recipients. Heterologous immunity is likely an important factor contributing to the different success rates of tolerance regimens in clinical transplantation versus experimental models that typically use young, specific-pathogen-free animals.
Homeostatic proliferation following therapies that cause massive T-cell depletion may represent a fourth barrier to tolerance (16). T cells from lymphopenic hosts undergo extensive proliferation. The phenotype and function of the re-emerging T cells is similar to that of memory T cells. Furthermore, memory T cells have been reported to be relatively resistant to depletion (17). Thus, following massive T-cell depletion, recipients may be selectively repopulated by homeostatically proliferating memory or memory-like T cells that are resistant to maneuvers that typically inhibit naïve responses. Taken together, these four factors produce barriers that vary not only between similar individuals, but also vary over time in the same individual. Thus, the route to tolerance is highly variable.
Mechanisms of Tolerance
Based upon our current approach to organ transplantation, tolerance appears an infrequent occurrence that requires the appropriately timed disruption of numerous immune mechanisms. However, tolerance is the default response to a multitude of self and environmental antigens, and many of the mechanisms that maintain self-tolerance may also be capable of promoting allograft tolerance. Tolerance mechanisms can be broadly classified as either central or peripheral. Central tolerance refers to the deletion within the thymus of T cells whose affinity for self antigens is inappropriately high and thus likely to result in autoimmunity. The tolerance displayed by neonatal mice to a transplanted organ demonstrates the robustness of these central mechanisms. Similarly, central deletion is an important mechanism promoting tolerance to organ allografts in mice displaying mixed hematopoietic chimerism following bone marrow transplantation (18).
In addition to central deletion, a number of mechanisms operating in the periphery have been reported to contribute to the tolerant state. Peripheral mechanisms for maintaining tolerance include ignorance (9), anergy (19), regulation or suppression (20–22), and apoptosis or peripheral deletion (23, 24).
Ignorance as a mechanism mediating tolerance was demonstrated by Lakkis et al. who showed that naïve mice lacking secondary lymphoid organs accepted skin or heart allografts indefinitely (9). Impaired rejection in this model resulted from the failure of T cells to be primed when they encountered donor antigens outside of lymphoid organs. The findings that in some settings recipient T cells can be primed by alloantigens encountered within the allograft (25) and that memory T cells can be reactivated after encountering antigen outside of secondary lymphoid organs (26) suggest that the role of this mechanism may be limited.
Active, antigen-specific suppression of immune responses was first reported in the 1970s (27,28). Though interest in this phenomenon waned, it was rekindled by the demonstration that CD4+CD25+ T cells from rats bearing long-term surviving cardiac allografts could transfer tolerance to untreated recipients (29). A growing body of experimental and clinical evidence suggests a role for regulatory T cells in the induction and maintenance of tolerance (reviewed in 21). While CD4+CD25+ cells constitute the most widely recognized phenotype of regulatory cells, cells of other lineages including NK1.1, C8+CD28-, and CD3+CD4-CD8- cells may display regulatory properties (30–32). Other markers of regulatory T cells may include CD45RB, GITR, CTLA4, CD103, and FOXP3 (33–41).
At least three mechanisms appear to be important for tolerance mediated by regulatory T cells. Preclinical and clinical evidence suggests that TR1, TH3, and NKT cells mediate regulatory effects at least in part via production of the cytokines IL-10 and TGFβ (33, 42–45). The function of CD4+ CD25+ T reg also appears to be affected by the expression of the cell surface molecules GITR (36, 37) and CTLA4 (42) which may contribute to the contact dependent effects of regulatory cells (reviewed in 21,22). Finally, anergic CD4+ T cells may mediate their regulatory effects by inhibiting the maturation and function of dendritic cells (46).
Although regulation has been extensively studied in experimental systems, far less is known about its role in clinical tolerance. Salama et al. reported that CD25+ regulatory cells developed as early as three months after renal transplantation and persisted for years (47). Furthermore, evidence of regulation in vitro as detected by ELISPOT analysis was more commonly observed in rejection-free recipients than those who had experienced rejection. Analysis of a limited number of operationally tolerant transplant recipients using the trans-vivo DTH assay has also suggested a role for regulation that was dependent upon the production of TGFβ and/or IL-10 and the expression of CTLA4 (48, 49).
Although transplant tolerance may develop as a result of spontaneous regulatory mechanisms, clinical application of regulation is likely to require interventions that purposely produce regulatory cells. Many of these approaches (i.e., DST and in vitro generation of T reg) depend upon exposure to donor antigen, and are facilitated by the demonstration that recipient T cells need not be tolerized to each foreign antigen expressed by the transplanted organ. Rather, linked, dominant suppression has been described (50–52). In rodent and large animal models, the expression of one MHC molecule recognized by regulatory T cells effectively suppresses the response to other mismatched MHC molecules. A final consideration pertinent to the clinical application of regulatory mechanisms is the possible effect of currently used immunosuppressive agents on T regs. Although still controversial, some agents such as calcineurin inhibitors may inhibit the development of tolerance by preventing the activation or function of regulatory T cells (53).
In addition to ignorance and regulation, peripheral deletion of alloreactive T cells may contribute to tolerance. This can occur in the setting of chronic alloantigen stimulation or when alloantigen is encountered under suboptimal conditions for T-cell activation. Clonal exhaustion has been reported after liver transplantation as an example of how chronic stimulation may induce peripheral T cell deletion (54). Alternatively, the blockade of costimulatory signals such as CD28 or CD154 at the time T cells encounter alloantigens has been reported to result in incomplete T-cell activation followed by anergy and apoptosis (55). Several groups have now demonstrated that peripheral deletion mediated by apoptosis is an important event contributing to long-term allograft acceptance (23, 24). Apoptosis of T cells can be mediated either by cell surface death receptors or cytokine withdrawal. The death receptors mediating apoptosis are comprised of TNF receptor superfamily members that have a cytoplasmic death domain that binds cell-signaling proteins such as TRADD, FADD, FLICE, and caspase-3 leading to apoptosis. Cytokines promoting T-cell survival include the common γ chain cytokines IL-2, IL-4, IL-7, and IL-15. Their absence predisposes cells to active, apoptotic death.
From this review, it should be obvious that there are multiple barriers to tolerance and multiple mechanisms that, in the correct setting, may promote tolerance. For example, both genetic factors such as CCR5 gene polymorphisms (56) and acquired factors such as the development of allo-reactive memory populations will differ between recipients and fundamentally affect the nature of the immune response to transplanted organs. It is also likely that the organ transplanted affects the nature of the immune response and the probability of developing tolerance (47, 57). How these multiple variables interact will determine the outcome of transplantation. The multiplicity of possible interactions explains our current inability to predict the outcome of transplantation in seemingly similar recipients.
In addition to variations between recipients in anti- and pro-tolerogenic factors, it is increasingly accepted that tolerance may be dependent upon the sequential development of more than one mechanism (58,59). For example, profound T-cell depletion eliminates the large population of cells with direct alloantigen specificity. However, other pro-tolerant mechanisms are required to maintain tolerance as recipient T cells re-emerge. This is exemplified by the finding that tolerance in NOD and IL-2−/− recipients treated with sirolimus, an agonist IL-2 and antagonist IL-15-related fusion protein was associated with the early deletion of alloreactive T cells followed by the development of CD4+CD25+ regulatory T cells (60). Deletion of donor-reactive T cells has also been shown to occur in recipients rendered tolerant to organ allografts following bone marrow transplantation (61). However, even in this model it may not be accurate to ascribe the development of donor-specific tolerance entirely to deletion. Tolerance developing after the infusion of donor bone marrow in mice treated with busulfan and short-term blockade of CD28 and CD154 is initially dependent upon CD4+ T cells (62). Although tolerance is eventually associated with deletion of donor-reactive T cells, this process requires months for completion, implying that mechanisms other than depletion are responsible for early graft acceptance. Using the trans-vivo DTH assay, we have recently demonstrated the existence of regulatory cells that produce IL-10 and/or TGFβ at early time points following transplantation (63). Interestingly, these regulatory cells were not detected at later time points, suggesting that they may be deleted together with alloaggressive recipient T cells. Thus, the mechanisms which induce tolerance may be distinct from those that maintain tolerance. Finally, mechanisms that protect organ allografts at one point in time may be harmful at later times. For instance, treatment of mice with an anti-CD4 antibody or gallium nitrate allows the long-term acceptance of heart allografts. This effect is mediated by regulatory cells that produce TGFβ (64). However, with time, heart grafts from anti-CD4 and gallium nitrate treated recipients develop chronic rejection, which has been postulated to be the result of the pro-fibrotic effects of TGFβ (65).
How Can We Converge on the Target?
The Impact of Early Tolerance Studies
Our current concept of transplantation tolerance remains heavily influenced by the original studies of acquired tolerance (66). These studies predicted that tolerance should be achievable following a brief intervention that fundamentally changes the immune system. Ideally, this intervention would be initiated at the time of transplantation and result in permanent, antigen-specific organ acceptance. However, it is important to recall that even in the landmark study of Billingham et al., only two of the five recipients were tolerant (two developed acute rejection and one developed chronic rejection). Nevertheless, the view that tolerance could be acutely induced has been reinforced by experiments demonstrating that prolonged, if not indefinite, allograft survival could be achieved following brief treatments with agents such as CTLA4-Ig, anti-CD154, or anti-CD4 (65, 67).
Moving Transplantation Tolerance to the Clinic
For some time, nonhuman primate (NHP) models have been used to evaluate the safety and efficacy of the most promising tolerogenic regimens developed in rodent models prior to their use in humans. However, when compared to the results obtained using rodents, virtually all tolerogenic strategies have proven far less effective in NHPs (68). This may be the result of the homogeneity of rodent models (i.e., uniform age, environmental exposure, and genetic background) and the reductionist design of studies aimed at determining the role of a specific pathway/molecule. Attempts to closely replicate these approaches in large animal preclinical or clinical transplantation have been largely unsuccessful, likely due to the more diverse environmental exposure and genetic background of primate transplant recipients.
One of the more promising approaches tested in NHPs is brief therapy with an antibody against the CD3 molecule coupled to a diphtheria-derived immunotoxin (69) that mediates profound but reversible T cell depletion. Approximately 40% of the treated animals develop operational tolerance following pretransplant T-cell depletion, as indicated by the absence of rejection and the presence of donor-specific hyporesponsiveness following T cell repopulation. This approach was made more reproducible by the addition of deoxyspergualin, a polyamine antibiotic that inhibits APC function (70). Based on this approach, a number of groups have undertaken clinical trials utilizing early recipient T-cell depletion. Several investigators have used alemtuzumab (an anti-CD52 monoclonal antibody) to induce profound T-cell depletion. Despite achieving depletion that is equivalent to that obtained using anti-CD3-immunotoxin with respect to kinetics, magnitude, and effectiveness within the secondary lymphoid tissues, treatment with alemtuzumab alone or in combination with deoxyspergualin is not sufficient to induce tolerance in adult humans (71, 72). This experience has led other groups to combine the T-cell depletion with other agents such as sirolimus (73). Although this approach remains promising, initial studies have shown a significant incidence of acute rejection (73) and an unusually high incidence of antibody-mediated rejection. The failure of these T-cell-centric approaches suggests that other components of the immune system, such as B cells, NK cell, or monocytes, may need to be specifically targeted to achieve tolerance.
Chimerism and Tolerance
Combined nonmyeloablative bone marrow and solid organ transplantation has been shown to induce robust tolerance in rodents (18, 62). In humans, successful bone marrow transplantation allows the acceptance of subsequent organ allografts from the same donor in the absence of immunosuppression (74). These observations form the basis for clinical trials, sponsored by the Immune Tolerance Network, of combined bone marrow and kidney transplantation. Using a nonmyeloablative conditioning regimen (cyclophosphamide, thymic irradiation, and antithymocyte globulin) and a short course of cyclosporine, two patients have been reported to display functional tolerance and sustained antitumor responses at 2 and 4 years following combined bone marrow and kidney transplantation from the same donor (75). To date, this approach has proven less effective for non-HLA identical donor and recipient pairs. The effectiveness of this approach may be in large part related to its ability to target multiple mechanisms of rejection and tolerance. There is experimental evidence to suggest that these types of regimens control alloreactive recipient T cells by central deletion and peripheral mechanisms including anergy, deletion, and regulation (18, 76, 77). Despite targeting multiple mechanisms, combined bone marrow/solid organ transplantation does not invariably prevent the development of chronic allograft vasculopathy (78, 79). This suggests that in addition to inhibiting multiple mechanisms that promote acute rejection, tolerogenic strategies will also need to target a discreet group of alloimmune responses that cause chronic allograft injury, or alternatively promote protective responses that suppress injurious responses.
A New Mind-Set Towards Tolerance Induction
Fifty years after the initial description of acquired transplant tolerance, the occurrence of tolerance in clinical transplantation remains largely accidental and unpredictable. However, the argument in favor of continued efforts to design, test, and implement tolerogenic regimens in transplantation remains as compelling today as at any time in the past. The heterogeneous nature of clinical transplantation together with the redundancy and plasticity of the immune system conspire against the acquisition of tolerance, and suggest that when tolerance does develop, it may be impermanent. In this review, we propose that successful tolerogenic strategies will need to inhibit multiple destructive immune responses while promoting immune responses that protect the transplanted organ. Furthermore, a single such regimen might not prove to be uniformly effective as the targeted mechanisms may vary significantly between recipients and within a given recipient over time. The clinical observation that transplantation tolerance may be lost, often after several years or even decades of drug-free allograft acceptance, supports the notion that the immune response of tolerant patients mutates over time. Thus, the timing, as well as the nature, of tolerance-promoting interventions may need to be varied. Consequently, as we converge on tolerance, we are not focusing on one static target, but many moving targets. If long-term allograft acceptance requires a series of discreet immunologic responses or nonresponses, it seems unlikely that a single, brief intervention will result in long-term allograft acceptance. Rather, a longer period of treatment sequentially targeting developing immune responses as they occur may afford the greatest likelihood of achieving drug-free allograft acceptance. If we are to apply such an approach to transplantation, we will need to develop a battery of monitoring techniques that are capable of quantifying the balance of destructive and protective mechanisms following transplantation.
The authors thank Lisa Carlson for critical reading of this manuscript.
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