Frontiers in Nephrology
Authors: Anna Valujskikh, Xian Chang Li
Journal: Journal of the American Society of Nephrology (2007)

Immunologic memory, or the ability to respond more rapidly and effectively to the previously encountered pathogens, represents a fundamental feature of the adaptive immune system. Such memory functions are carried out by subsets of specialized memory cells that are long-lived as a population. Although memory cells are important constituents of protective immunity, the enhanced host protection turns into a liability in transplantation because memory responses against donor antigens compromise survival of life-saving organ or tissue transplants. In most models, memory response is a collaborative effort between humoral and cellular immunity, but this review focuses specifically on memory T cells in transplant models. We start by introducing the basic concept of T cell memory, followed by a discussion of alloreactive memory T cells and their contribution to allograft rejection process. Next, we summarize the known effects of existing graft prolonging therapies on memory T cells. Finally, we outline several promising approaches to targeting memory T cells in transplant settings and some future challenges in this area. MEMORY T CELLS: GENERATION, PHENOTYPE, AND CHARACTERISTICS After initial exposure to antigens, naïve T cells proliferate extensively and differentiate into effector T cells capable of infiltrating inflamed tissues and destroying invading microorganisms (Figure 1). After the pathogen is cleared, most effector T cells undergo apoptotic cell death, leaving behind a population of antigen-experienced memory T cells.1–3 The ability to produce a robust memory response seem to be imprinted in the course of initial T cell priming. Thus, CD4 T cells help or CD40/CD154 interactions during activation phase are required for functional secondary CD8 T cell responses to viral antigens.4,5 Co-stimulation through CD134 and 4-1BB promotes capacity for secondary expansion of CD8 T cells.6 Similarly, development of CD4 T cell memory response is dependent on the presence of CD134/CD134 interactions during priming phase.7 In contrast, the lack of CD28 co-stimulation does not entirely abrogate protective antiviral CD8 T cell memory, despite the lower magnitude of primary response.8 The mechanisms controlling the transition of effector T cells to a memory phenotype are still poorly understood. Because effector T cells have to escape from apoptosis to become memory cells, acquisition of survival advantage in a timely manner is critical to memory development. To this end, IL-7 has been identified as a critical factor for survival of primary effector T cells and their progression to a memory stage.9,10 The roles of IL-2 may differ in the formation and in the sustaining T cell memory. On the one hand, IL-2 promotes CD4 T cell memory by enhancing expression of IL-7 receptor and increases the size of CD8 T cell memory population if present during T cell activation.11,12 On the other hand, IL-2 interferes with homeostatic proliferation of memory CD8 T cells,11 and the CD8 memory T cell pool is maintained by another growth factor, IL-15.13,14 The factors controlling survival and homeostasis of fully differentiated memory T cells seem to be different for CD4 and CD8 subsets and were recently reviewed in great detail.15–17 From a functional point of view, the magnitude of antigen-specific memory T cell responses is several-fold higher than that of naïve T cell subsets. In addition, some intrinsic properties of memory T cells make them much better responders compared with naïve T cells. As a result, re-exposure to the same antigen leads to faster and stronger recall responses and rapid pathogen clearance. In the current literature, memory T cells are often defined on the basis of the expression of cell surface molecules, activation markers, and tissue homing receptors (Figure 2). However, some traditionally used markers of naïve T cells, such as CD62L and higher molecular weight isoforms of CD45, can be re-expressed on late memory T cells.18–20 Furthermore, most markers are shared by different T cell subsets. For example, high levels of CD44 are expressed by both memory and effector T cells,21 whereas low expression of the IL-2R α chain CD25 is typical for both naïve and memory T cells.22,23 Therefore, the best approach to identify reliably a memory T cell population is to use a constellation of cell surface criteria in combination with functional characteristics. What are the features that make memory T cells distinct from their naïve counterparts? The obvious strength of memory T cell responses is in numbers. After the primary response has subsided, the host immune repertoire contains many more antigen-specific T cells than before antigen exposure.3,24 The increased frequency of memory T cells translates into amplified effector functions after re-infection with the same pathogen. T cell receptor (TCR) transgenic models made possible the precursor frequency-independent analysis of antigen-specific T cells at distinct stages of differentiation. This direct comparison demonstrated that memory T cells can be triggered by lower doses of antigen than naïve cells.25,26 Furthermore, whereas conventional co-stimulatory signals are essential for activation of naïve T cells, memory T cells are less dependent on CD28 and CD154 co-stimulation.18,27,28 As a result, memory T cells can recognize antigens presented by nonprofessional antigen-presenting cells, such as endothelial or parenchymal cells, outside of secondary lymphoid organs.27,29 Once reactivated, memory T cells readily differentiate into effector cells.30–33 Importantly, memory T cells are more resistant to activation-induced cell death, presumably because of their increased expression of antiapoptotic genes such as members of the Bcl family.1,17,34 Another distinct feature of memory T cells is their in vivo trafficking patterns. Whereas naïve T cells circulate predominantly through lymphoid compartments, memory T cells are present in both lymphoid and nonlymphoid tissues.2,3,35–37 On the basis of expression of homing receptors, tissue distribution, and functional properties, a dichotomy within memory T cell population has been proposed.3,24,38 In this model, some memory T cells express high levels of homing receptors CD62L and CCR7 and migrate preferentially to secondary lymphoid organs, where they may undergo homeostatic proliferation and give rise to a second wave of effector T cells ("central memory"). Another subset of memory T cells is CD62LloCCR7lo and is typically found in nonlymphoid tissues ("effector memory"), where it presumably performs pathogen surveillance. Importantly, these extra lymphoid memory T cells are capable of immediate effector functions independent of secondary lymphoid tissues.37,39,40 However, the division between central and effector memory T cells is not absolute. For example, some studies demonstrated that both central and effector memory CD8 T cells have similar potential to produce cytokines and exhibit cytotoxicity. In addition, expression of CD62L does not seem to correlate with preferential migration of memory CD4 T cells into lymphoid tissues.41–44 Nevertheless, the division into central and effector subsets is still widely used to describe the anatomic location of memory T cells. There is no doubt that memory T cells are well equipped and uniquely positioned to mount readily immune attacks on antigen re-encounter. The specific features of memory T cells in general apply to alloreactive memory T cells as well.45,46 The obvious difference is that unlike microbial infections, the sensitization to alloantigens is an extraordinary event. Nevertheless, the immune repertoire of many humans contains allospecific T cells with memory phenotype.47–49 Some individuals may develop such T cells through the direct alloantigen exposure during previous transplants, blood transfusions, and pregnancies. Another source of allospecific memory T cells is so-called heterologous immunity, whereby T cells, initially primed against infectious agents and environmental antigens, cross-react with allogeneic MHC molecules.50–53 In addition, recent studies in rodents suggested that memory T cells can be generated during homeostatic proliferation after lymphoablative therapies, and some proportion of these cells is potentially alloreactive.54,55 Thus, in humans who have a normal history of vaccinations and infections, donor-specific memory T cells are likely to be present even without previous alloantigen exposure. The unusual features of memory T cells that are developmentally programmed to enhance host protection against pathogens may become a liability in transplant settings. Upon exposure to alloantigens, both CD4 and CD8 memory T cells rapidly proliferate and differentiate into effector cells that are capable of inflicting damage to the graft tissue. Notably, this expansion and differentiation can occur outside of secondary lymphoid organs.39,40 It has also been proposed that memory T cells, particularly CD8 subset, can directly interact with donor vascular endothelium before the expansion phase. Such interactions lead to upregulation of adhesion molecules and chemokines by endothelial cells, thereby promoting infiltration of the recipient's immune cells into the graft.56–58 In addition, memory CD4 T cells provide help for naïve donor-reactive T cells and B cells.59,60 Even though this list of effector mechanisms may be incomplete, alloreactive memory T cells can certainly contribute to the graft rejection through multiple pathways (Figure 3). In this view, recipients with an immune repertoire enriched for donor-specific memory T cells should exhibit poor allograft survival. This assumption was corroborated decades ago by an observation that animals that have previously rejected an allograft reject the subsequent grafts from the same donor with accelerated kinetics (so-called "second set rejection").61,62 Importantly, some of these early experiments used model systems in which donor-reactive alloantibodies are not induced, and the accelerated rejection is mediated exclusively by T cells. Even though cause-and-effect relationships are harder to establish in human studies, a large body of data implicates memory T cells as a barrier to long-term allograft survival in humans. The method of immune monitoring in transplant patients evolved from the detection of T cells with memory phenotype in peripheral blood and biopsies to measuring donor-specific direct and indirect alloresponses by limiting dilution assay and IFN-γ or granzyme B enzyme-linked immunosorbent spot assays.47,48,63–67 Similar to the findings in animal models, the presence of detectable alloreactive T cell memory in humans correlates with poor graft outcome as evidenced by the incidence and severity of acute rejection episodes, inferior graft function, and the development of chronic rejection.46–49,68 MEMORY T CELLS AND EXISTING ANTIREJECTION THERAPIES Lymphoablation Because the presence of donor-specific T cell memory is often associated with poor allograft outcome, nonspecific deletion or at least reduction in numbers of memory T cells should enhance graft survival. The majority of peritransplantation lymphoablative strategies in clinic are based on the use of whole-body irradiation, antibody-mediated cell lysis, or immunotoxins. Although these approaches certainly have merit in promoting engraftment and lowering the risk for early acute rejection, recent studies in rodents, nonhuman primates, and human transplant patients have raised a number of concerns regarding the efficacy of lymphoablation on a memory T cell pool. First, there are several lines of evidence that, compared with naïve T cells, memory T cells are more difficult to deplete, presumably because of their survival advantages over naïve T cells.46 Analysis of T cell phenotypes in renal transplant patients revealed that memory CD4 T cells survive treatment with anti-CD52 mAb (Campath-1) or polyclonal rabbit anti-thymocyte globulin.69 Second, memory T cells that survive after lymphoablation are likely to have faster kinetics of homeostatic proliferation compared with naïve T cells.14 Third, homeostatic proliferation of naïve T cells in an "empty" host results in generation of T cells with a memory-like phenotype. Although the true functional identity of these cells remains to be determined, they certainly constitute a hurdle for the induction of transplantation tolerance, similar to the antigen-induced memory T cells.54,55,70,71 As a result, the recipient's immune repertoire will be enriched for memory T cells, including alloreactive memory T cells. The likelihood of this scenario was recently demonstrated by Pearl et al.,69 who reported that after nonspecific T cell depletion in renal allograft recipients, CD4 T cells with memory phenotype expand and predominate during acute rejection episodes. Conventional Immunosuppression Transplant patients rely on life-long immunosuppressive drugs to prevent T cell activation and graft loss. However, despite effective control of early rejection episodes with better immunosuppressive protocols, the long-term outcome of organ transplants has not improved much in the past few decades.72 Given the unique features of memory T cells and their apparent involvement in rejection, it is important to determine whether commonly used immunosuppressive drugs interfere with the development and effector functions of alloreactive memory T cells. The immunosuppressive drugs commonly used in clinic are designed specifically to target key checkpoints that are critical to activation and clonal expansion of naïve T cells (Table 1).73 Considering their unique features, memory T cells may be less susceptible to the effects of conventional immunosuppression. Indeed, memory T cells can develop and persist in transplant patients who receive immunosuppression therapy as primary, and booster immunizations are generally beneficial in these individuals.74–78 However, global immunosuppression can compromise T cell memory, as evidenced by lower vaccination efficiency in transplant patients compared with healthy control subjects and by the reactivation of latent viruses, such as cytomegalovirus, that are normally contained by antigen-specific memory T cells.74,79 Among other contributing factors, such as variability of immune responses between individuals, characteristics of particular virus strains, and differences in viral load, this paradox may be related in part to the heterogeneity of memory T cells. Different subsets of memory T cells at specific anatomic locations may have different susceptibility to immunosuppressive treatments. Alternatively, various immunosuppressive drugs may have distinct effects on generation, reactivation, or effector functions of memory T cells. Recent in vitro studies provide a compelling argument that most of the commonly used immunosuppressive drugs can inhibit the generation of memory CD8+ T cells from human naïve CD8+ T cells activated through TCR engagement with anti-CD3 antibody.80 Because the initial clonal burst is crucial to memory development, the effects of immunosuppressive drugs on memory T cell generation may be related to the suppression of early T cell proliferation. It is interesting that only calcineurin inhibitors—but not rapamycin, mycophenolic acid, 6-mercaptopurine, or hydrocortisone—blocked the release of proinflammatory cytokines by established CD8+ memory T cells after re-stimulation with anti-CD3 antibody.80 Similar results were observed during in vitro re-stimulation of CD4+ T cells with memory phenotype obtained from patients who were treated with anti-thymocyte globulin or alemtuzumab (Campath-1H). FK506 and, to a lesser degree, CsA inhibited proliferation and cytokine production by such postdepletion memory CD4+ T cells in response to staphylococcal enterotoxin B, whereas rapamycin, steroids, or deoxyspergualin had no effect.69 These findings are in agreement with the general belief that TCR stimulation is mandatory to the activation of memory T cells and that calcineurin inhibitors specifically target the TCR-triggered signaling events.73 As mentioned, not all memory responses are sensitive to calcineurin inhibitors, especially in vivo. Moreover, when present at low levels during T cell priming, CsA has been shown to promote the T cell commitment to a memory state.81 In addition, in models in which transplant rejection is mediated by donor-specific memory T cells (e.g., in presensitized recipients), calcineurin inhibitors have minimal effects in preventing the rejection response.82 In kidney transplant recipients who receive aggressive depletion therapy, rejection still occurs despite concurrent CsA treatment, even though postdepletion memory-like CD4+ T cells are sensitive to calcineurin inhibitors in vitro.69 These results indicate that in vitro measurement of memory T cell functions on the basis of the release of effector cytokines may not accurately portray their effector functions in vivo. In addition, it is likely that alloreactive memory T cells use other effector mechanisms besides cytokine release in damaging the transplants. Thus, multiparameter analysis of memory T cells should facilitate better understanding of factors controlling generation and effector functions of memory T cells. Conventional Co-Stimulatory Blockade Targeting co-stimulatory pathways to prolong allograft survival stems from the observation that optimal T cell activation, differentiation, and survival require engagement of several accessory cell surface molecules in addition to TCR signaling.83,84 The terms "conventional" and "classical" co-stimulation are generally used to describe the two most prominent and best studied co-stimulatory pathways: CD28/B7 and CD154/CD40. Multiple studies in rodents demonstrated that interference with either one or both of these pathways inhibits donor-specific T cell responses and facilitates prolonged graft survival or even tolerance.85 However, the same therapies were much less efficacious when applied to large animals and humans.86–88 Among many other differences between primates and laboratory rodents, the former are likely to harbor higher frequencies of memory T cells (including alloreactive memory T cells) than mice and rats housed in a pathogen-free environment. To test the hypothesis that memory T cells are responsible for co-stimulatory blockade–resistant rejection, several groups generated mice containing allospecific memory T cells and used them as recipients of skin or cardiac allografts. In some studies, T cell memory was induced through rejection of a previous allograft.59,82,89 Others have explored the role of alloreactive T cells primed through cross-reactive heterologous immunity to infections such as lymphocytic choriomeningitis virus or Leishmania major.50,52,90 In addition, the study by Wu et al.54 evaluated memory-like T cells generated during lymphopenia-induced proliferation. Various strategies were used to facilitate graft survival, including monotherapy with anti-CD154 mAb, anti-CD154 mAb in combination with either CTLA4-Ig or donor-specific cell transfusion, and even potent protocols based on bone marrow transplantation and mixed chimerism.50 Despite the differences in model systems, the conclusions reached from such studies are similar: (1) Co-stimulatory blockade–based approaches that are capable of prolonging graft survival in naïve animals fail to do so in the presence of donor-specific memory T cells; (2) both CD4 and CD8 memory T cells can precipitate co-stimulatory blockade–resistant allograft rejection, although the effector mechanisms leading to graft destruction may be distinct; and (3) the numbers of donor-specific memory T cells must reach a certain threshold to overcome the graft-prolonging effects of co-stimulatory blockade.50–52,82,89 Overall, these experiments validated the concern that alloreactive memory T cells in many transplant patients may interfere with the classical co-stimulatory blockade strategies in clinical settings. NEW APPROACHES TARGETING MEMORY T CELLS Alternative Co-Stimulation Molecules Cell surface structures with co-stimulatory properties are not confined to CD28 and CD154, and a number of new T cell co-stimulatory molecules have been recently described (referred to here as "novel" or "alternative" co-stimulatory molecules; Figure 4). Most of these alternative co-stimulatory molecules are expressed only upon T cell activation, suggesting a particularly important role in the effector/memory phases of T cell response rather than the initial T cell priming. Indeed, recent studies from several laboratories provide compelling evidence that memory T cells may rely on alternative co-stimulation for activation, survival, or effector functions. For example, the ICOS/ICOSL co-stimulatory pathway is involved in both CD4+ and CD8+ memory responses, the 4-1BB/4-1BBL pathway plays a particularly important role in CD8+ T cell recall responses in certain models, and the OX40/OX40L pathway may significantly contribute to memory CD4+ T cell responses (reviewed by Yamada et al.91). Accumulating data in transplantation implicate these alternative T cell co-stimulatory pathways in allograft rejection. In models of transplant rejection mediated primarily by memory T cells, prolonged allograft survival can be achieved by blocking the OX40/OX40L pathway in combination with CD28 blockade.92,93 In a different model of skin allograft rejection, blockade of CD27/CD70 co-stimulatory interactions limits clonal expansion and/or survival of donor-specific memory CD8 T cells, thereby leading to indefinite allograft acceptance.94 Finally, treatment with blocking anti-ICOS mAb synergizes with donor-specific transfusion plus anti-CD154 antibody therapy and prolongs murine cardiac allograft survival despite the presence of donor-reactive memory CD4 T cells (Zhang et al., unpublished observation). Taken together, these data show that memory T cell reactivation and effector functions require co-stimulation, but the co-stimulatory molecules that are involved in memory responses may differ from those that are critical for naïve T cell activation. This makes alternative co-stimulatory molecules an attractive therapeutic target for improving allograft outcome. However, despite recent progress in rodent studies, the clinical significance of targeting these alternative co-stimulatory molecules requires further investigations. Growth Factor Limitation It is believed that homeostasis of memory T cells is largely controlled by the common γc-dependent cytokines IL-7 and IL-15. Although these cytokines display overlapping functions, IL-7 is important for maintaining cell survival, whereas IL-15 is necessary to drive homeostatic proliferation of memory CD8+ T cells.9,10,13–15,17 In contrast, IL-7 seems to play a more important role than IL-15 for maintenance of CD4+ T cell memory.16 Because IL-7 and IL-15 are members of the common γc family, the question arises whether other γc cytokines have a similar function. The role of IL-2 in memory T cell generation and persistence is controversial. On the one hand, injection of IL-2 at the end of the primary immune response can prevent death of CD122hi CD8+ effector cells. On the other hand, the injection of neutralizing anti–IL-2 mAb induces a marked expansion of memory CD8+ T cells. The interpretation of such data is complicated because, in addition to IL-2 neutralization, anti–IL-2 antibody may either restrict IL-2–dependent CD25+CD4+ T regulatory cell (Treg), releasing memory T cells from the Treg-mediated suppression,95 or enhance the in vivo biologic activity of preexisting IL-2.96 The involvement of the growth factors in homeostasis of memory T cells opens an additional avenue for therapeutic intervention in transplant recipients. Indeed, blocking the common γc of cytokine receptors has been shown to inhibit memory T cell–mediated destruction of islet allografts.97 The potential caveat to this approach is that targeting the graft-destructive memory T cells may occur at the expense of losing protective T cell memory. Thus, the timing and duration of growth factor inhibition are key issues in blocking memory T cells in transplant models. Alteration of Memory Cell Trafficking Another emerging strategy to control memory T cells is to prevent their infiltration into the graft. In theory, this can be achieved by targeting chemokine receptors that are responsible for T cell recruitment into inflamed tissues such as CCR5 or CXCR3.98 However, the role of these receptors for memory T cell recruitment has not been directly tested. Recent studies in mice showed lymphoid sequestration of memory T cells with sphingosine 1–phosphate receptor 1 agonist FTY720.60 Similar to its effect on naïve T cells, FTY720 reversibly retained donor-reactive memory CD4 T cells in secondary lymphoid organs, thereby prolonging survival of cardiac allografts. Nonetheless, despite limited trafficking, memory CD4 T cells remained capable of providing help for the induction of antidonor CD8 T cell and alloantibody responses. These results indicate that although FTY720 may limit memory CD4 T cell trafficking and attenuate their direct contribution to allograft rejection, additional control of donor-reactive humoral immunity may be required. FUTURE CHALLENGES Recent advances in experimental transplantation demonstrate that T cell memory ultimately compromises the development of donor-specific unresponsiveness or tolerance. However, studies in other areas of immunology suggest that not all memory T cells are inherently resistant to tolerance induction. For example, memory CD4+ T cells that are reactive to a model antigen can be rendered tolerant by low-avidity engagement of their TCR.99 Furthermore, memory CD8+ T cells that are specific to an influenza hemagglutinin peptide can be readily tolerized in vivo by administration of high dosages of soluble peptide or by cross-presentation of the peptide by dendritic cells.99,100 Studies in this regard may provide key mechanistic insights into memory T cell tolerance. Another group of strategies involves targeting membrane ion channels or signaling molecules specific to activated or resting memory T cells. To this end, selective inhibition of the potassium channel Kv1.3 has been successfully used to treat memory T cell–mediated experimental autoimmune encephalomyelitis in rats.101,102 Finally, memory T cell–mediated rejection can be inhibited by the presence of CD4+Foxp3+ Treg in some transplant models,103 although the mechanism of Treg-mediated suppression remains poorly defined. Another important line of investigation may involve combinatorial approaches that have been successfully used in human transplant patients to establish tolerance through partial or full lymphoablation, bone marrow transplantation and co-stimulatory blockade.104 Even though each of the individual treatment components may fail to control preexisting memory T cells, it would be of utmost clinical importance to know the relationship among these therapeutic protocols and donor-reactive T cell memory. Finally, in the view of heterologous immunity, the requirement for donor-specific tolerance in allograft recipients needs to be reassessed. Ideally, preventing donor-reactive memory responses should spare previous immune history of the recipient and not interfere with the memory responses against pathogens against which the immune cells were originally raised. Similarly, although generating new allograft-reactive T cell memory is highly undesirable, the ability to develop immunologic memory against cross-reactive pathogens should not be undermined after transplantation. Keeping a balance between destructive and protective immunologic memory will be one of the most challenging future goals of transplant immunology. CONCLUSION Donor-specific tolerance is often referred to as the "Holy Grail" of transplant immunology. It is widely recognized that memory T cells are present in human transplant recipients and can circumvent the effects of immunosuppressive drugs and conventional co-stimulatory blockade treatments, thereby thwarting our efforts to achieve a tolerant state. On the basis of the current knowledge of immunobiology of memory T cells and their interactions with the existing graft-prolonging therapies, the possibility of creating a magic cure that will control all aspects of alloreactive memory without compromising protective immunity is highly unlikely. Therefore, the success of future tolerance-inducing therapies will demand rational development of combinatorial strategies that target generation, homeostasis, reactivation, and various effector functions of memory T cells. DISCLOSURES None.Figure 1: Kinetics of T cell response.Figure 2: Donor-reactive memory T cells contribute to allograft rejection through multiple pathways.Figure 3: Cell surface phenotype of naïve, effector, and memory T cells.Figure 4: Co-stimulatory molecules.Table 1: Commonly used immunosuppressive drugsaA.V. is supported by the National Institutes of Health. X.C.L. is supported by the National Institutes of Health and the Juvenile Diabetes Research Foundation International.