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This review details the role of memory T cells in physiologic and allospecific immunity, and summarizes the effects of immunosuppressive agents used to manipulate their function in the context of organ transplantation.
Memory T cells are lymphocytes with characteristics that are thought to promote anamnestic immune responses. They have a unique capacity to generate rapid effector functions upon secondary exposure to a pathogen, and this is achieved through truncated requirements for antigen presentation, reduced activation thresholds, and enhanced trafficking and adhesion mechanisms. In general, these same mechanisms also appear to evoke improved efficiency in mediating allograft rejection. The phenotype of these cells has been increasingly well defined and associated with a characteristic pattern of susceptibility to immunosuppressive agents. This knowledge is now being exploited in the development of immune therapeutic regimens to selectively mollify T memory cell effects.
A specific targeting of memory T cells has potential to prevent allograft rejection in a more precise manner that current means of immunosuppression. However, these benefits will be balanced by the reciprocal risk of susceptibility to recurrent infection.
Immunological memory, or the ability to generate increasingly efficient antigen-specific protective immune responses with subsequent antigenic exposures, is a fundamental hallmark of adaptive immunity in higher vertebrates. The effect of an initial exposure to an environmental antigen is imprinted on a host organism’s immune cell repertoire in such a way so as to increase the magnitude and rapidity of antigen clearance following re-exposure to that antigen. In particular, antigen-experienced T cells take on characteristics indicative of prior activation and give rise to a population of cells collective referred to a memory T cells (TMs). These cells mediate enhanced protection against invading pathogens and are thought to convey an evolutionary survival advantage. However, in the context of transplantation, the presence of cells with prejudiced reactivity against donor antigens increases the likelihood of immune mediated rejection such that adaptive immunity becomes counter-adaptive. While the precise pathways and cellular interactions that shape TM function rejection remain to be fully elucidated, emerging evidence suggests that these cells play a critical role in rejection. In this review we describe fundamental characteristics of TMs, discuss their role in allograft rejection, and relate their unique traits to existing and emerging immune therapeutic agents.
T cells emerge from the thymus with a naïve or non-activated phenotype characterized by relatively high T cell receptor (TCR) density and limited adhesion molecule expression. This phenotype persists until the cell becomes primed. Priming requires repetitive binding of a cell’s TCRs to major histocompatibility complex (MHC) molecules presenting the T cell’s cognate peptide antigen in the context of sufficient costimulatory signals, accessory molecules and adhesion molecules to induce cell division. Following several rounds of division, naïve T cells differentiate into an activated, effector T cell population that then mediates antigen elimination. Most of these cells undergo apoptosis in the conduct of their effector function, leading to population contraction with antigen elimination. However, some cells persist as a pool of long-lasting antigen-specific TMs. Two models have been suggested to describe the generation of TMs from naïve precursors: a linear progression model postulating that memory populations arise from a pool of previously primed effectors, and a parallel progression model stipulating that memory populations develop as a separate lineage alongside the population of short-lived effectors [1–3]. In addition, recent evidence suggests that the development of TMs may be influenced by antigen-specific T cell precursor frequency, the extent of antigenic stimulation, and/or the cytokine milieu present at the time of priming [4–6]. Antigen-specific T cell memory is maintained within the host by a basal homeostatic turnover that is thought to be supported independent of antigen by cytokines including IL-15 [7–11].
As compared to naïve T cells, TMs possess distinct phenotypic, functional, and homing properties (Figure 1) [9, 10]. They produce cytokines faster than naïve T cells, potentially from decreased activation thresholds , and possess direct cytolytic function in vivo following reencounter with antigen [13, 14]. They also express higher levels of CD2, CD11a, and CD44 compared with their naïve counterparts and in humans express the RO isoform of CD45 as opposed to the RA isoform [10, 15–18]. Numerous groups have demonstrated that altered expression of selectins, integrins, and chemokine receptors on TMs are likely responsible for their unique homing properties including residence in peripheral tissues, allowing them more immediate access to peripheral antigen including alloantigen following transplantation [19–27]. While TMs are heterogeneous, two well-described subsets exist within most antigen-specific memory populations. Central memory T cells (TCM; CCR7+ CD62Lhi) migrate primarily to secondary lymphoid tissues (e.g. lymph node and spleen) and are responsible for generating a burst of new effectors following recall. Effector-memory T cells (TEM; CCR7- CD62Llo) migrate to non-lymphoid tissues and provide immediate effector function at peripheral sites [19, 26, 27]. Whether these two populations derive from one another or have distinct origins is unclear, and there is evidence to support both paradigms [4, 28, 29].
In unsensitized transplant recipients, two unique mechanisms for the generation of donor-reactive TMs have been described. First, heterologous immunity is the phenomenon whereby previous exposures to environmental pathogens influence the course of future immune responses to seemingly unrelated antigens [30–32]. Once thought to be exquisitely specific for a single peptide:MHC complex, TCRs are now appreciated to possess inherent degeneracy with regard to their recognition of antigen, such that a T cell recognizing one antigen can also respond to other antigens, albeit with altered affinity. Heterologous alloimmunity thus results when a TM population primed by self-MHC presenting an environmental antigen generates cross-reactive TMs responsive to allo- or self-MHC presenting an allopeptide. Recently, dual receptor T cells have been described as being over-represented in alloreactive T cell populations, raising the possibility that if a dual receptor T cell is activated via one pathogen-specific TCR, it could later respond as a TM if its second, alloreactive TCR were to encounter donor antigen .
Donor-reactive TMs can also be generated via homeostatic proliferation, a process whereby transient lymphopenia caused by viral infection, or in the case of transplantation, therapeutic T cell depletion, induces the proliferation and differentiation of naïve T cells into cells with true phenotypic and functional characteristics of TMs as well as cells that appear to be TMs but fail to have robust effector functions [34–36]. Therefore, a proportion of the naïve alloreactive T cell pool is likely stochastically converted into alloreactive TMs throughout life, giving most patients some degree of allo-responsive memory, although they have not had exposure to alloantigen.
A growing body of evidence exists to suggest that TMs may play a critical role in inhibiting allograft acceptance. For example, TMs have recently been shown to have the ability to migrate into allograft tissues and secrete inflammatory cytokines well before the 4–6 days required for donor-specific priming in the spleen . Furthermore, tolerance achieved in cardiac allografts using anti-CD154 are rejected when alloreactive TMs are generated in recipients via sensitization with donor-type skin grafts [39, 40]. In addition, regimens to induce tolerance have been highly successful in rodents, but less so in non-human primates (NHPs). One possible explanation for this is that longer lived, socially housed animals such as NHPs have, in general, greater exposure to environmental pathogens as compared to rodents housed in specific-pathogen free facilities, and therefore possess an increased opportunity to generate a diverse repertoire of TMs [37, 38]. Evidence supporting this hypothesis includes experiments in rodents where tolerance was achievable in naïve animals treated with CTLA4-Ig, anti-CD40L mAb, busulfan and donor bone marrow, but not in animals infected with LCMV, vaccinia virus, and vesicular stomatitis virus. Even after anti-viral immune responses were allowed to resolve to memory, the mice resisted tolerance induction. Stimulation of splenocytes isolated with these recipients with donor tissue resulted in increased frequency of cytokine producing cells, thus implicating the virus-elicited allo-crossreactive TMs in the prevention of tolerance induction in this model .
Similar inhibition of tolerance has been demonstrated in rodents by donor-reactive TMs generated through homeostatic proliferation. In an experimental model where recipients were devoid of endogenous T cells, adoptive transfer of naïve syngeneic splenocytes resulted in the rapid homeostatic expansion of the transferred cells and acquisition of a memory phenotype. Importantly, when these animals were challenged with allogeneic skin grafts, the transplants were rejected despite treatment with costimulation blockade . These data provide direct experimental evidence that donor-reactive memory T cells can be generated by homeostatic mechanisms.
Given that TEM have unique properties, it is not surprising that they also have characteristic sensitivities to various immune therapeutics that distinguish the from naïve cells.
Many agents are used in clinical transplantation to intentional evoke global T cell depletion. These include polyclonal antibody preparations such as anti-thymocyte globulins (ATGs) and monoclonal antibodies specific for CD3 (muromonab) or CD52 (alemtuzumab). These agents mediate depletion through a variety of mechanisms [42, 43] and while the T cell depletion that occurs following treatment with these drugs is profound, emerging evidence suggests that TMs may have some degree of resistance to depletional therapies. For example, treatment of human transplant recipients with alemtuzumab resulted in >90% T cell depletion; however, the cells that remain have been shown to contain a predominance of CD4+ CD45RO+ CD62Llo TEMs. The origin of the TM predominance likely stems from a combination of TM resistance to antibody mediated depletion and resultant homeostatic activation of non-depleted cells. The proliferating population is likely derived from naïve cells as these cells, being less terminally differentiated, should be expected to have greater proliferative capacity. Based on these and other studies, it is accepted that T cell depletional therapy will increase the overall frequency of TMs, both due to the relative resistance of TMs as well as the likely conversion of naïve to TMs via homeostatic activation [44, 45, 46]. Recent studies have also suggested that alemtuzumab may decrease the requirement for immunosuppression by down-regulating the CD4+ TEM population associated with rejection . In a human cohort receiving alemtuzumab for kidney transplantation, CD8+ T cells recovered to their baseline population in 6 months, while the recovery of CD4+ T cells was delayed until approximately 15 months. Furthermore, the CD8+ T cells that repopulated the peripheral T cell compartment by homeostatic proliferation were of immunosenescent CD28−/CD8+ phenotype, which the investigators postulated may compete for space with or even may suppress the proliferation of CD4+ TEM cells . Alternatively, the absence of CD28 may render these cells resistant to costimulation blockade based therapies while remaining sensitive to calcineurin inhibitors. Importantly, post-depletional T cells are TEM skewed and clearly capable of mediating rejection despite exceptionally low numbers of cells without some adjuvant immunosuppressive therapy [48, 49]. Thus, while their long-term characteristics may be altered in ways that influence their sensitivity to immunosuppressants, they remain immunocompetent.
Costimulation is required for optimal activation of naïve antigen-specific T cells. The role of costimulation in the activation of TMs is dependent on both the type of TM and the costimulatory molecule. The CD28 pathway is one of the most important and well studied of the T cell costimulatory pathways. CD28 binds to its ligands CD80 or CD86, and propagates a positive costimulatory signal into the T cell. Agents have been developed to target this pathway. These include CTLA4-Ig, a fusion protein containing the extracellular domain of the CTLA-4 molecule, which associates tightly with CD80 and CD86 and therefore prevents CD28 ligation, and LEA29Y, a second-generation derivative of CTLA-4 Ig with increased affinity for CD86 . However, evidence from several studies exists to suggest that TMs are relatively independent of CD28-mediated costimulation for recall responses, thus refractory to the effects of CD28 pathway blockers. In rodents deficient in CD28, this costimulatory pathway is not necessary for the generation or recall of TMs [7, 51, 52]. Furthermore, tolerance induction protocols based on CTLA-4 Ig have been found to be ineffective in recipients that possess cross-reactive virus-elicited donor-reactive TMs. Rejection in these recipients was characterized by a lack of attenuation of donor-reactive CD8+ T cell responses; however, CD4+ donor-reactive TM responses were significantly reduced following this costimulation blockade based regimen . In another study, CTLA4-Ig was found to inhibit proliferation and expansion of memory CD4 T cells in response to peptide antigen challenge, with no effect on early activation . Taken together, these data suggest that the type and character of the donor-reactive TM population may influence its relative requirement for CD28-mediated costimulation upon recall. In humans and NHPs, which contain a large population of TMs in their peripheral T cell repertoires, CTLA4-Ig and LEA29Y have been shown to prolong graft survival, but not to the extent evident in mouse models. Interestingly, the TEM population in NHPs is largely void of CD28 and thus unlikely to be sensitive to agents targeting this pathway . In humans, many CD8+ TEMs are CD28− while CD4+ TEMs tend to retain CD28 expression. Although systematic measurement of the effect of CD28 pathway blockade in humans and NHPs has not been performed, it is clear that adjuvant immune modulatory agents are required to complement the effect of costimulation blockade [55, 56].
The CD154/CD40 costimulation pathway is also intimately involved in the activation of T cells. In rodent models, inhibition of CD154 with anti-CD154 blocking antibodies has shown to be effective in the prevention of rejection in pre-sensitized hosts, but ineffective in sensitized hosts, indicating that CD154 may not be necessary for the activation of memory T cells [39, 40, 57]. Initial CD154 blocking agents did not undergo development, and their brief investigation in human transplantation suggested a lack of efficacy . As such, other modes of blocking CD154, such as blocking CD40 have been attempted, and have shown promise in long-term kidney allograft tolerance in NHPs . Recent success using a fully human CD40 specific monoclonal antibody suggest that this approach has promise, but remains dependent on adjuvant therapy [60, 61]. However, the direct effects of blocking CD40 and CD154 on donor-reactive TMs have varied depending on the model used. Using mice infected with LCMV, investigators have demonstrated that the CD4 TM response was downregulated compared to the CD8 response when given anti-CD154 agents . In contrast, using a murine cardiac allograft model, the TM response was unchanged with anti-CD154 therapy . These findings suggest that the type and rate of antigen exposure, combined with the heterogeneity of the host TM population may demonstrate variable sensitivity to blockade of the CD154/CD40 pathway.
Another costimulatory molecule that has been shown to be important in the activation of memory T cells is the OX40 pathway, a member of the TNF receptor superfamily. This pathway has been implicated in synergistically driving the proliferation of TMs along with CD28-mediated costimulation . In murine models, TM mediated graft rejection was prevented when OX40 blockade was given combined with CD28/CD40 blockade, while grafts were rejected when OX40 blockade was given alone .
While there is a growing body of evidence to suggest that TMs are relatively resistant to costimulation blockade, other agents have been designed to specifically target and deplete the memory compartment based on unique enhanced expression of certain integrins, specifically CD11a and CD2 [66–68]. LFA-3-IgG1 fusion protein (Alefacept) binds to CD2 and has been shown to both prevent the activation of TMs and induce apoptosis, thereby decreasing the TM population [68, 69]. Alefacept is currently approved for clinical treatment of psoriasis, and its therapeutic effect is linked to its ability to deplete TM [69, 70]. Recently, alefacept has been shown to extend kidney allograft survival in NHPs when added to a CTLA4-Ig-based regimen . In this study, CD4+ and CD8+ TEM were shown to be specifically depleted by alefacept, and this appeared to be related to the increased expression of CD2, the target of alefacept, on TEM populations. Further in vitro studies of this model examined the effect of alefacept on alloreactive cytokine producing cells and demonstrated responding alloreactive CD4+ and CD8+ T cells also exhibited increased CD2 expression, thus providing an increased available target for the effects of alefacept. This study was the first to specifically target TMs with the prospective intent on neutralizing cells resistant to costimulation blockade .
The most commonly used immunosuppressants, the calcineurin inhibitors (CNIs) (cyclosporine A and tacrolimus), target TCR-mediated signaling. These agents prevent the nuclear translocation of NFAT that is required for gene transcription of IL-2, which is important for optimal expansion and survival of T cells. The reduced requirements of TMs for costimulation focus a greater reliance on the TCR, and as such, CNIs have been shown to be unique among the clinically available immunosuppressants in preventing TM proliferation and cytokine production . This has been suggested to be a predominant reason why CNIs have been such important contributors to the prevention of rejection in humans. However, while TCR signal inhibition prevents TM activation, it also may inhibit TM apoptosis and inhibit response contraction . Thus, the CNI approach appears to have exquisite ability to prevent rejection, but may also prevent regulation and TM contraction through apoptosis.
Inhibiting non-TCR signals in the T cell may have paradoxical influence of TM function and in fact enhance some aspects of immunity. A recent example involves sirolimus, a clinically used immunosuppressant known to attenuate downstream signaling events through mTOR, thereby preventing G1→S transition required for T cell proliferation and population expansion. Sirolimus has been used as a replacement for CNIs with one rationale being to specifically allow TCR signaling to foster activation induced cell death. While there are few studies assessing the direct effects of these agents on the memory cell population despite their common use, it has recently been demonstrated in an infection model that sirolimus alone acts to actually increase the frequency of antigen-specific T cells that differentiate into the memory lineage . In this study, mice given low-dose sirolimus following acute LCMV infection demonstrated enhanced quality and quantity of virus-specific TMs compared with untreated controls. Similarly, NHPs given sirolimus after modified vaccinia (MVA) vaccination exhibited an increased frequency of memory T cells.
Like sirolimus, the CD25-specific monoclonal antibodies daclizumab and basiliximab also interfere with the IL-2 pathway, by binding the high-affinity α chain of the IL-2 receptor (CD25) . Although CD25 is up-regulated in both naïve and TMs upon encounter with antigen, TMs up-regulate CD25 faster, and have been shown to subvert the blocking effects of anti-CD25 monoclonal antibodies through higher expression of the low affinity IL-2 receptor subunits (CD122 and CD132) that may support TM proliferation and activation by ligating IL-15 in addition to IL-2 . Thus, the extent to which TMs are relevant to an individual’s clinical outcome may significantly influence the relative success of CNI-sparing regimens in general, and sirolimus or anti-CD25-based regimens specifically.
Janus kinases (JAKs) are a family of cytoplasmic tyrosine kinases that regulate gene expression after signaling by cytokines. JAK3, expressed primarily in hematopoietic cells, is downstream of the common γ chain (CD132). Therefore blocking JAK3 has the potential to interrupt the signals of a broader array of cytokines used by TMs such as IL-2, IL-7, and IL-15. Indeed, JAK3 kinase inhibitors have been shown to prevent allograft rejection in both murine and NHP models [75, 76]. Further studies have proposed that prolonged graft survival observed following administration of JAK3 inhibitors could be due to blockade of signaling through the IL-7 and IL-15 receptors and thus target TM methods for by-passing a requirement for CD25. For instance, recent investigations have suggested that IL-7 is critical both to the generation of TMs following homeostatic proliferation, to aid the survival of TMs, and that IL-15 is required for the generation and maintenance of anti-viral CD8 TMs . Currently one such inhibitor of the JAK3 pathway (CP-690550) is in phase II clinical trials of renal transplantation . It has yet to be specifically investigated for its role in thwarting allospecific memory, but may offer a means of targeting TMs without TCR inhibition.
Given the role of TEMs in initiating effector function in the periphery and the role of TCMs in rapidly deriving new effectors, interruption of trafficking could have selectively potent effects of TM function. As discussed above, inhibition of LFA-3 interactions with CD2 appear to effectively target TMs and facilitate costimulation-based therapies. Several other agents currently under study for use in transplantation target similar processes. One such treatment is FTY720, which binds sphingosine-1-phosphate (S1P) receptor as an agonist and disables the SIP receptor from performing its function of allowing lymphocyte migration from the thymus and peripheral lymphoid tissues, effectively sequestering T cells in the lymph nodes and inhibiting them from trafficking to peripheral graft sites [26, 79, 80]. Blockade of CD11a also may hold promise as an agent that targets the trafficking of TMs. For example, recent work has demonstrated in murine models of transplantation that anti-LFA-1 monoclonal antibodies result in attenuation of donor-reactive memory recall responses and decreased T cell trafficking to the allograft following graft placement [81, 82]. The initial phase 2 testing of the anti-LFA-1 agent efalizumab in renal transplantation suggests that this agent indeed has potent inhibitory effects on protective memory in that its combination with a standard immunosuppressive regimen evoked a higher rate of EBV-associated malignancy . Thus, memory inhibition is likely to require cautious paring with other agents to avoid pathologic inhibition of protective immunity.
Memory T cells can pose a critical barrier to successful organ transplantation. The population of TMs of a given individual may vary based on the prior immune history of that patient, including the type and frequency of environmental exposures to pathogens. The alloreactivity of the TM population may also vary based on heterologous cross-reactivity or direct prior alloantigen exposure. Regardless, the importance of a thorough understanding of the mechanisms of donor-specific TM generation and the effects of immunosuppressive agents on these heterogeneous populations is becoming increasingly apparent. Closer attention to memory responses will likely enhance the potential to tailor immunomodulatory strategies a given transplant recipient.
ADK is supported by the National Institutes of Health (1U01AI079223-01A1), the Georgia Research Alliance, the McKelvey Foundation, and the Juvenile Diabetes Research Foundation. MLF is supported by the National Institutes of Health (AI 079409).
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