CD8 T-Cell Memory Differentiation during Acute and Chronic Viral Infections

Kalia V, Sarkar S, Ahmed R.

Publication Details

CD8 T-cell responses play an important role in protection against intracellular pathogens. Memory CD8 T cells mediate rapid clearance of pathogens upon secondary infection owing to their elevated frequency, ready localization to peripheral sites of infection and their ability to rapidly expand and mount effector functions. Such potent long-lasting protective memory CD8 T cells develop in acute infections where antigen is effectively cleared. In contrast, chronic infections with persistently high viral loads are characterized by CD8 T-cell dysfunction. In this chapter we present our current understanding of signals and mechanisms that regulate the development of functional long-lived memory CD8 T cells during acute infections. This is discussed in the context of proposed models of memory differentiation and compared with CD8 T-cell exhaustion and altered T-cell homeostasis, as occurs during persistent viral infections.

Introduction

Immunological memory is a cardinal feature of adaptive immunity, whereby the first encounter with a pathogen is imprinted indelibly into the immune system. Subsequent exposure to the same pathogen then results in accelerated, more robust immune responses that either prevent reinfection or significantly reduce the severity of clinical disease. Protective immune memory can persist for many years after initial antigenic exposure, even up to the lifetime of an individual. Both humoral and cellular immune responses comprise important arms of immunological memory and have evolved to perform distinct complementary effector functions of tackling free pathogens versus infected cells. Humoral immunity includes preexisting antibody, memory B-cells and long-lived plasma cells. The antibodies provide the first line of defense by neutralizing or opsonizing free extracellular pathogens. CD4 T cells further provide help for antibody production and the generation and maintenance of CD8 T-cell memory. Memory CD8 T cells, unlike antibodies, cannot recognize free pathogens, but instead identify infected cells and exert effector functions including direct cytotoxic effects on target cells and/or release of cytokines to inhibit growth or survival of the pathogen. Thus, the development of CD8 T-cell responses is necessary for the control of a variety of intracellular bacterial and viral infections and tumors.

In this chapter, we will focus on our current understanding of how protective CD8 T-cell responses are generated and maintained following two major types of infection, acute and chronic. Viral infection is largely divided into two types: (i) acute, where virus is eliminated; and (ii) chronic, where virus persists. Chronic infections are further classified into two broad categories: (i) latent infections, where virus is usually dormant, but occasional viral replication may occur during periodic episodes of reactivation; and (ii) persistent infections, where viral replication continues to maintain persistent viremia. Acute viral infections usually result in effective antiviral immune responses. In contrast, chronic persistent infections are typically associated with compromised CD8 T-cell function. In this chapter we will first focus on CD8 T-cell immunological memory following acute infections and discuss the underlying mechanisms of memory CD8 T-cell differentiation and maintenance when antigen is effectively cleared from the system. We will then discuss how the CD8 T-cell differentiation program is altered during chronic infections, where viral loads are maintained at persistently high levels. Due to the plethora of information pertinent to this topic, we will primarily describe broad themes of CD8 T-cell differentiation that have emerged from studies in the mouse model system, with appropriate references to other higher order model systems, which are discussed extensively elsewhere.

CD8 T-Cell Responses following Acute Infection

Primary infection results in the activation and proliferation of a subset of naïve CD8 T cells that have the capability of specifically responding to the invading pathogen. By some estimates, a mouse contains 50-200 naïve CD8 T cells specific for any one epitope.1 Following activation, naïve cells go through as many as 15-20 cell divisions and expand their numbers by up to 50,000 fold.2-4 At the population level, these expanded CD8 T cells express effector molecules such as perforin, granzymes and antiviral cytokines that aid in the elimination of infected host cells and are typically referred to as cytotoxic T-lymphocytes (CTL). After clearance of the pathogen, most pathogen-specific CD8 T cells die, but a small fraction (5-10%) of the cells survive long-term and form the memory pool of CD8 T cells (Fig. 1), which provides rapid protection to the host in case of reinfection with the pathogen (Table 1).5,6

Figure 1.. Antiviral CD8 T-cell responses.

Figure 1.

Antiviral CD8 T-cell responses. Illustration of the kinetics of CD8 T-cell response following infection of a mouse with a virulent pathogen such as Listeria monocytegenes, LCMV, VSV or vaccinia. Antigen-specific T cells clonally expand during the first (more...)

Table 1. Defining characteristics of memory T cells.

Table 1

Defining characteristics of memory T cells.

A typical primary CD8 T-cell response to acute infection is classically divided into three phases based on the kinetics of accumulation of antigen-specific T cells, as well as specific functional and phenotypic properties that are associated with them (Fig. 1): (i) the effector phase, when naïve CD8 T cells get primed, undergo dramatic expansion, differentiate into potent killer cells (cytotoxic T-lymphocytes, CTL) by acquiring a host of effector functions (such as: antiviral cytokine production, cytotoxicity, chemokine production and the ability to migrate to peripheral sites of infection to mediate pathogen clearance); (ii) the contraction phase, when majority of the effector CD8 T-cell population dies, with about 5-10% cells of the original burst size surviving to form the long-lived memory pool; (iii) the memory differentiation and maintenance phase, when surviving CD8 T cells progressively acquire hallmark memory characteristics and stable memory CD8 T-cell numbers are maintained via homeostatic proliferation for up to the lifetime of the mouse. Memory T cells differ from both naïve and effector T cells and show a range of differentiation states defined by phenotype (Table 2), function, anatomic localization and contribution to protection from reinfection. How and when is it decided which pathogen-specific CD8 T cells die after clearance of the pathogen (terminal effectors, TE) and which become long-lived memory is a question that is being vigorously studied and actively debated. In the context of this question, we will next discuss our current knowledge of memory CD8 T-cell differentiation as occurs following an acute infection.

Table 2. Markers that distinguish between naïve, effector and memory T cells.

Table 2

Markers that distinguish between naïve, effector and memory T cells.

Programming during the Expansion Phase

CD8 T-cell responses are initiated when a naïve cell encounters antigen. In vivo, T-cell activation involves the transmission of two distinct inductive signals from APCs to naïve precursors. Signal 1 is antigen-specific and delivered via stimulation of the T-cell antigen receptor (TCR) by peptide-MHC class I complexes on the APC surface. Signal 2 (CD28) is costimulatory and serves to amplify or modify signal 1 by lowering the threshold required for responsiveness. A common theme that has emerged over recent years is that of "programming" of T-cell responses.7 Several studies demonstrate that following initial antigenic instruction, the ensuing CD8 T-cell proliferation and effector cell differentiation events occur in a programmed fashion without further need for antigen.8-12 Moreover, the onset and kinetics of contraction and memory differentiation are also programmed during the early stages of an immune response.8,13 While these studies underscore the importance of instructive priming events in T-cell expansion, effector differentiation, contraction and memory generation, it is important to bear in mind that during an acute infection, other environmental cues are also collectively and progressively integrated during the course of CD8 T-cell immune responses to modulate CD8 T-cell differentiation.14

In addition to TCR and CD28 signals, recent data implicate the participation of a third signal in promoting strong CD8 T-cell expansion, development of effector functions and survival of the effector cells in vivo. This includes adjuvants, IL-12,15-20 or Type-I interferons.21,22 Using Type-I interferon receptor deficient CD8 T cells, it was observed that in the absence of direct Type-I interferon signals there was 99% reduction in CD8 T-cell expansion and memory generation. In recent years, it has been shown that CD8 T-cell expansion and cytotoxicity are also impaired in the absence of IL-21 signals.23 Whether IL-21 signals can replace the third signal provided by IL-12 or Type-I interferons, or if IL-21 (and possibly another cytokine) is needed as an independent (fourth?) signal remains to be determined. Other factors that are dynamically regulated during an acute infection and have been implicated in impacting CD8 T-cell differentiation include the strength and duration of antigenic stimulation, type of costimulatory signal (CD40, CD30, CD27, OX-40, 4-1BB, inducible costimulatory molecule ICOS), the complex cytokine milieu of inflammatory cytokines and growth factors, the type of antigen-presenting cells and interaction with other cell-types like CD4 T cells.24-26 Signals through the inhibitory receptors (cytotoxic T-lymphocyte antigen 4 CTLA-4, B and T-lymphocyte attenuator BTLA and programmed death 1 PD-1, killer lectin-like receptor KLRG-1, 2B4, etc.) are also proposed to act to control the extent of expansion and effector differentiation and prevent immunopathology by blunting the immune response.5,6,14

Thus, collective assimilation of these signals directs the acquisition of key effector properties such as production of antiviral cytokines (IFN-γ TNF-α), downregulation of lymphoid homing molecules (CD62L, CCR7) to enable peripheral tissue migration and cytolysis of infected target cells. This generation of potent CTLs is responsible for efficient pathogen clearance via direct effects on the pathogen and cytolysis of infected target cells.

Selective Survival of Memory Precursors during the Contraction Phase

After elimination of the pathogen, 90-95% of pathogen-specific CTL die by apoptosis during the contraction phase. Apoptosis primarily occurs by Fas and Bim pathways,27 leaving behind a pool of surviving pathogen-specific cells that differentiate into long-lived memory cells. Towards distinguishing pathogen-specific CD8 T cells that are destined to die (terminal effectors, TE) from those that will live long-term to comprise the memory pool (memory precursors, MP), several studies were conducted. Based on these, we now know that the pathogen-specific CTL population is heterogenous and is marked by differential expression of various cell surface (CD62L, IL-7Rα, KLRG-1) and intracellular (IL-2, serine protease inhibitor 6, Spi6) markers.28-31 Furthermore, differential expression of these markers is associated with diverse cell fates, in certain cases. For example, in acute infections, higher level of expression of IL-7Rα, Spi6 and IL-2 and lower expression of KLRG-1 by a subset of effector CD8 T cells correlates with selective survival and differentiation of this subset into long-lived memory cells.32-36 Such phenotypic distinction of effector CD8 T cells into two subsets: one that preferentially survives following antigen clearance (memory precursors) and one that will predominantly die (terminal effectors) has opened up new lines of experimental pursuit to dissect the signals that drive their selective generation. Moreover, this observation suggests that the ability to survive during the contraction phase and differentiate into long-lived memory cells is actively programmed during the priming and expansion phase, whereby enhanced survival potential does not result from a passive, stochastic survival of a sub-population of effectors due to limitation of growth factors in the face of largely increased T-cell numbers.

However, little is known about the cell intrinsic and cell extrinsic mechanisms that control contraction. While IL-2 and Spi6 expression directly correlates with memory precursors, their precise role in mediating selective survival of memory precursors during the contraction phase is unclear. It is proposed that by inhibiting granzyme activity, Spi6 protects effector CTL from damage during target cell killing. The availability of growth factors including IL-15, IL-7 and IL-2 have been proposed to play a crucial role during the contraction phase and memory T-cell maintenance.37-39 Rapid contraction is indeed observed in the absence of IL-15 signals, but the size of the resultant memory pool in IL-15-/- mice is similar to that in wild-type mice.40,41 This suggests that selective survival of memory precursors is independent of IL-15 signals. While higher IL-7Rα effectively distinguishes memory precursors during an acute infection, studies involving augmented delivery of IL-7 signals did not lead to enhanced memory generation,42-45 suggesting that IL-7 signals are not sufficient to drive the preferential survival of memory precursors. Additionally, studies showing similar contraction in the setting of an acute infection where CD8 T cells lacked IL-2Rα expression45,46 are suggestive of IL-2 independent survival of memory precursors during the contraction phase. Knowledge of various cell intrinsic and extrinsic factors that regulate contraction will aid in the manipulation of the kinetics and quantity of CD8 T-cell memory.

Memory CD8 T-Cell Differentiation and Heterogeneity

After antigen is cleared, terminal effector cells are eliminated during contraction, leaving behind memory precursors that give rise to the long-lived memory pool. Differentiation of memory CD8 T cells is a progressive process wherein key genotypic, phenotypic and functional properties are acquired over several weeks following antigen clearance.47-49 In the absence of antigen, virus-specific CD8 T cells return to a resting phenotype by downregulating the expression of certain effector molecules such as granzyme B, while progressively acquiring key memory properties of rapid proliferation upon exposure to antigen and antigen independent homeostatic proliferation in response to IL-7 and IL-15 cytokines. It is noteworthy that not all effector functions are downregulated during transition of effector cells into memory; memory CD8 T cells retain the ability to rapidly produce IFN-γ and TNF-α upon reexposure to antigen. Memory cells can also quickly reacquire cytotoxic activity upon secondary antigen encounter. Combined with elevated frequencies of virus-specific memory cells compared to naïve cells (upto 1000-fold higher frequencies), the ability to rapidly mount effector functions renders memory cells more efficacious than naïve CD8 T cells at combating infection (Table 1).

Processes involved in conversion from effector to memory stage are largely unknown. Two recent studies in Nature by Araki et al. and Pearce et al49a, 49b, demonstrate for the first time that metabolic changes in T cells may be crucial for T cell memory generation. Using the immunosuppressive drug, rapamycin, which inhibits mTOR signaling, Araki et al.49a showed that treating mice with rapamycin during the expansion phase led to enhanced generation of memory precursors and long-lived memory T cells. Furthermore, treatment during the contraction phase sped up the conversion of effector T cells to long-lived memory cells with superior recall ability. It was also shown that mTOR functioned in a T cell intrinsic manner to regulate memory cell differentiation. In a parallel study, Pearce et al49b, found compromised memory generation in TRAF-6 deficient mice. In microarray analyses they found that in the absence of TRAF-6, which is a negative regulator of T cell signaling, several metabolic pathways such as fatty acid oxidation were defective. Compromised memory T cell generation in TRAF-6 deficient mice could be reversed by treatment with anti-diabetic drug metformin, or by rapamycin. Both these drugs affect cellular metabolism; while metformin activates AMP kinase, an enzyme that inhibits mTOR activity, rapamycin directly inhibits mTOR. Typically, mTOR is activated by antigen-induced TCR signaling and growth factors, and regulates various cellular processes including cell growth and metabolism, autophagy, etc. While these studies suggest that an alteration of metabolic state via mTOR inhibition may be crucial for effector to memory conversion, how a change in metabolic state of a T cell could enhance memory T cell numbers and function remains to be determined. Moreover, manipulation of mTOR and key downstream signaling molecules holds promise for improving future vaccine strategies.

The memory CD8 T-cell compartment is characterized by significant heterogeneity with respect to surface protein expression, gene expression, effector functions, proliferative potential and contribution to protection from reinfection and trafficking. Two main cell-types involved in CD8 T-cell memory are effector memory (TEM) and (CD62L-/CCR7-) central memory (TCM) (CD62L+/CCR7+) cells.48,50,51 TCM cells are concentrated in secondary lymphoid tissues and have little or no immediate effector functions. Instead, they respond to antigen by rapidly dividing and differentiating into effector cells. Moreover, they possess stem cell like qualities of self-renewal in response to homeostatic cytokines including IL-7 and IL-15. TEM cells, on the other hand, can migrate to peripheral tissues and mount a more pronounced immediate cytolytic activity compared to TCM cells. TEM cells undergo modest proliferation upon antigenic stimulation, albeit to lower levels than TCM cells. Together, both TEM and TCM cells contribute to protective immunity depending on the nature and route of infection. Besides this well-defined TEM/TCM dichotomy of recirculating memory CD8 T cells, additional levels of complexity in memory CD8 T-cell phenotypes exist between distinct peripheral tissues and in different infectious models; for example, pathogen-specific lymphocytes residing in the gut, lung-airways or brain retain a distinguishing CD69 expression.6 Such functional, anatomic and phenotypic heterogeneity in the CD8 T-cell memory pool has important consequences for immunity and the factors that govern this cell fate decision are of major interest.

Molecular Basis of Optimal Memory Functions

Accelerated, more efficacious recall responses of memory cells result from a reprogramming of gene expression profile by epigenetic changes involving DNA methylation, histone modifications and reorganization of chromatin structure.52,53 Moreover, accelerated demethylation of the IFN-γ promoter by a putative enzymatic factor specifically active in memory cells may present an additional novel mechanism of differential gene expression.54 In addition to epigenetic changes, heritable programs of gene expression are also maintained by continued expression of certain transcription factors such as the tissue-specific T-box transcription factors T-bet and eomesodermin.55,56 Interestingly, mice with mutations of the genes encoding T-bet and eomesodermin exhibit defective effector cytotoxic programming, decreased expression of CD122 and are nearly devoid of IL-15 dependent memory CD8 T cells. These studies provide a molecular link between programming of effector and memory CD8 T cells and exemplify a framework in which transcription factors specifying lineage function can also specify responsiveness to growth signals. In addition, transcriptional repressor BCL6b has been shown to enhance magnitude of secondary response of memory CD8 T cells independent of primary responses.57 At a molecular level rapid proliferative responses of memory upon rechallenge are also attributed to modifications in TCR signal transduction machinery leading to more sensitive and rapid assimilation of stimulatory signals.58 Moreover, memory cells are precharged with several factors necessary for G1- to S-phase transitions,59,60 thereby suggesting that they may require a lower threshold of stimulation to enter cell cycle.

Models of Memory CD8 T-Cell Differentiation

The differentiation path followed by memory CD8 T cells is keenly studied. Whether memory cells arise as direct descendants of effector cells (linear differentiation model), or develop as a separate lineage from naïve cells (divergent differentiation model) has long been debated (Fig. 2). Technological advances are now making it possible to distinguish between these two possibilities more incisively, yet data supporting both models of memory T-cell generation exist. The conventional model of memory CD8 T-cell differentiation is the linear differentiation model, which proposes that memory cells are derived directly from effector cells. In fact, several studies have shown that T-cell activation and proliferation are tightly coupled to effector cell and eventually memory cell differentiation.47,61 The use of CRE/LOXP system in transgenic mice to indelibly 'mark' (via Cre-mediated recombination) pathogen-specific effector T cells that have activated a "signature" effector gene (granzyme B) promoter with a reporter molecule (alkaline phosphatase or a fluorescent molecule)62,63 showed that 'marked' effectors were maintained in the memory T-cell pool. Using this elegant system, it was found that effector cells that upregulated granzyme B expression form long-lived memory cells in both lymphoid and nonlymphoid compartments, indicating that both TCM and TEM cells are direct descendants of effector cells. However, this experimental system cannot distinguish between TE and MP cells and does not allow one to ask the question whether intrinsic differences between TE and MP cells, other than granzyme B expression, may be responsible for their diverse cell fates. With the ability to now phenotypically distinguish TE and MP cells, detailed protein expression, gene profiling and functional analyses of these subsets has demonstrated that memory precursors are remarkably similar to terminal effectors in their effector differentiation.35 This further supports the paradigm that memory T cells pass through an effector phase. However, whether transition through an effector stage is obligatory for memory generation is unclear from these studies.

Figure 2. . Models of memory cell differentiation.

Figure 2.

Models of memory cell differentiation. A simplistic illustration of the currently debated models of T-cell differentiation is presented. Model 1 represents the B-cell paradigm of divergent pathways followed by effector and memory T cells, such that following (more...)

The second model of memory differentiation proposes that memory T-cell development occurs in a nonlinear fashion without passing through a fully functional effector phase. Thus, asymmetric division after activation of a naïve T cell can lead to the formation of two distinct daughter cells with polarized terminal effector and memory cell fates, due to unequal partitioning of proteins during the first division.64 In certain cases (for e.g., activation with heat killed bacteria, or in vitro stimulation with high doses of IL-2 or IL-15 cytokines)65,66 memory T cells have been shown to develop without passing through an effector-cell stage. Depending on the priming milieu, it is proposed that antigen plus costimulation in the presence of an inflammatory milieu early during an infection (for e.g., IL-12, Type-I interferons and IL-21 signals) may favor differentiation of effector T cells, whereas antigen plus costimulation in the absence of inflammation (as antigen and infection are waning) may lead to memory T-cell differentiation.36,65-71 A recent study provides evidence that the quality of TCR signals can also determine effector versus memory development.72,73 By introducing point mutations in the transmembrane domain of TCR-β, which leads to poor polarization of the TCR to the immunological synapse without any evident effects on T-cell-APC interaction in vitro, the investigators found that the effector differentiation was unaltered, but pathogen-specific memory pool was largely abrogated. While this study clearly demonstrates that the quality of signal 1 from the TCR can direct transcriptional programs that are unique to effector versus memory development, it does not provide incontrovertible proof for the divergent model, as it is unclear whether memory precursors were generated in this system but failed to differentiate into functional memory cells. Also, whether the signaling defects that are associated with lack of memory generation in this study are recapitulated in vivo is unclear at present.

In summary, presently evidence in support of both linear and divergent models of memory differentiation exist and additional creative approaches are needed to resolve this issue. The key to understanding the differentiation path followed by memory T cells will be to identify true memory precursors at the earliest possible time during an immune response and to determine the signals required for their generation.

Mechanisms Regulating Memory CD8 T-Cell Heterogeneity

While developing complete models of memory T-cell differentiation, it is also important to consider the heterogeneous nature of memory T-cell pool. What is the source of memory T-cell heterogeneity? Is this continuum of differentiation states and/or lineages programmed via unique transcriptional regulation that is cell autonomous and can cell extrinsic factors be manipulated to dictate the final outcome of the differentiation process? As discussed above, early priming events strongly influence the number, location and functional properties (quality) of memory CD8 T cells. Although, antigen exposure is needed only briefly (20-24 hrs) to initiate T-cell development, the type of effector and memory CD8 T-cell responses eventually generated is further influenced by the duration and/or dose and the "context" of antigenic stimulation (for e.g., cytokine milieu, chemokines signals and costimulation, as determined by the nature and activation state of APCs). Distinct lymphoid environments have also been shown to program T cells to adopt different trafficking properties, thereby implicating unique environmental cues in possibly dictating memory outcome. Additionally, following emigration from secondary lymphoid tissue, inductive signals unique to distinct anatomical compartments may further regulate memory CD8 T-cell differentiation by providing a unique milieu of cytokines, costimulation, immune accessory cells and antigen persistence.

It is believed that the balance between effector and memory cells and the heterogeneity in memory population is directly related to the extent and frequency of TCR stimulation5,6,50,67,74-77 and the division history of the cells (likely conditioned by the dose of the antigen), such that functionally fit memory cells arise only under optimal stimulation conditions in which antigen load is effectively controlled. This is incorporated in the decreasing potential model of memory differentiation (Fig. 3, which proposes that the potential of effector CD8 T cells to differentiate into memory cells is progressively lost with increased antigenic stimulation.)74 Several studies have helped further refine this model to explain the generation of memory T-cell heterogeneity. It is proposed that whereas, suboptimal stimulation might lead to limited T-cell expansion and memory development, optimal TCR signal integration during activation of naïve cells leads to the generation of effector cells that have the potential to differentiate into memory cells.6 Whereas a short duration of antigenic stimulation favors TCM generation, longer stimulation promotes the generation of TEM and TE cells thereafter. In support of this, reducing antigen load, by antibiotics or by using higher CD8 T-cell precursor frequencies, was found to lead to a more rapid conversion to TCM phenotype (CD62L+).30,48,78-84 Since the expression pattern of CD62L on activated T cells appears to be regulated by antigenic stimulation (initial TCR stimulation results in rapid shedding of CD62L from cell-surface by proteolytic cleavage, but continued TCR stimulation leads to transcriptional silencing of CD62L encoding locus), it is interesting to speculate that differential modes of CD62L downregulation may relate to differential reexpression during memory development. The decreasing potential model of memory differentiation also explains CD8 T-cell dysfunction in chronic infections where antigen persists (Fig. 4). In this case effector T cells do not differentiate into durable memory cells and may survive in an antigen-dependent manner as dysfunctional cells (exhaustion), or may eventually die (deletion). The degree to which CD8 T cells become defective appears to correlate with antigen load and can range from partial loss of cytokine production to complete loss of cytolytic function and cytokine secretion as discussed below.

Figure 3.. The decreasing potential model of memory CD8 T-cell development.

Figure 3.

The decreasing potential model of memory CD8 T-cell development. Optimal antigenic stimulation triggers a developmental program of expansion and differentiation of naïve T cells into effectors, a fraction (5-10%) of which progressively differentiate (more...)

Figure 4.. CD8 T-cell differentiation during acute and chronic infections.

Figure 4.

CD8 T-cell differentiation during acute and chronic infections. Acute viral infections are characterized by clearance of virus and progressive differentiation of CD8 T cells into functional memory cells capable of IL-7 and IL-15 driven homeostatic proliferation (more...)

CD8 T-Cell Responses following Persistent Infection

CD8 T-cell differentiation pathway described above represents the paradigm for most acute infections. However, under conditions of chronic infections where antigen persists several aspects of a normal CD8 T-cell response are altered (Table 3).85 First, the hierarchy of epitope-specific CD8 T cells may be skewed such that subdominant specificities can predominate the virus-specific T-cell response, while immunodominant specificities may even be lost in certain cases.86,87 Second, the tissue distribution of virus-specific CD8 T cells may be altered, such that virus-specific cells preferentially localize in nonlymphoid tissues. This is likely driven by antigen localized in these compartments or by altered expression of homing molecules on virus-specific CD8 T cells. Third, chronic infections result in severely impaired T-cell function (functional exhaustion) and can also lead to physical elimination of responding T cells (deletion). Fourth, the molecular requirements for maintenance of virus-specific CD8 T cells during chronic infections are also altered.88 In the following sections, we will describe the altered CD8 T-cell responses observed during chronic infections and discuss our current understanding of the molecular basis of CD8 T-cell dysfunction.

Table 3. Comparison of CD8 T-cell differentiation in acute and chronic infections.

Table 3

Comparison of CD8 T-cell differentiation in acute and chronic infections.

Functional Exhaustion of CD8 T Cells during Chronic Infections

One of the key properties of memory CD8 T cells generated following acute infection is that they maintain the ability to reactivate antiviral effector functions upon antigenic stimulation. Exhaustion or loss of effector function was first reported in mice chronically infected with LCMV.86 During chronic LCMV infection, there is a hierarchical loss of the ability to perform effector functions, starting during the effector phase and becoming progressively more severe as the infection progresses.89 This exhaustion of effector functions occurs in a hierarchical manner. First, properties such as the ability to produce IL-2 and mount cytolysis and robust proliferation are lost at early stages of exhaustion. During this stage there may be loss of TNF-α production as well, which appears to be more resistant to exhaustion than IL-2. As infection progresses, IFN-γ production also begins to be compromised, ultimately leading to functionally inactive virus-specific cells that do not produce IL-2, TNF-α or IFN-γ and are incapable of ex vivo cytotoxicity. If antigen load in the form of MHC/peptide complexes presented in vivo is high, epitope-specific CD8 T cells can be physically deleted. During chronic LCMV infection, this is the case for two immunodomiant responses (Db/NP396 and Kb/GP34).87 This continuum of inactivation, with loss of function becoming progressively worse as either viral load or the duration of infection increases, is distinct from T-cell anergy, wherein acquisition of function is impaired to begin with following priming. Functional exhaustion is not limited to chronic LCMV infection, but is also observed in other animal models such as polyoma virus,90 Friend's leukemia virus,91 adenovirus,92 mouse hepatitis virus93 and SIV infection of macaques.94

Altered Memory CD8 T-Cell Homeostasis

A hallmark of memory CD8 T cells generated following acute infections is their ability to persist in the absence of antigen. Longevity of memory T cells, perhaps indefinite, is attributed to their stem cell-like quality of replenishing their numbers in the absence of antigen via homeostatic proliferation. This property of self-renewal in the absence of antigen distinguishes memory CD8 T cells from naïve and effector cells (Table 4). Clearly factors that enhance cell division (IL-15) or promote cell survival (IL-7) are important in maintaining the numbers of memory T cells in the absence of antigen. Bone marrow is the preferential homing site for memory T cells,95,96 where they proliferate more extensively than in secondary lymphoid organs in response to self-renewal signals, which are likely produced constitutively by specific cell-types within the bone marrow.

Table 4. T-cell homeostasis in acute and chronic infections.

Table 4

T-cell homeostasis in acute and chronic infections.

Contrary to acute infections, virus-specific CD8 T cells generated during chronic LCMV infection fail to persist when adoptively transferred into naïve mice.88,89,97-99 This defect in CD8 T-cell homeostasis correlates with decreased expression of CD127 and CD122, the receptors for homeostatic cytokines IL-7 and IL-15. Similar observations of loss in homeostatic proliferation in response to IL-15 was also reported for CD8 T cells generated in response to the murine γ-herpesvirus infection of mice100 and is likely not limited to mouse models of chronic viral infection. Although virus-specific CD8 T cells from chronically infected mice respond poorly to IL-7 and IL-15, they are maintained for long-periods in chronically infected mice. This maintenance is apparently dependent on the presence of infection since virus-specific cells decline when adoptively transferred into uninfected hosts. As opposed to the slow and steady homeostatic proliferation of antigen-independent memory cells generated following acute infections, CD8 T cells are maintained in chronic infections via extensive proliferation.99 This suggests an altered homeostatic regulation in persistent infections.

Mechanisms of CD8 T-Cell Exhaustion

As we continue to understand the underlying molecular causes of CD8 T-cell exhaustion, it is important to note that the functional programming of memory responses during persistent infection of mice is not hardwired during priming but is alterable and is impacted by continuous instruction from the antigenic environment. Through an elegant set of adoptive transfer experiments,101 it has been shown that removal of dysfunctional T cells from the infection and/or antigenic milieu bears the potential to rescue T-cell functionality. When dysfunctional CD8 T cells are transferred from a persistently infected mouse into a mouse that has cleared an acute infection, reversal of T-cell dysfunction is observed such that cells regain their ability to produce TNF-α and IL-2 and also upregulate the expression of survival molecules CD127 and Bcl-2. However, restoration of function was dependent on the extent of CD8 T-cell dysfunction, such that longer duration of persistent infection resulted in a progressive loss of functional recovery potential. This provides a basis for future therapeutic strategies to treat persistent viral infections.

Understanding the molecular basis of CD8 T-cell exhaustion is an area of intense research. CD8 T-cell exhaustion is marked by gene expression changes, such that the transcriptional profile of exhausted CD8 T cells differs from that of naïve CD8 T cells as well as functional effector and memory cells generated during an acute infection.102 Interestingly, exhausted CD8 T cells generated following chronic LCMV infection exhibit overexpression of several inhibitory receptors (PD-1, 2B4, CTLA-4, LAG-3, CD160, etc), which in certain cases is even nonredundant.103 Using the mouse model of chronic LCMV infection, programmed death-1 (PD-1), an inhibitory receptor in the CD28 superfamily, has been found to serve as an important negative regulator of T-cell function.104 Exhausted CD8 T cells express high levels of PD-1 compared to functional memory cells and blockade of PD-1/PD-L1 interactions results in enhanced T-cell function and viral control. HIV-1 and HCV-specific CD8 T cells also express high levels of PD-1 and in the case of HIV-1 patients, the level of PD-1 expression correlates directly with viral loads and inversely with CD4 T-cell counts.105-110 Furthermore, HIV-1 long-term nonprogressors expressed lower levels of PD-1 than progressors and PD-1 expression declined in viremic patients following initiation of HAART.108,111 In vitro blockade of the PD-1/PD-L1 pathway on human cells led to enhanced proliferation and improved function of HIV-specific CD4 and CD8 T cells as well as HCV-specific CD8 T cells.105-110 Whether different infections upregulate unique inhibitory receptors and whether different inhibitory receptors act cooperatively to downregulate T-cell responses in chronic infections are important questions that will guide development of therapeutic approaches specific to a particular pathogen. Additionally, the mechanisms by which CD8 T-cell function is restored by blockade of inhibitory receptors present another important question that will further our understanding of CD8 T-cell exhaustion.

IL-10 has also been recently implicated in limiting optimal T-cell responses during chronic infections.112-114 Mice lacking IL-10 or blockade of IL-10R led to efficient control of replication of chronic LCMV and development of functional T-cell responses.112,113 In contrast, control mice progressed to chronic infection. These results suggest that the IL-10/IL-10R pathway plays a key role in early events that determine whether an infection is rapidly cleared or becomes chronic with T-cell dysfunction. Additionally, Foxp3+ regulatory T cells (Tregs) can also influence the quality and potency of antiviral CD8 T cells directly by modulating CTL function and indirectly via production of immunoregulatory cytokines or inhibition of APC maturation. Given the potent ability of Tregs to suppress T-cell proliferation in vitro115,116 and their role in modulating the cytotoxicity of CD8 T cells in vivo,117 it is possible that different negative regulatory pathways such as IL-10, PD-1 and Tregs may regulate different effector T-cell properties during chronic infection.

Conclusion

In conclusion, recent years have seen major advances in the field of CD8 T-cell memory differentiation. With the molecular distinction of memory precursors and terminal effectors, we are now uniquely poised to ask important mechanistic questions pertaining to the generation of memory cells. For example, precisely when during an immune response are memory cells generated? What are the signals that regulate the generation and developmental program of memory cells? Is there a unique transcriptional signature comprising memory-specific genes? What are the precise mechanisms regulating T-cell exhaustion? Can dysfunctional CD8 T cells be rescued at any stage of exhaustion? Answers to these and many other exciting questions will help move the field forward towards a more rational design of vaccines that aim at inducing potent CD8 T-cell immunological memory to chronic viral infections and cancer.

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