Dendritic cells: driving the differentiation programme of T cells ... - Nature

Immunology and Cell Biology (2008) 86, 333–342 & 2008 Australasian Society for Immunology Inc. All rights reserved 0818-9641/08 $30.00 www.nature.com/icb

REVIEW

Dendritic cells: driving the differentiation programme of T cells in viral infections Frederick Masson1, Adele M Mount1,2, Nicholas S Wilson1,3 and Gabrielle T Belz1 Protective immunity against viral pathogens depends on the generation and maintenance of a small population of memory CD8+ T cells. Successful memory cell generation begins with early interactions between naı¨ve T cell and dendritic cells (DCs) within the inflammatory milieu of the secondary lymphoid tissues. Recent insights into the role of different populations of DCs, and kinetics of antigen presentation, during viral infections have helped to understand how DCs can shape the immune response. Here, we review the recent progress that has been made towards defining how specific DC subsets drive effector CD8+ T-cell expansion and differentiation into memory cells. Further, we endeavour to examine how the molecular signals imparted by DCs coordinate to generate protective CD8+ T-cell immunity. Immunology and Cell Biology (2008) 86, 333–342; doi:10.1038/icb.2008.15; published online 18 March 2008 Keywords: T cells; immunity; memory; virus; antigen presentation

Protective immunity to viral infection depends on the development of long-term protective immunity in response to pathogen invasion. Following viral infection, antigen-presenting cells (APCs) drive the activation and differentiation of T cells to acquire effector functions. While effector cells can expand rapidly and exhibit potent cytolytic function, only a small fraction of activated T cells will go on to become long-lived memory cells that can rapidly respond to reinfection. Dendritic cells (DCs) are a rare population of APCs that are required to activate naı¨ve T cells.1–5 They are able to activate both CD8+ and CD4+ T cells by virtue of their capacity to present antigens on major histocompatibility complex (MHC) class I and II molecules, respectively, combined with their ability to impart costimulatory signals to T cells. While other APC populations, like macrophages and B cells, also express MHC class II and some costimulatory molecules, their role in stimulating naı¨ve T cells appears to be limited in in vivo situations.5 The interaction between T cells and DCs can trigger multiple T-cell fates that can lead to tolerance or autoimmunity in response to peripheral self-antigens, or alternately, protective immunity by generation of effector or memory T cells in response to foreign- or pathogen-derived antigens. The nature of the synapse between the DC and T cell will ultimately dictate the magnitude, polarization and heterogeneity of the cells responding to an immune challenge. Over the past decade, the complexity of DC–T-cell interactions has begun to be unravelled. The signals generated during the intimate contacts between these two cell types play a crucial role in shaping the differentiation process of T cells in viral infections. In this review, we will discuss the potential role of different DC populations in present-

ing viral antigens and their differential roles in driving the amplification of naı¨ve and memory CD8+ T cells within the inflammatory milieu of lymphoid tissues. We will also review the control of the kinetics of antigen presentation and how this affects T-cell expansion to ensure broadly protective and robust immunity in the face of viral challenge. DRIVING THE T-CELL RESPONSE: PROGRAMMING, SPECIALIZATION AND PLASTICITY Multiple factors sculpt T-cell activation and differentiation during an immune response. These include (i) the number of DCs displaying MHC–peptide complexes, (ii) the maturation status of the presenting DCs, (iii) the kinetics of antigen presentation, (iv) the type of DC presenting MHC–peptide complexes, (v) the number of naı¨ve T cells displaying a TCR that can recognize MHC–peptide complexes and (vi) the intrinsic proliferative capacity of naı¨ve T-cell populations. The summation of these parameters has led to the term ‘programming’ to describe the differentiation fate of the T cells once activated.6,7 Naı¨ve T cells responding to immunogenic peptides presented on MHC molecules undergo discrete developmental phases that result in the generation of a population of ‘memory’ T cells. The development of memory begins with the proliferation of a small number of naı¨ve antigen-specific T-cell clones (generally comprising fewer than 200 cells per specificity of the total naı¨ve T-cell repertoire8,9), paralleled by the progressive acquisition of effector function, and finally contraction in which 80–95% of expanded cells die by apoptosis. The initial expansion of naı¨ve T cells is thought to be pre-programmed such that they undergo differentiation into effector and memory T cells follow-

1Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia and 2Department of Medical Biology, The University of Melbourne, Melbourne, Victoria, Australia Correspondence: Dr GT Belz, Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia. E-mail: [email protected] 3Current address: Department of Molecular Oncology, Genentech Inc, I DNA Way, South San Francisco, CA 94080, USA. Received 20 February 2008; accepted 21 February 2008; published online 18 March 2008

Dendritic cells and viral infection F Masson et al 334

ing a very brief priming period (o24 h).6,10,11 T-cell programming occurs during the initial DC–T-cell interactions and orchestrates changes in phenotype (e.g. L-selectin or CD62L, homing molecule CD44, interleukin-7 receptor or CD127, killer cell lectin-like receptor G1 or KLRG1, CD27 and chemokine receptors such as CCR7), gene expression (e.g. cytokines, perforin and granzymes, transcriptional regulators such as zinc-finger transcriptional repressor Blimp1/Prdm1, inhibitor of differentiation/DNA-binding (Id) proteins, T-box family of transcription factors and GATA-binding proteins) and chromatin structure leading to the emergence of epigenetic signatures. This has focused our attention on the idea that a discrete programme preceding the first cell division integrates differences in signal strength and inflammatory mediators into the decision to activate or tolerize, specific naı¨ve T-cell clones.6,10 Furthermore, it has been proposed that the initial interactions between T cells and DCs may lead to a predefined apoptotic programme in activated T cells.12 This intrinsic programming implies that T cells do not require additional instruction to reach a terminally differentiated state. It also suggests that their fate is independent of further changes in their microenvironment, such as subsequent DC interactions or changes in the inflammatory cytokines. It is generally accepted that stable interactions between the DCs and T cells are necessary for efficient activation and proliferation. However, it remains to be determined whether only the initial DC–Tcell interaction is sufficient to programme a T-cell clone, or whether naı¨ve T cells can ‘accumulate’ information from multiple DC interactions.13–16 Once formed, memory T cells exhibit certain pluripotent qualities that allows their fate to be re-defined during a secondary or recall immune response.17 This contrasts with effector CD8+ T cells that seem to have little proliferative potential beyond the period of pathogen infection and appear to be terminally differentiated.18,19 Several models have been proposed to describe how T-cell differentiation occurs, but there is no clear consensus on a precise pathway taken by T cells (reviewed in Kaech and Wherry20). Recent insights into the transcriptional regulation of CD8+ T-cell differentiation have begun to shed light on the specific T-cell checkpoints that must be negotiated during the differentiation process.18,21–24 These and future studies will be important in providing more detailed information on how phenotypic and qualitative heterogeneity emerge within T-cell populations. Nevertheless, the initial T-cell–DC interactions are critical in defining the nature of the T-cell developmental programme after infection. These early events are followed by progressive differentiation of T cells and the acquisition of T-cell memory over several weeks. The interplay of antigenic stimuli, inflammatory mediators and specific transcriptional regulation of T-cell fate coordinates this differentiation programme. INFLAMMATION: A KEY TO RECRUITMENT AND AMPLIFICATION Inflammation is considered to be a key trigger in driving immune responses. How inflammatory stimuli impact on the activation state of the DCs, and influence antigen presentation and DC recruitment is multifaceted. Activation of DCs by pathogen-derived products, such as ligands of the Toll-like family of receptors, induces a transient increase in their phagocytic capacity followed by rapid downregulation.25,26 This feature likely allows DCs to focus their antigen-presenting capacity on antigens captured at the time of sensing a pathogen-derived signal. During this process, DCs show dramatic increases in the surface expression of MHC molecules and T-cell costimulatory molecules.27–31 This ‘maturation programme’ appears to provide DCs with superior T-cell stimulatory capacity, giving rise to the Immunology and Cell Biology

paradigm that ‘immunogenicity’ is intrinsically linked to DC ‘maturation’.27,32 Despite this, it appears that the in vivo situation is more complex as there is a general poor correlation between the prototypical DC maturation and immunogenicity.27 During an immune response, the lymphoid organs become centre stage for a captive audience of naı¨ve T-cell clones to interrogate DCs for antigenic information and costimulatory signals to start their differentiation programme.33 In addition to antigenic peptides presented via MHC, naı¨ve CD8+ T cells require signals that include CD4+ T-cell help, and inflammatory cytokines, such as IL-2, IL-12, IL-18, interferon (IFN)-a, IFN-b and IFN-g.34–38 DCs directly produce a number of these immunogenic cytokines at varying levels; however, other lymphoid immune cells can also contribute soluble factors that enhance T-cell activation.39–45 IL-12 has been shown to be a key cytokine produced by DCs that can promote effector and memory T-cell differentiation.46–49 Recently, this cytokine has been shown to modulate the expression of the transcription factor T-bet and its graded expression helped to define memory T-cell development.18 The intimate synapse between T cells and DCs is likely to enhance the activity of DC-produced cytokines in influencing T-cell differentiation. A less well-defined aspect of successful T-cell priming is the importance of activating and recruiting innate immune cells into lymphoid organs. During viral infection, the cellularity of lymph nodes increases dramatically, promoting interactions among DC, T cells and other lymphoid immune populations. DC migration from peripheral sites of pathogen invasion into the draining lymph node is a key step in the initiation of the adaptive response. Recently, it has been discovered that recruitment of DCs into the lymph node is enhanced through remodelling of the lymph node lymphatic network, a process that unexpectedly involves the presence of B cells50 together with macrophages that produce lymphangiogenic factors51 or might be involved in lymphatic vessel formation.52 Further research is continuing into how DCs migrate into and traffic within the lymphatics, which will help define how DC migration is fine-tuned during immune responses.53 DCs ARE NECESSARY FOR AMPLIFICATION OF BOTH NAI¨VE AND MEMORY CD8+ T-CELL RESPONSES It is now accepted that DCs are the predominant APC population driving the formation of primary T-cell immune responses to viral pathogens in vivo. Compelling evidence demonstrating their role came with the development of mice where the CD11c-expressing cells (predominantly DCs) could be selectively ablated during antigenic or pathogen challenge.5,54 Using this elegant model, DC depletion in a range of viral models has revealed their exclusivity in priming antigenspecific CD8+ T-cell immunity.3–5 This outcome is somewhat surprising given the trophism of certain viral pathogens to infect a broad range of cell types, including APC populations like macrophages—all of which are capable of presenting endogenous viral antigens on MHC class I when infected in vitro.55 In contrast to other APC populations, DCs appear to have a unique capacity to present exogenously captured antigens on MHC class I—a process referred to as cross-presentation.56 CD8+ T cells generally cluster around macrophages (as defined by sialoadhesin or CD169 expression), which have also been found to retain antigens for capture by follicular B cells.57 In contrast to DCs, macrophages are often permissive to viruses for replication, leading some researchers to suggest that a ‘goal’ of macrophages is to become infected.16 Such a strategy may provide an abundant source of viral proteins for DC to ingest for subsequent cross-presentation allowing enhanced CD8+ Tcell responses.16,58


Studies that have used techniques to ablate DCs appear to imply that non-DC APC populations are not required for MHC class I antigen presentation to naı¨ve or memory CD8+ T cells. Certainly acute models of localized viral infection such as influenza virus, herpes simplex virus (HSV)-1 and -2 and vaccinia virus, which have relatively restricted cellular trophisms, support an obligate requirement for DCs.2,59–61 However, a separate analysis of systemic viral infections with vaccinia virus, vesticular stomatitis virus and lymphocytic choriomeningitis virus, which infect a wide variety of cell types, suggests that antigen presentation by parenchymal cells can also drive the expansion of effector CD8+ T cells.62 This adds another layer of complexity, in which antigen presentation by non-DCs can affect the overall quality and quantity of T cells generated during the antiviral response. Strikingly, it appears that a contribution by nonDCs can only occur after an initial DC priming event has occurred, suggesting that effector, but perhaps not long-term memory, T cells may be generated by this form of activation. Historically, the cardinal features of memory CD8+ T cells are that they have a lower threshold for activation, rapid proliferative capacity and less stringent requirements for costimulatory molecules than naı¨ve T cells.63–65 Consistent with this notion, it has been proposed that memory cells could readily be activated by parenchymal cells that lack costimulatory molecules but can provide viral peptides via MHC class I molecules. This implies that memory T cells are far more promiscuous than naı¨ve T cells in responding to antigen presented by non-DCs. From a teleological perspective, such a schema would provide the most efficient mechanism for eliminating infected cells at the site of viral invasion. Unexpectedly, a detailed analysis of the requirements of memory CD8+ T cells by our group3 and Zammit et al.4 revealed that DCs were also required to amplify memory CD8+ T cells in recall responses to viral infections. In addition, a key component of this DC-mediated memory T-cell amplification was a dependence on peripherally situated DCs to bring viral antigens from site of infection to DCs in the lymph nodes for MHC class I presentation3,4 One potential caveat to the schema was that amplification of effector CD8+ T cells was detected in the lung, the site of viral invasion, following intranasal influenza infection. Therefore, it remains to be determined if effector CD8+ T-cells trafficking to the lung following DC priming in the lymph node can be subsequently amplified by non-DCs within the lung. Alternately, this observed proliferation could be mediated by DCs residing in organized ectopic lymphoid structures, such as broncho-alveolar lymphoid tissue, developing in the lung in response to inflammation.66 In other nonlymphoid tissues, such as experimental reactivation of HSV-1 virus in the kidney, both DCs and CD4+ T-cell help were required to activate recently stimulated CD8+ T cells.67 DC activation of CD8+ T cells in non-lymphoid tissues does not appear to occur as a front line of defence, perhaps because the number of DC is small in the face of high viral loads, but it is likely to be important in augmenting T-cell development initiated in the lymph node and perhaps in providing immunosurveillance for persistent infections. Tissue-mediated defence is critical to contain infection, therefore it remains to be determined whether parenchymal cells support short-term amplification of effector CD8+ T cells or can act more broadly in driving protective memory T-cell development. Collectively, these studies provide a novel conceptual viewpoint of memory T cells, suggesting that DC– T-cell interactions are not only required for naı¨ve T-cell activation but that they are also necessary for optimal expansion of memory CD8+ T cells.3,4,68 The above studies raise several important questions. Do different rules or mechanisms guide DC activation of memory T cells versus

naı¨ve T cells? If memory T cells respond more readily than naı¨ve T cells, can naı¨ve T cells compete with memory cells to participate in a secondary infection? Participation of naı¨ve T cells in a secondary infection may be important where an earlier viral infection generates a weakly cross-reactive or non-protective memory T-cell response. Such a scenario is likely to be relatively rare for different species of virus but may be more common for infections with human immunodeficiency virus, lymphocytic choriomeningitis virus, hepatitis C virus and influenza virus, where the virus may mutate and memory T cells may no longer be effective in limiting the infection.69–72 Clearly, mechanisms that allow naı¨ve T cells to respond in the face of aggressive preformed memory cells would be important to ensure a broad and effective immune response. One solution could be that amplification of naı¨ve and memory T cells might be achieved through distinct preferences by naı¨ve or memory T cells for different DC populations67,68,73 (Table 1). Such a concept seems plausible given that DC subsets express a variety of surface and signalling molecules, such as toll-like receptors, CD70, CD205 or ICAM-1, that could define the mechanistic basis for differential activation of naı¨ve and memory T cells.73–76 At least one way in which such molecules determine the type of T cell generated is by promoting more stable DC–T-cell interactions, as observed with DCs expressing ICAM-1.74 These prolonged interactions appear to influence efficient memory T-cell formation. DC SUBSETS Dendritic cells are a rare population of cells comprising fewer than 1% of lymphoid cells. They are armed with specialized antigen-processing machinery together with costimulatory molecules that enable them to efficiently endocytose and present endogenous and exogenous antigens to T cells and provide them with the necessary accessory signals to ensure their effective activation. They are located in both lymphoid and non-lymphoid sites and represent a heterogeneous population that can be functionally delineated by the expression of surface antigens. Broadly, DCs can be divided into two categories—conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs Conventional DCs can be divided into three major DC populations.77–79 Specifically, these are (i) the migratory DCs that originate in the peripheral tissues, such as the skin and mucosal tissues and migrate into the regional draining lymph nodes, (ii) resident DCs that spend their entire lifespan within the lymphoid organs and (iii) monocytederived DCs that differentiate in response to inflammatory signals in tissues. Migratory DCs. Migratory DCs can be further subdivided into additional populations that are either derived from precursor cells Table 1 Role of DC populations in differential amplification of naı¨ve and memory T cells DC subset

CD8a DC Migratory DC

CD8+ T cells

CD4+ T cells

Naı¨ve

Memory

Naı¨ve

+++ ++/+

+++

++/+ ++

Inflammatory monocyte/DC

+

ND

pDC

ND

Abbreviations: DC, dendritic cell; pDC, plasmacytoid dendritic cell; ND, not determined; , little presentation detected. To date, the role of different DC subsets has not been extensively examined for memory CD4+ T cells.

Immunology and Cell Biology


that develop in the tissues, or precursor cells that seed the periphery from the bone marrow.80 A key feature of migratory DCs is their capacity to transport antigens from peripheral tissues to the draining lymph nodes. Langerhans cells, which develop from a precursor population situated within the peripheral tissues, are historically regarded as the prototypical DC and are located in the squamous epithelium of the skin. DCs migrating from the subcutaneous tissue (dermis/submucosa/lamina propria and interstitial layers underlying the epithelium) are collectively known as interstitial DCs.80–83 Both populations of migratory DCs constitutively migrate to lymph nodes via the afferent lymphatics, providing an elegant mechanism to provide continual antigenic information about the peripheral tissue environment. This flow of information is important for both steadystate immune homoeostasis and during infectious immune challenges.84–86 Resident DCs. These cells comprise around half the total DCs present in lymph nodes, and are the only DC populations in the spleen and thymus as these lymphoid tissues lack afferent lymphatic vessels. Resident DCs can be divided into functionally discrete subsets based on the expression of the CD4 and CD8 surface makers.77–79,87 Using these two markers, three populations can be discerned in the spleen: the CD8a+CD4 (CD8a DCs), the CD8aCD4+ and the CD8aCD4 (hereafter collectively referred to as non-CD8a DCs) DCs. Similarly, three resident DC populations can be shown in the lymph node and thymus. However, CD4 is not typically used as a marker to discriminate DC as the lymph node and thymic DCs display a propensity to nonspecifically acquire the CD4 antigen from CD4+ T cells.77,87 Resident DCs develop from bone marrow precursors that continuously seed the lymphoid organs via the blood.88 Of the cDC subsets, a role for CD8a DCs in viral antigen presentation has been most clearly established.56,89,90 The comparative role of other DC subsets in activating naı¨ve CD4+ T cell has only recently begun to be explored.75,91 Monocyte-derived or inflammatory DCs. Monocytes circulate in the blood and conventionally give rise to macrophage populations within tissues. Despite this, under inflammatory conditions, monocytes have the potential to generate DCs.88,92,93 This DC population is characterized by the expression of the Ly6C antigen and can comprise one of the major infiltrates in murine skin infected with Leishmania major and in a form of leprosy.94 The importance of inflammatory DCs has only recently become apparent in viral control.67 pDCs Plasmacytoid DCs, or IFN-producing cells, are characterized by their ability to rapidly produce IFN-a following pathogen exposure.42,95–98 While pDCs appear to share developmental similarities to cDCs, they are functionally distinct. Moreover, pDCs represent an immature DC population, as characterized by their low surface levels of costimulatory molecules and MHC class II molecules.99 To date, on a per cell basis, pDCs have been shown to, at best, weakly stimulate naı¨ve T cells.100 Rather, it appears that the primary function of pDCs is to help limit viral progression through the production of large amounts of type I IFNs.96,98,101–104 DCs AND ANTIVIRAL IMMUNITY Viral infection hijacks the biosynthetic machinery of the host cell and allows the virus to rapidly synthesize its own proteins. In this way, the dominant cytosolic proteins generated are of viral origin, thereby providing a reservoir of viral peptides to incorporate into MHC Immunology and Cell Biology

molecules. When DCs detect pathogen-derived products, they rapidly begin to mature. Full DC maturation is phenotypically characterized by the rapid upregulation of surface MHC class I and II and costimulatory molecules. Functionally, mature DCs are defined by their acquired capacity to stimulate naı¨ve T cells, coordinated with their ability to provide prolonged ‘antigenic memory’ of antigens sampled at the time of activation.26 Many viruses exert effects on DCs that could significantly impair the ability of DCs to initiate the early antiviral response if they become infected. For example, some viruses, such as influenza virus, do not directly interfere with antigen presentation but are cytopathic and can rapidly induce apoptosis.55,105,106 In contrast, persistent viruses can encode an array of molecules that interfere with antigen-processing pathways, cell death and recognition of ‘self’.107–110 Despite this, in most viral infections, a highly efficient virus-specific T-cell response is initiated. This implies other mechanisms, such as cross-presentation, may ensure initiation of the immune response regardless of direct viral effects on DCs (Figure 1). While direct infection of DC has been difficult to ascertain in vivo, one important way of ensuring that a CD8+ T-cell response is mounted, particularly when infection itself may disable DCs or DCs are poorly infected, is through the process of cross-presentation.56,111 Cross-presentation is the ability to ingest exogenous antigens, such as those derived from virally-infected cells, and present captured peptides on MHC class I molecules to naı¨ve CD8+ T cells. This pathway appears to be unique to the CD8a DC subset, although under certain circumstances migratory DC populations may also exhibit this feature.2,112,113 In contrast, all DC subsets can present peptides via MHC class II to CD4+ T cells. The pathway of cross-presentation is now well accepted as a key pathway for antigen presentation during the antiviral response.56,58,114 Despite this, we know little about the molecular regulation of this pathway. It can be demonstrated that in vitrogenerated DCs readily capture antigens from live and dying cells.115 In vivo, both CD8a and non-CD8a DC subsets can phagocytose antigens but only the CD8a DC subset is able to efficiently cross-present cellassociated or soluble antigens.116–118 The underlying basis for this apparent DC subset-specific specialization has been controversial. However, several experimental models support the view that the dominant role of CD8a DCs in viral infection resides in their constitutive ability to effectively cross-present viral antigens.111,114,117–124 DC SUBSETS IN VIRAL INFECTIONS The appreciation that multiple DC subsets exist in mice and man has led several groups to propose that these individual DC populations play specific roles in maintaining tolerance and in generating immunity. Understanding the interplay of specific DC populations—encompassing cDCs, pDCs, inflammatory monocytes—is clearly essential to understand how the immune response is coordinated, controlled and could possibly be manipulated in vaccine strategies. The generation of CD8+ T cells that can cytolytically eliminate infected cells, often depends on the coordinate activation of ‘helper’ CD4+ T cells via DCs.125–127 Analysis of which DC subset(s) present viral antigens and model antigens has made it possible to define a general dichotomy in the role of DC subsets in priming T cells: that is, amplification of naı¨ve CD8+ T cells is largely driven by CD8a DCs, while activation of naı¨ve CD4+ T cells relies largely on non-CD8 DC subsets.59,61,75,91,128–133 Classically, DCs migrating from peripheral tissues to the lymph node have been thought to play a central role in activating naı¨ve T cells. However, careful analysis of the antigen presentation by individual DC subsets during viral infections has challenged this notion. For


Figure 1 The interplay between migratory and resident dendritic cells (DCs) promotes T-cell activation during a viral infection. (a) During a viral infection, peripheral tissue DCs resident in tissues may become infected but this does not interfere with their migration or antigen presentation capacity. These DCs are able to load endogenously generated viral antigens into their major histocompatibility (MHC) class I and II molecules for presentation to CD8+ and CD4+ T cells. DCs residing within the lymph node itself may become infected in situ by virus draining from the infected site via the afferent lymphatics. In general, the migratory DCs preferentially present their antigens on MHC class II molecules to CD4+ T cells, while the lymphoid-resident CD8a DCs present their antigens via the MHC class I direct or cross-presentation pathway to CD8+ T cells. (b) In some circumstances, the migratory DCs themselves may not become significantly infected, but are able to take up exogenous antigens from the infected epithelial cells by cross-presentation. This could occur when epithelial cells are heavily infected with virus, thereby promoting apoptosis or cell death of the infected cell; or at low levels of viral infection where epithelial cells remain viable sources of antigen for migratory DCs. They then migrate to the lymph node draining the site of infection and present the viral antigens to T cells. (c) In other situations, DCs that are virally infected or have taken up exogenous antigen migrate to the lymph node where they present antigens to T cells, or alternately, they themselves act as a source of exogenous viral antigens available to lymphoid-resident CD8a DCs for cross-presentation to CD8+ T cells. The resident DCs may acquire their antigen either from live, or dying migratory DCs, or from cellular fragments of other cells carried in the afferent lymph. The relative contribution of direct (endogenous antigens) and cross-presentation (exogenous antigens) is at this stage unknown.

example, cutaneous infection of mice with HSV-1 demonstrated a lack of involvement of Langerhans cells in the activation phase of the response.61,134 By contrast, influenza infection in the lung revealed a specific migratory DC population that could both transport antigen to the regional lymph node and present antigen to naı¨ve CD8+ T cells.2 These studies support the view that even when migratory DCs do not prime naı¨ve T cells they perform an essential function of transporting antigen from the periphery to the lymph node, which is key to initiating T-cell responses135 (Figure 1). In the vast majority of viral infectious models, the central driver of CD8+ T-cell activation was the resident CD8a DC2,60,61,90,91,128,134,136 (Table 1 and Figure 1). The capacity of CD8a DCs to present virusderived peptides on MHC class I molecules appears to be contingent on their expression of specialized molecular machinery.75,118 This

distinct role of CD8a DCs in activating CD8+ T-cell contrasts with the diversity of DC populations that appear to be proficient at presenting antigenic peptides via MHC class II to CD4+ T cells.129,130,137,138 Unlike the subset specialization observed for MHC class I presentation, analyses of presentation by DC populations using model antigens like hen egg lysosome or ovalbumin has implicated resident CD8a and non-CD8a DCs in presenting antigen via MHC class II. However, consistently the non-CD8a DC population appears to be quantitatively more effective at MHC class II presentation than CD8a DCs.26,137–139 MHC class II presentation by migratory DCs has also been reported in the pathogen setting. For example, in the parasitic infection Leishmaniasis, which is largely controlled by CD4+ T cells, antigen presentation involves both interstitial DCs and lymphoid-resident CD8a DCs.131,133 In contrast, only nonImmunology and Cell Biology


CD8a DCs activate CD4+ T cells following vaginal infection with HSV-2.59 These studies suggest that migratory DCs may play an important role in CD4+ T-cell priming but do not discount the involvement of resident CD8a DCs. Why is there no clear consensus about which DC subset(s) present viral or model antigens on MHC in different settings? It has been difficult to assess whether the apparent differential role of DCs in CD8+ and CD4+ T-cell priming reflects specific roles of DC subsets, or simply variations in experimental approaches. To gain greater insight into the division of labour between DC populations in pathogen infection, we developed an experimental system allowing both CD4+ and CD8+ T-cell responses to be concurrently monitored in the context of the same viral antigen. Careful analysis has shown that both CD8a and non-CD8a DCs could prime CD4+ T cells, while CD8a DCs were the main drivers of CD8+ T-cell amplification.91 These findings are supported by Dudziak et al.75 who analysed how different DC subsets handled the model antigen, ovalbumin. They mapped the capacity of different subsets of DC to prime T-cell populations with the differential expression of genes important in MHC class I and II processing pathways. These studies further support the notion that the intrinsic properties of DCs to handle antigens guide the activation and differentiation of T-cell subsets in vivo. CONTROLLING THE NUMBER OF T CELLS AMPLIFIED Following viral infection, the magnitude and quality of the effector T-cell response are regulated by (i) the number of APCs presenting MHC–peptides, (ii) the kinetics of antigen presentation and (iii) the size of the antigen-specific naı¨ve T-cell pool. While it is attractive to speculate that a larger effector response will lead to improved immune protection, uncontrolled effector T-cell proliferation could potentially induce immunopathology. Therefore, a mechanism is required to finetune T-cell expansion to limit the emergence of potentially pathogenic cells. Conventionally, it has been assumed that the dominant mechanism regulating uncontrolled T-cell expansion is the ability of effector cells to not only remove virally infected host cells but also to provide a ‘feedback’ loop to rapidly kill DCs and limit continued antigen presentation.140 Although this negative regulatory loop controlling antigen presentation has been the accepted paradigm, emerging data suggest that regulation of DCs is not so simple. During a peripheral infection, the initial event required to stimulate naı¨ve T cells is the migration of antigen-bearing DCs from the periphery into the draining lymph node.2,61,141 The precise number, and the time taken to reach the peak number of DCs carrying antigen, will depend on the initial dose of pathogen inoculum and the time required to reach peak replication and infection of cells.142,143 Longterm antigen presentation appears to be a feature of primary immune responses and may provide the conditions for T cells to have prolonged interactions with DCs necessary for full activation and differentiation.2,15,142,144 In addition, it may also allow the recruitment of naı¨ve T cells that enter the lymph node late in the response.143,145 The precise mechanism by which long-term antigen presentation (up to 2 weeks following acute infection) is maintained remains to be elucidated. Understanding this feature is particularly confounding, given the half-life of DCs in vivo is generally short (t1/2¼2–3 days) and migration of tissue-derived DCs from the site of infection to the draining lymph node is substantially downregulated within days of initial pathogen challenge despite high levels of virus.2,141,146 It is conceivable that very small numbers of antigen-loaded DCs continue to feed into the lymph nodes after the initial burst and this is sufficient to prolong the period of antigen presentation. Alternately, dividing DCs may transfer antigen to progeny daughter cells within the Immunology and Cell Biology

lymphoid tissues.147,148 This extended time frame of antigen presentation suggests that DCs are not simply eliminated in a feedback loop by effector T cells.140 DCs appear to be able to at least partly circumvent the effects of cytotoxic effector CD8+ T cells by inducing protective molecules such as the serpin, Spi-6, and possibly pro-survival molecules, that blunt the effects of effector T-cell–DC interactions.149 Loss of DCs from the lymph node in primary infections appears to be largely attributable to the natural turnover of DCs, although FasL expression on lymph node DCs has been implicated as a regulator of DC destruction.150 More recently, induction of expression of the Fas receptor was detected on DCs following microbial stimuli, suggesting that Fas-dependent elimination of APCs is also an important pathway for the destruction of DCs during immune responses.151 The antigen presentation kinetics observed in secondary infections contrasts markedly with the prolonged antigen presentation detected in primary infection. Following a secondary influenza infection, DC antigen presentation decays precipitously within a few days of the recall response, implying that DCs are destroyed by extrinsic mechanisms such as those utilized by cytotoxic T cells.140,147,152 These observations also suggest that memory T cells, which have heightened levels of granzymes and cytotoxicity, can rapidly turn on effector functions within the lymph node and eliminate APCs. Interestingly, the phenotype acquired by memory cells following secondary activation (secondary memory cells) is distinct from primary memory T cells.17,153 It appears that secondary and tertiary memory cells maintain a CD62 L (L-selectin) low phenotype characteristic of effector CD8+ cells for a protracted period of time. Whether they acquire and maintain this phenotype through interactions in the lymph node or by secondary encounter with the DCs at the effector site is unclear. Despite this, understanding how the stimulation history impacts on the quality and quantity of the formation of these populations would be particularly important for the design of prime-boost vaccine regimens. EMERGENCE OF T-CELL HETEROGENEITY: IMPACT OF DECREASING ANTIGEN PRESENTATION DURING INFECTION A striking feature of an immune response is the vast heterogeneity of T-cell populations that emerge in response to an infection. Although broadly naı¨ve T cells are CD62LhighCD44low effector cells are CD62LlowCD44high and memory T cells express high levels of both CD62 L and CD44 surface markers, a plethora of additional markers (including CD27, CD127 and KLRG-1) highlight that numerous subsets of T cells can be generated following infection. Models of T-cell differentiation assume that naı¨ve T cells exhibit relatively uniform differentiation potential. This feature of naı¨ve T cells has not been examined in detail. Indeed, it has been proposed that the heterogeneity generated within the T-cell population depends on precisely when a T cell arrives in the lymph node draining the site of infection, apparently reflecting differences in the stimulatory capacity of APCs as the infection wanes. Support for this model is drawn from recent analyses of primary influenza infection in which it is proposed that early arriving cells typically differentiate into effector cells, while latecomers are more likely to become memory T cells.145,154,155 This contrasts with the recruitment of memory cells in a secondary response where the early phase of the immune response appears critical in activating T cells, with little contribution from those cells arriving later.73 Elegant experiments involving the transfer of a single naı¨ve CD8+ T cell suggest that the timing of arrival in the lymph node might not play such an important role as these cells developed into diverse effector and memory T-cell subsets regardless of when transfer occurred.156 These experiments trace the spectrum of responding CD8+ T cells that


develop from an individual or small number of cells, rather than diverse differences in the stimulation history of progeny daughter cells. Processes such as asymmetric division may result in daughter cells acquiring different cytosolic and surface-based proteins, which may define the differentiation potential of cells.157 Differences in subsequent antigenic experience and exposure to inflammatory mediators regulating transcription factors would significantly impact on the cellfate map that emerges for each cell, whether it arrives early or late, in an infection. Importantly, these approaches are providing the tools necessary to start to unravel how descendents arise from a single T cell, giving rise to distinct classes of effector and memory T cells in response to antigenic challenge. CONCLUSIONS The importance of heterogeneity in the DC network and the distinct, non-overlapping functions that DC subsets perform are only beginning to be elucidated. Carefully dissecting which DC subset or subsets are presenting antigen during an immune challenge has begun to unravel the quintessential interactions between DCs and T cells that are required to generate protective T-cell immunity. Not only have these studies shed light on how T-cell immunity develops, they also provide mechanistic insight into how uncontrolled or erroneous T-cell responses can result in pathologies like autoimmunity. Despite this, we are still only beginning to understand the mechanisms that regulate the kinetics of antigen presentation and the stability and cycling kinetics of pathogen-derived antigens. Models that simultaneously allow antigen uptake and MHC class I and II presentation to be measured will be essential to further elucidate the role of DC subsets in immune challenges. In the case of viral infections, tags such as antibodies or fluorescently-labelled proteins that allow visualization of infected cells will be necessary to discriminate between DCs that are infected and those that have acquired virus or viral proteins from infected cells for cross-presentation to CD8+ T cells. Understanding precisely how different DC subsets drive different outcomes for naı¨ve or memory T cells, and how T cells accumulate antigen information during initial and subsequent DC–T-cell interactions helps to define the T-cell differentiation pattern. We are only beginning to grasp how different DC subsets impact on the development of T-cell immunity. It is clear that additional in vivo models will need to be developed to fully define the cellular, molecular and transcriptional regulators of Tcell differentiation into effector and memory cells. It is tantalizing to speculate that advances in this area of immunology will lead to the design of therapeutic T-cell-based vaccines and the development of novel treatments of T-cell-mediated autoimmune disorders.

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ACKNOWLEDGEMENTS Our work was supported by grants from the National Health and Medical Research Council (Australia), the Wellcome Trust Foundation (UK), the Howard Hughes Medical Institute (USA) and the Viertel Foundation (Australia) (to GTB), a Swiss National Science Foundation (FM) and a University of Melbourne Postgraduate Scholarship (AMM).

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Conflict of interest We declare no conflicting financial interest.

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