Review

T cell mediated immunity to influenza: mechanisms of viral control Nicole L. La Gruta and Stephen J. Turner Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Victoria, 3010, Australia

Infection with influenza A virus (IAV) is a major cause of worldwide morbidity and mortality. Recent findings indicate that T cell immunity is key to limiting severity of disease arising from IAV infection, particularly in instances where antibody immunity is ineffective. As such, there is a need to understand better the mechanisms that mediate effective IAV-specific cellular immunity, especially given that T cell immunity must form an integral part of any vaccine designed to elicit crossreactive immunity against existing and new strains of influenza virus. Here, we review the current understanding of cellular immunity to IAV, highlighting recent findings that demonstrate important roles for both CD4+ and CD8+ T cell immunity in protection from IAV-mediated disease. T cells and immunity to IAV infection Worldwide, seasonal IAV infection is a major cause of morbidity and mortality, estimated to be responsible for 3–5 million cases of severe illness and 250 000–500 000 deaths worldwide per annum (WHO influenza centre website). IAV-specific immunity can be induced by vaccination that generates IAV-specific antibodies that limit or prevent IAV infection. However, the IAV vaccine needs to be reformulated on an annual basis because influenza viruses rapidly evolve, with new strains emerging that have lost or mutated the targets recognized by the preceding antibody response. Thus, an arms race ensues whereby vaccine-induced immune pressures select for new strains that are no longer recognized by vaccine induced IAV-specific antibodies, necessitating the production of updated IAV vaccines. CD8+ and CD4+ T cells have distinct but important roles in the control, and eventual clearance, of influenza virus infection [1]. Upon activation (Box 1), CD4+ T cells (or helper T cells) are thought to promote effective immunity primarily by providing the necessary secondary signals for optimal antibody responses, as well as producing antiviral and proinflammatory cytokines upon infection, although recent data indicate their role may extend beyond just cytokine production [2]. CD8+ T cells are often considered the ‘hit-men’ of the immune system because they locate and kill virus-infected cells in the body, thus limiting viral spread and contributing to the eventual Corresponding author: Turner, S.J. ([email protected]). Keywords: influenza A virus; CD8+ T cell; CD4+ T cell; immunological memory MHC class I MHC class II vaccination. 1471-4906/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2014.06.004

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clearance of infection. CD8+ T cells express a range of effector genes including granzymes and perforin, which mediate their signature cytotoxic capacity. Given that processing and presentation of viral peptide targets on the host cell surface can only occur after infection, unlike preformed antibody responses, pre-existing T cell immunity cannot prevent IAV infection per se; an issue that has, in the past, resulted in the dismissal of cellular immunity as a goal of effective vaccination. However, the utility of cellular immunity stems from the fact that unlike antibody responses, cellular immunity targets viral proteins that are more likely to be shared between different virus strains and subtypes [1,3], thereby offering a greater breadth of protection. Moreover, unlike for chronic viruses such as HIV or hepatitis B virus, where a primary goal of vaccination must be sterilizing immunity, for acute viruses such as IAV, the principal objective is the amelioration of infection-associated pathology until virus is cleared. Thus, it is widely acknowledged that a comprehensive vaccine against IAV must include the ability to elicit T cell immunity [4]. Here, we examine the current state of knowledge regarding IAVspecific T cell immunity and discuss how a greater understanding of factors that shape and promote IAV-specific cellular immunity will contribute to improved vaccine strategies capable of eliciting heterologous immunity. Targets of the T cell response during influenza infection The fact that IAV-specific memory T cells can target a broad range of peptides derived from proteins that are relatively conserved between different influenza strains and subtypes means that T cell immunity induced by one IAV strain has the potential to provide immunity against distinct IAV strains in the absence of neutralizing antibody (termed heterologous immunity). If we are to take full advantage of IAV-specific T cell immunity via development of a novel T cell based vaccine strategy, knowing the precise IAV peptide targets recognized by T cell immunity after infection will be key. Lee and colleagues [5] utilized ex vivo stimulation of human peripheral blood mononuclear cells (PBMCs) with overlapping peptides that spanned the whole IAV protein spectrum, to show that individuals who had not been exposed to the H5N1 virus (i.e., seronegative for the virus) had both CD4+ and CD8+ T cells that could recognize peptides derived from this highly pathogenic virus. This suggested that previous infection by seasonal influenza could generate T cell immunity that was capable of recognizing serologically unrelated IAV subtypes. Moreover, they also demonstrated that the major T cell targets were derived from the IAV

Review Box 1. Viral escape from cellular immunity: a paradox of acute infections DCs are a specialized subset of antigen-presenting cells that are key for alerting the host to infection and initiating T cell responses. DCs exist as two general populations; those located in peripheral tissues and those located within secondary lymphoid tissues such as the lymph nodes. At least within the murine system, DCs located within these locations can be further divided into distinct subsets, with each reported to have distinct roles in antigen presentation and priming of T cell responses [58]. DCs within the lymph nodes can be broadly separated into the CD11b+ CD8a or CD11b CD8a+ subsets, with the CD8a+ DCs being most efficient at presenting influenza antigens and activating naı¨ve, virus-specific T cells after infection [59]. Within peripheral tissues, DCs can be divided into CD103+ CD11b or CD103 CD11b+ DCs, with the CD103+ DCs capable of migrating most efficiently to the draining lymph nodes after IAV infection [60]. Importantly, in the context of IAV infection, both lungderived and lymph-node-derived DCs appear to play roles in the induction of T cell immunity to influenza [58,61]. For CD8+ T cell responses, both the CD8a+ CD11b (LN) and CD103+ CD11b (lung) DC subsets are important for priming [60–62]. Although priming of CD8+ T cell responses is essentially limited to CD8a+ (LN) and/or CD103+ (lung) derived DCs, a broader range of DC subsets are capable of presenting antigen to CD4+ T cells [63]. However, in the context of IAV infection, the migratory CD11b CD103+ DCs derived from the lung are capable of activating naı¨ve CD4+ T cell responses [62,64]. Although activated B cells can also present antigen to CD4+ T cells, the key purpose of this T–B interaction is the promotion of effective antibody responses, rather than initial priming of naı¨ve CD4+ T cell responses. Finally, although there is little information regarding the role of human DC subsets in priming IAV-specific T cell responses, subsets analogous to the mouse CD8a and CD103+ DC subsets have been identified in humans and are most efficient at priming naı¨ve CD8+ T cell responses [65,66]. Thus, determining whether these same DC subsets are key players in the initiation of IAV-specific T cell responses in humans after infection/vaccination is of great interest and relevance to T cell based vaccine design.

matrix protein (M)1 and nucleoprotein (NP). Studies that have also used the approach of ex vivo stimulation of human PBMCs with overlapping IAV peptides have since confirmed that peptides from the M1 and NP are the major targets for T cell immunity [6–8]. Thus, pre-existing IAV-specific T cell immunity induced by infection with one strain may have the capacity (through cross-reactivity with conserved epitopes from a limited number of viral proteins) to limit infection by different strains or subtypes, particularly in the absence of any neutralizing antibody responses. Moreover, novel T cell based vaccine strategies would only need to include a limited number of protein targets to ensure broad-based immunity and likely make vaccine formulation a more straightforward exercise than having to use something like whole inactivated IAV. In terms of precise peptide targets recognized by human memory T cells, there remains much to be learned. For example, a robust CD8+ T cell response against a peptide derived from the IAV matrix protein (M1, residues 58–66; M158–66) can be readily detected within HLA-A2+ individuals [9]. Given the repeated demonstration of this response across HLA-A2+ individuals, it is considered a dominant response. Are other such dominant responses prevalent within individuals with other MHC haplotypes, and what is the full repertoire of IAV peptides eliciting a CD8+ T cell response? Chen and colleagues addressed these questions by taking a systematic approach wherein peripheral human T lymphocytes from several healthy donors were cocultured with live IAV as

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an antigenic stimulus whereby infected PBMCs self presented IAV antigens to pre-existing memory T cells. They then screened T cell reactivity against individual influenza proteins, enabling them to narrow the candidate pool, so that they could then define the minimal peptide targets after in vitro stimulation with overlapping peptides [10]. As previously reported [11], the dominant CD8+ T cell responses from different individuals targeted the conserved matrix (M) and nucleoprotein (NP) proteins. Importantly, using this systematic approach, new peptide targets presented by an array of distinct HLA molecules were identified and found to be at times more prominent than the benchmark HLA-A2-M1 epitope. Interestingly, it was noted that at least three of the newly identified IAV-peptide targets identified were longer than typical MHC class I binding peptides, and hence would not have been identified using established epitope prediction algorithms [10]. These results suggest that a more systematic and direct approach, such as that outlined in the study by Chen and colleagues [10], is needed if peptide targets for a range of diverse MHC alleles are to be identified for possible inclusion in T cell based vaccine approaches. Although the use of specific peptides in vaccines is a direct way of targeting T cell response, the application of targeted peptide based vaccines will likely be limited by the fact that potential epitope targets will be missed, as well as by the difficulty of ensuring adequate coverage across numerous HLA subtypes. Moreover, there is an increasing appreciation that IAV-specific T cell immunity may in fact drive immune escape in targeted T cell epitopes (Box 2). Alternatively, vaccine strategies that incorporate whole protein antigens, rather than peptides, would ensure adequate antigenic coverage across different HLA types. Aside from ensuring broad T cell immunity, another advantage of whole protein vaccination against IAV would be the potential to maximize fully humoral responses either against conserved proteins, such as MP or NP [12], or conserved protein structures such as the stem region of the hemagglutinin (HA) [13]; both of which have shown promise in protection from heterologous challenge. It is important to note that whole protein vaccine strategies do not circumvent the need for high-resolution epitope identification. A major driver is the increasing need to be able to track antigen-specific T cell responses after vaccination by use of soluble pMHC tetramer reagents [14]. The identification of new IAV-specific pMHC complexes, combined with recent advances in flow cytometric techniques that enable multiple specificities/parameters to be measured from a single blood sample [15,16], means we are potentially at the beginning of an era that will provide an unprecedented level of information about the kinetics, function and persistence of IAV-specific T cell immunity. Role of T cell immunity against IAV infection: lessons from humans Although IAV-specific CD4+ and CD8+ T cells are readily identifiable in humans [5,17–20], their precise role in controlling IAV infection is unclear. A retrospective analysis demonstrated that prior symptomatic A(H1N1) infection was associated with increased protection from the 1957 A(H2N2) pandemic virus in adults but not children, suggesting an accumulation of heterologous immunity 397

Review Box 2. Viral escape from cellular immunity: a paradox of acute infections The acute nature of IAV infection is not typically considered to be a strong driver of mutational escape within targeted T cell epitopes because the duration of virus infection and the subsequent immune response is thought to be insufficient for the outgrowth of viral escape mutants. However, recent studies that examined the evolution of amino acid sequences within the relatively conserved NP from an array of different IAV isolates taken over the past 40 years reported a high frequency of mutation. It was subsequently shown that these amino acid changes within known NP-derived CD8+ T cell epitopes resulted in loss of CD8+ T cell recognition of IAV-infected cells [67]. Importantly, amino acid variation is not limited to a single CD8+ T cell epitope, with variations identified within a number of other known NP-derived CD8+ T cell peptides that bind numerous different human MHC class molecules [67–70]. In a C57BL/6J mouse model of IAV infection, it was recently demonstrated that viral variants containing mutations at the MHC class I position 5 anchor residue of the DbNP366 epitope emerged coincidently with the peak of the DbNP366-specific CD8+ T cell response. When these viral variants were used to inoculate MHCmismatched Balb/c mice (and were thereby relieved of T cell immune pressure) the variant IAVs reverted back to the wild type NP sequence. These data demonstrate that even a primary CD8+ T cell response to IAV has the capacity to exert sufficient immune pressure to select escape variants. Although not as extensively studied, there is also some evidence that mutation within CD4+ T cell epitopes can result in escape from IAV-specific CD4+ T cell recognition. Given that T cell epitopes tend to be more conserved between different IAV strains than those recognized by antibody immunity, these data emphasize the need to select carefully potential antigens for inclusion in any future vaccine strategy, so that although a range of T cell reactivates are included, it might be necessary to exclude antigens that, from an evolutionary perspective, appear to be targets of immune T cell selection.

with age [21]. Although the mechanism is unknown, the fact that protection was mediated in the absence of any crossreactive antibody responses (because it was a pandemic event), strongly suggests a key role for T cell mediated protection [21]. The earliest indirect evidence in humans that CD8+ T cell immunity is important for protection against influenzamediated illness came from a challenge study in which volunteers were intranasally infected with a live, attenuated IAV, and viral shedding was measured in clinical samples [20]. Decreased viral shedding was associated with a concomitant increase in IAV-specific CD8+ T cell responses in volunteers who lacked neutralizing, strain-specific antibodies. These findings implied that IAV-specific CD8+ T cell responses could effectively limit primary IAV infection. The recent 2009 H1N1 pandemic (2009 pdmH1N1) provided a unique opportunity to determine whether pre-existing CD8+ T cell immunity provides protection from heterologous IAV infection. In one particular study [7], a cohort of individuals that lacked pre-existing antibodies to the 2009 H1N1 IAV pandemic were followed during pandemic cycles to determine whether pre-existing circulating memory T cell populations correlated with less severe disease outcomes after 2009 pdmH1N1 infection. Individuals who developed mild or no symptoms after 2009 pdmH1N1 influenza infection were found to have higher circulating levels of pre-existing IAV-specific CD8+ effector memory T cells (defined by CD45RA+ CCR7 expression). Functionally, these effector memory CD8+ T cells exhibited 398

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the capacity to produce interferon (IFN)-g and were capable of direct cytotoxicity against infected target cells. Interestingly, there was no significant correlation between symptom severity (symptoms noted were runny nose, fever, and sore throat) and the presence of pre-existing IAVspecific CD4+ T cells. Thus, in the setting of natural infection, when antibody immunity is lacking, elevated influenza-specific CD8+ T cell immunity appears to help limit both disease symptoms and the spread of the virus. As noted earlier, both CD4+ and CD8+ T cell responses can target relatively conserved internal influenza proteins, implying that CD4+ T cells may have the potential to provide IAV-specific heterologous immunity [5]. To test this directly, Wilkinson and colleagues [8] challenged volunteers with either a wild type H1N1 or a H3N2 seasonal IAV strain, and then measured clinical symptoms and viral shedding over the course of infection. All volunteers were seronegative for their respective challenge strain, therefore, the levels of pre-existing memory T cell responses were measured to determine their relation with disease progression. Although no significant correlation was found between symptom severity and presence of pre-existing IAV-specific CD4+ T cells [7], Wilkinson et al. found a significant inverse correlation between disease severity and pre-existing levels of circulating IAVspecific CD4+ cells. It is not clear why these two recently published IAV challenge studies came to different conclusions about the respective roles of IAV-specific CD8+ and CD4+ T cells. It might be the fact that Sridhar and colleagues examined a natural experiment, whereas Wilkinson et al. analyzed heterologous IAV-specific T cell responses in the context of an experimental challenge of human volunteers. Alternatively, analysis of T cell populations found within the respiratory tract during IAV infection may provide stronger correlates of immunity. Although more studies like these are needed before any definitive answer, these findings nevertheless provide strong impetus for further developing an understanding of both CD8+ and CD4+ T cell effector functions and their role in IAV control. Mechanisms of CD8+ T cell dependent control of IAV infection Signature virus-specific CD8+ T cell effector functions include the ability to produce a variety of cytotoxic molecules such as perforin (Pfp) and granzymes (gzm), as well as being able to secrete a variety of potent inflammatory cytokines such as tumor necrosis factor (TNF)a and IFN-g (Figure 1). It is natural to expect that perhaps many, if not all, of these effector functions contribute to the limiting and eventual clearance of IAV infection. Pfp-deficient mice display an impaired capacity to clear IAV infection, suggesting that Pfp-dependent cytotoxicity plays a major role [22]. Unexpectedly, mice deficient in the major granzyme proteins, A and B, do not show heightened susceptibility and can control IAV infection as effectively as wild type mice [23]. This suggests that other cell death pathways, such as Fas–Fas ligand interactions mediated by activated T cells [22] or other death-domain-containing proteins such as TNF-related apoptosis inducing ligand (TRAIL) [24], may have a role. A more intriguing possibility is that other granzymes, such as grzK, can compensate for the loss of grzA and B and contribute to

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Figure 1. T cell effector mechanisms in the IAV infected lung. (A) Recently activated IAV-specific CD8 and CD4 T cells are recruited to infected lung tissue in a CCR5dependent manner. During a secondary response, recruitment of newly activated memory T cells supplements lung-resident memory T cells that remained in the lung after resolution of a primary infection. (B) IAV-specific CD8+ T cells recognize IAV-infected lung epithelial cells presenting MHC class I molecules presenting IAV-derived peptides. Upon T cell receptor recognition, effector CD8+ T cells contribute to viral control and elimination via a combination of mechanisms including: (i) delivery of cytotoxic moleucles such as perforin and granzymes; (ii) secretion of proinflammatory cytokines such as IFN-g and TNF-a; and (iii) expression of death domain receptors FasL and TRAIL that can initiate cell death after binding to their respective ligands. At later stages of infection, IAV-specific CD8+ T cells can also express IL-10 as a way of helping limit T cell dependent immunopathology in the lung. (C) IAV-specific effector CD4+ T cells contribute to viral control and elimination via secretion of either TH1 or TH17 proinflammatory cytokines. Upregulation of MHCII on inflammed epithelial cells means that CD4+ T cells can directly recognize infected cells, and there is a suggestion that lung effector CD4+ T cells may also mediate direct cell cytotoxicity via delivery of perforin and granzymes. (D) Activated effector CD4+ T cells can also trigger the secretion of innate cytokines such as IL1-b, CXCL9, and CCL2 helping contribute to the proinflammatory response in the infected lung. The cellular source of these cytokines is not known are likely to be lung resident macrophages. Abbreviations: CCL2, chemokine CC ligand 2; CCR5, chemokine CC receptor 5; CXCL9, chemokine CXC ligand 9; Gzm, granzyme; IAV, influenza A virus; FasL, Fas ligand; IFN, interferon; IL, interleukin; TH, T helper; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis inducing ligand.

limiting and eventual control IAV infection. grzK is expressed at high frequency by both mouse and human virus-specific CD8+ T cells [25–28], and is also expressed at high levels in IAV-specific CD8+ T cells in grzA/B-deficient mice [23]. It is possible that perhaps grzK is the key cytolytic molecule that mediates CD8+ T cell killing of virus-infected cells, and it remains to be determined whether grzK can compensate for the loss of grzA or B. IAV-specific CD8+ T cells can simultaneously produce a variety of proinflammatory cytokines in response to antigen activation [29]. Effector CD8+ T cells isolated from bronchoalveolar lavage exhibit a heightened functional capacity, particularly in terms of proinflammatory cytokine production [25,29]. Secretion of these mediators

preferentially occurs at sites of active infection where there is increased presentation of viral determinants and a preexisting inflammatory environment as a consequence of innate inflammatory mediators [30]. At least in mouse experimental systems, lung-resident memory T cells can persist long term after IAV infection, where they are thought to constitute a frontline defense against secondary challenge [31]. Following secondary IAV challenge, the lung-resident memory T cells are supplemented by chemokine CC receptor (CCR)5-dependent recruitment of circulating memory CD8+ T cell to the infected lung [32]. Both the resident memory and newly recruited IAV-specific CD8+ T cells can immediately secrete IFN-g upon antigen recognition and contribute to early virus elimination [33]. 399

Review The recruitment to the murine lung of these highly active memory CD8+ T cells expressing IFN-g has been shown to be key for protection against influenza infection [34]. The enhanced effector potential of lung localized CD8+ T cell effectors also increases the risk of damage to lung tissue due to excessive inflammation (reviewed in [35]). Thus, to mitigate damage to the sensitive lung tissue by potent T cell effector functions, a balance must be struck between ensuring effective antiviral potency while not causing immunopathology. It is intriguing that IAV-specific CD8+ effector T cells isolated from infected mouse lungs are capable of producing interleukin (IL)-10; a potent negative regulator of inflammation [36]. The ability of effector CD8+ T cells to produce IL-10 is dependent on migration into the inflamed lung [37], suggesting that there are signals specific to the infected lung microenvironment that trigger regulatory functions in otherwise proinflammatory IAV-specific CD8+ T cells. In this way, the immune response can balance the need for inflammation required to clear IAV infection, with the need to limit tissue injury by the inflammatory response. Mechanisms of CD4+ T cell control of viral infection Classically, activated CD4+ T cells are considered to be key for promotion of effective antibody responses via support of germinal center formation that results in affinity maturation and isotype switching [38–41]. This occurs through the provision of key co-stimulatory signals such as inducible T cell co-stimulator (ICOS), and the production of cytokines such as IL-21 [40,41]. The antibody response to IAV infection is critical for protection [42]; lack of neutralizing antibody levels in the population is a key factor controlling the emergence of IAV pandemics [43]. The finding that memory CD4+ T cell responses contribute to heterosubtypic immunity against a potential pandemic IAV [8] suggests that memory CD4+ T cells play a key role in the control of IAV infection, but the mechanisms involved are not clear. Adoptive transfer of a large number of ex vivo isolated memory IAV-specific CD4+ T cells into a mouse model of infection augmented both IAV-specific CD8+ and B cell responses against primary infection [44]. A more detailed analysis of how memory CD4+ T cells can promote primary IAV-specific B cell response has shown that establishment of NP-specific memory CD4+ T cells by peptide vaccination of mice promoted robust germinal center formation and a more rapid primary NP-specific antibody response after IAV infection, compared to unvaccinated mice [45]. Strikingly, memory NP-specific CD4+ T cells did not promote antibody responses to other viral proteins, including the HA protein, the major target of the antibody response. This suggests that both the antibody and T cell responses are linked to the same viral target; likely as a consequence of the ability of B cells to process and present CD4+ T cell epitopes from antigen captured and internalized via surface immunoglobulin receptors. Mouse models of IAV infection provide a tool whereby CD4+ T cell effector mechanisms can be delineated more precisely (reviewed in [46]). For example, recent studies have utilized adoptive transfer of T cell receptor (TCR) transgenic CD4+ T cells specific for an epitope of the HA 400

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protein of an H1N1 IAV [A/PR8/34 (HNT)] to determine the contribution of CD4+ T cells to protection from IAV infection [44]. Initial experiments demonstrated that adoptive transfer of CD4+ HNT T cells that were differentiated in vitro into proinflammatory T helper (TH)1 or TH17 lineages (Figure 1) were more capable of mediating clearance and protection from IAV infection, compared to uncommitted (TH0) or anti-inflammatory (TH2) CD4+ effectors [44]. This control was partly via augmentation of endogenous IAV-specific CD8+ T cell and B cell responses and partly via triggering expression of innate cytokines such as IL-1b, IL-6, chemokine CXC ligand (CXCL)9 and chemokine CC ligand (CCL)2, particularly within the infected lung [47]. The demonstration that at least in murine models, influenza infection can induce in vivo MHC class II expression on lung epithelial cells [48] highlights the possibility that CD4+ T cells could have a role in control of IAV infection by directly recognizing and eliminating virusinfected targets. In fact, protection from IAV infection conferred by adoptive transfer of memory HNT CD4+ T cells into immunodeficient mice was abrogated when these cells were deficient in either IFN-g or perforin [44]. Hence, aside from providing help to B cells and CD8+ T cells, IAVspecific CD4+ T cells have the capacity to target directly IAV-infected cells, thereby contributing to the control and elimination of IAV infection. Such unconventional mechanisms of T cell action must be appreciated for the strategic design of vaccines aiming to elicit effective cellular immunity. CD4+ T cell regulation of IAV-specific CD8+ T cell responses The precise role of CD4+ T cells in promoting and regulating CD8+ T cell responses induced by IAV infection was, until recently, enigmatic. This is partly because in mice, an effective primary CD8+ T cell response to IAV can be induced independently of CD4+ T cells [49]. In this case, direct activation of dendritic cells (DCs) via the engagement of Toll-like receptors (TLRs) by IAV circumvents the need for CD4+ TH-dependent CD40 ligand (CD40L) licensing of DCs to promote primary virus-specific CD8+ T cell responses [50]. However, memory CD8+ T cells that are primed in the absence of CD4+ T cells are reduced in number and show an inability to response to secondary infection, compared to memory CD8+ T cells primed in the presence of CD4+ T cells [49]. So, although dispensable for primary activation and expansion, CD4+ T cell help is crucial (during the initial priming phase) for programming optimal IAV-specific CD8+ T cell memory. The role of CD4+ T cell help in this case is the provision of co-stimulatory signals via CD40L–CD40-dependent interactions with DCs that lead to optimal priming of the IAV-specific CD8+ T cell response. These signals received from ‘licensed’ DCs then ensure the responding CD8+ T cells are capable of autocrine IL-2 production [51], which is crucial for their survival into memory. What remains unclear are the precise signals provided by CD4-dependent ‘licensing’ of DCs that ensure responding CD8+ T cells can establish effective memory populations. Uncovering these mechanisms would

Review provide information crucial to the design of any CD8+ T cell based vaccine strategy to promote optimal memory formation. CD4+ T regulatory (Treg) cells have been shown to limit effector CD8+ T cell differentiation in response to virus infection and immunization [52–54], and they have the capacity to suppress potently primary IAV-specific CD8+ T cell responses after infection of mice [55,56]. So, how is an effective primary CD8+ T response sustained in the face of CD4+ Treg cell mediated suppression? Recent work from Randall and colleagues suggests that activation of CD40L+ CD4+ TH cells early after IAV infection is key to ensuring appropriate DC activation that serves to limit the expansion and activation of Treg cells [55], which in turn limits Treg cell suppression of the primary CD8+ T cell response during the early phases of infection. As the infection is cleared and antigen becomes limiting, Treg cells begin to exert their suppressive effects and effectively promote the tapering of the effector CD8+ T cell response during the contraction phase [55]. In this way, Treg cells may limit potential damage caused by a prolonged CD8+ T cell response at later stages of infection. This mechanism is only recently described and raises several questions. For example, is the induction of Treg cells diminished in the case of highly pathogenic IAV infection, where increased immunopathology is associated with highly pathogenic H5N1 or the recent H7N9 infection of humans? The overall picture is that is that all arms of the adaptive response have a role to play in the control of IAV infection. B cell/antibody-mediated immunity plays the major role when it comes to preventing infection with antigenically matched strains. In cases where antibody reactivity is limiting or absent, there is mounting evidence that both CD8+ and CD4+ T cells have key roles in limiting IAV infection, particularly in the case of heterologous IAV challenge. What has begun to emerge, yet remains incompletely understood, is the range and redundancy of mechanisms utilized by T cells in both controlling infection and limiting immunopathology, as well as the precise interactions between the adaptive immune cell populations. When considering the development of new vaccines for engaging heterologous T cell immunity, it is essential that these features of T cell activity be understood to ensure establishment of an effective memory T cell population. Concluding remarks Although there has long been acknowledgement that cellular immunity to IAV plays a role in protection from infection, it is only with recent advances in the identification and isolation of IAV-specific T cells that this has been accepted as an important immunological correlate of protection from IAV infection. Given the extremely high mutation rate of the influenza proteins (NA and HA) typically targeted by antibodies, it is becoming clear that protection from IAV infection, and a broader range of infections such as HIV, hepatitis C virus, and malaria, will require vaccine strategies that induce robust and long-lived T cell responses [57]. The improved capacity to enumerate and isolate IAV-specific T cell responses has also allowed greater insight into the dynamics, location, gene expression, and genomic organization of IAV-specific T cell immunity at

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distinct stages of T cell immune response [57]. Although such analyses are greatly enhancing our understanding of both molecular regulation and immune mechanisms, the practical challenge of how best to manipulate both CD4+ and CD8+ T cell responses, particularly via vaccination, to achieve a measure of long-term, if partial, heterosubtypic protection is still ahead of us. Acknowledgments This work is supported by an Australian Research Council Future Fellowship (awarded to S.J.T.); a Sylvia and Charles Viertal Senior Research Fellowship (awarded to N.L.L.); Australian National Health and Medical Research Council (NHMRC) program grant 5671222 (awarded to S.J.T.) and NHMRC project grant AI1046333 (awarded to N.L.L.).

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