T cell mediated immunity to inuenza:

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 inuenza A virus (IAV) is a major cause
of worldwide morbidity and mortality. Recent ndings
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 mecha-
nisms that mediate effective IAV-specic cellular immu-
nity, especially given that T cell immunity must form an
integral part of any vaccine designed to elicit crossreac-
tive immunity against existing and new strains of inu-
enza virus. Here, we review the current understanding
of cellular immunity to IAV, highlighting recent ndings
that demonstrate important roles for bothCD4
+
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
35 million cases of severe illness and 250 000500 000
deaths worldwide per annum (WHO inuenza centre web-
site). IAV-specic immunity can be induced by vaccination
that generates IAV-specic antibodies that limit or prevent
IAV infection. However, the IAV vaccine needs to be refor-
mulated on an annual basis because inuenza viruses
rapidly evolve, with new strains emerging that have lost
or mutated the targets recognized by the preceding anti-
body response. Thus, an arms race ensues whereby vac-
cine-induced immune pressures select for new strains that
are no longer recognized by vaccine induced IAV-specic
antibodies, necessitating the production of updated IAV
vaccines. CD8
+
and CD4
+
T cells have distinct but impor-
tant roles in the control, and eventual clearance, of inu-
enza 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 produc-
ing antiviral and proinammatory cytokines upon infec-
tion, 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
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 canonly 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 immu-
nity 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 protec-
tion. 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-associat-
ed pathology until virus is cleared. Thus, it is widely ac-
knowledged that a comprehensive vaccineagainst IAV must
include the ability to elicit T cell immunity [4]. Here, we
examine the current state of knowledge regarding IAV-
specic T cell immunity and discuss how a greater under-
standing of factors that shape and promote IAV-specic
cellular immunity will contribute to improved vaccine strat-
egies capable of eliciting heterologous immunity.
Targets of the T cell response during inuenza infection
The fact that IAV-specic memory T cells can target a broad
range of peptides derived from proteins that are relatively
conserved between different inuenza 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-specic T cell immunity via development of a novel T
cell basedvaccine 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 ex-
posed 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 inuenza could generate
T cell immunity that was capable of recognizing serological-
ly unrelated IAV subtypes. Moreover, they also demonstrat-
ed that the major T cell targets were derived from the IAV
Review
1471-4906/
2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2014.06.004
Corresponding author: Turner, S.J. (sjturn@unimelb.edu.au).
Keywords: influenza A virus; CD8
+
T cell; CD4
+
T cell; immunological memory MHC
class I MHC class II vaccination.
396 Trends in Immunology, August 2014, Vol. 35, No. 8
matrix protein (M)1 and nucleoprotein (NP). Studies that
have also used the approach of ex vivo stimulation of human
PBMCs withoverlappingIAV peptides have since conrmed
that peptides fromtheM1 andNP are themajor targets for T
cell immunity [68]. Thus, pre-existing IAV-specic T cell
immunity inducedby infectionwithone strainmay 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 vaccinestrategies wouldonlyneedto includea limited
number of protein targets to ensure broad-based immunity
and likelymake vaccineformulation a more straightforward
exercise than having to use something like whole inacti-
vated 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 5866;
M1
5866
) can be readily detected within HLA-A2
+
individuals
[9]. Given the repeated demonstrationof this response across
HLA-A2
+
individuals, it is considered a dominant response.
Are other suchdominant responses prevalent within individ-
uals with other MHC haplotypes, and what is the full reper-
toire of IAV peptides eliciting a CD8
+
T cell response? Chen
and colleagues addressed these questions by taking a sys-
tematic approach wherein peripheral human T lymphocytes
fromseveral healthy donors were cocultured with live IAV as
an antigenic stimulus whereby infected PBMCs self pre-
sented IAV antigens to pre-existing memory T cells. They
then screened T cell reactivity against individual inuenza
proteins, enabling themto narrowthe candidate pool, so that
they could then dene the minimal peptide targets after in
vitro stimulation with overlapping peptides [10]. As previ-
ously reported [11], the dominant CD8
+
T cell responses from
different individuals targeted the conserved matrix (M) and
nucleoprotein (NP) proteins. Importantly, using this system-
atic approach, new peptide targets presented by an array of
distinct HLA molecules were identied 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 identied IAV-peptide targets identied were
longer than typical MHC class I binding peptides, and hence
would not have been identied 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 identied for possible inclu-
sion in T cell based vaccine approaches. Although the use of
specic peptides in vaccines is a direct way of targetingT 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 difculty of ensuring
adequate coverage across numerous HLA subtypes. More-
over, there is an increasing appreciation that IAV-specic 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 vaccinationagainst 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]; bothof which have shown promise
in protection from heterologous challenge.
It is important to note that whole protein vaccine strat-
egies do not circumvent the need for high-resolution epi-
tope identication. A major driver is the increasing need to
be able to track antigen-specic T cell responses after
vaccination by use of soluble pMHC tetramer reagents
[14]. The identication of new IAV-specic pMHC com-
plexes, combined with recent advances in ow cytometric
techniques that enable multiple specicities/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-specic T cell immunity.
Role of T cell immunity against IAV infection: lessons
from humans
Although IAV-specic CD4
+
and CD8
+
T cells are readily
identiable in humans [5,1720], their precise role in
controlling IAV infection is unclear. A retrospective analy-
sis demonstrated that prior symptomatic A(H1N1) infec-
tion was associated with increased protection from the
1957 A(H2N2) pandemic virus in adults but not children,
suggesting an accumulation of heterologous immunity
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 nave, 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 lung-
derived 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 [6062]. 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 nave CD4
+
T cell responses
[62,64]. Although activated B cells can also present antigen to CD4
+
T cells, the key purpose of this TB interaction is the promotion of
effective antibody responses, rather than initial priming of nave
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 nave 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.
Review Trends in Immunology August 2014, Vol. 35, No. 8
397
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 pan-
demic event), strongly suggests a key role for T cell medi-
ated protection [21].
The earliest indirect evidence in humans that CD8
+
T cell
immunity is important for protection against inuenza-
mediated illness came from a challenge study in which
volunteers were intranasally infected with a live, attenuat-
ed IAV, and viral shedding was measured in clinical samples
[20]. Decreased viral shedding was associated with a con-
comitant increase in IAV-specic CD8
+
T cell responses in
volunteers who lacked neutralizing, strain-specic antibo-
dies. These ndings implied that IAV-specic 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 anti-
bodies 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 inuenza infection were found to have higher
circulating levels of pre-existing IAV-specic CD8
+
effector
memory T cells (dened by CD45RA
+
CCR7

expression).
Functionally, these effector memory CD8
+
T cells exhibited
the capacity to produce interferon (IFN)-g and were capa-
ble of direct cytotoxicity against infected target cells. In-
terestingly, there was no signicant correlation between
symptom severity (symptoms noted were runny nose, fe-
ver, and sore throat) and the presence of pre-existing IAV-
specic CD4
+
T cells. Thus, in the setting of natural infec-
tion, when antibody immunity is lacking, elevated inuen-
za-specic 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 inuenza proteins,
implying that CD4
+
T cells may have the potential to
provide IAV-specic heterologous immunity [5]. To test
this directly, Wilkinson and colleagues [8] challenged
volunteers with either a wild type H1N1 or a H3N2 sea-
sonal IAV strain, and then measured clinical symptoms
and viral shedding over the course of infection. All volun-
teers 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 signicant correlation
was found between symptom severity and presence of
pre-existing IAV-specic CD4
+
T cells [7], Wilkinson
et al. found a signicant inverse correlation between dis-
ease severity and pre-existing levels of circulating IAV-
specic CD4
+
cells. It is not clear why these two recently
published IAV challenge studies came to different conclu-
sions about the respective roles of IAV-specic CD8
+
and
CD4
+
T cells. It might be the fact that Sridhar and collea-
gues examined a natural experiment, whereas Wilkinson
et al. analyzed heterologous IAV-specic T cell responses in
the context of an experimental challenge of human volun-
teers. 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 denitive answer,
these ndings 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-specic 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 inammatory 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-decient mice display an
impaired capacity to clear IAV infection, suggesting that
Pfp-dependent cytotoxicity plays a major role [22]. Unexpect-
edly, mice decient 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 FasFas ligand
interactions mediated by activated T cells [22] or other
death-domain-containing proteins such as TNF-related apo-
ptosis inducing ligand (TRAIL) [24], may have a role. Amore
intriguing possibility is that other granzymes, such as grzK,
can compensate for the loss of grzA and B and contribute to
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 [6770].
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 D
b
NP
366
epitope emerged
coincidently with the peak of the D
b
NP
366
-specific CD8
+
T cell
response. When these viral variants were used to inoculate MHC-
mismatched 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 perspec-
tive, appear to be targets of immune T cell selection.
Review
Trends in Immunology August 2014, Vol. 35, No. 8
398
limiting and eventual control IAVinfection. grzK is expressed
at high frequency by both mouse and human virus-specic
CD8
+
T cells [2528], and is also expressed at high levels in
IAV-specic CD8
+
T cells in grzA/B-decient 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-specic CD8
+
T cells can simultaneously produce a
variety of proinammatory cytokines in response to anti-
gen activation [29]. Effector CD8
+
T cells isolated from
bronchoalveolar lavage exhibit a heightened functional
capacity, particularly in terms of proinammatory cyto-
kine production [25,29]. Secretion of these mediators
preferentially occurs at sites of active infection where there
is increased presentation of viral determinants and a pre-
existing inammatory environment as a consequence of
innate inammatory 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 chemo-
kine CC receptor (CCR)5-dependent recruitment of circu-
lating memory CD8
+
T cell to the infected lung [32]. Both
the resident memory and newly recruited IAV-specic
CD8
+
T cells can immediately secrete IFN-g upon antigen
recognition and contribute to early virus elimination [33].
(A)
Lung airways
Cytokine
T
H
1 T
H
17
Cytotoxic
FasL
Trail
MHCI
MHCII
IL-10
Perforin
Gzm A, B K
Perforin
Gzm A, B
IL-2
IFN
IFN
IL-17A
IL-6
IL-21
TNF
TNF-
IL1-
CXCL9
CCL2
Lung parenchyma
CCR5+
CCR5+
Blood vessels
(B)
(C)
(D)
TRENDS in Immunology
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 CCR5-
dependent 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 T
H
1 or T
H
17
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; T
H
, T helper; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis inducing ligand.
Review Trends in Immunology August 2014, Vol. 35, No. 8
399
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 inuenza 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 inammation (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-spe-
cic CD8
+
effector T cells isolated from infected mouse
lungs are capable of producing interleukin (IL)-10; a potent
negative regulator of inammation [36]. The ability of
effector CD8
+
T cells to produce IL-10 is dependent on
migration into the inamed lung [37], suggesting that
there are signals specic to the infected lung microenvi-
ronment that trigger regulatory functions in otherwise
proinammatory IAV-specic CD8
+
T cells. In this way,
the immune response can balance the need for inamma-
tion required to clear IAV infection, with the need to limit
tissue injury by the inammatory 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 afnity matura-
tion and isotype switching [3841]. 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 infec-
tion 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 nding that memory CD4
+
T cell responses contrib-
ute to heterosubtypic immunity against a potential pan-
demic 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-specic CD4
+
T cells into a
mouse model of infection augmented both IAV-specic
CD8
+
and B cell responses against primary infection
[44]. A more detailed analysis of how memory CD4
+
T cells
can promote primary IAV-specic B cell response has
shown that establishment of NP-specic memory CD4
+
T cells by peptide vaccination of mice promoted robust
germinal center formation and a more rapid primary
NP-specic antibody response after IAV infection, com-
pared to unvaccinated mice [45]. Strikingly, memory
NP-specic 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 immuno-
globulin 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 specic for an epitope of the HA
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 differen-
tiated in vitro into proinammatory T helper (TH)1 or
T
H
17 lineages (Figure 1) were more capable of mediating
clearance and protection from IAV infection, compared to
uncommitted (T
H
0) or anti-inammatory (T
H
2) CD4
+
effectors [44]. This control was partly via augmentation
of endogenous IAV-specic 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,
inuenza infection can induce in vivo MHC class II expres-
sion 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 virus-
infected targets. In fact, protection from IAV infection
conferred by adoptive transfer of memory HNT CD4
+
T
cells into immunodecient mice was abrogated when these
cells were decient in either IFN-g or perforin [44]. Hence,
aside from providing help to B cells and CD8
+
T cells, IAV-
specic CD4
+
T cells have the capacity to target directly
IAV-infected cells, thereby contributing to the control and
elimination of IAV infection. Such unconventional mecha-
nisms of T cell action must be appreciated for the strategic
design of vaccines aiming to elicit effective cellular immu-
nity.
CD4
+
T cell regulation of IAV-specic CD8
+
T cell
responses
The precise role of CD4
+
T cells in promoting and regulat-
ing 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 engage-
ment of Toll-like receptors (TLRs) by IAV circumvents the
need for CD4
+
T
H
-dependent CD40 ligand (CD40L) licens-
ing of DCs to promote primary virus-specic 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-spe-
cic CD8
+
T cell memory. The role of CD4
+
T cell help in
this case is the provision of co-stimulatory signals via
CD40LCD40-dependent interactions with DCs that lead
to optimal priming of the IAV-specic CD8
+
T cell re-
sponse. These signals received from licensed DCs then
ensure the responding CD8
+
T cells are capable of auto-
crine IL-2 production [51], which is crucial for their surviv-
al 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
Trends in Immunology August 2014, Vol. 35, No. 8
400
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 [5254], and they have the
capacity to suppress potently primary IAV-specic 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
+
T
H
cells early after IAV infection is key to ensuring
appropriate DC activation that serves to limit the expan-
sion 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 re-
sponse at later stages of infection. This mechanism is only
recently described and raises several questions. For exam-
ple, is the induction of Treg cells diminished in the case of
highly pathogenic IAV infection, where increased immu-
nopathology 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 incom-
pletely understood, is the range and redundancy of mech-
anisms utilized by T cells in both controlling infection and
limiting immunopathology, as well as the precise interac-
tions 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 estab-
lishment of an effective memory T cell population.
Concluding remarks
Although there has long been acknowledgement that cel-
lular immunity to IAV plays a role in protection from
infection, it is only with recent advances in the identica-
tion and isolation of IAV-specic T cells that this has been
accepted as an important immunological correlate of pro-
tection from IAV infection. Given the extremely high mu-
tation rate of the inuenza 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-specic T cell responses has also allowed great-
er insight into the dynamics, location, gene expression, and
genomic organization of IAV-specic T cell immunity at
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.).
References
1 Doherty, P.C. and Christensen, J.P. (2000) Accessing complexity: the
dynamics of virus-specic T cell responses. Annu. Rev. Immunol. 18,
561592
2 Strutt, T.M. et al. (2013) Multipronged CD4
+
T-cell effector and
memory responses cooperate to provide potent immunity against
respiratory virus. Immunol. Rev. 255, 149164
3 Thomas, P.G. et al. (2006) Cell-mediated protection in inuenza
infection. Emerg. Infect. Dis. 12, 4854
4 Doherty, P.C. et al. (2006) Inuenza and the challenge for immunology.
Nat. Immunol. 7, 449455
5 Lee, L.Y. et al. (2008) Memory T cells established by seasonal human
inuenza A infection cross-react with avian inuenza A (H5N1) in
healthy individuals. J. Clin. Invest. 118, 34783490
6 Grant, E. et al. (2013) Nucleoprotein of inuenza A virus is a major
target of immunodominant CD8
+
T-cell responses. Immunol. Cell Biol.
91, 184194
7 Sridhar, S. et al. (2013) Cellular immune correlates of protection
against symptomatic pandemic inuenza. Nat. Med. 19, 13051312
8 Wilkinson, T.M. et al. (2012) Preexisting inuenza-specic CD4
+
Tcells
correlate with disease protection against inuenza challenge in
humans. Nat. Med. 18, 274280
9 Gotch, F. et al. (1987) Cytotoxic T lymphocytes recognize a fragment of
inuenza virus matrix protein in association with HLA-A2. Nature 326,
881882
10 Wu, C. et al. (2011) Systematic identication of immunodominant
CD8
+
T-cell responses to inuenza A virus in HLA-A2 individuals.
Proc. Natl. Acad. Sci. U.S.A. 108, 91789183
11 Nayak, J.L. et al. (2010) Analyses of the specicity of CD4 Tcells during
the primary immune response to inuenza virus reveals dramatic
MHC-linked asymmetries in reactivity to individual viral proteins.
Viral Immunol. 23, 169180
12 Alam, S. et al. (2014) CD4 Tcell help is limiting and selective during the
primary B cell response to inuenza virus infection. J. Virol. 88, 314
324
13 Corti, D. et al. (2010) Heterosubtypic neutralizing antibodies are
produced by individuals immunized with a seasonal inuenza
vaccine. J. Clin. Invest. 120, 16631673
14 Guillaume, P. et al. (2009) Soluble MHC-peptide complexes: tools for
the monitoring of T cell responses in clinical trials and basic research.
Cancer Immun. 9, 7
15 Hadrup, S.R. et al. (2009) Parallel detection of antigen-specic T-cell
responses by multidimensional encoding of MHC multimers. Nat.
Methods 6, 520526
16 Newell, E.W. et al. (2012) Cytometry by time-of-ight shows
combinatorial cytokine expression and virus-specic cell niches
within a continuum of CD8
+
T cell phenotypes. Immunity 36, 142152
17 Forrest, B.D. et al. (2008) Correlation of cellular immune responses
with protection against culture-conrmed inuenza virus in young
children. Clin. Vaccine Immunol. 15, 10421053
18 Kreijtz, J.H. et al. (2007) Primary inuenza A virus infection induces
cross-protective immunity against a lethal infection with a
heterosubtypic virus strain in mice. Vaccine 25, 612620
19 Kreijtz, J.H. et al. (2008) Cross-recognition of avian H5N1 inuenza
virus by human cytotoxic T-lymphocyte populations directed to human
inuenza A virus. J. Virol. 82, 51615166
Review Trends in Immunology August 2014, Vol. 35, No. 8
401
20 McMichael, A.J. et al. (1983) Cytotoxic T-cell immunity to inuenza. N.
Engl. J. Med. 309, 1317
21 Epstein, S.L. (2006) Prior H1N1 inuenza infection and susceptibility
of Cleveland Family Study participants during the H2N2 pandemic of
1957: an experiment of nature. J. Infect. Dis. 193, 4953
22 Topham, D.J. et al. (1997) CD8
+
T cells clear inuenza virus by perforin
or Fas-dependent processes. J. Immunol. 159, 51975200
23 Jenkins, M.R. et al. (2008) Granzyme K expressing cytotoxic T
lymphocytes protects against inuenza virus in granzyme AB
-/-
mice. Viral Immunol. 21, 341346
24 Brincks, E.L. et al. (2008) CD8 Tcells utilize TRAILto control inuenza
virus infection. J. Immunol. 181, 49184925
25 Jenkins, M.R. et al. (2007) Heterogeneity of effector phenotype for
acute phase and memory inuenza A virus-specic CTL. J. Immunol.
179, 6470
26 Jenkins, M.R. et al. (2008) Cell cycle-related acquisition of cytotoxic
mediators denes the progressive differentiation to effector status for
virus-specic CD8
+
T cells. J. Immunol. 181, 38183822
27 Mintern, J.D. et al. (2007) Cutting edge: tissue-resident memory CTL
down-regulate cytolytic molecule expression following virus clearance.
J. Immunol. 179, 72207224
28 Smith, C. et al. (2012) Endogenous antigen presentation impacts on T-
box transcription factor expression and functional maturation of CD8
+
T cells. Blood 120, 32373245
29 La Gruta, N.L. et al. (2004) Hierarchies in cytokine expression proles
for acute and resolving inuenza virus-specic CD8
+
T cell responses:
correlation of cytokine prole and TCRavidity. J. Immunol. 172, 5553
5560
30 Stinchcombe, J.C. and Grifths, G.M. (2007) Secretory mechanisms in
cell-mediated cytotoxicity. Annu. Rev. Cell Dev. Biol. 23, 495517
31 Kohlmeier, J.E. and Woodland, D.L. (2008) Immunity to respiratory
viruses. Annu. Rev. Immunol. 27, 6182
32 Kohlmeier, J.E. et al. (2008) The chemokine receptor CCR5 plays a key
role in the early memory CD8
+
T cell response to respiratory virus
infections. Immunity 29, 101113
33 Kohlmeier, J.E. et al. (2010) Type I interferons regulate cytolytic
activity of memory CD8
+
T cells in the lung airways during
respiratory virus challenge. Immunity 33, 96105
34 Cerwenka, A. et al. (1999) Naive, effector, and memory CD8 T cells in
protection against pulmonary inuenza virus infection: homing
properties rather than initial frequencies are crucial. J. Immunol.
163, 55355543
35 La Gruta, N.L. et al. (2007) A question of self-preservation:
immunopathology in inuenza virus infection. Immunol. Cell Biol.
85, 8592
36 Sun, J. et al. (2009) Effector T cells control lung inammation during
acute inuenza virus infection by producing IL-10. Nat. Med. 15, 277
284
37 Palmer, E.M. et al. (2010) IFNgamma-producing, virus-specic CD8
+
effector cells acquire the ability to produce IL-10 as a result of entry
into the infected lung environment. Virology 404, 225230
38 Breitfeld, D. et al. (2000) Follicular B helper T cells express CXC
chemokine receptor 5, localize to B cell follicles, and support
immunoglobulin production. J. Exp. Med. 192, 15451552
39 Lee, S.K. et al. (2011) B cell priming for extrafollicular antibody
responses requires Bcl-6 expression by T cells. J. Exp. Med. 208,
13771388
40 Linterman, M.A. et al. (2010) IL-21 acts directly on B cells to regulate
Bcl-6 expression and germinal center responses. J. Exp. Med. 207, 353
363
41 Zotos, D. et al. (2010) IL-21 regulates germinal center B cell
differentiation and proliferation through a B cell-intrinsic
mechanism. J. Exp. Med. 207, 365378
42 Gerhard, W. et al. (1997) Role of the B-cell response in recovery of mice
from primary inuenza virus infection. Immunol. Rev. 159, 95103
43 Wrammert, J. et al. (2011) Broadly cross-reactive antibodies dominate
the human B cell response against 2009 pandemic H1N1 inuenza
virus infection. J. Exp. Med. 208, 181193
44 McKinstry, K.K. et al. (2012) Memory CD4
+
T cells protect against
inuenza through multiple synergizing mechanisms. J. Clin. Invest.
122, 28472856
45 Alam, S. and Sant, A.J. (2011) Infection with seasonal inuenza virus
elicits CD4 Tcells specic for genetically conserved epitopes that can be
rapidly mobilized for protective immunity to pandemic H1N1 inuenza
virus. J. Virol. 85, 1331013321
46 Swain, S.L. et al. (2012) Expanding roles for CD4
+
T cells in immunity
to viruses. Nat. Rev. Immunol. 12, 136148
47 Strutt, T.M. et al. (2010) Memory CD4
+
T cells induce innate responses
independently of pathogen. Nat. Med. 16, 558564
48 Brown, D.M. et al. (2012) Multifunctional CD4 cells expressing gamma
interferon and perforin mediate protection against lethal inuenza
virus infection. J. Virol. 86, 67926803
49 Belz, G.T. et al. (2002) Compromised inuenza virus-specic CD8
+
-T-
cell memory in CD4
+
-T-cell-decient mice. J. Virol. 76, 1238812393
50 Johnson, S. et al. (2009) Selected Toll-like receptor ligands and viruses
promote helper-independent cytotoxic T cell priming by upregulating
CD40L on dendritic cells. Immunity 30, 218227
51 Feau, S. et al. (2011) Autocrine IL-2 is required for secondary population
expansion of CD8
+
memory T cells. Nat. immunol. 12, 908913
52 Fulton, R.B. et al. (2010) Foxp3
+
CD4 regulatory T cells limit
pulmonary immunopathology by modulating the CD8 T cell
response during respiratory syncytial virus infection. J. Immunol.
185, 23822392
53 McNally, A. et al. (2011) CD4
+
CD25
+
regulatory T cells control CD8
+
T-
cell effector differentiation by modulating IL-2 homeostasis. Proc. Natl.
Acad. Sci. U.S.A. 108, 75297534
54 Suvas, S. et al. (2003) CD4
+
CD25
+
T cells regulate virus-specic
primary and memory CD8
+
T cell responses. J. Exp. Med. 198, 889901
55 Ballesteros-Tato, A. et al. (2013) CD4
+
T helper cells use CD154-CD40
interactions to counteract T reg cell-mediated suppression of CD8
+
T
cell responses to inuenza. J. Exp. Med. 210, 15911601
56 Haeryfar, S.M. et al. (2005) Regulatory T cells suppress CD8
+
T cell
responses induced by direct priming and cross-priming and moderate
immunodominance disparities. J. Immunol. 174, 33443351
57 Pulendran, B. et al. (2010) Systems vaccinology. Immunity 33, 516529
58 Heath, W.R. and Carbone, F.R. (2009) Dendritic cell subsets in primary
and secondary T cell responses at body surfaces. Nat. Immunol. 10,
12371244
59 Belz, G.T. et al. (2004) Cutting edge: conventional CD8 alpha
+
dendritic
cells are generally involved in priming CTL immunity to viruses. J.
Immunol. 172, 19962000
60 Ho, A.W. et al. (2011) Lung CD103
+
dendritic cells efciently transport
inuenza virus to the lymph node and load viral antigen onto MHC
class I for presentation to CD8 T cells. J. Immunol. 187, 60116021
61 Waithman, J. et al. (2013) Resident CD8
+
and migratory CD103
+
dendritic cells control CD8 T cell immunity during acute inuenza
infection. PLoS ONE 8, e66136
62 Belz, G.T. et al. (2004) Distinct migrating and nonmigrating dendritic
cell populations are involved in MHC class I-restricted antigen
presentation after lung infection with virus. Proc. Natl. Acad. Sci.
U.S.A. 101, 86708675
63 Banchereau, J. and Steinman, R.M. (1998) Dendritic cells and the
control of immunity. Nature 392, 245252
64 GeurtsvanKessel, C.H. et al. (2008) Clearance of inuenza virus from
the lung depends on migratory langerin
+
CD11b
-
but not plasmacytoid
dendritic cells. J. Exp. Med. 205, 16211634
65 Poulin, L.F. et al. (2010) Characterization of human DNGR-1
+
BDCA3
+
leukocytes as putative equivalents of mouse CD8alpha
+
dendritic cells.
J. Exp. Med. 207, 12611271
66 Henri, S. et al. (2010) CD207
+
CD103
+
dermal dendritic cells cross-
present keratinocyte-derived antigens irrespective of the presence of
Langerhans cells. J. Exp. Med. 207, 189206
67 Berkhoff, E.G. et al. (2007) The loss of immunodominant epitopes
affects interferon-gamma production and lytic activity of the human
inuenza virus-specic cytotoxic T lymphocyte response in vitro. Clin.
Exp. Immunol. 148, 296306
68 Berkhoff, E.G. et al. (2004) A mutation in the HLA-B*2705-restricted
NP383-391 epitope affects the human inuenza A virus-specic
cytotoxic T-lymphocyte response in vitro. J. Virol. 78, 52165222
69 Berkhoff, E.G. et al. (2007) Assessment of the extent of variation in
inuenza A virus cytotoxic T-lymphocyte epitopes by using virus-
specic CD8
+
T-cell clones. J. Gen. Virol. 88, 530535
70 Boon, A.C. et al. (2002) Sequence variation in a newly identied HLA-
B35-restricted epitope in the inuenza A virus nucleoprotein
associated with escape from cytotoxic T lymphocytes. J. Virol. 76,
25672572
Review
Trends in Immunology August 2014, Vol. 35, No. 8
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