Invariant NKT Cells: Regulation and Function during Viral Infection

Abstract

Natural killer T cells (NKT cells) represent a subset of T lymphocytes that express natural killer (NK) cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT), express a highly restricted T cell receptor (TCR) and respond to CD1d-restricted lipid ligands. iNKT cells are now appreciated to play an important role in linking innate and adaptive immune responses and have been implicated in infectious disease, allergy, asthma, autoimmunity, and tumor surveillance. Advances in iNKT identification and purification have allowed for the detailed study of iNKT activity in both humans and mice during a variety of chronic and acute infections. Comparison of iNKT function between non-pathogenic simian immunodeficiency virus (SIV) infection models and chronic HIV-infected patients implies a role for iNKT activity in controlling immune activation. In vitro studies of influenza infection have revealed novel effector functions of iNKT cells including IL-22 production and modulation of myeloid-derived suppressor cells, but ex vivo characterization of human iNKT cells during influenza infection are lacking. Similarly, as recent evidence suggests iNKT involvement in dengue virus pathogenesis, iNKT cells may modulate responses to a number of emerging pathogens. This Review will summarize current knowledge of iNKT involvement in responses to viral infections in both human and mouse models and will identify critical gaps in knowledge and opportunities for future study. We will also highlight recent efforts to harness iNKT ligands as vaccine adjuvants capable of improving vaccination-induced cellular immune responses.

Full text

Review
Invariant NKT Cells: Regulation and Function during Viral
Infection
Jennifer A. Juno
1.
, Yoav Keynan
1,2,3,4.
, Keith R. Fowke
1,2,3
*
1Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada, 2Department of Community Health Sciences, University of Manitoba,
Winnipeg, Manitoba, Canada, 3Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya, 4Department of Internal Medicine, University of Manitoba,
Winnipeg, Manitoba, Canada
Abstract: Natural killer T cells (NKT cells) represent a
subset of T lymphocytes that express natural killer (NK)
cell surface markers. A subset of NKT cells, termed
invariant NKT cells (iNKT), express a highly restricted T
cell receptor (TCR) and respond to CD1d-restricted lipid
ligands. iNKT cells are now appreciated to play an
important role in linking innate and adaptive immune
responses and have been implicated in infectious disease,
allergy, asthma, autoimmunity, and tumor surveillance.
Advances in iNKT identification and purification have
allowed for the detailed study of iNKT activity in both
humans and mice during a variety of chronic and acute
infections. Comparison of iNKT function between non-
pathogenic simian immunodeficiency virus (SIV) infection
models and chronic HIV-infected patients implies a role
for iNKT activity in controlling immune activation. In vitro
studies of influenza infection have revealed novel
effector functions of iNKT cells including IL-22 production
and modulation of myeloid-derived suppressor cells, but
ex vivo characterization of human iNKT cells during
influenza infection are lacking. Similarly, as recent
evidence suggests iNKT involvement in dengue virus
pathogenesis, iNKT cells may modulate responses to a
number of emerging pathogens. This Review will
summarize current knowledge of iNKT involvement in
responses to viral infections in both human and mouse
models and will identify critical gaps in knowledge and
opportunities for future study. We will also highlight
recent efforts to harness iNKT ligands as vaccine
adjuvants capable of improving vaccination-induced
cellular immune responses.
Introduction
The immune response to invading pathogens requires the
successful activation of innate immunity, which informs the
development of the subsequent adaptive immune response. A
small subset of T lymphocytes expressing surface markers
characteristic of both T cells and natural killer (NK) cells are
now appreciated to form an important link between the innate
and adaptive immune responses. These NKT cells can be
activated in both antigen-dependent and independent manners
and respond with robust Th1 and Th2 cytokine production,
allowing them to exhibit remarkable functional plasticity with
both pro-inflammatory and immunoregulatory characteristics.
NKT cells can be grouped into several subsets (Table 1), but the
most commonly described group is the Type 1 or invariant NKT
(iNKT) subset, which is the focus of this Review. iNKTs are
highly conserved among mouse, non-human primate (NHP)
species, and humans [1–4] and are so named due to the
expression of a highly restricted T cell receptor (TCR) repertoire.
In humans and NHPs, iNKT cells are characterized by
expression of a TCR comprised of Va24-Ja18 paired with
Vb11 (reviewed in Porcelli [5]), while mouse iNKTs express
Va14-Ja18 paired with one of Vb8.2, Vb7, or Vb2 [6]. The
majority of iNKTs express CD161 (NK1.1 in mice) and all
respond to lipid ligands through CD1d restriction. Despite the
low frequency of the iNKT population in the periphery (0.01%–
1% of CD3+lymphocytes in humans), iNKT activity is now
appreciated to play important roles in infectious disease, allergy,
autoimmunity, and tumor surveillance. This review will focus on
the current understanding and gaps in knowledge regarding
iNKT function during human viral infection. A description of
iNKT function during viral infection in mouse models has
previously been reviewed by Diana et al. [7].
iNKT Thymic Selection and Development
Current knowledge regarding iNKT thymic selection has
recently been thoroughly reviewed by Hu et al. [8]. Like
conventional T cells, iNKTs develop in the thymus from
CD4+CD8+thymocytes. Expression of the iNKT TCR is
selected by reactivity with CD1d-presented endogenous lipid,
which directs cells to the iNKT lineage; the contribution of high-
affinity ligand negative selection to iNKT development is still
unclear but may also play a role [9,10]. Signaling from both the
TCR and signaling lymphocyte-activation molecule (SLAM)
receptors is required for iNKT development. Maturation and
proliferation of iNKT cells can occur either in the periphery or
the thymus, with mature iNKT cells requiring IL-15 for
maintenance [11]. Determinants of iNKT maturation are not
fully understood, but were recently shown to involve microRNA-
150 expression in mice [12,13].
Citation: Juno JA, Keynan Y, Fowke KR (2012) Invariant NKT Cells: Regulation and
Function during Viral Infection. PLoS Pathog 8(8): e1002838. doi:10.1371/
journal.ppat.1002838
Editor: Tom C. Hobman, University of Alberta, Canada
Published August 16, 2012
Copyright: ß2012 Juno et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: KRF is supported by the Canadian Institutes for Health Research (CIHR).
JAJ is supported by the CIHR International Infectious Disease and Global Health
Training Program. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests
exist.
* E-mail: fowkekr@cc.umanitoba.ca
.These authors contributed equally to this work.
PLOS Pathogens | www.plospathogens.org 1 August 2012 | Volume 8 | Issue 8 | e1002838

iNKT Activation by Ligand-Dependent and Independent
Mechanisms
iNKT TCR–mediated responses are restricted by CD1d, a
member of the non-polymorphic CD1 antigen presenting protein
family [5], which promotes the presentation of endogenous [14]
and pathogen-derived [15–26] lipid antigens to the TCR [27].
Although no viral-associated lipid iNKT antigens have been
described, iNKT activation in the absence of a pathogen-derived
lipid antigen can occur in a CD1d-dependent or independent
manner (reviewed in Brigl et al. [22] and Matsuda et al. [28]).
iNKT activation by antigen presenting cell (APC)-mediated lipid
antigen presentation involves IL-12 production and is strongly
dependent on CD40/CD40L interactions [29], with low levels of
CD40L being detectible ex vivo on the surface of iNKT cells
[30,31] and intracellular, pre-formed CD40L mobilized upon
activation [32]. Numerous pathogen-derived lipid antigens have
now been identified from bacterial species (reviewed in [33]) and
the endogenous lipid b-D-glucopyranosylceramide was recently
shown to accumulate in APCs following infection and to activate
mouse and human iNKTs [14]. Additionally, both gram negative
and gram positive bacteria are capable of activating iNKT cells via
TLR stimulation of, and IL-12/IFNa/bproduction by, APCs
[26,34–37]. This mechanism appears to require CD1d-restricted
presentation of endogenous lipid. Finally, non-specific CD1d-
independent iNKT activation can occur in the context of
lipopolysaccharide (LPS)-induced APC production of IL-12 and
IL-18 [38]. Given the lack of viral lipid antigens available for
CD1d presentation, the capacity to be activated by APC cytokine
production allows the iNKT subset to respond to viral infections as
well as bacterial and parasitic infections. Indeed, new evidence
demonstrates that weak TCR stimulation by endogenous lipids
temporarily ‘‘primes’’ iNKT cells to rapidly respond to cytokine
activation signals, emphasizing the broad, innate responsiveness of
the iNKT subset during infection [39].
iNKT Subsets and Functional Capacity
Human iNKTs express CD4 and CD8a[40,41], allowing
iNKT subsets to be defined as CD4+, CD42CD82(DN), or
CD8+. Subset-specific differences in surface marker expression
have been described (Figure 1) [30], with CD4+iNKTs exhibiting
lower expression of CCR5 but increased expression of CCR4
compared to the CD42subset, which characteristically expresses
CCR1, CCR6, CXCR6, and NKG2D (reviewed in Kim et al.
[42]). All iNKTs express high levels of CXCR3 and CXCR4 and
typically exhibit an effector/memory phenotype [43,44]. The
CD42subset tends to express low levels of CD62L but higher
levels of CD11a, suggesting a tissue-infiltrating phenotype, while
the CD4+subset preferentially expresses CD62L and therefore
exhibits a lymph node homing phenotype [45]. CD4 and CD8 are
both expressed on thymic iNKT cells, but the CD4+subset
predominates. Expansion in the periphery therefore appears to
account for the increased proportion of CD8+/DN iNKTs
observed outside the thymus [46]. While CD4 expression has
known functional consequences during iNKT activation [47,48], a
similar functional impact for CD8 expression has not been
described.
A hallmark of iNKT activation is the rapid production of a vast
array of cytokines and chemokines [49,50] including IFNc, TNFa,
TGFb, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-
21, RANTES, Eotaxin, MIP-1a, and MIP-1b(reviewed in
Matsuda et al. and Tessmer et al. [28,49]). CD4+iNKTs produce
both Th1 and Th2 cytokines, while CD42iNKTs generally
produce only Th1 cytokines [30,51] (reviewed in Kim et al. [42]).
Other iNKT effector functions include perforin/granzyme release
[28,51,52], and Fas/FasL-mediated cytotoxicity [28,49]. iNKTs
can play an important role in the activation and regulation of
multiple immune cell subsets (Figure 2), including NK, T cell,
regulatory T cell, and B cell activation [53–55]. Stimulation of
iNKT cells in conjunction with soluble T cell antigen enhances
both CD4+and CD8+antigen-specific responses via a mechanism
involving CD40 signaling [56]. Similarly, iNKT activation
improves antibody titres, substitutes for CD4+T cell help to B
cells, and enhances B cell memory in mice [57].
The functional plasticity of iNKT cells, combined with their
ability to modulate activation of other immune cell subsets,
suggests that they may play both a protective role in controlling
viral infection and a detrimental role in enhancing viral
pathogenesis. Here, we review the current understanding of the
roles of iNKT cells in human viral infections, with particular focus
on HIV and influenza infection (summarized in Table 2).
Chronic Viral Infections
Human Immunodeficiency Virus
CD4
+
iNKT depletion. iNKT cell frequency is significantly
reduced among HIV-1 positive individuals [45,58], with a specific
depletion of the CD4+iNKT subset compared to the CD42
subset [45]. Longitudinal analysis of pre-seroconversion and 1 year
and 5 year post-seroconversion samples demonstrated significant
Table 1. Human and mouse CD1d-restricted NKT cell subsets [130–134].
NKT Cell Subset Mouse Human
Type I TCR Va14-Ja18; Vb8.2/7/2 Va24-Ja18; Vb11
Subsets CD4+, DN CD4+, CD8+,DN
Ligand aGalCer aGalCer
Restriction CD1d CD1d
NK receptors NK1.1+/2CD161+/2
Type II TCR Va3.2-Ja9orVa8; Vb8 Diverse
Subsets CD4+, DN CD4+, CD8+
Ligand Sulfatide, lysosulfatide, lysophosphatidylcholine Sulfatide, lysosulfatide, lysophosphatidylcholine
Restriction CD1d CD1d
NK receptors NK1.1+/2CD161+
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iNKT loss within the first year of infection, with continual declines
by 5 years [58]. Expression of CCR5 and CXCR4 on CD4+
iNKTs [43,45,59] makes them susceptible to infection with R5-
tropic, X4-tropic, and primary isolate viruses [43,45,59], resulting
in the preferential depletion of CD4+iNKT cells during in vitro
infection [45]. The lack of change in CD42iNKT populations
during in vitro infection suggests that loss of the CD4+subset is not
largely due to CD4 downregulation. Replication of similar studies
in a number of cohorts has largely confirmed these initial
observations [60–63], although the impact of highly active
antiretroviral therapy (HAART) on iNKT cell reconstitution
remains controversial [58,64–66] and the kinetics of iNKT
reconstitution appear to be slower than that of conventional
CD4+T cells [62,67].
Although it is now agreed that iNKT cells, particularly CD4+
iNKTs, are depleted during HIV-1 infection, less data is
available to clarify the impact of this depletion on disease
progression and viral pathogenesis. While it appears that the
iNKT subset is involved in the host response to viral infection, it
is unknown whether iNKT activation could control HIV
replication and immune activation, or what role the iNKT
subset might play in anti-tumor responses and prevention of
opportunistic infections in immunocompromised hosts [68].
One study to date has demonstrated iNKT cell culture
supernatant inhibition of HIV p24 production during in vitro
CD4+T cell infection, which was shown to be IFNc-dependent
[61]. In a study of risk factors involved in developing cancer
among HIV-1 positive women, NKT cell frequency was
associated with a reduced risk of cancer [69]. While further
studies are required to assess the increased risk of progression or
co-infection, if any, associated with iNKT decline, it is clear that
both iNKT number and function are affected by HIV infection,
as discussed below.
iNKT dysfunction. Even among individuals with minimal
iNKT depletion during HIV-1 infection, the iNKT subset displays
functional impairment [61,64,70]. Both CD4+and CD42iNKTs
exhibit reduced proliferation and IFNc, TNFa, and IL-4 secretion
in response to aGalCer/IL-2/PMA stimulation [61,64,70], with
variable restoration among HAART recipients. Increased iNKT
expression of exhaustion marker programmed death (PD)-1 was
reported among HIV-1 positive individuals in one study [64], but
PD-1 levels did not significantly correlate with IFNcproduction or
proliferative capacity and PD-1 blockade did not restore iNKT
function. While Moll et al. suggest that this implies an irreversibly
exhausted phenotype, the expression and functional impact of
other inhibitory receptors such as 2B4, Tim-3, and LAG-3 on the
iNKT subset during HIV-1 infection remains unknown. As
evidence now suggests that the function of Tim-3 differs between
T cell and NK cell subsets [71,72], the precise impact of
exhaustion marker regulation on iNKT cells during infection
must be determined. Additionally, in the Vasan et al. study,
stimulations were carried out on iNKT-enriched PBMC cultures
that were B cell– and CD8+T cell–depleted [61]. Given that the
unique functional properties of CD8+iNKT cells and the ability of
B cells to present lipid antigen via CD1d are now appreciated, the
depletion of these subsets could influence the cytokine production
of the iNKT population. More data is also required to address the
dysfunction of CD8+versus DN iNKT subsets during HIV
infection, as studies to date have often failed to differentiate these
subsets.
Non-human primates and SIV infection. Other clues as to
the importance of iNKT activation during HIV-1 infection may
come from NHP studies of SIV infection. In vivo infection of
macaques with SHIV
mn229
and SIV
mac251
resulted in CD4+iNKT
depletion similar to human HIV-1 infection, and iNKT depletion
was tightly correlated to CD4 decline [73]. Animals capable of
Figure 1. Surface marker and cytokine expression of human iNKT cell subsets. Both subsets express CD161, a4b7, and high levels of
CXCR4. CD4+iNKTs preferentially express CCR4 and CD62L, and produce both Th1 and Th2 cytokines. CD42iNKTs preferentially express chemokine
receptors CCR1, CCR6, and CXCR6, as well as CD11a and NKG2D. This subset secretes predominately Th1 cytokines and more quickly secretes perforin
than the CD4+subset.
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viral control exhibited reduced CD4+iNKT decline, and iNKT
levels were inversely correlated with viral load. The similarities in
iNKT depletion between HIV and SIV infection provide a model
to investigate iNKT activation during natural control of infection.
Unlike macaques, Sooty mangabeys (SM) control immune
activation during chronic SIV infection and do not exhibit
progressive immunodeficiency. SM iNKT cells are either CD8+or
CD42CD82and express neither CD4 nor CCR5 [74]. As a
result, the iNKT subset is maintained following infection and
exhibits no impairment in IFNcproduction. Given the capacity of
SM iNKTs to produce IFNc, TNFa, IL-2, IL-13, and IL-10 and
to degranulate in response to aGalCer stimulation, the authors
speculate that iNKTs may play a role in controlling immune
activation in this model of natural infection. As murine iNKT IL-4
and IL-10 production can induce regulatory T cell (Treg)
development [75], the production of IL-10 by SM iNKTs is of
particular interest. Additionally, iNKT-pDC cross-talk during
mouse viral infection can induce naı
¨ve T cell differentiation into
Tregs, suggesting another potential mechanism of iNKT-mediated
Treg activation [76]. Maintenance of Tregs during SIV infection
is a characteristic of natural SIV control [77], and despite the low
frequency of peripheral iNKT cells, the role of iNKT activation in
promoting Treg maintenance and controlling immune activation
during infection should be further examined [74].
CD1d downregulation. While iNKT cells are depleted and
exhausted during HIV infection, CD1d expression is also
modulated by the virus itself. The HIV-1 protein Nef, responsible
for the downregulation of MHC-I A and B alleles [78], also
downregulates CD1d via a common tyrosine-based motif [79,80].
This downregulation was shown in vitro to reduce NKT activation
and IFNcsecretion after aGalCer stimulation [79,80].
Hepatitis
Murine NKT cells are highly enriched in the liver (comprising
10%–30% of T cells) [81,82], and murine models have clearly
demonstrated a crucial role for NKT cell activation in mediating
Figure 2. iNKT regulation of NK, T cell, and B cell activation. Presentation of lipid antigen to iNKT cells by DCs leads to iNKT activation and
upregulation of CD40L. CD40–CD40L interactions and iNKT cytokine secretion promotes DC activation and maturation, which in turn leads to antigen
cross-presentation and augmentation of CD4+and CD8+T cell responses. iNKT IFNcsecretion rapidly activates NK cells and induces further IFNc
secretion. iNKTs can substitute for CD4+T cell help in B cell activation through CD40–CD40L interactions, and iNKT activation improves antibody
titres and B cell memory responses. Finally, iNKT production of IL-2 induces regulatory T cell (Treg) proliferation, while Tregs can also inhibit iNKT
proliferation and functional responses.
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liver pathology in viral- and ConA-induced hepatitis [83]. While
many studies have focused on aGalCer-mediated iNKT activation
and autoimmune-like models of hepatitis, less is known about the
role of NKT and iNKT cells in control of acute and chronic
hepatitis B virus (HBV) and hepatitis C virus (HCV) infection in
humans. Although human iNKTs do not appear to be highly
enriched in the liver compared to peripheral blood, further
characterization of human hepatic iNKT subsets is required [84].
Unlike studies of stringently defined iNKT cells in HIV infection,
mouse hepatitis studies include a range of NKT subsets and
definitions, making it more difficult to draw comparisons from
study to study.
Hepatitis B virus. Transgenic mouse models of HBV
infection have suggested iNKT control of HBV replication
through hepatic IFNa/b/cinduction and NK activation
[85,86]. aGalCer-activated Va14+iNKT cells also enhance the
generation of HBV-specific cytotoxic T lymphocytes (CTLs)
following HBsAg-immunization [87], suggesting a potential
mechanism by which to promote viral clearance during chronic
infection. Studies of NKT function in human HBV infection are
currently lacking. Injection of aGalCer in a clinical trial resulted in
a significant decrease in iNKT (Va24+Vb11+) frequency following
treatment, but only one patient exhibited a sustained decrease in
HBV DNA levels [88]. Other HBV literature reports only on
NKT cells (defined as CD3+CD56+), a cell subset that does not
necessarily overlap with the iNKT subset. One group reported a
significant drop in NKT (CD3+CD56+) frequency in the first
weeks after hospital admission among acute hepatitis B patients,
and suggested trafficking of NKT cells to the liver as a potential
explanation [89], while a study in India reported significantly
increased NKT (CD3+CD56+/CD16+) frequency among acute,
but not fulminant, HBV cases [90]. Further characterization of
human NKT cells, including more specific delineation of iNKT/
NKT subsets, during acute and chronic HBV infection will be
required to understand their role in innate immune control of the
virus.
Hepatitis C virus. Description of peripheral and hepatic
iNKT cells during human HCV infection has been highly
inconsistent. One study reported significantly lower peripheral
blood iNKT (Va24+Vb11+) frequency among viremic compared
to aviremic HCV seropositive individuals and healthy controls
[91]. A similar depletion of hepatic Va24+iNKTs was observed
among cirrhotic HCV disease patients [92]. Conversely, other
studies reported no change in peripheral iNKT frequency between
healthy and seropositive individuals [93,94], nor any correlation
with serum HCV RNA titre [94]. Longitudinal analysis showed no
change in iNKT frequency following antiviral therapy, or
differences between responders and nonresponders [93].
Functional data suggests that iNKT cells may traffic to the liver
during HCV infection and acquire a fibrogenic cytokine
producing profile. CXCR3 is upregulated on iNKT cells among
HCV+patients [94], possibly due to the increased hepatic levels of
IP-10 and MIG during infection [95,96]. Following expansion of
iNKTs derived from HCV+individuals, IFNcproduction
negatively correlated and IL-4 positively correlated with serum
RNA titre, indicating a potential role for iNKTs in control of
HCV replication. Interestingly, iNKTs from HCV+patients
produced more IL-13 and tended toward greater Th2 cytokine
production [94]. Given that iNKT cells contribute to liver fibrosis
during chronic viral hepatitis via production of IL-4 and IL-13
[97–100], this data supports the idea of iNKT functional
modification toward a pathogenic cytokine secretion profile.
Latent/relapsing viruses. HSV. In the context of herpes
virus infections, evidence is emerging to support viral interference
of iNKT function. Kaposi’s sarcoma-associated herpesvirus
(KSHV) was the first to be shown to possess the ability to
downregulate CD1d expression, an effect mediated by the viral
modulation of immune recognition proteins MIR1 and MIR2
[101]. Similarly, herpes simplex virus type I (HSV-1) infection of
human peripheral monocytes and immature dendritic cells results
in rapid downregulation of CD1d expression via glycoprotein B
and US3 [102,103]. This downregulation results in decreased DC-
mediated activation of human NKT cell lines and is thought to
facilitate viral evasion of the iNKT-mediated immune response.
Interestingly, HSV infection of keratinoctyes does not induce
CD1d downregulation, but, through a contact-dependent mech-
anism, inhibits iNKT cytokine secretion and induces an anergic-
like iNKT phenotype [104]. The mechanism of inhibition was not
determined, but was not mediated by iNKT PD-1 or Tim-3
expression, suggesting the involvement of an additional, dominant
inhibitory pathway. Given the lack of effect of PD-1 or Tim-3
blocking in restoring iNKT function during viral infection (HSV
Table 2. Summary of iNKT studies in viral pathogenesis.
Mouse Human
Frequency Function Frequency Function
HIV N/A N/A Depletion of total and CD4+
subset [45,58]; variable recovery
after HAART [58,60,64–66]
Inhibition of IFNc, IL-4 and proliferation
[61,64,70]; iNKT cells demonstrate
anti-HIV activity [61]
HBV Increase in hepatic
type II NKT cells during
acute hepatitis [135]
Activation enhances HBV-specific
T cell responses [87]; promote IFNc-
dependent viral inhibition [85]
N/A N/A
HCV N/A N/A Variable depletion following
infection in viremic individuals
[91–94]
CXCR3 upregulation [94]; greater
Th2 cytokine production after
expansion [94]
HSV N/A Required for viral load control,
protection from mortality [105,106]
N/A CD1d downregulation reduces
iNKT activation [102]
Influenza N/A Activation promotes effective NK and
CD8+response [113]; control of viral
titre [111]
N/A Activation reduces the suppressive
capacity of MDSCs, improves
antigen-specific responses [110]
HAART, highly active antiretroviral therapy; MDSC, myeloid-derived suppressor cell.
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and HIV), it remains to be seen whether multiple inhibitory
receptors contribute in each case. The effect of NKT cells in the
early response to infection has been studied using a zosteriform
model of HSV-1 infection. iNKT-deficient mice have been shown
to suffer increased morbidity, enhanced spread of the virus in the
nervous system, and diminished ability to clear the virus [105].
In murine models, CD1d
2/2
mice exhibit significantly higher
HSV-1 viral load within dorsal root ganglia, larger skin lesions,
and greater neuronal death than wild-type mice, indicating that
efficient early viral control requires intact iNKT cells [106]. These
results could not, however, be replicated by another group using a
different viral strain [107], although the differences in virulence
between the viruses used in these studies is worth noting [108].
Similarly, susceptibility of mice to intravaginal challenge with
HSV-2 has been studied in several naı
¨ve knockout mouse strains.
The NKT-deficient mice exhibited intermediate mortality and a
10-fold lower lethal dose compared to wild-type mice [109].
Acute Viral Infection
Influenza
iNKT cells in host response to influenza infection. A
novel role for iNKT cells in modulating immune activity was
discovered when De Santo et al. reported the identification of
myeloid-derived suppressor cells (MDSCs) that could inhibit
influenza-specific immune responses and result in increased viral
titres and mortality [110]. The group demonstrated that in both
mice and humans, iNKT cells functioned to reduce the
suppressive capacity of the MDSCs and improved influenza-
specific responses (Figure 3). Similarly, activation of iNKT cells
boosted early innate immune responses and reduced viral titre
[111]. Although iNKTs have not previously been reported to
produce IL-22, Paget et al. recently reported that activation of DC
TLR7 and RIG-I during murine H3N2 infection results in IL-1b-
and IL-23-mediated signals that induce iNKT IL-22 secretion
[112]. While IL-22 production was not found to affect viral
replication, it did protect epithelial cells from damage in vitro.
Influenza infection of CD1d-deficient mice also suggests that
iNKT-mediated IFNcproduction is required for full NK and
CD8+T cell activation and antiviral activity [113], although these
results are inconsistent with other studies of CD1
2/2
mice [114].
In a high pathogenicity model of murine influenza infection,
iNKT cells were implicated in the control of infiltrating
inflammatory monocytes. Activated iNKT cells were also shown
to directly lyse infected monocytes in vitro [115]. Increased
consistency in the virulence of strains used in challenge
experiments and the genetic background of mouse strains will be
required in order to conclusively determine the effects of iNKT
activation during influenza infection. To our knowledge, only one
study has examined iNKT frequency during human influenza
infection, but did report a 20% decrease in absolute NKT counts
among severe cases of pandemic H1N1 infection [116].
Figure 3. iNKT modulation of myeloid-derived suppressor cells (MDSCs) elicited during influenza A infection. Influenza infection leads
to the expansion of the MDSC population (comprised of immature dendritic cells, immature macrophages, and granulocytes), which can inhibit T cell
proliferation in vivo and in vitro. iNKT cells suppress both the expansion of the MDSCs and the suppressive effect of MDSCs in a CD40-CD40L-
dependent manner [110].
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Use of aGalCer as a vaccine adjuvant to activate iNKT
cells. The vast majority of literature on iNKT cells in influenza
infection focuses on the role of aGalCer and iNKT activation as a
vaccine adjuvant in mouse models. Initially, it was shown that
nasal administration of aGalCer with the antigen PR8 HA
(influenza virus A/PR/8/34 (PR8, H1N1)) induced high levels of
systemic IgG and mucosal S-IgA Abs, high levels of IFN-cand IL-
4 both locally and systemically, and Ag-specific CTLs. These
responses were associated with complete protection against an
influenza viral challenge [117]. Subsequent studies documented
augmented influenza antibody responses induced by co-adminis-
tration of vaccine with aGalCer [57,118]. The study by Galli et al.
reported an increased influenza-specific CD4
+
T cell response
after co-administration of vaccine with the adjuvant. They
demonstrated that the adjuvant led to activation of iNKT cells,
which in turn resulted in antibody responses even in the absence of
CD4+T cells (MHC class II knockout mice), an effect not
reproduced by T cell adjuvants such as alum. The authors
concluded that iNKT cells can compensate for the absence of
CD4+T cell help [57].
Several lines of evidence also suggest that the stimulation of
iNKT cells influences the subsequent cell mediated response to
influenza. Administration of aGalCer with a high dose of an
inactivated, non-replicating virus had a strong iNKT activating
effect; however, this was accompanied by diminished peak CD8+
response to the immunodominant nucleoprotein epitope (NP
366
).
Interestingly, increased NP
366
-specific memory CD8+responses
were demonstrated after 6 weeks in the group that received the
adjuvant. Taken together, this study indicates a blunted antigen-
specific effector CTL response that is followed by an enhanced
CD8+recall [119]. Similarly, injection of aGalCer during murine
cytomegalovirus infection also resulted in increased CD8+central
memory cell frequency, further supporting a role for iNKT
activation in antigen-specific memory responses [120].
The potential for boosting of both antibody responses and
CD8+memory by stimulation of iNKT cells is appealing in the
context of providing cross-protection against emerging strains of
influenza. Use of aGalCer as an adjuvant for a live attenuated
NS1truncated vaccine has been shown to increase IgG, IgG1, and
IgG2a antibodies as well as IFN-csecreting CD8+T cells, in an
iNKT-dependent manner [121]. Indeed, cross-protection induced
by mucosal influenza vaccine along with iNKT cell adjuvant was
illustrated by high levels of nasal IgA and cross-protection against
a challenge with a non-vaccine strain [122]. Similarly, Lee et al.
used two aGalCer analogues with different cytokine release
profiles along with inactivated influenza vaccine and were able
to induce antibody responses and achieve better cellular immune
responses; however, the ability to induce cross-protection was not
directly studied [123]. Overall, the use of aGalCer as a vaccine
adjuvant to stimulate iNKT cell activation may result in an
enhanced mucosal antibody response, improved generation of
CD8+memory, and greater responses to recall antigen. aGalCer
may be a particularly useful adjuvant for mucosal immunizations,
as mucosal iNKT cells do not become anergic following activation,
in contrast to some cases of peripheral iNKT activation [124].
Biochemical modifications of CD1d ligands to produce aGalCer
analogues that elicit specific iNKT cytokine secretion profiles will
further enhance the utility of iNKT activation as immunotherapy
[125,126]. The fine-tuning of this technique to induce robust
memory and cross-protection against emerging influenza strains is
promising, and provides a new avenue for vaccine research.
Conclusions and Future Directions
Since the identification of iNKT cells just over a decade ago,
better characterization of CD4+and CD8+subsets and descrip-
tion of the growing list of roles they play in bridging innate and
adaptive responses has led to appreciation of their importance in
the orchestrated response to viral infections (summarized in
Table 2). Perhaps most impressive is the amount of information
that has been collected in the HIV field with ample evidence of the
targeting of these cells by the virus and specific viral effects on
CD1d expression, leading to early depletion and dysfunction of the
iNKT population. Many questions remain, however, with regards
to the kinetics of these changes immediately after acquisition of
HIV and the true potential of antiretroviral therapy to reverse
dysfunction. NHP studies may play an important role in
illuminating whether iNKT cells can contribute to protection
from infection at mucosal surfaces or to the control of immune
activation and disease progression. Determining whether iNKT
cells play a similar role in chronic HBV and HCV infections will
require a focus on studies of human infection and improved
consistency in the detection and definition of iNKT populations.
In contrast to the plethora of research in the context of HIV as
well as other chronic and persistent infections, a paucity of data is
available in the context of acute, resolving infections. The vast
majority of studies are based on murine models with obvious
limitations in their applicability to humans. An accumulation of
excellent studies focused on the ability of adjuvants directed at
activation of iNKT cells, and co-administered with influenza
vaccine formulations, to lead to the generation of a robust humoral
and cell mediated immunity is intriguing. Most of these studies use
mouse models but hold promise by demonstrating a mechanism
that may improve influenza vaccine’s ability to result in long
lasting CD8+memory and potentially lead to better cross-
protection against newly arising viral strains. As an appreciation
of the impact of iNKT activity on viral immunity continues to
increase, iNKT cells will likely be found to contribute to host
defence in a number of other viral infections. CD1d downregu-
lation appears to be a common immune evasion tactic among
viruses, and has also been identified in human papillomavirus
(HPV) infection [127]. Some evidence suggests the mast cell–
mediated recruitment of NKT cells to sites of dengue virus
infection [128] and a potentially detrimental role during patho-
genesis in mouse models of infection [129]. As we better
understand the mechanisms by which iNKT cells contribute to
viral immunity, the therapeutic potential of modulating their
activation and function will drive new research avenues.
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