You are on page 1of 11

REVIEWS

Harnessing invariant NKT cells in


vaccination strategies
Vincenzo Cerundolo, Jonathan D. Silk, S. Hajar Masri and Mariolina Salio

Abstract | To optimize vaccination strategies, it is important to use protocols that can


jump-start immune responses by harnessing cells of the innate immune system to assist the
expansion of antigen-specific B and T cells. In this Review, we discuss the evidence indicating
that invariant natural killer T (iNKT) cells can positively modulate dendritic cells and B cells,
and that their pharmacological activation in the presence of antigenic proteins can enhance
antigen-specific B- and T-cell responses. In addition, we describe structural and kinetic
analyses that assist in the design of optimal iNKT-cell agonists that could be used in the
clinical setting as vaccine adjuvants.

Primeboost vaccination
Repeated immunizations that
are administered when a single
application of a vaccine is
insufficient, using the same
vaccine preparation
(homologous primeboost)
or using different vaccine
preparations (heterologous
primeboost) to sequentially
stimulate a stronger immune
response. Prior exposure to
one vaccine strain can elicit
antibody and Tcell responses
to shared epitopes following
exposure to a second vaccine
strain, increasing the efficacy
of heterologous primeboost
regimens.

Tumour Immunology Group,


Weatherall Institute of
Molecular Medicine,
University of Oxford,
Oxford, OX3 9DU, UK.
Correspondence to V.C.
email: vincenzo.cerundolo@
imm.ox.ac.uk
doi:10.1038/nri2451
Published online
12 December 2008

Current vaccination strategies that are based on the use of


recombinant viruses are failing to elicit Tcell responses
that are similar to those generated by natural infections.
The limited success of recombinant viruses as delivery
vectors for vaccines is mainly due to their high immuno
genicity, which results in an overwhelming immune
response to the viralvector proteins rather than the
recombinant protein antigens. Although heterologous
primeboost vaccination strategies are designed to overcome
the immunodominance of vectorspecific Tcell responses,
current priming strategies, such as DNA priming, are
proving ineffective for the generation of large numbers of
antigenspecific Tcell responses in humans13.
Advances in molecular technology have allowed the
design of synthetic protein vaccines, which provide a level
of specificity that has not been possible with traditional
vaccines that are based on live attenuated pathogens or
whole inactivated organisms. Such specificity is offering
a basis for the design of Tcell therapies for infectious
diseases and cancer. Optimization of such vaccination
strategies to ensure the induction of immune responses
that are specific for recombinant protein antigens is of
paramount importance and requires a better understand
ing of the signals that coordinate immune responses
to infection. In an effort to overcome the limitation
of immunodominant virusspecific Tcell responses,
compounds that mimic these signals and activate innate
immune responses should be used as adjuvants for
vaccinations and in combination with subunit vaccines.
Initiation of dendritic cell (DC) activity during
infection occurs through the integration of a series of
instructive signals: from the pathogen itself, through
receptors for pathogenassociated molecular patterns

(PAMPs), such as Tolllike receptors (TLRs), and from


other antigenresponsive cells in the environment that
provide additional activating signals, such as the ligation
of CD40. Over the past few years, it has been shown
that invariant natural killer T (iNKT) cells, a subset of
Tcell receptor (TCR)+ T cells that are restricted by
CD1d molecules, can modulate DC and Bcell activity,
and can increase DCinduced B and Tcell responses
(FIG. 1a). It has also been shown that iNKT cells can
amplify TLRderived signals. So, it is thought that com
binations of compounds that activate iNKT cells and
compounds that stimulate immune cells through TLRs,
could potentially provide effective vaccine adjuvants.
In this Review, we summarize the evidence indicat
ing that the activation of iNKT cells bridges innate and
adaptive immune responses, resulting in the expansion
of antigenspecific B and Tcell responses, properties
that could be used to optimize vaccination strategies.

Activation of iNKT cells


iNKT cells are a unique population of T cells with
immunomodulatory properties that link innate and
adaptive immune responses (BOX 1; FIG. 1a). iNKT cells
develop in the thymus from haematopoietic precursors
and are selected by CD1d molecules on the surface of
CD4+CD8+ double positive (DP) cortical thymocytes.
iNKTcell development follows a pathway that branches
from mainstream Tcell development and leads to the
acquisition of a memoryactivated phenotype and the
capacity to rapidly secrete cytokines following engage
ment of their TCR. The molecular mechanisms of the
unique iNKTcell developmental pathway are being
revealed and have been extensively reviewed4,5.

28 | jANuARy 2009 | vOLuMe 9

www.nature.com/reviews/immunol
2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
a

Antibody secretion

iNKT cell
B cell

CD1d
Lipid

TCR

IFN and IL-4

CD40
CD40L

Microbial-derived
lipid

CD40

CD40L

CD1d

Lysosome

TCR

CD1d

TCR

DC

DC

iNKT cell

IL-4?

Microorganism

CD40L
CD27 CD70

CD40

TCR

OX40

OX40L

CD1d
MDSC

NOS2 production
Arginase 1 production

IFN

IL-12

NK cell

iNKT cell

CD8+
T cell

CD4+
T cell

IL-12, IL-18
and type I IFN

TCR

Endogenous
lipid
CD1d

TLR

Lytic function
IFN production

Proliferation
Lytic function
Cytokine production

DC

Lysosome

Figure 1 | Natural killer T cells interact with and modulate the function of many different cell types. a | Invariant
natural killer T (iNKT) cells directly and indirectly modulate the function of many other cell types, such as NK cells and
T cells. These interactions are bidirectional, as iNKT cells receive signals from antigen-presenting
cellsReviews
(APCs) and
vice
Nature
| Immunology
versa. Signals can be received through cell-surface receptors, such as T-cell receptors (TCRs) recognizing glycolipidCD1d
complexes, co-stimulatory receptors, such as CD40, CD27 and OX40 recognizing their ligands CD40L, CD70 and OX40L,
respectively, as well as through soluble mediators, such as cytokines. Activated iNKT cells inhibit the function of
myeloid-derived suppressor cells (MDSCs), which suppress immune responses with nitric oxide synthase 2 (NOS2) and
arginase 1. b | APCs infected with microorganisms can process microbial glycolipids and present them by CD1d molecules,
which results in iNKT-cell activation. c | Signalling through Toll-like receptors (TLRs) leads to the presentation of
endogenous glycolipids by CD1d molecules that, together with the secretion of interleukin-12 (IL-12), activate iNKT cells.
TLR-induced release of soluble mediators, such as IL-12, IL-18 and type I interferons (IFNs), can also activate iNKT cells in a
CD1d-independent manner. DC, dendritic cell.

unlike conventional CD4 + and CD8 + T cells,


iNKT cells are activated by endogenous or exog
enous lipid ligands (FIG. 1b,c), for example, those of
Sphingomonas spp. and Borrelia burgdorferi 610, which
are recognized directly by the iNKTcell TCR when
bound to CD1d. (FIG. 1b). This recognition is independ
ent of TLRmediated activation of antigenpresenting
cells (APCs) or interleukin12 (IL12) secretion.
Sphingomonas spp. are Gramnegative bacteria and
have a cell wall that lacks lipopolysaccharide (LPS)
but is rich in linked glycosphingolipids, which can
vary in the complexity and sequence of the linked
sugar, the acyl chain and the sphingoid base of the
ceramide lipid11. It has been suggested that the balance
between immunogenic glycosphingolipids, such as
glucuronosylceramides, galacturonosylceramides
(including the Sphingomonas cellwall antigen PBS30)7,8,

and nonimmunogenic glycosphingolipids, such as


tetrasaccharidecontaining glycosphingolipids 11, in
the cell wall of any given Sphingomonas strain may
serve as an immuneevasion mechanism 11. During
B. burgdorferi infection the lipid antigens that are recog
nized by iNKT cells are galactosyl diacylglycerols (FIG. 2).
Similar to results obtained with different Sphingomonas
strains, B. burgdorferidependent iNKTcell activation
varies depending on the length and saturation of the
galactosyl diacylglycerol acyl chains6. Other microbial
lipids that have been reported to activate subsets of
iNKT cells are the Leishmania donovani surface glyco
conjugate lypophosphoglycan12 and the Mycobacterium
leprae phosphatidylinositol tetramannoside (PIM4) 13
(FIG. 2), although iNKTcell recognition of PIM4 has
been shown only with purified PIM4 (ReF. 13) and not
with synthetic PIM4 (ReF. 6).

NATuRe RevIewS | immuNology

vOLuMe 9 | jANuARy 2009 | 29


2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
Box 1 | Phenotype of invariant natural killer T cells
Natural killer T (NKT) cells are a heterogeneous population of cells that express a Tcell receptor (TCR) and are restricted
by the CD1d molecule98. Most NKT cells (known as invariant NKT cells; iNKT cells) express an invariant TCR chain
(V14J18 in mice and the homologous V24J18 in humans) that is paired with a semiinvariant TCR chain (V2,
V7 or V8.2 in mice and V11 in humans). iNKT cells can be identified by staining with multimeric complexes of CD1d
molecules loaded with the lipid galactosylceramide (GalCer)99101. In mice iNKT cells are either CD4+ or double
negative (DN) for coreceptor expression, whereas in humans a CD8+ subset of iNKT cells also exists. It has been shown
that these subsets have slightly different functional properties: human DN and CD8+ iNKT cells are highly cytolytic and
produce T helper 1 (TH1)type cytokines, whereas CD4+ iNKT cells produce both TH1 and TH2type cytokines102,103 and
differentially regulate dendriticcell activity104. In addition, the antitumour activity of iNKT cells has been shown to apply
mainly to the liver DN subset in two mouse models105.
Although iNKTcell maturation in the periphery has previously been associated with the acquisition of NK1.1 expression,
a population of mature NK1.1 iNKT cells has recently been reported106. Furthermore, a CD4NK1.1 subset that produces
interleukin17 (IL17) and constitutively expresses IL23 and retinoicacidreceptorrelated orphan receptort (RORt) has
been identified58,107. IL17 secretion may contribute to their pathogenic role in several diseases108,109.
iNKT cells are found in abundance in the thymus, spleen, liver and bone marrow, which may be explained by their
expression of a chemokine receptor profile that is similar to that of TH1 cells which home to inflamed tissues. Few
iNKT cells express lymphnode homing receptors110113.

In addition to direct recognition of bacterial lipids,


human and mouse iNKT cells can be activated indirectly
(for example, during Salmonella enterica infection) by
stimulatory cytokines, such as IL12 and type I interfer
ons (IFNs). These are released by DCs following TLR
signalling 8,1416 and amplify the weak activating signals
that are generated from the ligation of self glycolipids
(BOX 2; FIG. 1c). APCderived cytokines can also acti
vate iNKT cells irrespectively of TCR engagement by
lipidCD1d complexes17.
In addition to bacterial infections and TLRdependent
signalling events, activation and expansion of iNKT cells
can be achieved with pharmacological compounds that
bind to CD1d molecules and are recognized by the
iNKTcell TCR. The first compound to be identified
as an iNKTcell activator was galactosylceramide
(GalCer) (FIG. 2), which is an active component of
glycosphingolipid extracts from the marine sponge Agelas
mauritianus and has been shown to have antitumour
activity in the mouse B16 melanoma model18,19.
In the past few years several analogues of GalCer
have been synthesized that can activate iNKT cells
both in vivo and in vitro (reviewed in ReF. 20) (FIG. 2).
The results of experiments carried out using these
compounds revealed that pharmacological activa
tion of iNKT cells is a more rapid and efficient way
to activate iNKT cells than the use of TLR agonists or
microbial injection68,21. However, synthetic compounds
that activate iNKT cells should be used cautiously, as
overstimulation of iNKT cells can result in iNKTcell
anergy 22,23 and lysis of GalCerloaded APCs following
TCR engagement 24,25.
B16 melanoma
A widely used experimental
mouse melanoma. B16
melanoma is poorly
immunogenic and therefore is
difficult to eliminate. Largely
because of this, B16
melanoma is a good model
for testing cancer
immunotherapies.

iNKT cells bridge innate and adaptive immunity


Recently, it has become clear that iNKT cells have
an important role in bridging innate and adaptive
immune responses, and that activation of iNKT cells
results in rapid DC and Bcell maturation and NKcell
activation (FIG. 1a). Initial studies showed that injection
of GalCer could promote the survival of mice with
melanoma9. It was subsequently found that iNKT cells
are important mediators of cancer immunosurveillance,

as shown by the increased susceptibility of iNKT


celldeficient mice to methylcholanthreneinduced
tumours; these mice have both an earlier onset and
a higher incidence of tumours 26. Although there is
compelling evidence that iNKT cells have an impor
tant role in immunosurveillance, the mechanisms
by which iNKT cells control tumour growth remain
unclear. Indeed, direct recognition of tumour cells by
iNKT cells is not required for rejection, as tumours
that lack CD1d expression can be rejected in wildtype
mice27, which indicates that iNKT cells might promote
tumour rejection indirectly. It is probable that the
iNKTcelldependent production of T helper 1 (TH1)
type cytokines18, which favour NKcell activation28 and
inhibit tumour angiogenesis29, support tumour elimi
nation. Recent results showing that iNKT cells can
counterbalance the suppressive activity of myeloid
derived suppressor cells (MDSCs)30 provide further
insights into the mechanisms by which iNKT cells
might contribute to tumour immunosurveillance
(BOX 3). Indeed, higher numbers of MDSCs are found
in the spleen and ascites fluid of tumourbearing
iNKTcelldeficient mice than wildtype mice31.
Stimulation of iNKT cells through recognition
of the GalCerCD1d complex results in the rapid
production of TH1 and TH2type cytokines, such as
IFN and IL4 (ReFs 18,32), and the increased expres
sion of CD40 ligand (CD40L)33 (FIG. 1a), which induces
DC maturation34,35. As DC maturation is required for
the initiation of adaptive immunity, it was proposed
that coinjection of iNKTcell agonists and antigens
could be used to expand antigenspecific responses.
Consistent with this hypothesis, we and others showed
that stimulation of iNKT cells with GalCer in vivo
significantly increases immune responses to coinjected
antigens3539. Cytokines, such as tumournecrosis factor
(TNF), type I IFNs and IFN, further enhance DC
maturation in trans 34,38 (FIG. 1a). It has recently been
shown that upregulation of the expression of CD70
(ReF. 40) and OX40 ligand41 by mature DCs is impor
tant for costimulating iNKT cells and for promoting
antigenspecific CD8+ Tcell responses.

30 | jANuARy 2009 | vOLuMe 9

www.nature.com/reviews/immunol
2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
a Natural ligands

O
HO

HN
OH
O

HO
HO

OH

OH

OH
O

OH
O

OH
O

HO
O

O
OH

HO

OH

HO

OH

OH

-Glucuronosylceramide (Sphingomonas spp.)

HO
HO

O
O

O
OH

HO

Glycolipid II (B. burgdorferi)

O
O

HO
HO

HO

Phosphatidylinositol tetramannoside
(M. leprae)

OH
O

HO
HO

HN

OH

HO

HO
HO

OH

HO

OH

HO

HO O
O

Isoglobotrihexosylceramide (endogenous)

O HO

OH
O

OH
O

b Synthetic ligands
O
HN
OH
O

OH

HN

OH

HO

OH

HO

-Galactosylceramide (synthetic or
marine sponge)
O
HN
OH
O

O
OH

HO

OH

HO

C20:2

OH

Threitolceramide

HO

OH

OH

HO

O
HN

OH
O

O
OH

HO

OCH9

OH

-C-galactosylceramide
O

OH

OH
O

OH

HO

OH

H2
C

HN

OH

OH

HO

HO

Figure 2 | Examples of invariant-natural-killer-T-cell agonists. a | Numerous invariant natural killer T (iNKT)-cell


natural agonists have been characterized. Isoglobotrihexosylceramide is an endogenous glycolipid115. Examples of
microbial ligands include Borrelia burgdorferi -galactosyldiacylglycerol (glycolipid II), Sphingomonas spp.
-glucuronosylceramide (PBS-30) and Mycobacterium leprae phosphatidylinositol tetramannoside (PIM4)68,13. b | Synthetic
Nature Reviews | Immunology
ligands include -galactosylceramide, which can also be obtained from the marine sponge Agelas mauritianus18, -C61
25
galactosylceramide , OCH9 (ReF. 59), C20:2 (ReF. 63) and threitolceramide .

The adjuvant effect of iNKTcell stimulation is further


increased by the addition of TLR agonists38,42, suggesting
that signals transmitted by iNKT cells to DCs can amplify
the effect of pathogeninduced signalling. Indeed, TLR
and CD40 signalling seem to be highly coordinated, as
triggering of CD40 in vivo with CD40specific antibod
ies in the absence of microbial stimulation results in low
levels of IL12p70 production. Similarly, inflammatory
signals in the absence of TLR stimulation fail to prime
functional CD4+ Tcell responses43. Clearly, the timely
provision of iNKTcellderived signals is important for
optimizing DC and, consequently, Tcell responses.
In addition to promoting the generation of potent
antigenspecific CD4+ and CD8+ Tcell responses35,3739,
iNKTcell activation can also provide help to B cells,

inducing Bcell maturation, higher antibody titres and


expansion of the Bcell memory pool25,42,44,45. More
recently, Bcell receptor (BCR)mediated uptake of
CD1drestricted antigens was shown to be an effec
tive means of enhancing iNKTcelldependent Bcell
responses in vivo46,47. Targeting of iNKTcell ligands to
antigenspecific B cells can be achieved by using anti
gen and iNKTcell agonists bound to particles, such
as silica beads46, or by synthesizing Bcell antigens
linked to GalCer 47. This mediates the production
of high titres of specific IgM and early classswitched
antibodies. These results indicate that iNKT cells
can facilitate Bcell activation in response to particu
late iNKTcell agonists, thereby enhancing specific
antibody responses.

NATuRe RevIewS | immuNology

vOLuMe 9 | jANuARy 2009 | 31


2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
Box 2 | Invariant-natural-killer-T-cell endogenous ligands
The nature of the antigen (or antigens) mediating invariant natural killer T (iNKT)cell selection in the thymus is still
uncertain. Evidence from a glucosylceramidesynthasedeficient cell line that cannot stimulate V14+ iNKT cells
suggested that the natural ligand is a glycosphingolipid114. The endogenous glycosphingolipid isoglobotrihexosyl
ceramide (iGb3) (FIG. 2), an agonist for human and mouse iNKT cells115, was initially considered to be the only natural
selecting ligand for these cells, as mice deficient in the lysosomal enzyme hexosaminidase, which generates iGb3 from
iGb4, had impaired iNKTcell development115.
However, subsequent work revealed that mice lacking other lysosomal enzymes that are involved in glycosphingolipid
catabolism and in cholesterol transport have severe defects in iNKTcell development (reviewed in ReF.116). It is probable
that altered lipid trafficking and lysosomal processing, which are common to all these mutant mice, account for the
defect in iNKTcell development. Indeed, analysis of iGb3synthasedeficient mice did not reveal any anomaly in the number
or function of iNKT cells117. iGb3 was not detected by a sensitive highperformance liquid chromatography in the thymi
of these mice or in their dendritic cells (DCs)118, despite an earlier report that it may mediate the recognition of
lipopolysaccharidematured DCs8. Finally, iGb3 was not detected in any human tissue118, which is consistent with the
observation that humans lack a functional iGb3 synthase owing to several mutations in the gene that encodes it119.
More recently, however, the presence of iGb4 has been detected in the human thymus using mass spectrometry120.
Therefore, the biochemical identification of iGb3 remains an open question.
The identity of the lipid (or lipids) that mediates iNKTcell activation in the periphery is also under investigation. It has
recently been shown that Tolllike receptor (TLR)mediated activation of DCs enhances the expression of transcripts of
several glycosyltransferases that are involved in the biosynthesis of glycosphingolipids15,16, and mouse TLRstimulated
DCs produce a charged linked glycosphingolipid that can activate iNKT cells together with type I interferon15. Other
endogenous antigens that have been reported to activate subsets of mouse iNKT cells are phospholipids121 and the
gangliosides GD3 and GM3, which are often overexpressed in melanomas and other tumours of neuroectodermal origin122.

Cytokine storm
A strong systemic immune
response that results in the
release of high levels of
inflammatory mediators (such
as cytokines, oxygen free
radicals and coagulation
factors). Both proinflammatory
cytokines (such as
tumournecrosis factor,
interleukin1 (IL1) and IL6)
and antiinflammatory
cytokines (such as IL10 and
IL1 receptor antagonist) are
increased in the serum of
patients experiencing a
cytokine storm.

The activation of iNKT cells with GalCer and the


subsequent expansion of antigenspecific B and T cells
following injection of recombinant proteins is achiev
able only within a short time period; maximal efficacy
is observed only when the antigen and GalCer are
administered simultaneously 35. Consistent with a role
for DCs in enhancing iNKTcell activation in vivo, it was
also shown that the duration of cytokine production fol
lowing GalCer injection was significantly increased by
injecting GalCerloaded bonemarrowderived DCs,
compared with the injection of free GalCer 48,49.
iNKTcelldependent induction of antigenspecific
B and Tcell responses can be observed following the
injection of GalCer through several routes, including
intranasal50,51 and oral administration38,52. By contrast,
because of the dichotomy between rapid iNKTcell
dependent DC maturation and the length of time that
is required for the expression of antigens encoded by
plasmid DNA, it remains unclear whether intramuscular
injection of plasmid DNA with GalCer can result in
the enhancement of antigenspecific immune responses.
However, recent results indicate that intramuscular co
administration of GalCer and plasmid DNA encoding
the HIv1 protein Gag can facilitate the expansion of
Gagspecific immune responses53.
In addition to the ability of iNKT cells to facilitate
DC maturation and Bcell activation, we have recently
shown that iNKT cells can increase antigenspecific
immune responses by regulating the suppressive activity
of CD11b+GR1+ MDSCs54, which increase in number
during inflammation30. we observed greater numbers
of MDSCs during infection with influenza A virus in
iNKTcelldeficient and CD1ddeficient mice than
wildtype mice, suggesting that iNKT cells have a role in
regulating the frequency and activity of MDSCs during
infection with this virus30. Consistent with these find
ings, we showed that adoptive transfer of iNKT cells to

iNKTcelldeficient mice abolished the suppressive activity


of MDSCs by reducing the activity of nitric oxide synthase
and arginase 1, thereby rescuing the ability of these mice
to clear influenza A virus infection. we also showed that
myeloid cells with a suppressive phenotype were present
in the peripheral blood of individuals that were infected
with influenza A virus and that iNKT cells could abol
ish their suppressive phenotype. These results highlight
a new role for iNKT cells in modulating immunological
suppressive mechanisms that are activated during acute
inflammatory processes30.

Cytokine secretion and iNKT-cell anergy


Activation of iNKT cells results in TCR downregula
tion, proliferation and prolonged cytokine secretion5557.
In addition to the secretion of IL4 and IFN, activated
iNKT cells have also been shown to produce IL2, IL3,
IL5, IL6, IL9, IL10, IL13, IL17, IL21, TNF, trans
forming growth factor (TGF) and granulocyte/
macrophage colonystimulating factor (GMCSF), as well
as a wide range of chemokines5,58. It is becoming clear
that the repertoire of TH1 and TH2type cytokines that
is produced by iNKT cells is modulated by the strength
of iNKTcell TCR signalling events24,5961 and by the type of
APC presenting the iNKTcell agonists49,62,63. Activation
of iNKT cells by strong agonists such as GalCer, which
has a high affinity for the iNKTcell TCR and a long half
life24,64,65, results in high levels of cytokine production.
This is further amplified by iNKTcelldependent DC
maturation and NKcell activation. As such an exces
sive production of cytokines could potentially cause a
cytokine storm, the activation of iNKT cells needs to be
tightly regulated.
Importantly, overstimulation of iNKT cells by strong
agonists results in DC lysis24,25 and activationinduced
iNKTcell anergy (defined as unresponsiveness to sub
sequent stimuli). iNKTcell anergy has been observed

32 | jANuARy 2009 | vOLuMe 9

www.nature.com/reviews/immunol
2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
Box 3 | Non-invariant natural killer T cells
A second population of natural killer T (NKT) cells exists (known as type II NKT cells),
which is CD1d restricted but does not express an invariant Tcell receptor (TCR) and
lacks reactivity to galactosylceramide. Type II NKT cells were first described in 1995
(ReF. 123), but their characterization has been limited because of the lack of specific
markers and agonistic reagents. More recently, a fraction of type II NKT cells has been
shown to be reactive to the selfglycolipid sulphatide and has been detected by the
ability of these cells to bind sulphatideloaded CD1d tetramers124. Type II NKT cells
have an important immuneregulatory role in infection, autoimmune disease and
tumour immunosurveillance (recently reviewed in ReF.125). In contrast to invariant
iNKT cells, type II NKT cells suppress antitumour immunity, thereby promoting the
activation of myeloidderived suppressor cells through the production of interleukin13.

after injection of GalCer 22,23, of bacteria that can


stimulate iNKT cells and of certain TLR ligands21,66.
The molecular mechanisms that control iNKTcell
unresponsiveness are still ill defined. However, iNKT
cell unresponsiveness is similar to conventional Tcell
anergy67 in that it is long lasting (up to 46 weeks), it
can be reversed by the administration of IL2 and it is
cell intrinsic2123,66. IL12 is required for bacterium but
not GalCerinduced iNKTcell unresponsiveness21,
and alteration of the expression of NKcell receptors
may contribute to the hyporesponsive phenotype
that is observed in some experimental systems 21,68.
Following restimulation of anergic iNKT cells, there is
a bigger reduction in the secretion of IFN than of IL4
(ReFs 21,22), which may explain the efficacy of repeated
injection of GalCer in ameliorating autoimmunity in
disease models that are driven by a TH1cell response,
such as type 1 diabetes.
It is worth noting that, in contrast to the injection of
free GalCer, repeated injections of GalCerloaded
DCs do not seem to induce iNKTcell unresponsiveness
in mice48 and humans69.

T-cell anergy
A state of Tcell
unresponsiveness to
stimulation with antigen.
Tcell anergy can be induced
by stimulation with a large
amount of specific antigen
in the absence of the
engagement of costimulatory
molecules.

Structure and function of iNKT-cell agonists


Combined structural, kinetic and functional studies on
the molecular mechanisms of iNKTcell activation are
paving the way for the identification of optimal iNKT
cell agonists that could be used as vaccine adjuvants. The
crystal structures of human and mouse CD1d in complex
with GalCer 70 (FIG. 3) or the variant agonist PBS25
(ReF. 71), which has an 8carbon acyl chain instead of the
long 26carbon fattyacid chain of GalCer, revealed
a narrow and highly hydrophobic antigenbinding
groove that is characterized by two channels, A and F.
GalCer saturates the binding capacity of the CD1d
binding groove, the 26 carbon atoms of the acyl chain
filling the A channel and the 18 carbon atoms of the
phytosphingosine chain that fits into the Fchannel70,72
(FIG. 3c). The structure of the CD1d binding groove is
markedly different from that of the shallower, wider
and multipocketed grooves of the classical MHC mol
ecules (FIG. 3d), which only accommodate the anchoring
sidechain residues of presented peptide antigens73.
In addition to GalCer 70,72 and PBS25 (ReF. 71), the
structures of CD1d in complex with iNKTcell ago
nists galacturonosylceramide74 and the endogenous
ligand isoglobotrihexosylceramide (iGb3)75 provide a

thorough characterization of different binding surfaces,


all of which can be recognized by the semiinvariant
TCRs of iNKT cells.
Borg and colleagues72 crystallized a human iNKT
cell TCR with an GalCerCD1d complex, providing
insight into the interactions between the molecules and
the chain residues that are involved in the process. They
described a unique lockandkey mode of interac
tion, whereby the iNKTcell TCR docked in parallel
to the Fchannel end of the CD1d antigenbinding
pocket 72 (FIG. 3a). This is different to the interaction
that has been described for TCRpeptideMHC
complexes, in which both the hypervariable segments
complementaritydetermining region 3 (CDR3)
and CDR3 of the TCR adopt a more central position
relative to the binding groove (FIG. 3b). The CDR2 loop
of the iNKTcell TCR mainly interacts with the CD1d
backbone, and germlineencoded residues of both the
CDR1 and CDR3 loops contribute to the recognition
of GalCer by interacting with CD1d and the bound
GalCer molecule. This form of recognition resembles
that of a patternrecognition receptor and explains
the use of the invariant v24j18 segments by all
iNKTcell TCRs (v14j18 in mice)72,76,77.
Several research groups have carried out a series of
kinetic and functional experiments to assess the role
of the polar head and the length and saturation of the
GalCer alkyl chains in controlling the rate of dis
sociation of lipids that are bound to CD1d molecules
and the affinity of binding of lipidspecific TCRs. The
knowledge derived from the crystallographic studies
has provided a framework to understand the properties
of the different compounds. In the next sections, we sum
marize some of the conclusions that have been drawn
from these experiments.
Modifications of GalCer lipid chains. The results from
experiments carried out using either GalCer analogues
or bacteriumderived iNKTcell agonists that had modi
fied lipid tails suggested three conclusions. First, the
length of the acyl and phytosphingosine chains affects
the stability of the lipidCD1d complex 6,11,24,7882. Second,
the level of saturation of the alkyl chains influences the
rate of lipid dissociation from CD1d molecules24,63.
Third, the length of the lipid chain occupying the
F channel modulates the affinity of the iNKTcell TCR
for the glycolipidCD1d complex 24,59,8082.
The effect of lipid length and saturation on the stability
of the lipidCD1d complex is consistent with structural
features of the A channel, which follows a curved path
circumventing a pole formed by residues cysteine 12
and phenylalanine 70 (ReFs 7072)(FIG. 3c). These results
suggest that a preformed kink in the acyl chain caused
by the presence of unsaturated bonds might stabilize
binding of the lipid by favouring the tightly curved con
formation that is required for binding to the A channel.
Indeed, compounds with unsaturated bonds in the acyl
chain occupying the A channel can be directly loaded
onto cellsurfaceexpressed CD1d molecules, resulting in
their presentation by nonprofessional APCs and in a bias
towards the secretion of TH2type cytokines63,83,84.

NATuRe RevIewS | immuNology

vOLuMe 9 | jANuARy 2009 | 33


2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
b

a
C

TCR

iNKT-cell TCR

V
-GalCer

Peptide

CD1d

HLA-A2

2m

2m

c
Lipid chains
F70

CDR
loops

F84

Peptide
131
C12
A channel

V98
F114

V116
F channel

e -GalCer

f Threitolceramide
F29

CDR3

R95

HLA-A2

CDR1
G96

F29

CDR2

S30

S30 F51

G96

3'-OH
4'-OH

2'-OH

R95
W153

F51

3'-OH
4'-OH

2'-OH
W153

Figure 3 | Structural features of the interactions between the T-cell receptors


of invariant natural killer T cells and lipidCD1d complexes.
| Comparison
of
Naturea,b
Reviews
| Immunology
the structure of the complex comprising galactosylceramide (-GalCer)CD1d and the
invariant natural killer T (iNKT)-cell T-cell receptor (TCR) with the structure of the complex of
the JM22-TCR with peptide 5866 from influenza virus matrix protein in the binding groove
of an HLA-A2 molecule. Significant differences were revealed in TCR recognition of the
antigen. c | The side view of -GalCer bound to the CD1d groove; channels A and F are
indicated in grey, -GalCer is indicated in orange and key residues of CD1d are indicated in
blue. d | A side view of peptide 157165 from the NY-ESO-1 tumour antigen (yellow) in the
binding groove of the HLA-A2 molecule (grey) is shown with complementarity-determining
region (CDR) loops from the cognate TCR above the peptide. Structures c and d highlight
the depth of the channels in CD1d molecules that anchor the fatty-acid tails (c) compared
with the shallow peptide-binding groove in MHC class I molecules (d). e | Hydrogen bonds
that stabilize the TCR-GalCerCD1d complex are shown. f | Molecular modelling of the
TCRthreitolceramideCD1d complex is shown25, based on the structure shown in part e.
The numbers of stabilizing hydrogen bonds with F29, S30 and G96 residues in the TCR are
shown in parts e and f. The human CD1d molecule is indicated in green, the ligands are
indicated in magenta and CDR1 and CDR3 of the iNKT-cell TCR are indicated in yellow
and cyan, respectively. 2m, 2-microglobulin; C, constant region; V, variable region.
Part a is reproduced, with permission, from ReF.72 (2007) Macmillan Publishers Ltd.
All rights reserved. Part b is reproduced, with permission, from ReF. 126 (2003) Macmillan
Publishers Ltd. All rights reserved. Part c is reproduced, with permission, from ReF.70
(2005) Macmillan Publishers Ltd. All rights reserved. Part d is reproduced, with permission,
from ReF. 127 (2005) The Rockefeller University Press. Parts e and f are reproduced, with
permission, from ReF. 25 (2008) The American Association of Immunologists.

The effect of variations in the length of the lipid tail


that occupies the F channel on iNKTcell TCRbinding
affinity is consistent with results from analyses using
sitedirected mutagenesis of human 77 and mouse 76
iNKTcell TCRs. These analyses revealed that the region
directly above the F channel provides a binding hot
spot for the iNKTcell TCR. These results suggests that
iNKT cells recognize both the group head and the length
of lipid antigens, thereby ensuring greater specificity of
antigen recognition.
In agreement with the results that highlight the impor
tance of the length of the lipid chains occupying the
F channel, stimulation of mouse iNKT cells with the com
pound OCH9 (FIG. 2), which has a shorter phytosphingo
sine chain, induced higher levels of IL4 secretion than
stimulation with GalCer and was effective at treating
mouse models of T H1cellmediated autoimmune
disorders, such as experimental autoimmune encephalo
myelitis59,85. Although OCH9 does not alter the cytokine
profile of human iNKT cells24 and does not skew antigen
specific Tcell responses towards IL4 secretion when
used to promote iNKTcelldependent Tcell activation
in mice38, in other experimental systems it has been shown
that early secretion of IL4 by iNKT cells is required to
prime naive CD4+ T cells to differentiate into TH2 cells86.
In summary, these results indicate that the length
of lipid chains occupying the Fchannel should be 18
carbons and the acyl chain occupying the A channel
should be 26 carbons and saturated to obtain optimal
stability of the loaded CD1d molecule and fully exploit
its binding capacity. This ensures prolonged duration
of iNKTcell stimulation and optimal presentation by
professional APCs.
Modifications of the GalCer polar head. Three hydro
gen bonds between human CD1d and GalCer at the
junction of the two alkyl chains and the polar head group
serve to anchor GalCer in a distinct orientation and
position it within the lipidbinding groove70. The 2OH
of the galactose ring, which is crucial for the antigenic
ity of GalCer, forms a hydrogen bond with aspartic
acid 151. The 3OH on the sphingosine chain forms a
hydrogen bond with aspartic acid 80 and the glycosidic
linkage 1O of GalCer forms the third hydrogen bond
to threonine 154. Surprisingly, a Cglycosidic variant of
GalCer (CGalCer), in which the glycosidic link
age 1O of GalCer is replaced by CH2 (FIG. 2), was a
more potent adjuvant than GalCer in mouse models
of malaria and metastatic melanoma61 and could gener
ate more prolonged immune responses and greater levels
of IFN secretion. This was possibly because its binding
to CD1d in DCs was more stable and it also induced
increased NKcell activation61,87. Although no kinetic
measurements of CGalCer have been carried out, it
is possible that the Cglycosidic linkage may provide a
more rigid glycosyl head group and/or increase resist
ance to enzymatic degradation, which would make its
association with CD1d more stable61. Further studies are
therefore warranted to clarify the mechanisms that are
responsible for this increased potency of the Cglycosidic
variant of GalCer.

34 | jANuARy 2009 | vOLuMe 9

www.nature.com/reviews/immunol
2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
Initial experiments carried out to establish the con
tribution of the glycosidic portion of GalCer to iNKT
cell activation showed that substitution of galactose with
glucose reduced activation and that mannosylceramide
or GalCer were not recognized by iNKT cells 18.
Although more recent results have shown that linked
glycosphingolipids, such as some forms of anomeric
GalCer, can stimulate iNKT cells88,89, it remains unclear
whether these results were due to the potential contami
nation by linked glycosphingolipid that can occur
during the synthesis of these compounds. Additional
studies correlating structure with function indicated
that the glycolipidCD1dTCR interaction tolerates a
small molecule at the carbon 6 position on the sugar of
GalCer, but that iNKTcell activation is inhibited by
other substitution patterns (reviewed in ReF. 20).
until recently, all the known iNKTcell agonists
contained a glycosidic group in the polar head. we have
now shown that iNKT cells can also recognize com
pounds that lack a sugar head and have nonglycosidic
linkages between the hydrophilic head and the ceramide
moiety 25 (FIGs 2,3f). These compounds comprise a new
class of iNKTcell agonists, and this broadens the range
of compounds that can be recognized by these cells. Of
particular interest is threitolceramide, which is similar to
GalCer in that it stably binds CD1d molecules owing to
the optimal length of its lipid chains and it is likely to form
the three hydrogen bonds with CD1d molecules because
of the presence of 4OH, 3OH and 2OH residues in the
head group25 (FIG. 3f). we have shown that threitolceramide
can induce optimal DC maturation and antigenspecific
B and Tcell priming, but does not cause extensive iNKT
celldependent lysis of DCs, as is observed with GalCer.
This is probably because of its weaker binding affinity to
the iNKTcell TCR25. Furthermore, iNKT cells that have
been activated by threitolceramide recover more quickly
from anergy than those stimulated by GalCer 25.

Harnessing iNKT cells in the clinic


The evidence indicating that the harnessing of mouse
iNKT cells increases antigenspecific immune responses
by bridging innate and adaptive immunity provides the
basis for designing immunotherapeutic protocols to
potentiate immune responses against pathogens and
tumours, and possibly to modulate autoimmune responses
(reviewed in ReF. 90). In this section, we summarize the
clinical trials that have been carried out so far.
Intravenous injection of GalCer. A doseescalation
study using GalCer was carried out in patients
with solid tumours, who were injected intravenously with
504,800 mg per m2 GalCer 91. Consistent with results
obtained in mouse models, the frequency of iNKT cells
in the blood decreased following injection, presumably
as a result of activationinduced TCR downregulation
or of their migration to surrounding tissues following
activation by GalCer. GalCerdependent activa
tion of iNKT cells led to increased levels of IFN, IL12
and GMCSF in the serum of a few of the patients, and
this depended on the frequency of iNKT cells in the
prevaccination samples91.

Injection of GalCerloaded DCs. So far, four Phase I


clinical trials have been carried out using DCs pulsed
with GalCer. In the first Phase I trial autologous
immature monocytederived DCs that had been pulsed
with GalCer were administered intravenously to
patients with metastatic tumours 92. Similarly to the
results obtained from intravenous immunization with
GalCer 91, the frequency of iNKT cells transiently
decreased 12 days after treatment. A transient decrease
in the frequency of NK, B and T cells was also observed.
Increased serum levels of IFN and IL12 were detected
after vaccination, along with the activation of NK and
T cells. Interestingly, although adoptively transferred
DCs were initially found in the lungs, they migrated
to the liver and spleen within 24 hours92. A decrease
in tumour markers in the serum of two patients was
also reported.
In the second clinical trial, patients with nonsmall
cell lung cancer were immunized intravenously up to
four times with more than 5 x 107 immature DCs per m2
that were loaded with GalCer 93. Injections were gen
erally well tolerated and a significant increase in the
frequency of iNKT cells after two injections of DCs was
reported in one patient. The levels of mRNA encoding
IFN in circulating iNKT cells were also increased.
A marked increase in iNKTcell number was
observed in another Phase I clinical trial, in which five
patients with myeloma were immunized three times
intravenously with mature DCs69. The first injection
used unpulsed DCs to establish a baseline for the fre
quency of iNKT cells, and subsequent injections were
carried out with GalCerloaded DCs. Injection of
GalCerloaded DCs led to an increase of more than
100fold in the number of iNKT cells in some patients,
which was detectable for up to 6 months after vaccina
tion. In addition, the investigators detected increased
serum levels of the cytokines that are associated with
DC maturation, such as IL12, and an increase in the
frequency of cytomegalovirus (CMv)specific CD8+
T cells. These data indicated that injection of DCs loaded
with GalCer can lead to the activation of APCs in vivo,
resulting in enhanced antigenspecific Tcell responses,
and suggested that iNKTcell agonists may also facilitate
the expansion of antigenspecific responses in humans,
as was previously described in mice. This study also
showed that following immunization with GalCer
loaded DCs, three patients had clinically relevant
decreases in the level of monoclonal immunoglobulins
(M protein) in the serum or urine and one patient
showed stabilization of the disease69.
Finally, patients with head and neck cancer were
vaccinated with GalCerloaded autologous imma
ture DCs, which were administered into the nasal sub
mucosa94. The immunizations induced the expansion
of iNKT cells and enhanced NKcell activity in some
patients. In this study, the cells used were a mixture of
APCs that were derived from peripheralblood mono
nuclear cells, and the percentage of DCs administered
in each case varied from 30% to 55%. A partial reduction in
tumour size was reported in one patient and stabilization
of disease was observed in five out of nine patients.

NATuRe RevIewS | immuNology

vOLuMe 9 | jANuARy 2009 | 35


2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
Immunization with in vitro expanded iNKT cells.
Similarly to the clinical trials that have been carried
out using in vitro expanded peptidespecific T cells for
immunotherapy (reviewed in ReF. 95), a clinical trial
was performed using adoptively transferred iNKT cells.
Patients with nonsmallcell lung cancer received two
intravenous injections of up to 5 x 107 cells per m2 in vitro
activated (with GalCer and IL2) iNKT cells96. There
were no severe adverse events in any of the patients,
and in some cases increased frequencies of iNKT cells
were detected in blood samples postimmunization.
Administration of activated iNKT cells also induced IFN
production in vivo, mainly from NK cells. Nevertheless,
none of the patients had any notable positive clinical
response to this immunotherapy.

Conclusions and future perspectives


Since the discovery of iNKT cells over 20 years ago, it
has become clear that iNKT cells have extremely ver
satile responses to activation. The challenge remains to
understand how to activate and manipulate the differ
ent subpopulations of iNKT cells and how to translate
these results into the clinical setting to control the
presentation of iNKTcell agonists by different APCs
in different microenvironments both quantitatively
and qualitatively.
To date, no clinical trials have been carried out in
which iNKTcell agonists are coinjected with subunit
vaccines and only one clinical trial has looked indirectly
at the ability of GalCer to enhance the response of
the adaptive immune cells in the clinic69. Clinical trials

Donnelly, J. J., Wahren, B. & Liu, M. A. DNA vaccines:


progress and challenges. J. Immunol. 175, 633639
(2005).
2.
Hanke, T., Goonetilleke, N., McMichael, A. J. &
Dorrell, L. Clinical experience with plasmid DNAand modified vaccinia virus Ankara-vectored human
immunodeficiency virus type 1 clade A vaccine
focusing on T-cell induction. J. Gen. Virol. 88,
112 (2007).
3.
Smith, C. L et al. Recombinant modified vaccinia
Ankara primes functionally activated CTL specific for
a melanoma tumor antigen epitope in melanoma
patients with a high risk of disease recurrence.
Int. J. Cancer 113, 259266 (2005).
4.
Godfrey, D. I. & Berzins, S. P. Control points in
NKT-cell development. Nature Rev. Immunol. 7,
505518 (2007).
5.
Matsuda, J. L., Mallevaey, T., Scott-Browne, J. &
Gapin, L. CD1d-restricted iNKT cells, the Swiss-Army
knife of the immune system. Curr. Opin. Immunol. 20,
358368 (2008).
6.
Kinjo, Y. et al. Natural killer T cells recognize
diacylglycerol antigens from pathogenic bacteria.
Nature Immunol. 7, 978986 (2006).
7.
Kinjo, Y. et al. Recognition of bacterial
glycosphingolipids by natural killer T cells.
Nature 434, 520525 (2005).
8.
Mattner, J. et al. Exogenous and endogenous
glycolipid antigens activate NKT cells during microbial
infections. Nature 434, 525529 (2005).
9.
Mattner, J. et al. Liver autoimmunity triggered by
microbial activation of natural killer T cells. Cell Host
Microbe 3, 304315 (2008).
10. Sriram, V., Du, W., Gervay-Hague, J. &
Brutkiewicz, R. R. Cell wall glycosphingolipids of
Sphingomonas paucimobilis are CD1d-specific
ligands for NKT cells. Eur. J. Immunol. 35,
16921701 (2005).
References 68 and 10 show for the first time that
most iNKT cells are activated by glycosphingolipid
and diacylglycerol of bacterial origin.
1.

11.
12.

13.

14.

15.

16.

17.

18.
19.
20.
21.

should be carried out to assess whether results that have


been obtained in animal models, which show that iNKT
cell activation can enhance antigenspecific B and Tcell
responses, can be extended to humans.
Although numerous synthetic CD1dbinding ligands
have been described, it is important to ensure that the
iNKTcell agonists used in the clinic can be repeatedly
injected into patients without inducing iNKTcell over
activation, resulting in iNKTcell anergy and cytokine
storm. This could be achieved by identifying iNKTcell
agonists that have a reduced affinity for the iNKTcell TCR
and at the same time ensure optimal DC maturation.
The next generation of iNKTcell agonists should be
capable of targeting specific APC populations to ensure
preferential binding to DCs and/or antigenspecific
B cells. These properties could be achieved by either
modifying the structure of the synthetic iNKTcell
agonists to facilitate loading by either DCs or B cells
(as it was initially shown in ReF. 63) or by coupling the
iNKTcell agonists to celltypespecific antibodies.
Consistent with the latter possibility, recent data showed
that targeting of iNKTcell ligands to antigenspecific
B cells can be enhanced by linking them to known Bcell
antigens. This method uses BCRmediated uptake and
subsequent presentation of the glycolipid antigen in the
context of CD1d and enhancement of Bcell activity 46,47.
In addition, it has been shown that sustained activation
and tumourcell targeting of iNKT cells using a fusion
protein consisting of GalCerCD1d and scFv antibody
fragment specific for human epidermal growthfactor
receptor 2 induces an antitumour effect 97.

Kinjo, Y. et al. Natural sphingomonas glycolipids vary


greatly in their ability to activate natural killer T cells.
Chem. Biol. 15, 654664 (2008).
Amprey, J. L. et al. A subset of liver NK T cells is
activated during Leishmania donovani infection by
CD1d-bound lipophosphoglycan. J. Exp. Med. 200,
895904 (2004).
Fischer, K. et al. Mycobacterial phosphatidylinositol
mannoside is a natural antigen for CD1d-restricted
T cells. Proc. Natl Acad. Sci. USA 101, 1068510690
(2004).
Brigl, M., Bry, L., Kent, S. C., Gumperz, J. E. &
Brenner, M. B. Mechanism of CD1d-restricted natural
killer T cell activation during microbial infection.
Nature Immunol. 4, 12301237 (2003).
Paget, C. et al. Activation of invariant NKT cells by
Toll-like receptor 9-stimulated dendritic cells requires
type I interferon and charged glycosphingolipids.
Immunity 27, 597609 (2007).
Salio, M. et al. Modulation of human natural killer
T cell ligands on TLR-mediated antigen-presenting cell
activation. Proc. Natl Acad. Sci. USA 104,
2049020495 (2007).
Nagarajan, N. A. & Kronenberg, M. Invariant
NKT cells amplify the innate immune response to
lipopolysaccharide. J. Immunol. 178, 27062713
(2007).
References 1417 describe the different modes
of activation of iNKT cells: direct (refs 15, 16),
indirect (ref. 14) and CD1d independent (ref. 17).
Kawano, T. et al. CD1d-restricted and TCR-mediated
activation of v14 NKT cells by glycosylceramides.
Science 278, 16261629 (1997).
Morita, M. et al. Structureactivity relationship of
-galactosylceramides against B16-bearing mice.
J. Med. Chem. 38, 21762187 (1995).
Savage, P. B., Teyton, L. & Bendelac, A. Glycolipids for
natural killer T cells. Chem. Soc. Rev. 35, 771779
(2006).
Kim, S. et al. Impact of bacteria on the phenotype,
functions, and therapeutic activities of invariant

36 | jANuARy 2009 | vOLuMe 9

22.
23.

24.

25.
26.
27.

28.

29.

30.

NKT cells in mice. J. Clin. Invest. 118, 23012315


(2008).
Parekh, V. V. et al. Glycolipid antigen induces longterm natural killer T cell anergy in mice. J. Clin. Invest.
115, 25722583 (2005).
Uldrich, A. P. et al. NKT cell stimulation with glycolipid
antigen in vivo: costimulation-dependent expansion,
Bim-dependent contraction, and hyporesponsiveness
to further antigenic challenge. J. Immunol. 175,
30923101 (2005).
McCarthy, C. et al. The length of lipids bound to
human CD1d molecules modulates the affinity
of NKT cell TCR and the threshold of NKT cell
activation. J. Exp. Med. 204, 11311144
(2007).
Silk, J. et al. Non-glycosidic CD1d lipid ligands
activate human and murine invariant NKT cells.
J. Immunol. 180, 64526456 (2008).
Smyth, M. J. et al. Differential tumor surveillance by
natural killer (NK) and NKT cells. J. Exp. Med. 191,
661668 (2000).
Crowe, N. Y., Smyth, M. J. & Godfrey, D. I. A critical
role for natural killer T cells in immunosurveillance of
methylcholanthrene-induced sarcomas. J. Exp. Med.
196, 119127 (2002).
Carnaud, C. et al. Cutting Edge: cross-talk between
cells of the innate immune system: NKT cells rapidly
activate NK cells. J. Immunol. 163, 46474650
(1999).
Hayakawa, Y. et al. IFN--mediated inhibition of tumor
angiogenesis by natural killer T-cell ligand,
-galactosylceramide. Blood 100, 17281733
(2002).
De Santo, C. et al. Invariant NKT cells reduce the
immunosuppressive activity of influenza A virusinduced myeloid-derived suppressor cells in mice and
humans. J. Clin. Invest. 13 Nov 2008 (doi: 10.1172/
JCI36264).
This work highlights a new role for iNKT cells that
is based on their ability to relieve the suppressive
activity of MDSCs.

www.nature.com/reviews/immunol
2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
31. Renukaradhya, G. J. et al. Type I NKT cells protect
(and type II NKT cells suppress) the hosts innate
antitumor immune response to a B-cell lymphoma.
Blood 111, 56375645 (2008).
32. Spada, F. M., Koezuka, Y. & Porcelli, S. A. CD1drestricted recognition of synthetic glycolipid antigens
by human natural killer T cells. J. Exp. Med. 188,
15291534 (1998).
33. Vincent, M. S. et al. CD1-dependent dendritic cell
instruction. Nature Immunol. 3, 11631168 (2002).
34. Fujii, S., Liu, K., Smith, C., Bonito, A. J. & Steinman,
R. M. The linkage of innate to adaptive immunity via
maturing dendritic cells in vivo requires CD40 ligation
in addition to antigen presentation and CD80/86
costimulation. J. Exp. Med. 199, 16071618 (2004).
35. Hermans, I. F. et al. NKT cells enhance CD4+ and
CD8+ T cell responses to soluble antigen in vivo
through direct interaction with dendritic cells.
J. Immunol. 171, 51405147 (2003).
36. Gonzalez-Aseguinolaza, G. et al. Natural killer T cell
ligand -galactosylceramide enhances protective
immunity induced by malaria vaccines. J. Exp. Med.
195, 617624 (2002).
37. Fujii, S., Shimizu, K., Smith, C., Bonifaz, L. &
Steinman, R. M. Activation of natural killer T cells
by -galactosylceramide rapidly induces the full
maturation of dendritic cells in vivo and thereby acts
as an adjuvant for combined CD4 and CD8 T cell
immunity to a coadministered protein. J. Exp. Med.
198, 267279 (2003).
References 3537 show that co-injection of
-GalCer with antigenic proteins increases
antigen-specific B- and T-cell responses.
38. Silk, J. D. et al. Utilizing the adjuvant properties of
CD1d-dependent NK T cells in T cell-mediated
immunotherapy. J. Clin. Invest. 114, 18001811
(2004).
39. Stober, D., Jomantaite, I., Schirmbeck, R. & Reimann, J.
NKT cells provide help for dendritic cell-dependent
priming of MHC class I-restricted CD8+ T cells in vivo.
J. Immunol. 170, 25402548 (2003).
40. Taraban, V. Y. et al. Invariant NKT cells promote
CD8+ cytotoxic T cell responses by inducing CD70
expression on dendritic cells. J. Immunol. 180,
46154620 (2008).
41. Zaini, J. et al. OX40 ligand expressed by DCs
costimulates NKT and CD4+ Th cell antitumor
immunity in mice. J. Clin. Invest. 117, 33303338
(2007).
42. Hermans, I. F. et al. Dendritic cell function can be
modulated through cooperative actions of TLR ligands
and invariant NKT cells. J. Immunol. 178,
27212729 (2007).
43. Reis e Sousa, C. Dendritic cells in a mature age.
Nature Rev. Immunol. 6, 476483 (2006).
44. Galli, G. et al. CD1d-restricted help to B cells
by human invariant natural killer T lymphocytes.
J. Exp. Med. 197, 10511057 (2003).
45. Galli, G. et al. Invariant NKT cells sustain specific
B cell responses and memory. Proc. Natl Acad. Sci.
USA 104, 39843989 (2007).
46. Barral, P. et al. B cell receptor-mediated uptake
of CD1d-restricted antigen augments antibody
responses by recruiting invariant NKT cell help
in vivo. Proc. Natl Acad. Sci. USA 105, 83458350
(2008).
47. Leadbetter, E. A. et al. NK T cells provide lipid
antigen-specific cognate help for B cells. Proc. Natl
Acad. Sci. USA 105, 83398344 (2008).
References 4547 describe the ability of
iNKT cells to directly provide help to B cells.
References 46 and 47 describe two new protocols
to promote antibody responses in vivo by recruiting
iNKT cells.
48. Fujii, S., Shimizu, K., Kronenberg, M. & Steinman, R. M.
Prolonged IFN--producing NKT response induced
with -galactosylceramide-loaded DCs. Nature
Immunol. 3, 867874 (2002).
49. Schmieg, J., Yang, G., Franck, R. W., Van Rooijen, N. &
Tsuji, M. Glycolipid presentation to natural killer
T cells differs in an organ-dependent fashion.
Proc. Natl Acad. Sci. USA 102, 11271132 (2005).
50. Ko, S. Y. et al. -galactosylceramide can act as a nasal
vaccine adjuvant inducing protective immune
responses against viral infection and tumor.
J. Immunol. 175, 33093317 (2005).
51. Kamijuku, H. et al. Mechanism of NKT cell
activation by intranasal coadministration of
-galactosylceramide, which can induce crossprotection against influenza viruses. Mucosal
Immunol. 1, 208218 (2008).

52. Chung, Y., Chang, W. S., Kim, S. & Kang, C. Y. NKT cell
ligand -galactosylceramide blocks the induction of
oral tolerance by triggering dendritic cell maturation.
Eur. J. Immunol. 34, 24712479 (2004).
53. Huang, Y. et al. Enhancement of HIV DNA vaccine
immunogenicity by the NKT cell ligand,
-galactosylceramide. Vaccine 26, 18071816 (2008).
54. Gabrilovich, D. I. et al. The terminology issue for
myeloid-derived suppressor cells. Cancer Res. 67,
425 (2007).
55. Crowe, N. Y. et al. Glycolipid antigen drives rapid
expansion and sustained cytokine production by
NK T cells. J. Immunol. 171, 40204027 (2003).
56. Wilson, M. T. et al. The response of natural killer
T cells to glycolipid antigens is characterized by
surface receptor down-modulation and expansion.
Proc. Natl Acad. Sci. USA 100, 1091310918 (2003).
57. Harada, M. et al. Down-regulation of the invariant
V14 antigen receptor in NKT cells upon activation.
Int. Immunol. 16, 241247 (2004).
58. Coquet, J. M. et al. Diverse cytokine production
by NKT cell subsets and identification of an
IL-17-producing CD4NK1.1NKT cell population.
Proc. Natl Acad. Sci. USA 105, 1128711292
(2008).
59. Miyamoto, K., Miyake, S. & Yamamura, T. A synthetic
glycolipid prevents autoimmune encephalomyelitis by
inducing TH2 bias of natural killer T cells. Nature 413,
531534 (2001).
60. Chang, Y. J. et al. Potent immune-modulating and
anticancer effects of NKT cell stimulatory glycolipids.
Proc. Natl Acad. Sci. USA 104, 1029910304
(2007).
61. Schmieg, J., Yang, G., Franck, R. W. & Tsuji, M.
Superior protection against malaria and melanoma
metastases by a C-glycoside analogue of the natural
killer T cell ligand -galactosylceramide. J. Exp. Med.
198, 16311641 (2003).
62. Bezbradica, J. S. et al. Distinct roles of dendritic cells
and B cells in Va14Ja18 natural T cell activation
in vivo. J. Immunol. 174, 46964705 (2005).
63. Yu, K. O. et al. Modulation of CD1d-restricted
NKT cell responses by using N-acyl variants of
-galactosylceramides. Proc. Natl Acad. Sci. USA 102,
33833388 (2005).
64. Gadola, S. D. et al. Structure and binding kinetics of
three different human CD1d--galactosylceramidespecific T cell receptors. J. Exp. Med. 203, 699710
(2006).
65. Sidobre, S. et al. The T cell antigen receptor
expressed by V14i NKT cells has a unique mode of
glycosphingolipid antigen recognition. Proc. Natl
Acad. Sci. USA 101, 1225412259 (2004).
66. Chiba, A., Dascher, C. C., Besra, G. S. & Brenner, M. B.
Rapid NKT cell responses are self-terminating during
the course of microbial infection. J. Immunol. 181,
22922302 (2008).
67. Schwartz, R. H. T cell anergy. Annu. Rev. Immunol. 21,
305334 (2003).
68. Ota, T. et al. IFN--mediated negative feedback
regulation of NKT-cell function by CD94/NKG2.
Blood 106, 184192 (2005).
69. Chang, D. H. et al. Sustained expansion of NKT cells
and antigen-specific T cells after injection of
-galactosyl-ceramide loaded mature dendritic cells in
cancer patients. J. Exp. Med. 201, 15031517 (2005).
70. Koch, M. et al. The crystal structure of human CD1d
with and without -galactosylceramide. Nature
Immunol. 6, 819826 (2005).
71. Zajonc, D. M. et al. Structure and function of a potent
agonist for the semi-invariant natural killer T cell
receptor. Nature Immunol. 6, 810818 (2005).
72. Borg, N. A. et al. CD1d-lipid-antigen recognition by
the semi-invariant NKT T-cell receptor. Nature 448,
4449 (2007).
73. Godfrey, D. I., Rossjohn, J. & McCluskey, J. The
fidelity, occasional promiscuity, and versatility of
T cell receptor recognition. Immunity 28, 304314
(2008).
74. Wu, D. et al. Design of natural killer T cell activators:
structure and function of a microbial glycosphingolipid
bound to mouse CD1d. Proc. Natl Acad. Sci. USA
103, 39723977 (2006).
75. Zajonc, D. M., Savage, P. B., Bendelac, A., Wilson, I. A.
& Teyton, L. Crystal structures of mouse CD1diGb3
complex and its cognate V14 T cell receptor suggest
a model for dual recognition of foreign and self
glycolipids. J. Mol. Biol. 377, 11041116 (2008).
76. Scott-Browne, J. P. et al. Germline encoded
recognition of diverse glycolipids by NKT cells.
Nature Immunol. 8, 11051113 (2007).

NATuRe RevIewS | immuNology

77. Wun, K. S. et al. A minimal binding footprint on


CD1dglycolipid is a basis for selection of the
unique human NKT TCR. J. Exp. Med. 205,
939949 (2008).
References 72, 76 and 77 describe the unique
model of antigen recognition by the iNKT-cell TCR.
78. Brossay, L. et al. Structural requirements for
galactosylceramide recognition by CD1-restricted
NK T cells. J. Immunol. 161, 51245128 (1998).
79. Naidenko, O. V. et al. Binding and antigen
presentation of ceramide-containing glycolipids by
soluble mouse and human CD1d molecules. J. Exp.
Med. 190, 10691080 (1999).
80. Oki, S., Chiba, A., Yamamura, T. & Miyake, S. The
clinical implication and molecular mechanism of
preferential IL-4 production by modified glycolipidstimulated NKT cells. J. Clin. Invest. 113,
16311640 (2004).
81. Goff, R. D. et al. Effects of lipid chain lengths in
-galactosylceramides on cytokine release by natural
killer T cells. J. Am. Chem. Soc. 126, 1360213603
(2004).
82. Stanic, A. K. et al. Another view of T cell antigen
recognition: cooperative engagement of glycolipid
antigens by Va14Ja18 natural T(iNKT) cell receptor.
J. Immunol. 171, 45394551 (2003).
83. Denkberg, G. et al. Phage display-derived
recombinant antibodies with TCR-like specificity
against -galactosylceramide and its analogues
in complex with human CD1d molecules.
Eur. J. Immunol. 38, 829840 (2008).
84. Yu, K. O. et al. Production and characterization of
monoclonal antibodies against complexes of the
NKT cell ligand -galactosylceramide bound to
mouse CD1d. J. Immunol. Methods 323, 1123
(2007).
85. Miyake, S. & Yamamura, T. NKT cells and autoimmune
diseases: unraveling the complexity. Curr. Top.
Microbiol. Immunol. 314, 251267 (2007).
86. Yoshimoto, T., Bendelac, A., Watson, C., Hu-Li, J. &
Paul, W. E. Role of NK1.1+ T cells in a TH2 response
and in immunoglobulin E production. Science 270,
18451847 (1995).
87. Fujii, S. et al. Glycolipid -C-galactosylceramide is a
distinct inducer of dendritic cell function during innate
and adaptive immune responses of mice. Proc. Natl
Acad. Sci. USA 103, 1125211257 (2006).
88. Ortaldo, J. R. et al. Dissociation of NKT stimulation,
cytokine induction, and NK activation in vivo by the
use of distinct TCR-binding ceramides. J. Immunol.
172, 943953 (2004).
89. Parekh, V. V. et al. Quantitative and qualitative
differences in the in vivo response of NKT cells to
distinct - and -anomeric glycolipids. J. Immunol.
173, 36933706 (2004).
90. Van Kaer, L. -Galactosylceramide therapy for
autoimmune diseases: prospects and obstacles.
Nature Rev. Immunol. 5, 3142 (2005).
91. Giaccone, G. et al. A phase I study of the natural killer
T-cell ligand -galactosylceramide (KRN7000) in
patients with solid tumors. Clin. Cancer Res. 8,
37023709 (2002).
92. Nieda, M. et al. Therapeutic activation of
V24+V11+ NKT cells in human subjects results in
highly coordinated secondary activation of acquired
and innate immunity. Blood 103, 383389 (2004).
93. Ishikawa, A. et al. A phase I study of
-galactosylceramide (KRN7000)-pulsed dendritic
cells in patients with advanced and recurrent
non-small cell lung cancer. Clin. Cancer Res. 11,
19101917 (2005).
94. Uchida, T. et al. Phase I study of -galactosylceramidepulsed antigen presenting cells administration to the
nasal submucosa in unresectable or recurrent head
and neck cancer. Cancer Immunol. Immunother. 57,
337345 (2008).
95. Rosenberg, S. A., Restifo, N. P., Yang, J. C., Morgan,
R. A. & Dudley, M. E. Adoptive cell transfer: a clinical
path to effective cancer immunotherapy. Nature Rev.
Cancer 8, 299308 (2008).
96. Motohashi, S. et al. A phase I study of in vitro
expanded natural killer T cells in patients with
advanced and recurrent non-small cell lung cancer.
Clin. Cancer Res. 12, 60796086 (2006).
97. Stirnemann, K. et al. Sustained activation and tumor
targeting of NKT cells using a CD1danti-HER2scFv
fusion protein induce antitumor effects in mice.
J. Clin. Invest. 118, 9941005 (2008).
98. Godfrey, D. I., MacDonald, H. R., Kronenberg, M.,
Smyth, M. J. & Van Kaer, L. NKT cells: whats in a
name? Nature Rev. Immunol. 4, 231237 (2004).

vOLuMe 9 | jANuARy 2009 | 37


2009 Macmillan Publishers Limited. All rights reserved

REVIEWS
99. Benlagha, K., Weiss, A., Beavis, A., Teyton, L. &
Bendelac, A. In vivo identification of glycolipid antigenspecific T cells using fluorescent CD1d tetramers.
J. Exp. Med. 191, 18951903 (2000).
100. Karadimitris, A. et al. Human CD1d-glycolipid
tetramers generated by in vitro oxidative refolding
chromatography. Proc. Natl Acad. Sci. USA 98,
32943298 (2001).
101. Matsuda, J. L. et al. Tracking the response of natural
killer T cells to a glycolipid antigen using CD1d
tetramers. J. Exp. Med. 192, 741754 (2000).
102. Gumperz, J. E., Miyake, S., Yamamura, T. & Brenner,
M. B. Functionally distinct subsets of CD1d-restricted
natural killer T cells revealed by CD1d tetramer
staining. J. Exp. Med. 195, 625636 (2002).
103. Lee, P. T., Benlagha, K., Teyton, L. & Bendelac, A.
Distinct functional lineages of human V24 natural
killer T cells. J. Exp. Med. 195, 637641 (2002).
104. Liu, T. Y. et al. Distinct subsets of human invariant
NKT cells differentially regulate T helper responses
via dendritic cells. Eur. J. Immunol. 38, 10121023
(2008).
105. Crowe, N. Y. et al. Differential antitumor immunity
mediated by NKT cell subsets in vivo. J. Exp. Med.
202, 12791288 (2005).
106. McNab, F. W. et al. Peripheral NK1.1 NKT cells are
mature and functionally distinct from their thymic
counterparts. J. Immunol. 179, 66306637
(2007).
107. Rachitskaya, A. V. et al. Cutting Edge: NKT cells
constitutively express IL-23 receptor and RORt and
rapidly produce IL-17 upon receptor ligation in an
IL-6-independent fashion. J. Immunol. 180,
51675171 (2008).
108. Michel, M. L. et al. Identification of an IL-17producing NK1.1neg iNKT cell population involved in
airway neutrophilia. J. Exp. Med. 204, 9951001
(2007).
109. Yoshiga, Y. et al. Invariant NKT cells produce IL-17
through IL-23-dependent and -independent pathways
with potential modulation of Th17 response in
collagen-induced arthritis. Int. J. Mol. Med. 22,
369374 (2008).

110. Kim, C. H., Johnston, B. & Butcher, E. C. Trafficking


machinery of NKT cells: shared and differential
chemokine receptor expression among V 24+V11+
NKT cell subsets with distinct cytokine-producing
capacity. Blood 100, 1116 (2002).
111. Thomas, S. Y. et al. CD1d-restricted NKT cells express
a chemokine receptor profile indicative of Th1-type
inflammatory homing cells. J. Immunol. 171,
25712580 (2003).
112. Johnston, B., Kim, C. H., Soler, D., Emoto, M. &
Butcher, E. C. Differential chemokine responses and
homing patterns of murine TCR NKT cell subsets.
J. Immunol. 171, 29602969 (2003).
113. Geissmann, F. et al. Intravascular immune surveillance
by CXCR6+ NKT cells patrolling liver sinusoids.
PLoS Biol. 3, e113 (2005).
114. Stanic, A. K. et al. Defective presentation of the
CD1d1-restricted natural Va14Ja18 NKT lymphocyte
antigen caused by -D-glucosylceramide synthase
deficiency. Proc. Natl Acad. Sci. USA 100,
18491854 (2003).
115. Zhou, D. et al. Lysosomal glycosphingolipid recognition
by NKT cells. Science 306, 17861789 (2004).
116. Silk, J. D., Salio, M., Brown, J., Jones, E. Y. &
Cerundolo, V. Structural and functional aspects of lipid
binding by CD1 molecules. Annu. Rev. Cell Dev. Biol.
24, 369395 (2008).
117. Porubsky, S. et al. Normal development and function
of invariant natural killer T cells in mice with
isoglobotrihexosylceramide (iGb3) deficiency.
Proc. Natl Acad. Sci. USA 104, 59775982 (2007).
118. Speak, A. O. et al. Implications for invariant natural
killer T cell ligands due to the restricted presence of
isoglobotrihexosylceramide in mammals. Proc. Natl
Acad. Sci. USA 104, 59715976 (2007).
119. Christiansen, D. et al. Humans lack iGb3 due to the
absence of functional iGb3-synthase: implications for
NKT cell development and transplantation. PLoS Biol.
6, e172 (2008).
120. Li, Y. et al. Sensitive detection of isoglobo and globo
series tetraglycosylceramides in human thymus by ion
trap mass spectrometry. Glycobiology 18, 158165
(2008).

38 | jANuARy 2009 | vOLuMe 9

121. Gumperz, J. E. et al. Murine CD1d-restricted T cell


recognition of cellular lipids. Immunity 12, 211221
(2000).
122. Park, J. E. et al. Fine specificity of natural killer T cells
against GD3 ganglioside and identification of GM3 as
an inhibitory natural killer T-cell ligand. Immunology
123, 145155 (2008).
123. Cardell, S. et al. CD1-restricted CD4+ T cells in major
histocompatibility complex class II-deficient mice.
J. Exp. Med. 182, 9931004 (1995).
124. Jahng, A. et al. Prevention of autoimmunity by
targeting a distinct, noninvariant CD1d-reactive T cell
population reactive to sulfatide. J. Exp. Med. 199,
947957 (2004).
125. Berzofsky, J. A. & Terabe, M. NKT cells in tumor
immunity: opposing subsets define a new
immunoregulatory axis. J. Immunol. 180,
36273635 (2008).
126. Stewart-Jones, G. B., McMichael, A. J., Bell, J. I.,
Stuart, D. I. & Jones, E. Y. A structural basis for
immunodominant human T cell receptor recognition.
Nature Immunol. 4, 657663 (2003).
127. Chen, J. L. et al. Structural and kinetic basis for
heightened immunogenicity of T cell vaccines.
J. Exp. Med. 201, 12431255 (2005).

Acknowledgements

We apologize to those colleagues whose data we have been


unable to cite owing to space limitations. We thank A. Stock
for critical reading of the manuscript.

DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
CD1d | CD40 | IFN | IL-4 | IL-12

FURTHER INFORMATION
Vincenzo Cerundolos homepage: http://www.imm.ox.ac.uk/
pages/research/human_immunology/enzo_cerundolo.htm
All liNkS ArE ACTivE iN ThE oNliNE pDf

www.nature.com/reviews/immunol
2009 Macmillan Publishers Limited. All rights reserved