Linking Inflammation to Natural Killer T Cell Activation
Mariolina Salio, Vincenzo Cerundolo*
Nuffield Department of Clinical Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
To help us fight infections, the immune system relies on the
coordinated activity of innate and adaptive immunity. Innate
immunity represents an evolutionarily conserved first line of
defense, kicking into action within hours of encountering
pathogens, while adaptive immunity involves a higher degree of
specificity for pathogen-encoded molecular determinants and
long-term memory responses. Components of the innate immune
system include epithelial barriers, antimicrobial compounds, and a
range of different cell types, including dendritic cells, macrophag-
es, neutrophils, and natural killer cells. Conversely, the main
components of adaptive immunity are B and T lymphocytes,
which survey billions of antigens via cell surface receptors that
undergo somatic DNA rearrangements to confer almost unlimited
specificities. Activation of innate responses, triggered by the
detection of conserved microbial molecules (such as viral nucleic
acids or bacterial cell wall components) by pattern-recognition
receptors (such as Toll-like receptors), is critical for eliciting
antigen-specific immune responses . Over the past ten years it
has emerged that a population of unconventional T lymphocytes,
called natural killer T cells (NKT cells), operates at the interface
between innate and adaptive immune responses; these cells express
rearranged antigen-specific surface T cell receptors (although with
limited diversity), but are also capable of a very rapid response
mode, resulting in the activation of antigen presenting cells and
facilitating the development of adaptive immunity.
Whereas most T lymphocytes recognize peptide antigens
presented by molecules of the major histocompatibility complex
(MHC), NKT cells recognize lipid antigens presented by CD1d
molecules, which are functionally related to MHC molecules .
Upon activation, NKT cells modulate the activity of CD1d-
expressing cells via the costimulatory molecule CD40 ligand and
induce interferon (IFN)-c-dependent activation of other cell types,
ultimately enhancing antigen specific B and T cell responses
(Figure 1) . Because of their effect on multiple cells of the
immune system, NKT cells contribute to the regulation of a
variety of processes, such as self-tolerance, tumor surveillance, and
anti-microbial responses .
Auto-Reactivity by Design
During maturation of T lymphocytes the majority of auto-
reactive cells are destroyed in the thymus to prevent autoimmu-
nity. However, a proportion of NKT cells expressing a semi-
invariant T cell receptor, hereafter referred to as invariant NKT
cells (iNKT cells), maintain the ability to recognize a range of
endogenous lipids in the context of CD1d molecules during both
inflammatory and non-inflammatory conditions. It has recently
been shown that in the absence of inflammation, iNKT-cell auto-
reactivity leads to a special activation state, characterized by
impaired calcium signaling, which leads to the secretion of GM-
CSF (granulocyte-macrophage colony-stimulating factor), with
limited amounts of inflammatory cytokines . In contrast, during
microbial infection, inflammatory cytokines, such as interleukin
(IL)-12 and IL-18, enhance basal iNKT-cell auto-reactivity and
promote secretion of IFN-c [6–9]. In addition, during inflamma-
tory responses, iNKT-cell activation can be influenced by
increased expression of surface CD1d molecules by activated
antigen presenting cells (APCs) [10,11] and/or by increased
expression of enzymes leading to the biosynthesis of endogenous
self lipids [7,8]. These multiple mechanisms influencing iNKT-cell
activation suggest an important link between inflammation and
iNKT cells. Indeed, recent results have highlighted the ability of
iNKT cells to abolish the suppressive activity of myeloid-derived
suppressor cells (MDSCs), which are expanded during tumor
growth and microbial infections and restore antigen-specific
immune responses during influenza virus infection (Figure 1) .
Although the identity of several bacteria-derived lipids capable
of activating iNKT cells has been determined [6,13,14], the
identity of the lipids modulating iNKT-cell auto-reactivity has
been elusive and is currently being investigated by several groups
. Identification of such lipids is important to a fuller
understanding of the rules regulating CD1d assembly and loading,
and also for the design of novel compounds that can selectively
modulate iNKT-cell activation [16,17]. Results published in this
issue of PLoS Biology , and in earlier issues of PLoS ONE 
and The Journal of Immunology , shed new light on the identity of
iNKT cell natural ligands and provide further support for the link
between inflammation and iNKT-cell activation.
A Closer Look at the Lipid Repertoire Bound to
Analysis of the ability of defined lipids to stimulate iNKT-cell
activation has revealed that a restricted number of murine iNKT-
cell clones can recognize phosphatidylinositol (PI) , phospha-
tidylethanolamine (PE), and phosphatidylglycerol (PG) and the
ganglioside GM3 . However, reactivity to these lipids is weak
and limited to a few iNKT-cell clones. To identify other
endogenous natural iNKT-cell agonists, one approach has relied
on the use of CD1d+cells with defined genetic defects in the lipid
biosynthetic pathway. The results of these experiments have
shown that a class of lipids known as glycosphingolipids (GSL),
Citation: Salio M, Cerundolo V (2009) Linking Inflammation to Natural Killer T Cell
Activation. PLoS Biol 7(10): e1000226. doi:10.1371/journal.pbio.1000226
Published October 27, 2009
Copyright: ? 2009 Salio, Cerundolo. 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: This work is funded by Cancer Research UK Program grant C399/A6199
to VC. 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
Abbreviations: APCs, antigen presenting cells; ER, endoplasmic reticulum; GM-
CSF, granulocyte-macrophage colony-stimulating factor; GSL, glycosphingolipid;
IFN, interferon; IL, interleukin; iNKT cells, invariant NKT cells; LPC, lysophospha-
tidylcholine; MDSCs, myeloid-derived suppressor cells; MHC, Major Histocompat-
ibility Complex; NKT cells, natural killer T cells; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol;
PLA2, phospholipase A2.
* E-mail: firstname.lastname@example.org
PLoS Biology | www.plosbiology.org1 October 2009 | Volume 7 | Issue 10 | e1000226
such as isoglobotrihexosylceramide (iGb3), contributes to the
pool of endogenous lipids that leads to human and murine
iNKT cell activation [6,23,24]. However, the presence of iGb3 in
dendritic cells and developing T lymphocytes remains controver-
sial [25–29]. Furthermore, it has become clear that experiments
carried out in mice or in cells with defective GSL catabolism need
to be carefully controlled, because impaired iNKT-cell positive
selection and activation could be due not to the absence of
activating lipids, but to the failure of lipid presentation resulting
from sequestering of lipids in the lysosomes of these APCs [30,31].
Consistent with this possibility, mice deficient in the lysosomal
enzyme b-hexosaminidase, which generates iGb3 from iGb4, have
impaired iNKT-cell development , whereas iGb3 synthase-
deficient mice have normal numbers of iNKT cells . Thus, the
role of iGb3 as a self antigen for iNKT cells remains unclear.
In order to better characterize the classes of self-lipids available
for recognition by iNKT cells, two groups have independently
employed a novel approach to analyze the repertoire of lipids
bound to CD1d molecules [18–20]. The Cresswell group
engineered human CD1d molecules that could be proteolytically
cleaved after recycling physiologically through the endocytic
pathway and then compared lipids that were associated with
CD1d molecules in different cellular compartments, that is, either
retained in the endoplasmic reticulum (ER), secreted in soluble
form, or cleaved . By using multiple mass spectrometry
methods to identify the major lipid species, the researchers
reported that the ER-retained form of CD1d was predominantly
loaded with phosphatidylcholine (PC), the most abundant
phospholipid in eukaryotic cells. The only detectable lipid
associated with the secreted CD1d molecules was sphingomyelin,
which is synthesized in the Golgi and is not present in the ER,
whereas the protease-cleaved CD1d molecules were loaded with
PC, sphingomyelin, and lysosphospholipids. In contrast, Gumperz
and colleagues focused their analysis on lipids associated with
secreted human CD1d molecules, as they had previously observed
that, in contrast to mouse iNKT cells, autoreactivity of human
iNKT-cell clones is largely independent of CD1d endosomal
trafficking . In the PLoS ONE paper, Gumperz and colleagues
found a large spectrum of lipids associated with soluble human
CD1d molecules . By a high-resolution analysis, a total of 177
lipid species were identified, comprising glycerophospholipids
(including common diacylglycerol species, plasmalogens, lysopho-
spholipids, and cardiolipins) and sphingolipids (including sphin-
gomyelins and GSL, such as the ganglioside GM3). Altogether,
these results highlight the possibility that, depending on the
cellular localization, CD1d molecules may be loaded with a
different spectrum of endogenous ligands.
Given that some of these lipids are known to be bioactive and
have been reported to play significant roles in cancer, autoimmune
disease, cellular signaling, and cell death [33,34], the Gumperz
group has investigated the ability of iNKT cells to recognize and
react to synthetic preparations of all the CD1d bound lipids - these
Figure 1. iNKT cells at the interface between innate and adaptive immunity. iNKT cells recognize lipids presented by CD1d molecules. Upon
activation, iNKT cells modulate the function of CD1d-expressing cells, such as APCs and B cells. This leads to the priming of antigen-specific T cells,
induction of antibody responses, and activation of natural killer cells, a subset of cells acting in the innate immune response. iNKT cells can also
inhibit the suppressive function of MDSCs.
PLoS Biology | www.plosbiology.org2 October 2009 | Volume 7 | Issue 10 | e1000226
findings are reported in this issue of PLoS Biology . Remarkably,
the mono acyl lysophosphatidylcholine (LPC) (Figure 2) was the
only antigenic species capable of activating both a panel of iNKT-
cell clones and lines and, albeit weakly, freshly isolated peripheral
LPC as an Inflammatory Lipid and Stimulating
Signal for iNKT Cells
LPC is produced by the phospholipase A2 enzymes (PLA2),
which can be localized to a number of intracellular and
extracellular sites. Activation of PLA2 by a variety of growth
factors, hormones, and cytokines can lead to the release of LPC
into the cytoplasm, the lysosome, or at the cell surface . At all
these locations, LPC could be available for loading onto CD1d
molecules (Figure 2). Of relevance is the observation from the
Gumperz group that blocking secreted PLA2 activity in monocytes
(and thereby reducing the levels of lipid ligand) with a polyclonal
antibody, led to reduced basal iNKT-cell activation without
affecting CD1d expression . Since LPC accumulates to high
concentrations in blood and other fluids during chronic inflam-
mation, and since Gumperz and colleagues showed that lysopho-
spholipids can bind to CD1d molecules previously loaded with
other cellular ligands or GSL , monitoring the levels of CD1d-
bound LPC could represent one of the mechanisms leading to
iNKT-cell activation and expansion. Interestingly, Dhodapkar and
colleagues previously reported an increase of LPC species in one
pathological setting - in the plasma of myeloma patients. This was
accompanied by an expansion of a subset of NKT cells ,
which, unlike iNKT cells, express a broader range of T cell
receptors. These results suggest that recruitment and expansion of
invariant and non-invariant NKT cells could occur more widely
in different inflammatory settings and eventually contribute
to immune pathology. Indeed, iNKT cells have a chemokine
receptor profile that allows them to preferentially ‘‘home’’ to
inflamed tissues . It is tempting to speculate that, during
inflammation, secreted LPC could be presented by APCs recruited
at inflammatory sites, resulting in iNKT-cell activation (Figure 2).
However, the efficiency of LPC presentation in vivo remains to be
defined. Gumperz and colleagues provide some support for this
Figure 2. iNKT-cell activation by lysosphospholipids. During inflammation cytoplasmic, membrane and secreted phospholipases (such as
PLA2) produce lysophospholipids (such as LPC) from cellular phospholipids. Lysosphospholipids can be loaded onto CD1d molecules at the cell
surface, in the lysosomes, or during intracellular trafficking through the ER and the Golgi. CD1d-LPC complexes elicit iNKT-cell activation in concert
with IL-12, IL-18, and type I IFN secreted by APCs during inflammatory reactions.
PLoS Biology | www.plosbiology.org3 October 2009 | Volume 7 | Issue 10 | e1000226
notion by showing very weak iNKT-cell stimulation with APCs
expressing wild-type CD1d molecules. In contrast, the authors
show that recognition of LPC by iNKT cells was enhanced using
CD1d molecules unable to recycle from the cell surface to the
lysosomes (where the acidic environment could lead to dissociation
of the CD1d-lipid complexes) . In addition, higher concen-
trations of LPC failed to activate iNKT-cell clones, showing an
unexplained inhibitory effect, which could be due to the formation
of micelles or less–CD1d-accessible structures .
It is known that the length of the lipid hydrocarbon chains
determines the stability of lipid binding to CD1d molecules, which
in turn influences iNKT-cell activation . Although further
studies are warranted to determine the binding affinity of lysopho-
spholipids for CD1d molecules and the half-life of these complexes,
it is likely that the mono alkyl chain LPC will have a higher rate of
dissociation from CD1d molecules than the dual alkyl chain PC.
Thus, the combination of limited presentation by recycling CD1d
molecules,with the inhibitoryeffectofhighLPCconcentrationsand
possibly the short half-life of CD1d-LPC complexes, could be
important features that help to fine-tune iNKT-cell responses in the
context of prolonged inflammatory processes.
The crystal structures of human and murine CD1d molecules
have revealed the presence of two hydrophobic channels, A9 and
C9, which are occupied by the lipid tails of iNKT-cell agonists
. Interestingly, the results by the Gumperz laboratory in this
issue of PLoS Biology have highlighted a dichotomy in the ability of
LPC and PC to stimulate iNKT cells . It will be, therefore,
very informative to carry out structural studies comparing CD1d
molecules loaded with either lysophospholipids or phospholipids
(e.g., mono versus di acyl lipids) to assess whether the differential
ability of LPC and PC to activate iNKT cells may be accounted
for by variations in the orientation of the polar head as a
consequence of different binding of their lipids chains to the A9
and C9 hydrophobic channels. This possibility would be consistent
with previously published CD1d structures, revealing the ability of
different phospholipids to bind CD1d molecules in different
In conclusion, the identification of LPC as an endogenous ligand
for iNKT cells is an important finding for the understanding of the
role that iNKT cells and other immune cells play during
inflammation. It will also be interesting to correlate iNKT-cell
numbers and activation with changes in activity of the PLA2
isoforms during different inflammatory conditions (for example,
upon microbial infections and Toll-like receptor-mediated activation
of APCs or during chronic inflammatory processes, such as cancer).
Future studies will reveal whether analogues of lysophospholipids
could be exploited as novel adjuvants to further harness iNKT cells’
ability to bridge innate and adaptive immune responses or to fine-
tune iNKT-cell autoreactivity during autoimmune diseases.
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