Download Role of NKT cells in the digestive system. IV. The role of canonical

Am J Physiol Gastrointest Liver Physiol 294: G1–G8, 2008.
First published October 18, 2007; doi:10.1152/ajpgi.00437.2007.
Themes
Role of NKT cells in the digestive system.
IV. The role of canonical natural killer T cells in mucosal immunity
and inflammation
Gerhard Wingender and Mitchell Kronenberg
La Jolla Institute for Allergy and Immunology, San Diego, California
Submitted 24 September 2007; accepted in final form 18 October 2007
innate immunity; colitis
TO BE OR NOT TO BE: THE MANY NAMES OF NKT CELLS
Natural killer T (NKT) cells are a unique subset of T
lymphocytes found in mice, humans, and other mammals. Like
natural killer (NK) cells, they exhibit features of innate immunity, but they also share properties with conventional T lymphocytes. They were originally defined by their coexpression
of an ␣␤ T cell antigen receptor (TCR) and NK cell receptors,
especially NK1.1 (NKR-P1C) in certain mouse strains and
CD161 (NKR-P1A) in humans. However, this classification is
an oversimplification, as conventional T cells can express NK
cell receptors, especially after activation. Moreover, the expression of NK cell receptors by NKT cells varies with their
maturity and activation state and, in mice, with the genetic
background. As knowledge of the properties of NKT cells has
increased, these cells have been subdivided according to their
TCR ␣-chain, the selecting antigen-presenting molecule, and
the expression of various surface molecules (for reviews, see
Refs. 30, 62, 68).
However, despite attempts, the lack of a generally accepted
nomenclature has made the field confusing. Here, we propose
Address for reprint requests and other correspondence: M. Kronenberg, La
Jolla Institute for Allergy and Immunology (LIAI), 9420 Athena Circle, San
Diego, CA 92037 (e-mail: [email protected], URL:http://www.liai.org).
http://www.ajpgi.org
a classification based on TCR usage and the antigen-presenting
molecule recognized. Thereby, four populations of NKT cells
can be distinguished (see Table 1). The first two NKT cell
populations have a canonical TCR and are specific for a
nonpolymorphic major histocompatibility complex (MHC)
class I-like molecule. “Canonical” and “invariant” have been
used synonymously, but historically the mouse V␣14 invariant
(V␣14i) NKT cells, along with their human homologs that
express a V␣24i TCR, have been termed invariant (iNKT).
Therefore, we will use the expression “canonical (c)NKT cell”
to include both the V␣14/V␣24i NKT cells and a second
population that expresses a fixed V␣7.2i TCR in mice and a
homologous V␣19i in humans, named here mucosal (m)NKT
cells. A third group, termed variant (v)NKT cells, encompasses
T lymphocytes reactive to CD1d but that do not have a
restricted TCR usage. The fourth group is more heterogeneous
because it combines all other T cells that express NK receptors.
This group has been termed xNKT cells or tgNKT cells (when
they express a TCR transgene). Not listed in Table 1 are T
lymphocytes with a ␥␦-TCR that express NK receptors. There
are also T cell subsets reactive with other MHC class I-like or
nonclassical class I molecules. Examples include T cells reactive with the human group I CD1 molecules, CD1a, -b, -c,
which also present lipid-containing antigens, and T cells specific for the mouse H2-M3 molecule, which present peptides
with a formylated amino terminus derived from bacteria. Although these cells may share some properties with the NKT
cells described above, they generally do not express NK
receptors. In this review, we focus on the two populations of
cNKT cells, particularly on their role in mucosal immunology.
THE JANUS-LIKE CHARACTER OF iNKT CELLS
The largest and best-studied fraction of NKT cells carries a
canonical V␣14 to J␣18 TCR rearrangement (V␣14i) in mice
and an orthologous V␣24-J␣18 TCR chain (V␣24i) in humans.
These are coexpressed with a limited set of V␤-chains, predominantly V␤8.2 in mice and V␤11 in humans, although
these have highly diverse rearrangements to J␤-segments.
V␣14i and V␣24i iNKT cells recognize glycolipid structures,
presented by CD1d, a nonpolymorphic MHC class I homolog.
There is a surprising degree of interspecies cross-reactivity,
with mouse V␣14i NKT cells recognizing human CD1d and
vice versa. The recently described trimolecular structure of a
glycosphingolipid (GSL) bound to human CD1d, which is
recognized by a V␣24i TCR (6), illustrates the importance of
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Wingender G, Kronenberg M. Role of NKT cells in the
digestive system. IV. The role of canonical natural killer T cells in
mucosal immunity and inflammation. Am J Physiol Gastrointest
Liver Physiol 294: G1–G8, 2008. First published October 18,
2007; doi:10.1152/ajpgi.00437.2007.—Lymphocytes that combine
features of T cells and natural killer (NK) cells are named natural
killer T (NKT) cells. The majority of NKT cells in mice bear highly
conserved invariant V␣ chains, and to date two populations of such
canonical NKT cells are known in mice: those that express V␣14 and
those that express V␣7.2. Both populations are selected by nonpolymorphic major histocompatibility complex class I-like antigen-presenting molecules expressed by hematopoietic cells in the thymus:
CD1d for V␣14-expressing NKT cells and MR1 for those cells
expressing V␣7.2. The more intensely studied V␣14 NKT cells have
been implicated in diverse immune reactions, including immune
regulation and inflammation in the intestine; the V␣7.2 expressing
cells are most frequently found in the lamina propria. In humans,
populations of canonical NKT cells are found to be highly similar in
terms of the expression of homologous, invariant T cell antigenreceptor ␣-chains, specificity, and function, although their frequency
differs from those in the mouse. In this review, we will focus on the
role of both of these canonical NKT cell populations in the mucosal
tissues of the intestine.
Themes
G2
cNKT, canonical natural killer T (NKT); iNKT, invariant NKT; mNKT, mucosal NKT; vNKT, variant NKT; DN, double negative; DP, double positive; ␣GalCer, ␣-galactosylceramide; ␣ManCer,
␣-mannosylceramide; BbGL, Borrelia burgdorferi glycolipid; GSL, glycosphingolipid; iGb3, isoglobotrihexosylceramide; MHC, major histocompatibility complex; nd, not determined; TCR, T cell antigen
receptor.
NK1.1
Effector/memory
nd
Liver, spleen
NK1.1
CD56, CD161
Effector/memory
IL-15
Liver, thymus, spleen, bone marrow
NK1.1
Common NK cell receptor
Peripheral phenotype
Peripheral requirements
Prominent steady-state location
CD161
Effector/memory
MR1 on B cells, gut flora
Lamina propria, lung, liver
MR1
␣ManCer
Thymus hematopoietic cell
DN, few CD4
DN, few CD4, CD8a␣
Restriction
Reactivity
Positive selection
Coreceptors
CD1d
␣GalCer, GSL, BbGl2, iGb3
DP thymocytes
CD4 or DN
CD4 or DN, CD8a␣
CD1d
Sulfatide and nd
Thymus, cell unknown
CD4 or DN
Diverse (xNKT),
transgenic (tgNKT)
MHC I and II
Diverse peptides
Thymus, cell unknown
CD4, CD8␣ (mostly
CD8a␣) or DN
DX5 and/or NK1.1
Naive and effector/memory
nd
Spleen, bone marrow, liver
Diverse
Invariant V␣24-J␣18
Invariant V␣14-J␣18
Invariant V␣7.2-J␣33
Invariant V␣19-J␣33
Human
Mouse
Human
Mouse
iNKT Cells (also known as V␣14i or V␣24i,
NKT type I or classical NKT cells)
mNKT Cells (also known as V␣19i or V␣7.2i NKT,
mucosal-associated invariant T cells or NKT-like cells)
cNKT Cells
Table 1. Features of different NKT cell subsets
TCR repertoire
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contacts made by the J␣18 segment with the antigen protruding
form the CD1d groove. V␣14i iNKT cells have the highest
frequency in the liver and the bone marrow (⬎10%), followed
by significant numbers in thymus, peripheral blood, and spleen
(1–3%), whereas they are relatively rare in other lymphoid
organs (⬍0.5%). Interestingly, in humans the frequency of
iNKT cells is generally much lower, although a high degree of
interindividual variability has been reported.
As noted above, there is inherent ambiguity in the term
“NKT cell.” However, since the development of CD1d tetramers loaded with ␣-galactosylceramide (␣GalCer), it has been
possible to unequivocally identify the iNKT cell subpopulation
(33). As summarized in Fig. 1, ␣GalCer is a synthetic GSL
compound that is able to strongly activate iNKT cells when
bound to CD1d. Furthermore, researchers have been able to
probe the functions of these cells using iNKT cell-deficient
mice, either J␣18⫺/⫺ mice, which lack only iNKT cells, or
Cd1d⫺/⫺ mice, which lack iNKT and the vNKT cells that have
more diverse TCRs. Even as they differentiate in the thymus,
iNKT cells express a pattern of cell surface markers (CD69⫹,
CD44high, CD11ahigh, CD62Llow, CD122⫹) typically associated with activated or memory T cells. After activation, iNKT
cells rapidly gain cytotoxic activity and produce both T helper
type 1 (TH1) cytokines, such as IFN-␥ and TNF, and T helper
type 2 (TH2) cytokines, such as IL-4, IL-10, and IL-13. Recent
evidence suggests that subsets of iNKT cells can also produce
IL-17 (34).
Because of their rapid initiation of effector functions, iNKT
cells have been reported to be crucially involved in the early
phases of a dazzling variety of different immune reactions,
ranging from self-tolerance and development of autoimmunity
to include responses to pathogens and tumors (30, 62). In the
different studies, iNKT cell have been shown to be either
beneficial or harmful, depending on whether they polarize the
immune response either toward a TH1 or TH2 direction, and the
beneficial or detrimental effects of such a cytokine polarization
in the context of different models. For this reason, others have
referred previously to their “Janus-like” character.
Fig. 1. Pictorial representation of some of the main features of canonical
natural killer T (cNKT) cells. DN, double negative; ␣GalCer, ␣-galactosylceramide; iNKT, invariant natural killer T cells; ␣ManCer, ␣-mannosylceramide;
mNKT, mucosal natural killer T cells.
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vNKT Cells (also known as
type II or nonclassical NKT cells)
xNKT or tgNKT Cells (also
known as NKT-like cells)
CANONICAL NKT CELLS IN MUCOSAL IMMUNITY AND INFLAMMATION
Themes
CANONICAL NKT CELLS IN MUCOSAL IMMUNITY AND INFLAMMATION
iNKT CELL ACTIVATION BY SELF ANTIGENS
iNKT CELL ACTIVATION BY MICROBIAL ANTIGENS
Abundant evidence has established an important role for
iNKT cells in the host defense against several pathogens,
especially in the early phases of infection (30, 62, 68). Before
the discovery of iGb3, however, the only compound presented
by CD1d known to activate most iNKT cells was ␣GalCer (see
Fig. 1) and its derivatives. ␣GalCer, originally derived from a
marine sponge, contains an unusual ␣-anomeric linkage of the
sugar to the ceramide lipid, and this ␣-linkage is crucial for its
stimulatory capacity. Most GSLs have ␤-linked sugars, which
do not form an epitope for iNKT cells because the hexose ring
of the sugar is oriented differently. Notwithstanding the highly
conserved recognition of the apparently marine sponge-derived
␣GalCer, a prevalent hypothesis was that iNKT cells recognize
natural glycolipid structures derived from pathogens. However,
as with the endogenous iNKT cell antigens, the first reports of
exogenous iNKT cell antigens, in purified components derived
from Leishmania donovani and from Mycobacterium bovis,
stimulated only a minority (⬍2%) of V␣14i NKT cells. Only
recently have bacterial antigens that stimulate the majority of
iNKT cells been characterized, including glycolipids from
environmental bacteria, Sphingomonas spp., and from Borrelia
burgdorferi, the causative agent of Lyme disease (for review,
see Refs. 30, 62, 68). We expect that additional bacterial
species will be found to have natural iNKT cell antigens,
although some bacteria, such as Salmonella spp., apparently do
not have antigens.
Activation of iNKT cells by TCR-mediated recognition of a
foreign antigen bound to CD1d has been referred to as direct or
foreign antigen-dependent activation. Apart from this, a second
pathway of iNKT cell activation is currently recognized. iNKT
cells can be activated by proinflammatory cytokines, such as
type I IFN, IL-12, and IL-18, alone or in combination, which
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are produced early after viral or bacterial infections by dendritic cells and macrophages. In some cases, it has been shown
that iNKT cells additionally require the recognition of endogenous or self-ligands presented by CD1d for activation in this
cytokine-dependent context (7). This type of activation leads to
the production of IFN-␥ but not IL-4 by the stimulated iNKT
cells. These pathways of iNKT cell activation that are independent of foreign antigen have been termed indirect activation.
NKT CELLS IN THE INTESTINE
Many of the studies reporting the presence of NKT cells in
the intestine have relied on the coexpression of the TCR/CD3ε
complex and NK cell receptors, such as NK1.1 in mice and
CD161 in humans, which does not allow for the distinction of
the four NKT cell populations shown in Table 1. An unequivocal identification is to date only possible for the cNKT cells,
either by measuring mRNA for the canonical ␣-chain TCRs
(mNKT and iNKT cells) or by flow cytometry using CD1d
tetramers loaded with ␣GalCer (iNKT cells).
Up to 4% of mouse small intestine (SI) intraepithelial
lymphocytes (IELs) (4, 18, 25, 39), 8 –10% of large intestine
(LI) IELs (4, 25), and 7% of LI lamina propria lymphocytes
(LPLs) (20) have been reported to coexpress the TCR-CD3ε
complex and NK cell receptors. Most of these cells were
CD8␣⫹, and two-thirds thereof were CD8␣␣⫹ (4, 25). It has
been noted that a significant portion of these CD3ε/NK1.1⫹
cells were ␥␦ T cells (25) and therefore not part of the four
NKT cell populations defined here. Furthermore, most of these
cells were CD1d independent (4, 25). With the use of CD1d
tetramers loaded with ␣GalCer, a few (1%) iNKT cells have
been detected in SI IELs (33), but they were more prevalent
(2%) in LPLs (49). Furthermore, cells derived from colitic LI
LPLs responded to ␣GalCer (20). Interestingly, 80% of the
iNKT cells were NK1.1⫺ (33). However, in other studies, no
tetramer-positive cells, invariant mRNA, or responsiveness to
␣GalCer could be detected in IELs (49). From these reports,
one might conclude that the majority of NKT cells in the
mouse intestine are CD1d independent (xNKT cells) and that
iNKT cells are mainly found in LPL.
In humans, the values for NKT cells have been expressed as
percentages of CD3ε⫹ cells, rather than total lymphocytes. The
following proportions of T cells were also positive for CD161:
50 –70% for SI IELs (23, 41), 40 – 45% for LI IELs (23, 41),
and 9% for LI LPLs (14). Of these cells, 60 – 80% were
CD8␣⫹ (23, 41). The dependence on CD1d could not be
addressed; however, only 1.6 –1.7% of the CD3ε/CD161⫹ IEL
cells expressed V␣24 (23, 41), and immunhistochemistry data
suggested that the majority of the V␣24⫹ cells in the SI are
actually located in the lamina propria (16). Furthermore, the
majority of these V␣24⫹ cells did not express the V␤11 TCR
chain associated with the invariant TCR (41). Therefore, the
great majority of CD161⫹ T cells in human intestine are not
iNKT cells, similar to results obtained by analysis of human
peripheral blood mononuclear cells (PBMCs). Together with
␣GalCer-loaded CD1d tetramer data from LPL (14), one can
estimate that the proportion of iNKT cells in the human
intestine is ⬍0.4% of all T cells and that they are, like in the
mouse, mainly localized in the lamina propria. Nonetheless,
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The effector or memory phenotype of iNKT cells and their
constitutive expression of mRNA for IL-4 and IFN-␥ suggest
that they undergo a strong antigenic stimulation during their
differentiation. This is consistent with the hypothesis that true
TCR agonists mediate their positive selection (30). In line with
this idea is the observation that mature iNKT cells can, under
some circumstances, recognize endogenous glycolipids bound
to CD1d. However, elucidation of the glycolipids involved in
this endogenous recognition process has proven to be quite
challenging. Several suggestions have been put forward (for
reviews, see Refs. 30, 68); however, for these cases, only a
minor fraction of the iNKT cells were responsive. The best
candidate to date for a more general endogenous ligand or
autoantigen for iNKT cells is isoglobotrihexosylceramide
(iGb3), a GSL found in the lysosomes of some cell types. The
majority of iNKT cells can recognize iGb3 when bound to
CD1d (69). The inability of mutant mice to synthesize iGb3 in
lysosomes, through catabolism of a tetrasaccharide precursor,
has been linked to V␣14i NKT cell deficiency (69). However,
recent data have raised doubts about whether iGb3 is the only
endogenous antigen driving iNKT cell development (31). Particularly striking is the finding that mice deficient for iGb3
synthase have normal iNKT cell number and function (47). It
seems possible that several different self-antigens can drive
CD1d-restricted iNKT cell development.
G3
Themes
G4
CANONICAL NKT CELLS IN MUCOSAL IMMUNITY AND INFLAMMATION
these iNKT cells were functionally active and produced cytokines after ␣GalCer stimulation (41).
CD1d EXPRESSION IN THE MUCOSAL IMMUNE SYSTEM
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NKT CELLS IN INFLAMMATORY BOWEL DISEASE
Most studies addressing the role of iNKT and vNKT cells in
the intestine were concerned with inflammatory bowel disease
(IBD) model systems. IBD denotes a variety of what are likely
to be T cell-dependent diseases of different parts of the intestine, as a consequence of an inflammatory response to antigens
derived from the gut lumen. This inflammation may follow
perturbation of the epithelial layer, either through genetic
alterations or the administration of exogenous agents. NKT
cells have been implicated in several animal models of IBD,
and, although the data are not entirely consistent, the findings
correlate with a protective role for NKT cells in TH1-mediated
IBDs and a deleterious one in TH2 IBDs.
Crohn disease is characterized by a chronic and discontinuous inflammation of both the SI and LI, with high levels of TH1
cytokines. Clinical studies have reported a significant reduction
of iNKT cells, measured as V␣24/V␤11⫹ or ␣GalCer-loaded
CD1d tetramer-positive cells, in the peripheral blood of affected patients (17, 65) and reduced V␣24 mRNA levels and
V␣24-expressing cell numbers in the intestine (17). A decrease
of iNKT cells in the PBMC and of V␣24⫹ T cells in the
intestine has also been reported for patients with celiac disease
(16). However, as noted above, the majority of these V␣24⫹
cells were not iNKT cells, as judged by the absence of V␤11
expression (41).
For three mouse colitis models characterized by a TH1
immune response, the involvement of NKT cells has been
reported, including 1) adoptive cell transfer of naive CD4 T
cells into immune-deficient hosts, 2) oral challenge with dextran sodium sulfate (DSS), and 3) rectal instillation of the
hapten trinitrobenzene sulfonic acid (TNBS). In the transfer
model of colitis, cotransfer of T cells expressing the NK
receptor DX5 with the pathogenic CD4 T cells ameliorated
disease, and this effect could be blocked by injection of an
anti-CD1d antibody (21). However, because only a minority of
DX5⫹ NKT cells are CD1d dependent (45), it is not certain
which NKT cell population was responsible for the protection,
and, given the induction of IL-10 by anti-CD1d, it is possible
that the antibody triggered CD1d functions independent of
their interactions with subsets of NKT cells. DDS-induced
colitis could be ameliorated by treatment with the iNKT cell
agonists ␣GalCer (38, 50) or the closely related compound
OCH (63). OCH has been reported to shift the immune response stimulated by iNKT cell activation to the TH2 cytokine
pattern (36); consistent with this, the protection in the DSS
colitis model was directly linked to the increased production of
IL-4 and IL-10 and decreased production of IFN-␥ (63). A
similar protection by OCH treatment was observed for TNBSinduced colitis; importantly, treatment with OCH was even
effective at ameliorating an ongoing disease in the DSS model
(63). Although in one study a single injection of ␣GalCer led
to a shift to a protective TH2 response (50), two later studies
reported that repetitive challenges were necessary (38, 63).
This is in line with the reported TH2 shift by repetitive ␣GalCer
treatment (9). These data clearly demonstrate the protective
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As noted above, an important role for iNKT cells is possible
even in the absence of CD1d expression within the local
environment because of cytokine-mediated or indirect activation. However, CD1d expression obviously is a prerequisite for
the local, antigen-specific activation of iNKT and vNKT cells.
Because most hematopoietic cells express CD1d, CD1d also is
likely to be found on antigen-presenting cells in the intestine.
An example of biologically relevant CD1d expression on
antigen-presenting cells in the intestine is provided by studies
of mesenteric lymph node B cells. It had been found that colitis
in TCRa⫺/⫺ mice is exacerbated when these mice are also
deficient for B lymphocytes (37). There also was an increase in
colitis when the TCRa⫺/⫺ mice were Cd1d⫺/⫺, and a subset of
B cells in the mesenteric lymph node increased their expression
of CD1d under inflammatory conditions (37). Transfer of this
B cell subset to TCRa⫺/⫺ mice that were also B cell deficient
prevented the increased colitis observed in these mice (37).
Transfer of mesenteric lymph node B cells also prevented
colitis induction in the Gai2⫺/⫺ model (66); however, in both
of these cases, it is uncertain whether T cell reactivity to the
CD1d abundantly expressed by the B cells was required for
their beneficial effect.
Conflicting reports have been made regarding the expression
of CD1d on human and mouse intestinal epithelial cells (IECs).
One reason for the controversy is the unusual form of CD1d
that IECs were reported to express. Interestingly, the majority
of the CD1d expressed by IECs is nonglycosylated and not
associated with ␤2-microglobulin, and it is localized mainly
inside the cells, with surface expression restricted to the apical
surface (2, 28, 29, 56). Its function remains to be elucidated,
and there is no evidence for the recognition of this form of
CD1d by iNKT cells, although it has been suggested that some
T cells, presumably vNKT cells, recognize ␤2-microglobulinindependent CD1d molecules (3, 10, 43). Mouse monoclonal
antibodies that recognize native CD1d made by several groups
do not detect this ␤2-microglobulin-independent form of
CD1d; consequently, they do not detect CD1d expression by
IECs, contributing to the above-mentioned controversy. The
native ␤2-microglobulin-associated CD1d molecule is weakly
expressed by human IECs with a preference for the basal
surface (56). IECs have been reported to bind (50) and present
␣GalCer (64), suggesting that this low amount of native CD1d
expression is significant, but IECs could not present derivatives
requiring carbohydrate antigen processing in lysosomes (64).
Contradictory data regarding the expression of CD1d in
patients with colitis have been reported. Whereas one study
reported higher expression of CD1d in affected tissue (42), two
other reports found lower expression (15, 46). The production
of the anti-inflammatory cytokine IL-10 has been linked to
CD1d expression by IEC lines and primary cells, and this
required an intact cytoplasmic domain of CD1d, implying a
signaling function for this molecule (11, 37). Furthermore,
mice lacking microsomal triglyceride transfer protein, an endoplasmic reticulum resident lipid transfer protein, display a
defective loading and presenting capability of CD1d and were
largely protected against oxazolone-induced colitis (8). This
protection was linked to IECs, as microsomal triglyceride
transfer protein within the intestine is preferential expressed by
IECs; however, the authors did not directly address the role for
CD1d expressed by IEC.
Themes
CANONICAL NKT CELLS IN MUCOSAL IMMUNITY AND INFLAMMATION
NKT CELL RESPONSES DURING INTESTINAL INFECTIONS
There is evidence for a role for iNKT and/or vNKT cells
after oral infections with several pathogens. Listeria monocytogenes, the causative agents of listeriosis, is a Gram-positive,
facultative, intracellular pathogen. It is acquired by food-borne
infection, and it is cleared by the immune system via a TH1
immune response. After intragastric infection of L. monocytogenes in mice, iNKT cells produced IFN-␥ but not IL-4 within
1–2 days, and they stimulated IFN-␥ production by NK cells
(1, 48). Although iNKT cells were not essential for survival,
they participated in the protective immune response, as indicated by the reduced hepatic bacterial load and increased
systemic IFN-␥ in mice that have iNKT cells (1, 48), and this
protection could be transferred by purified iNKT cells (48).
Furthermore, CD1d-deficient mice displayed higher levels of
pathology in the intestine and to a lesser extent in the liver and
spleen (1), suggesting that iNKT cells were important in
limiting the spread of bacteria. However, another study reported the opposite effect of iNKT cell deficiency, as J␣18deficient mice displayed a higher resistance to L. monocytogenes infection (13).
A protective contribution of iNKT cells after an intravenous
or intraperitoneal challenge with Salmonella spp. has been
established (62, 68). However, after intragastric challenge, no
major impact of iNKT cell deficiency on the course of infection
could be found, despite activation and cytokine production
by iNKT cells (5). Note that activation of iNKT cells after
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Salmonella infection is by the indirect route, as these bacteria
do not have antigens that engage the V␣14i TCR (7).
Toxoplasma gondii is an obligate intracellular protozoan
parasite acquired by ingestion of parasitic cysts or oocysts
contained in food or water. In immune-competent hosts, this
infection is reliably controlled by a TH1 immune response.
However, in infected C57BL/6 mice, but not in other strains,
this TH1 immune response leads to lethal acute ileitis, due to
overwhelming IFN-␥ production. The potential of iNKT cells
to produce copious amounts of IFN-␥ after stimulation makes
them a candidate for the source of the pathogenic IFN-␥ in
T. gondii-infected C57BL/6 mice. In accordance with this, it
has been shown that J␣18⫺/⫺ C57BL/6 mice are less susceptible to ileitis after infection with T. gondii (49). The deleterious effect of iNKT cells in wild-type C57BL/6 mice could
almost entirely be blocked by pretreatment with ␣GalCer (49).
Systemic challenge of the mice with ␣GalCer 1 day before T.
gondii infection led to a pronounced TH2 cytokine shift of the
iNKT cells, with markedly reduced IFN-␥ and increased
amounts of IL-4 and IL-10 (49). IL-10, produced mainly by
iNKT cells and by CD4/CD25⫹ regulatory T cells, was shown
to be the main agent for protection from runaway inflammation
(49). Surprisingly, however, the same report also showed that
Cd1d⫺/⫺ mice had entirely the opposite phenotype compared
with the J␣18⫺/⫺ mice. After T. gondii infection, the Cd1d⫺/⫺
animals had an increased mortality (49), and similar data on
Cd1d⫺/⫺ C57BL/6 mice were reported by another group (55).
Both reports are examples of the surprising differences between J␣18⫺/⫺ animals, specifically lacking iNKT cells, and
Cd1d⫺/⫺ mice, which not only lack all CD1d-reactive iNKT
and vNKT cells but also other potentially important CD1ddependent pathways, for example those based on CD1d-mediated signal transduction (see above).
Despite the clear involvement of iNKT and vNKT cells in
many different systemic and intestinal infections and their
rapid activation by pathogenic and even nonpathogenic microbes such as Sphingomonas spp., iNKT cell-deficient mice
have not been reported to be more susceptible to spontaneous
infections with what are normally commensal bacteria. Furthermore, iNKT cells themselves are not dependent on commensal bacteria, as even in germ-free animals iNKT cells are
phenotypically activated/memory T cells that function normally after antigenic stimulation (44).
mNKT CELLS
Less is known about a second population of canonical NKT
cells found in humans and mice. As noted, these cells express
an invariant V␣19-J␣33 (mouse)- or V␣7.2-J␣33 (human)rearranged TCR ␣-chain (for reviews, see Refs. 60, 61). The
invariant TCR contains only a few N-nucleotide additions, and
the coexpressed V␤ usage is skewed to V␤13 and V␤2 in
human and V␤8.1/8.2 and V␤6 in mice (51, 57).
Although mNKT cells have been detected in blood, lymph
nodes, liver, spleen, and bone marrow (27, 51, 53, 58), they are
most prominent under steady-state conditions in mucosal sites
of the gut and the lung (27, 59 – 61), hence the appellation
mNKT cells, also called mucosal-associated invariant T
(MAIT) cells (59). By PCR analysis for the invariant rearrangement, mNKT cells are especially enriched in the lamina
propria and mesenteric lymph node and virtually absent in the
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potential of iNKT cells that have been activated by potent
agonists to shift the immune response from the deleterious TH1
cytokine pattern in these diseases to a protective TH2 response.
There is also a profound lack of IL-4 production by mononuclear cells purified from the affected intestines of patients with
Crohn disease (26, 67). However, it is important to note that
the depletion of NK and NKT cells had no effect on the course
of TNBS-induced colitis (20), and, in the absence of activation
by potent synthetic agonists, it remains to be demonstrated in
these models whether the normal functioning of NKT cells
influences colitis.
Oxazolone-induced colitis is an IBD model in which TH2
cytokines induce pathology. The mucosal immune response
following oxazolone exposure is initially dominated by IL-4,
which is soon superseded by an IL-13 response (20). The IL-13
derived from iNKT cells has been shown to be crucial for
disease development (20), and interfering with iNKT cell
activation prevented disease development (8, 20). However, in
patients with ulcerative colitis, it was found that vNKT cells,
rather than iNKT cells, were the major producers of the
deleterious IL-13 (14). Specifically, CD4/CD161⫹ T cells
accumulated in patients’ lamina propria and these cells were
capable of secreting IL-13 in vitro in a CD1d-dependent
fashion (14). However, these cells did not express V␣24 and
did not respond to ␣GalCer (14). Notwithstanding the absence
of functional data implicating iNKT cells in disease causation
in humans with IBD, iNKT cells were decreased in the PBMCs
of patients with ulcerative colitis (17, 65). The differing requirements in mice and humans for iNKT cells and vNKT
cells, in colitis characterized by IL-13 production by T cells in
the intestine, could be due in part to the lower frequency of
iNKT cells in most humans.
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CANONICAL NKT CELLS IN MUCOSAL IMMUNITY AND INFLAMMATION
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mNKT cells are ⬃5–10 times more frequent in humans than
in mice, accounting for ⬃15% of all double-negative T cells in
PBMCs (i.e., 0.1– 0.2% of all T cells) and for 10% of doublenegative T cells in the intestine (59 – 61). Because of their
paucity in mice, the generation of V␣19i transgenic animals
has greatly facilitated the investigation of this subpopulation
(27, 40), complemented by the generation of MR1-deficient
mice (59). Although the high evolutionary conservation of
mNKT cells and their prominence in the intestine suggest an
important role in the mucosal immune system, no nonredundant function of mNKT cells in the intestinal mucosa has yet
been described. For example, the frequency of V␣7.2i NKT
cells is not altered in patients with Crohn disease or ulcerative
colitis (61).
Similar to iNKT cells, mNKT cells rapidly produce effector
cytokines like IL-4, IL-5, IL-10, IL-17, and IFN-␥ after activation via MR1 or ␣CD3ε antibody in vitro and in vivo (12, 27,
52, 53, 59). mNKT cells also have the capacity to migrate to
sites of inflammation, where their cytokines could influence the
inflammatory process. It has been reported that they accumulate in some central nervous system lesions and in the cerebral
fluid of patients with multiple sclerosis, as well as in peripheral
nerve samples of patients with chronic inflammatory demyelinating polyneuropathy (24). The same group demonstrated a
protective role of V␣19i NKT cells during experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis (12). Overexpression of V␣19i NKT cells ameliorated the
disease, whereas depletion of V␣19i NKT cells exacerbated it
(12). Furthermore, donor V␣19i NKT cells could transfer
protection (12). The data indicate that the interaction of
NK1.1⫹ V␣19i T cells and B cells via ICOS/ICOS-L, and to a
lesser extent via CD40/CD154, induced the production of
protective IL-10 by both cell populations (12). In this setting, the
interaction of iTCR and MR1 seemed not to be required (12).
SUMMARY
For iNKT and mNKT cells, there are a number of striking
similarities. Both are selected in the thymus by nonpolymorphic MHC class I-like molecules expressed by hematopoietic
cells. Interestingly, CD1d and MR1 are encoded in close
proximity on chromosome 1 (19), perhaps suggesting a similar
evolutionary selection and function. Both cNKT cell populations display a phenotype characteristic of activated or memory
cells, they rapidly secrete effector cytokines, and they exhibit
a degree of autoreactivity. Both cNKT cell populations have
been reported to be specific for glycolipids, although the
antigens recognized by mNKT cells are not yet well characterized. Additionally, both populations are prominent in sites
other than the lymph node, such as the intestine for mNKT
cells or the liver, which collects circulation from the intestine,
for iNKT cells. iNKT cells require CD1d in the periphery for
their final differentiation, and mNKT cells depend on MR1
expression on B cells and the gut flora for their homeostasis
and localization to the intestine. Selection by hematopoietic
cells in the thymus might imprint the properties that cNKT
cells share. In this context, it is of interest to note that the
forced selection of MHC class II-reactive CD4 T cells in
transgenic mice that express MHC class II only on thymocytes
also led to the development of T cells bearing an effector/
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intestinal epithelium (27, 59, 60). In this context, the observation that V␣19i mNKT cells are absent in germ-free mice (59,
60) is quite interesting because it suggests that microbially
derived stimuli, either directly or indirectly, are required for
their selection and/or expansion.
V␣19i mNKT cells are absent in athymic nu/nu (nude) mice
(58), and hematopoietic cells mediate their thymic-positive
selection, rather than the cortical epithelial cells that positively
select conventional T cells (59). Furthermore, V␣19i mNKT
cells are absent in B cell-deficient mice and in patients with a
mutation in Bruton’s tyrosine kinase, which leads to a profound B cell deficiency (59). Innate-like B1-B cells and plasma
cells are not involved, however, in forming the mNKT cell
population (59, 60). Whether this dependency reflects a requirement for the few thymic B cells to select V␣19i mNKT
cells in the thymus, or perhaps more likely their expansion,
homeostasis, or homing in the periphery, is so far unresolved
(59 – 61). In line with the latter hypothesis, the few intestinal
IgA⫹ B cells that develop in ␮-heavy chain-deficient
(␮MT⫺/⫺) mice were sufficient for the development of V␣19i
mNKT cells (59).
V␣19i mNKT cells are restricted by MR1 (59), a ␤2mdependent nonpolymorphic MHC class I-like molecule encoded by a gene linked to the Cd1d gene (Fig. 1). Considering
the entire family of antigen-presenting molecules, MR1 shows
the highest degree of conservation between mouse and human
(19, 35). Although MR1 mRNA is ubiquitous (19), cellular
expression of the protein is still incompletely characterized. In
MR1-transfected cell lines, most of the protein is retained in
the endoplasmic reticulum; therefore, it has been suggested
that attainment of a native conformation leading to MR1
surface expression might be limited by the availability of a
specific MR1-binding ligand(s) (35). Consistent with this hypothesis, addition of a surplus of serum components could
increase the surface expression of MR1 (22, 35). This and other
indirect evidence (22, 35) make it very likely that MR1 acts as
an antigen-presenting molecule. Furthermore, because some
V␣19i mNKT cell hybridomas were activated spontaneously
by MR1-transfected cell lines (22, 59), there may be a substantial degree of autoreactivity by V␣19i mNKT cells, similar
to their CD1d-dependent iNKT cell counterparts. Endogenous
MR1-binding ligands for potentially self-reactive mNKT cells
are not known so far, but it has recently been reported that
␣-mannosylceramide and particularly some related compounds
with modifications of the ceramide lipid can activate V␣19i
NKT cells after binding to MR1 (40, 52–54).
Consistent with their potential autoreactivity, mNKT cells display an effector/memory phenotype in the periphery characterized
by the expression of CD27⫹, CD44high, CD45RA⫺, and CD57⫺
in humans (59) and CD25low, CD44high, CD45RBlow, CD62L⫺,
and CD69⫹ in mice (27). Most mNKT cells are CD4⫺CD8⫺
(double negative) (27, 58), ⬃60 – 80% in mice (27). Although
in mice V␣19i mNKT cells are never CD8␣⫹ (27, 58), some
human V␣7.2i NKT cells express CD8␣ (58). Furthermore,
mNKT cells express several costimulatory molecules such as
CD28, CD154, and inducible costimulator (ICOS) (12, 27, 51,
59), as well as NK cell receptors such as NK1.1 in mice and
CD161 in humans (12, 24, 27, 51, 53, 59). Finally, V␣7.2i
mNKT cells have been shown to express the integrin ␣4␤7, which
is required for gut homing (59).
Themes
CANONICAL NKT CELLS IN MUCOSAL IMMUNITY AND INFLAMMATION
ACKNOWLEDGMENTS
The authors thank Barbara A. Sullivan and Dirk M. Zajonc for critical
review of the manuscript (B. A. Sullivan) and for helpful discussions (B. A.
Sullivan, D. M. Zajonc).
Because of space constraints, we had to limit citations of the primary
literature.
GRANTS
This work was supported by National Institute of Allergy and Infectious
Diseases Grants AI-71922 and AI-45053 (M. Kronenberg) and a Marie Curie
Foundation fellowship (G. Wingender). This is manuscript number 944 from
the La Jolla Institute for Allergy and Immunology.
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