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The Biology of NKT Cells

Albert Bendelac,1 Paul B. Savage,2
and Luc Teyton3
Howard Hughes Medical Institute, Committee on Immunology and Department of
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Pathology University of Chicago, Chicago, Illinois 60637;

Department of Chemistry, Brigham Young University, Provo, Utah 84602;
email: paul
Department of Immunology, Scripps Research Institute, La Jolla, California 92037;

Annu. Rev. Immunol. 2007. 25:297–336 Key Words

First published online as a Review in natural killer T cell, lymphocyte development, innate immunity,
Advance on December 6, 2006
α-proteobacteria, Sphingomonas, Ehrlichia, Salmonella, glycolipid,
The Annual Review of Immunology is online CD1d, antigen presentation

This article’s doi: Abstract

Recognized more than a decade ago, NKT cells differentiate from
Copyright  c 2007 by Annual Reviews. mainstream thymic precursors through instructive signals emanat-
All rights reserved
ing during TCR engagement by CD1d-expressing cortical thymo-
0732-0582/07/0423-0297$20.00 cytes. Their semi-invariant αβ TCRs recognize isoglobotrihexo-
sylceramide, a mammalian glycosphingolipid, as well as microbial
α-glycuronylceramides found in the cell wall of Gram-negative,
lipopolysaccharide-negative bacteria. This dual recognition of self
and microbial ligands underlies innate-like antimicrobial functions
mediated by CD40L induction and massive Th1 and Th2 cytokine
and chemokine release. Through reciprocal activation of NKT cells
and dendritic cells, synthetic NKT ligands constitute promising new
vaccine adjuvants. NKT cells also regulate a range of immunopatho-
logical conditions, but the mechanisms and the ligands involved
remain unknown. NKT cell biology has emerged as a new field
of research at the frontier between innate and adaptive immunity,
providing a powerful model to study fundamental aspects of the
cell and structural biology of glycolipid trafficking, processing, and

ANRV306-IY25-12 ARI 11 February 2007 12:20

INTRODUCTION tion of autoimmune, allergic, and tumor con-

ditions of broad clinical interest, where the
Several lines of research led to the identifi-
function of NKT cells remains speculative or
Natural killer T cation of NKT cells as a separate lineage of
(NKT) cell: a T cell controversial.
T lymphocytes. The first sightings included
expressing a (a) the identification of a canonical Vα14-
Jα18 ( Jα18 was previously known as Jα281 or
lipid-specific T cell DEFINITION
receptor combining Jα15) rearrangement in a set of hybridomas
a canonical derived from mouse KLH (keyhole limpet NKT cells are narrowly defined as a T cell
Vα14-Jα18 α chain hemocyanin)-specific suppressor T cells lineage expressing NK lineage receptors, in-
with a variable Vβ8, (1–3), and later in cDNA extracted from lym- cluding NK1.1 in the C57BL/6 background,
-7, or -2 β chain in
phoid organs of unimmunized mice (4, 5); in addition to semi-invariant CD1d-restricted
mouse or
Vα24-Jα18/Vβ11 in (b) the identification of a subset of mouse αβ TCRs. More than 80% of these TCRs
human CD4− 8− double-negative (DN) T cells with are Vα14-Jα18/Vβ8, Vβ7, and Vβ2 in mouse
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CD1: a family of a Vβ8 usage bias (6, 7); and (c) the identi- (or Vα24-Jα18/Vβ11 in human), with the
MHC-like molecules fication of a recurrent Vα24-Jα18 rearrange- remaining representing a collection of rare
that specialize in ment in human DN peripheral blood lympho- but recurrent Vα3.2-Jα9/Vβ8, Vα8/Vβ8,
presenting lipid cytes (8, 9). These observations were pieced and other TCRs (19, 20). Whereas both
antigens to
together when a subset of CD4 and DN the Vα14 and the non-Vα14 NKT cells
T lymphocytes
IL-4-producing thymocytes co-expressing exhibit autoreactivity to CD1d-expressing
NK lineage receptors was independently cells, particularly thymocytes, their antigen
glycolipids that identified and shown to express a biased set specificities do not overlap. Thus, mVα14
substitute for LPS in of Vβ8, Vβ7, and Vβ2 T cell receptor (TCR) and hVα24 NKT cells, irrespective of their
the cell wall of β chains (10–13) combined with a canonical Vβ-Dβ-Jβ chain usage, recognize a ma-
Gram-negative, Vα14-Jα18 in mouse (14) and with the ho- rine sponge–derived α-galactosylceramide
mologous Vα24-Jα18/Vβ11 pair in human (αGalCer) (21, 22) and closely related micro-
bacteria such as
Sphingomonas (14, 15). The finding that the mouse and hu- bial α-glycuronylceramides (23–25), as well
man NKT cells were autoreactive to cells as the self antigen isoglobotrihexosylceramide
expressing CD1d (15–18), a member of the (iGb3) (26). In contrast, the self and foreign
CD1 family of MHC-like molecules, com- antigens recognized by non-Vα14 NKT cells
pleted the initial characterization of this lin- remain to be identified. A striking, generic dif-
eage and raised modern questions relating to ference between Vα14 and non-Vα14 NKT
their development, specificity, and function. cells is that the natural Vα14 NKT ligands,
These issues have been treated in more including iGb3, require endosomal traffick-
than 1500 reports over the past 10 years, more ing of CD1d and intact lysosomal functions
than 300 of which were published in the past for presentation at the cell surface, whereas
year alone. We attempt to organize a critical the non-Vα14 ligands are normally presented
understanding of the general biology of NKT by a tail-truncated CD1d, which is defective
cells, mainly of the predominant mVα14 and in endosomal trafficking and likely presents
hVα24 subsets, on the basis of recent fun- antigens loaded in the secretory pathway or at
damental advances and newly emerging con- the cell surface (27). These CD1d-restricted
cepts. Owing to space limitations, it is not pos- NKT cells should be distinguished from
sible to exhaustively review or mention all the CD1d-restricted T cells that express nonin-
studies, many of which suggest new roles of variant TCRs and from a variety of other non-
NKT cells in various diseases and remain rel- CD1d-restricted T cells that express NK lin-
atively preliminary or isolated. We focus on eage receptors (28, 29). Although some studies
bacterial infections where the role of NKT have recently implicated non-Vα14 CD1d-
cells is well established and examine a selec- restricted T cells in various diseases, this

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review focuses mainly on the canonical and the source of the various lipids that
mVα14 and hVα24 NKT cells. bind naturally to CD1d remain poorly elu-
cidated. Early studies of CD1d immunopre-
cipitates obtained from cell detergent lysates
SPECIES AND TISSUE suggested a predominance of phospholipids—
DISTRIBUTION particularly glycosylphosphatidylinositols, an
Vα14 NKT cells have been well characterized anchor for various surface proteins, and phos-
in mouse, where they represent ∼0.5% of the phatidylinositols (45, 46). However, because
T cell population in the blood and peripheral these early studies used detergents that could
lymph nodes, ∼2.5% of T cells in the spleen, potentially displace natural lipids bound to
mesenteric, and pancreatic lymph nodes, and CD1d, or soluble forms of CD1d that did
up to 30% of T cells in the liver. Although not traffic through the endosome and might
their precise distribution within the lymphoid have acquired irrelevant lipids from mem-
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organs is still unknown, they reside within the brane compartments or culture medium, their
liver sinusoids, which they appear to patrol. interpretation is uncertain. Future studies of
Their expression of CXCR6 matches the ex- CD1d molecules engineered to express an
pression of CXCL16 on the endothelial cells enzymatic cleavage site at the membrane-
lining the sinusoids and appears to be impor- proximal portion of their extracellular domain
tant for survival rather than for migration (30). constitute an attractive approach to reexam-
NKT cell frequency in the whole thymus is ining this fundamental issue. Despite a lack
∼0.5%, but they represent up to 5% of the of direct biochemical studies of CD1-bound
recent thymic emigrants found in the spleen lipids, combinations of genetic, cell biological,
(31, 32). Although the tissue distribution is and chemical approaches have nevertheless
less well studied in humans, Vα24 NKT cells uncovered some key NKT ligands discussed
appear to be ∼10 times less frequent in all below.
these locations. However, high and low NKT
cell expressors exist in mice and in humans,
and NKT cell frequency appears to be a sta- Marine Sponge αGalCer
ble phenotype under the genetic control of The first NKT ligand emerged from studies
at least two recessive loci in mouse (33, 34). initiated at Kirin Pharmaceuticals to identify
Low Vα14 NKT cell expressors in mice in- natural anticancer medicines. Extracts from
clude NOD and SJL (35–37). The range of Agelas mauritianus, a marine sponge collected
frequencies found in human blood varies by in the Okinawan sea, prolonged survival of
up to 100-fold between individuals but is un- mice bearing B16 melanoma (47). The struc-
der strict genetic control, as shown by identi- ture of the active principle was identified as an
cal twin studies (38). Similar frequencies have α-branched galactosylceramide and slightly
been found in nonhuman primates (39). Vα14 modified for optimal efficacy to produce a
NKT cells are present in rats (40, 41), and, compound termed KRN7000, also commonly
based on genomic and functional studies of referred to as αGalCer (Figure 1) (48). The
CD1d, they may be absent in cows (42). lipid nature of this compound, its strong ef-
fect on liver metastasis, and its activation of
dendritic cells (DCs) independent of MHC
NKT LIGANDS class I or class II (49) led to the identifi-
Although disputed initially (43), there is now cation of Vα14 NKT cells as their target
a general consensus that CD1d, like other (21). As a surrogate ligand of very high ac-
CD1 family members, evolved to present tivity in vitro and in vivo, in the picomo-
lipids to T cells (44). However, the nature lar range αGalCer has been used broadly in • Biology of NKT Cells 299

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O O C2'
αGalCer HO C1"
(KRN7000) OH
O C3
C2 C4

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GSL- 2 O
GSL- 4 O


iGb3 OH O HN

Figure 1
Self and microbial glycosphingolipid ligands (GSL) of NKT cells. (a) Marine sponge αGalCer
(KRN7000) with carbon atom number assignments on sphingosine (C), acyl (C ), and carbohydrate (C );
(b) Sphingomonas GSL-1 through GSL-4; and (c) mammalian isoglobotrihexosylceramide (iGb3), or
Galα1,3Galβ1,4Glcβ1,1Cer. Note that the proximal glucose of the mammalian glycosphingolipid has a
β-anomeric linkage to ceramide, in contrast with the α-branched galactose of αGalCer or glucuronyl of
Sphingomonas GSLs.

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various functional assays and to generate the

first CD1d tetramers specific for mouse and α-PROTEOBACTERIA
human NKT cells. The affinity of interaction
between CD1d-αGalCer and mouse TCRs is α-proteobacteria constitute one of the most ubiquitous classes
one of the highest ever recorded for natural of Gram-negative bacteria on Earth. They exhibit a wide
TCR/ligand pairs with a Kd ∼100 nM, owing range of lifestyles, from free-living to obligate intracellular
to a slow off rate, for several Vα14-Jα18/Vβ8 pathogens, and are found in marine and soil environments.
combinations examined (50, 51) and may Obligate intracellular organisms include the Rickettsiales, with
be significantly lower in the human system lethal tick-borne pathogens such as Rickettsia and Ehrlichia,
(∼7 μM) (52). Although the expression of this agents of the ancient plague epidemic typhus, Rocky Moun-
ligand in marine sponges could not be linked tain spotted fever, and other severe febrile and typhus-like
with any physiologically relevant function, the syndromes. Whereas some of the Rickettsiae express LPS,
striking properties of αGalCer have provided the Ehrlichiae lack the genes required for LPS and pepti-
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early support for the hypothesis that the con- doglycan synthesis, and the composition of its cell wall is
served TCRs of NKT cells evolved to rec- mysterious. Mitochondria represent the ultimate example of
ognize conserved lipids. More than 95% of α-proteobacteria that have established an obligate relation-
cloned mouse and human NKT cells recog- ship with eukaryotic hosts. Bartonella and Brucella (an LPS
nize αGalCer, irrespective of their variable expressor) belong to a group phylogenetically related to the
CDR3 β sequence, and the mouse CD1d- Rickettsiales. Sphingomonas is a ubiquitous bacterium found in
αGalCer tetramers stain human and nonhu- marine (e.g., sponges and corals) and terrestrial environments
man primate NKT cells as well (22, 39), at- that is actively studied by industrial microbiologists because
testing to the high degree of conservation of of its ability to degrade xenobiotic aromatic compounds. Its
this recognition system. cell wall contains α-glycuronylceramide ligands of NKT cells,
instead of LPS. Sphingomonas was detected by PCR in stool
samples of 25% of healthy human beings and can cause acute
Microbial Ligands infections, particularly in immunocompromised individuals.
The lack of physiological relevance of Intriguingly, on the basis of the presence of a specific anti-
αGalCer should be revisited with the re- body response in patients’ sera, it has been implicated in the
cent discovery that closely related struc- etiopathogeny of primary biliary cirrhosis, a chronic autoim-
tures that substitute for lipopolysaccharide mune disease targeting intrahepatic bile ducts.
(LPS) are found in the cell wall of Sph-
ingomonas, a Gram-negative, LPS-negative
member of the class of α-proteobacteria (53, son and location and because these sponges
54). These glycosphingolipids are responsi- are often colonized by α-proteobacterial sym-
ble for the strong stimulation of NKT cells bionts, particularly by Sphingomonas (58), the
and their role in clearing infection (23–25, 55). marine sponge αGalCer may in fact have orig-
The most abundant glycosphingolipids have inated from bacterial symbionts.
only one sugar, galacturonyl or glucuronyl,
α-anomerically branched to the ceramide
backbone (Figure 1, GSL-1). Thus, they dif- Self Ligand iGb3
fer from the stimulating αGalCer or αGlcCer Although the discovery of bacterial NKT lig-
mainly by the carboxyl group in C6 , a po- ands provides a fascinating new perspective on
sition permissive to NKT cell recognition the evolutionarily relevant functions of NKT
(56, 57). Other more complex but less abun- cells, considerable attention has also focused
dant glycosphingolipids include GSL-2, -3, on self ligands. Indeed, mouse and human
and -4 (Figure 1). Because in general it NKT cells exhibit conspicuous low-level au-
is known that extracts from A. mauritianus toreactivity to various CD1d-expressing cell
have different properties depending on sea- types (15, 17, 59). This autoreactivity and • Biology of NKT Cells 301

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the presence of IL-12, triggered by Toll- their ability to process and present iGb4—the
like receptor (TLR) signaling, are required precursor to iGb3—or GalNAcβ1,4GalαCer,
for the commonly observed IFN-γ secre- both of which require removal of the outer,
tion by NKT cells during immune responses β-branched hexosamine for NKT cell recog-
against Gram-negative, LPS-positive bacte- nition. In addition, the Griffonia simplicifolia
ria (23, 60). Autoreactivity may also under- isolectin B4 (IB4) specific for the terminal
lie the thymic development of NKT cells Galα1,3Gal blocked CD1d-mediated presen-
(18), which includes an expansion phase af- tation of both exogenous iGb3 and endoge-
ter positive selection (31) and the acquisi- nous ligand (natural autoreactivity), but not
tion of a memory phenotype independent of αGalCer. These studies suggest that iGb3 is
microbial exposure or TLR signaling (61). an important physiological ligand of NKT
Recent findings demonstrate that the gly- cells. Additional findings reviewed below sug-
cosphingolipid iGb3 (Figure 1), both natu- gest that iGb3 may also be the natural ligand
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ral and synthetic, could activate a majority of activating NKT cells during Gram-negative,
mouse Vα14 and human Vα24 NKT cells, LPS-positive infections. These results are
irrespective of their Vβ chain, upon presen- therefore consistent with the requirement for
tation by DCs or plastic-bound CD1d/iGb3 endosomal trafficking of CD1d (27, 64) and
preformed complexes (26, 62, 63). iGb3 ap- the role of lysosomal saposins functioning as
pears to be a weaker agonist than αGalCer, glycosphingolipid exchange proteins in the
requiring ∼30- to 100-fold higher concen- presentation of the NKT ligand in vivo (65,
trations to achieve the same level of stim- 66). It should be noted, however, that the pres-
ulation. This may explain the failure to ence of iGb3 among CD1d-bound lipids re-
stain NKT cells using CD1d/iGb3 tetramers. mains to be demonstrated and that iGb3 itself
However, solubility issues and more strin- has not yet been directly identified in human
gent requirements for professional antigen- or mouse tissue, a task complicated by the rar-
presenting cells (APCs) may contribute to its ity of iGb3 and the dominance of the regioiso-
lower apparent activity, and the affinity of mer Gb3. Furthermore, other than the enzy-
CD1d/iGb3-TCR interactions remains to be matic pathways of synthesis and degradation,
measured directly, particularly to dissect the little is known about the general biology of
contribution of on and off rates. iGb3, its subcellular location, or its function.
Different lines of experiments suggest
that iGb3 is an important physiological
NKT ligand. β-hexosaminidase-B-deficient Other NKT Ligands
mice, which lack the ability to degrade α-galactosyldiacylglycerols expressed by
iGb4 into iGb3 in the lysosome, exhibited Gram-negative LPS-negative Borrelia
a 95% decrease in thymic NKT cell pro- burgdorferi, the agent of Lyme disease,
duction, and β-hexosaminidase-B-deficient resemble α-galactosylceramide and could
thymocytes could not stimulate autoreactive directly stimulate NKT cells (67). However,
Vα14 NKT cell hybridomas (26). Notably, recognition of intact or heat-killed bacteria
unlike other mutations of enzymes or trans- could not be demonstrated, and only one iso-
porters involved in lipid metabolism and as- lated report has suggested defective bacterial
sociated with lipid storage, the defect in clearance in vivo (68).
β-hexosaminidase-B-deficient cells appeared Purified phosphatidylinositolmannoside
to be specific in that β-hexosaminidase-B- PIM4, a mycobacterial membrane phospho-
deficient bone marrow–derived DCs nor- lipid, was reported to elicit IFN-γ but not
mally presented several complex derivatives IL-4 production from a fraction of mouse and
of αGalCer that required lysosomal process- human NKT cells, and PIM4-loaded CD1d
ing prior to NKT cell recognition, but lost tetramers showed weak staining of a fraction

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of NKT cells (69). However, CD1d-deficient (76) also did not exhibit NKT cell defects, the
mice did not reveal defects in mycobacterial physiological relevance of these observations
clearance (70), and a synthetic PIM4 failed to remains intriguing.
stimulate NKT cells (67). Because multiple In summary, despite some exciting break-
components of the mycobacterial cell wall throughs, this difficult and essential area of
are strong activators of TLR expressed study is somewhat controversial and remains
by APCs, contaminating lipids associated a work in progress. Owing to an array of cri-
with the PIM4 preparation may cause in- teria, including stimulation or staining by re-
direct stimulation of NKT cells through combinant CD1d complexed with synthetic
presentation of their endogenous ligand ligands, lack of TLR signaling requirement,
and amplification of IFN-γ production by stimulation of proliferation and cytokine se-
TLR-induced IL-12 (see Dual Reactivity to cretion by large populations of fresh NKT
Self and Microbial Ligands: A Paradigm for cells in mouse and human, and genetic or
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NKT Cell Activation and Function During functional indications of relevance in vivo dur-
Bacterial Infections). ing physiological processes and diseases, iGb3
Purified phospholipids originally extracted and microbial α-glycuronylceramides repre-
from tumors, such as phosphatidylinosi- sent the most compelling NKT ligands iden-
tol, phosphatidylethanolamine, and phos- tified so far. Their identification considerably
phatidylglycerol, weakly stimulated some reinforces the view that NKT cells and their
Vα14 and non-Vα14 NKT hybridomas when canonical mVα14-Jα18/hVα24-Jα18 TCRs
loaded onto recombinant CD1d, but there is evolved to recognize conserved ligands and
little support at present for their physiologi- to perform innate-like rather than adaptive
cal importance because neither the tumor nor functions. The significance of other reported
the synthetic lipids could expand or activate individual specificities without functional cor-
fresh NKT cells in vivo or in vitro (71). An- relates remains uncertain.
other report suggested the presence of CD1d-
restricted phosphatidylethanolamine-specific
αβ and γδ T cells in the blood of patients STRUCTURAL BIOLOGY OF
with pollen allergies, although few clones ex- GLYCOLIPID RECOGNITION
pressed the canonical Vα24 TCR (72, 73). Recent reports of the crystal structure of sev-
Human melanomas overexpress the gan- eral CD1d/lipid complexes have far-reaching
glioside GD3, and, on the basis of CD1d/ implications. The lipid-binding pocket of
GD3 tetramer staining, immunization with CD1d is particularly well adapted to bind
the human melanoma SK-MEL-28 was re- self and microbial glycosphingolipids, with
ported to expand a very small subset of Vα14 the acyl chain in the A hydrophobic pocket
NKT cells in mice in vivo (74). These studies, and the sphingosin chain in the F hydropho-
however, did not demonstrate a role for NKT bic channel (77–79). For αGalCer and the
cells in rejection of GD3-overexpressing closely similar α-glycuronylceramides, the α1
tumors. helix Arg79 and Asp80 establish hydrogen
Another common glycosphingolipid, bonds with the hydroxyl groups of the sph-
β-galactosylceramide, was shown to induce ingosine. The α2 helix Asp153 stabilizes the
downregulation of NKT cell numbers and galactose through hydrogen bonds with the
TCR surface level in whole spleens examined 2 and 3 hydroxyl group, solidly anchor-
in vivo and in vitro (75). These effects were ing the protruding sugar in a position paral-
relatively modest even at high concentrations lel to the plan of the α helices and explain-
of lipids, and a direct stimulation or expansion ing the exquisite stimulatory properties of
of cloned NKT cells could not be observed. several hydroxyl groups (Figure 2). Because
Because mice lacking β-galactosylceramide α-anomeric glycosylceramides do not exist in • Biology of NKT Cells 303

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Figure 2
Crystal structure of CD1d/αGalCer. (a) Transparent pocket view where the outer surface (light gray) of
CD1d has been partially removed to expose the binding groove inside (dark gray). The short αGalCer
PBS25 is found with the short C8 acyl chain in the A pocket and with the C18 sphingosine in the F
pocket. Note the deeply buried spacer C16 lipid at the bottom of the A pocket, likely originating from
the fly cell culture system where mouse recombinant CD1d was produced. (b) View of the α-anomeric
galactose sitting flat atop the groove. Molecular surfaces are presented with electrostatic potentials (red,
electronegative; blue, electropositive). The charged residues (Asp80, Arg79, and Asp153) involved in
hydrogen bonding with the hydroxyl groups of the carbohydrate and the sphingosine are indicated.

mammals, this structure represents a signa- and β loops (81), suggesting an unusual mode
ture of microbial invasion. of recognition of the trisaccharide within this
Notably, CD1d produced in fly cells in- TCR cavity. Future crystallographic studies of
cluded a spacer lipid present at the bottom of CD1d-iGb3 and ternary complexes with the
the A pocket, which preempted the loading TCR should clarify these fundamental issues
of full-length mammalian glycosphingolipid and illuminate novel aspects of carbohydrate
and explained why in general short lipids recognition by immune receptors.
have proven easier to load onto CD1d in the
absence of lipid transfer proteins. However,
lipids with long and short (C8 ) acyl chain CELL BIOLOGY OF LIPID
produced identical conformations when com- PRESENTATION BY CD1d
plexed with CD1d, and they bound the TCR CD1d is prominently and constitutively ex-
with similar on and off rates (77, 80). pressed by APCs such as DCs, macrophages,
CD1d-iGb3 complexes have not yet been and B cells (82, 83), particularly marginal zone
reported, but modeling suggests that the B cells (82), with relatively modest changes as-
β-linked sugar should emerge orthogonal to sociated with TLR activation and inflamma-
the plan of the α helices (77), which raises tory cytokines (84). CD1d is also strikingly
the general issue of how the TCR will recog- expressed on cortical thymocytes, where it is
nize two radically different structures and, in essential for NKT cell development (18), and
particular, accommodate the three protruding on Kupffer cells and endothelial cells lining
sugars. Intriguing insights have come from a liver sinusoids, where the highest frequen-
report that the human Vα24/Vβ11 TCR dis- cies of NKT cells are found in mice (30).
plays an unusual cavity between the CDR3 α Hepatocytes express CD1d constitutively in

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mouse and upon disease induction in human, pes virus, causing downregulation from the
for example, in the context of hepatitis C (85). cell surface without degradation (95). Inter-
CD1d expression in the liver is not required, estingly, another herpes virus, herpes simplex
however, for NKT cell homing (86), and nei- virus-1 (HSV-1), induces CD1d downregu-
ther is CXCR6 expression by NKT cells, al- lation from the cell surface, but the mecha-
though CXCR6/CXCL16 interactions are es- nism appears to be distinct, involving lysoso-
sential for survival in this organ (30). CD1d mal retention through impaired recycling to
is upregulated on microglial cells during in- the plasma membrane (96).
flammation (87). Similar to the MHC class
II system, most other solid tissue cells and
non-antigen-presenting hematopoietic cells Intersection of CD1d and Lipids
express low or undetectable levels of CD1d. in Late Endosome and Lysosome
Tail-truncated CD1d molecules fail to access
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the late endosome and lysosome, causing a

Trafficking of CD1d profound disruption of CD1d-mediated anti-
The intracellular trafficking of CD1d has gen presentation in vitro in cell lines and in
been studied thoroughly (Figure 3). Biosyn- vivo in knockin mice. Particularly affected are
thesis of the heavy chain associated with the presentation of the NKT endogenous lig-
β2-microglobulin involves the endoplasmic and (27) and, consequently, the thymic gen-
reticulum chaperones calnexin and calretic- eration of Vα14 NKT cells (64). The pre-
ulin and the thiol oxidoreductase ERp57 (88). sentation of diglycosylated αGalCer variants
It is logical to assume that endogenous lipids requiring processing prior to NKT cell recog-
in the endoplasmic reticulum would fill the nition, an important tool for research (56), or
groove of CD1d, and one study suggested the of iGb4, which requires processing into iGb3
presence of phosphatidylinositol (45), with prior to recognition, is also abolished (26).
the caveat that contamination by membrane However, other lipids that do not require
phospholipids could not be formally excluded. processing still exhibit variable requirements
CD1d rapidly reaches the plasma membrane for the late endosome and lysosome traf-
within 30 min after biosynthesis and under- ficking of CD1d, either partial in the case
goes extensive internalization and recycling αGalCer (three- to fivefold shift in dose re-
between the plasma membrane and endoso- sponse) or substantial in the case of iGb3
mal/lysosomal compartments in a manner de- (>10-fold shift). Recent studies of lipid up-
pendent upon a tyrosine motif encoded in the take, trafficking, and loading have begun to
CD1d cytoplasmic tail (89–91). The tyrosine shed some light on these observations.
motif in the cytoplasmic tail primarily binds
adaptor protein (AP)-2 and AP-3 in mouse
(92, 93), where the bulk of CD1d accumu- Lipid Uptake and Trafficking
lates in the lysosome, and AP-2 in humans, Lipids in the circulating blood or in cul-
where CD1d tends to reside in the late en- ture medium are bound to lipoproteins, and a
dosome (94). Additional but largely redun- dominant role for VLDL in the serum and its
dant contributions by the invariant chain or receptor, the LDL receptor, at the cell surface
invariant chain/MHC class II complexes that has been proposed for the clathrin-mediated
bind weakly to CD1d have been documented uptake of some lipids into endosomal com-
in mouse and human (89, 90). The CD1d partments (Figure 3) (97). Other extracellular
intracytoplasmic tail also expresses a lysine lipids can be captured by the mannose re-
targeted for ubiquitination by the MIR pro- ceptor langerin (98, 99) or can insert them-
teins of the Kaposi sarcoma–associated her- selves directly in the outer leaflet of the • Biology of NKT Cells 305

ANRV306-IY25-12 ARI 11 February 2007 12:20

iGb3 Exogenous
Vα14 TCR
Vα14 TCR



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Golgi Late endosome/lysosome



Figure 3
Intracellular trafficking and lipid loading of CD1d. Newly biosynthesized CD1d molecules, likely
containing lipid chains, reach the plasma membrane and are internalized through an AP-2/AP-3
clathrin-dependent pathway to late endosomal/lysosomal compartments, where lipid exchange is
performed by saposins. The endogenous ligand iGb3 is produced through lysosomal degradation of iGb4
by β-hexosaminidase. CD1d extensively recycles between lysosome and plasma membrane, allowing
further lipid exchange. Exogenous lipids bound to lipoproteins may enter the cell with VLDL (very low
density lipoprotein) particles through the LDL receptor pathway, whereas microbial lipids can be
released in the lysosome after fusion with the microbial phagosome. Additional lipid exchange proteins
may be involved in these processes, particularly during biosynthesis, when a role for microsomal
triglyceride transfer protein (MTP) has been proposed.

plasma membrane and undergo endocytosis reached the late endosome and were rapidly
through clathrin-dependent or -independent sorted to the endoplasmic reticulum and
pathways (100). the Golgi. In contrast, a prodan-conjugated
Glycosphingolipids tagged with a flu- (on carbohydrate C6 ) αGalCer accumu-
orochrome, BODIPY, on the acyl chain lated selectively in the lysosome (102). These

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pathways overlap only partially with those with the lipid in the groove (107). In contrast,
governing the trafficking of endogenous gly- during biological processes, membrane lipids
cosphingolipids, which are synthesized in the are extracted and transported by lipid ex-
lumenal part of the Golgi and thought to change proteins (108). Prosaposin is a protein
reach the plasma membrane first, then the precursor to four individual saposins, A, B,
endosome, through clathrin-dependent and C, and D, released by proteolytic cleavage in
-independent endocytosis until they are de- the lysosome. Prosaposin-deficient mice pro-
graded in the lysosome (103). How exoge- vided the first genetic link between NKT cells
nously administered or endogenous intracel- and lipid metabolism, as they lacked NKT
lular lipids choose between these pathways cells and exhibited greatly impaired ability
and the consequence for antigen presenta- to present various endogenous and exoge-
tion are questions that are just beginning to nous NKT ligands (65, 66). In cell-free as-
be addressed and may depend on intrinsic says, recombinant saposins readily mediated
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properties such as length or insaturation of lipid exchange between liposomes and CD1d
alkyl chains (104), composition of the po- in a nonenzymatic process requiring equimo-
lar head, and solubility in aqueous environ- lar concentrations of CD1d and saposins (65).
ments, as well as extrinsic variations in the Although they exhibited some overlap in lipid
mode of administration such as use of deter- specificity, individual saposins differed in their
gents, liposomes, or lipid-protein complexes. ability to load particular lipids. More de-
The development of new methodologies, ge- tailed studies of the effects of these and other
netic manipulation, and reagents will be re- lipid exchange proteins such as NPC2 and
quired to address these essential questions. In the GM2 activator are required to under-
addition, recognition of microbial lipids in the stand their function individually or cooper-
context of infection most likely involves dif- atively at different phases of lipid processing
ferent pathways because the uptake of bacteria and loading. In addition, the structural basis
is governed by different sets of cell surface re- of the lipid exchange mechanism and its rel-
ceptors and the release of cell wall lipids would ative specificity for lipid subsets remain to be
occur through degradation of the microor- elucidated.
ganism in the lysosome before processing and Another lipid transfer protein expressed
loading onto CD1d. in the endoplasmic reticulum, microsomal
triglyceride transfer protein (MTP), assists in
the folding of apolipoprotein B by loading
Lipid Exchange Proteins lipids during biosynthesis. Coprecipitation of
Although an intrinsic, pH-dependent mecha- MTP with CD1d suggested that MTP might
nism appears to favor the acquisition of some play a similar role for CD1 molecules (109).
lipids by CD1 proteins, perhaps through a Indeed, genetic or drug-induced inhibition of
conformational change (105, 106), lipid ex- MTP was associated with defects in lipid anti-
change now appears to be regulated by spe- gen presentation (109, 110). MTP was sug-
cialized lipid transfer proteins. By using vari- gested to transfer phosphatidylethanolamine
ous detergents, early studies of lipid binding to onto CD1d in a cell-free assay, but the ef-
CD1 molecules tacitly dealt with the fact that ficiency of this process remains to be estab-
in general lipids are insoluble in water, form- lished, and cell biological studies are required
ing micelles that cannot transfer monomeric in vivo to fully understand the role of MTP in
lipids onto CD1. These detergents, however, CD1d-mediated lipid presentation.
also tended to dislodge lipids bound to CD1, CD1e is a member of the human CD1 fam-
as shown directly in the crystal structure of ily that is not expressed at the plasma mem-
CD1b complexed with phosphatidylinositol, brane but is instead found as a cleaved soluble
where two molecules of detergent cohabited protein in the lysosome. Recent experiments • Biology of NKT Cells 307

ANRV306-IY25-12 ARI 11 February 2007 12:20

have shown that CD1e could assist the lacking β-hexosaminidase B, α-galactosidase
enzymatic degradation of phosphatidylinos- A, or galactosylceramidase did not show
itolmannoside, suggesting that this protein much alteration of general lipid functions
may have diverged from other CD1 molecules because they conserved their ability to pro-
to perform ancillary functions rather than cess several complex diglycosylated deriva-
to carry out direct antigen presentation tives of αGalCer for presentation to NKT
(111). cells (26, 56, 65), although a divergent re-
port was recently published (101). In contrast,
β-galactosidase-deficient cells exhibited more
Membrane Transporters general defects than expected from the speci-
NPC1 is a complex membrane multispan pro- ficity of the mutated enzyme ( J. Mattner and
tein present in the late endosome that is mu- A. Bendelac, unpublished data, and Reference
tated in Niemann-Pick type C1 disease and 101).
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associated with a lipid storage phenotype sim-

ilar to NPC2, a soluble lipid transfer protein
present in the lysosome. NPC1-mutant mice Cathepsins
exhibited broad defects of NKT cell develop- Paradoxically, studies of cathepsin-mutant
ment and CD1d-mediated lipid presentation, mice led to the first reports of defects in NKT
which could be attributed in part to an arrest cell development and CD1d-mediated lipid
of lipid transport from late endosome to lyso- antigen presentation. This is particularly well
some (102). The precise function of NPC1 established for cathepsin L because mutant
remains unknown, and it is unclear how this thymocytes, but not DCs (perhaps owing to
putative flippase translocating lipid between the redundancy of other cathepsins), failed to
leaflets of the membrane bilayer could induce stimulate Vα14 NKT hybridomas in vitro and
general alterations of lipid trafficking. consequently failed to select NKT cells in vivo
(112). Although its target remains to be identi-
fied, cathepsin L may be directly or indirectly
Other Glycosidases and Lipid required for thymocytes to process prosaposin
Storage Diseases into saposins.
Mutations of several proteins involved in
glycosphingolipid degradation or transport
are accompanied by lipid storage within dis- NKT CELL DEVELOPMENT
tended lysosomal vesicles, the impact of which Based on their canonical TCR receptors and
depends on the enzyme, the cell type, the antigenic specificities, their unusual expres-
mouse strain, and the age at which cells sion of NK lineage markers, their peculiar tis-
are examined (100, 101). This lipid accu- sue distribution, and their functional proper-
mulation may disrupt rate-limiting steps of ties independent of environmental exposure
lipid metabolism and indirectly alter CD1- to microbes, NKT cells constitute a sepa-
mediated lipid antigen presentation through rate lineage. Two models that explained the
defective lipid trafficking or lipid competi- basis of the NKT cell lineage were initially
tion for loading CD1d. For example, while opposed. One model suggested that NKT
NPC1-mutant cells showed a block in lipid cells originated from precursors committed
transport from late endosome to lysosome, prior to TCR expression (committed precur-
this block could be partially reversed by in- sor model), whereas the other model proposed
hibitors of glycosphingolipid synthesis such that the lineage was instructed after TCR ex-
as N-butyldeoxygalactonojirimycin, presum- pression and interaction with NKT ligands
ably through alleviation of the lipid overload (TCR instructive model). The first model was
(102). Bone marrow–derived DCs from mice based on a report suggesting the presence

308 Bendelac · Savage · Teyton

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of cells expressing the canonical Vα14 TCR As cells progress to the mature CD24low
at day 9.5 of gestation (113), well before a stage, three more stages are described: first
thymus was formed, but these data have not a CD44low NK1.1neg stage (naive), then a
been reproduced with the new, more specific CD44high NK1.1neg (memory) stage, and fi-
CD1d tetramer reagents. Instead, the TCR nally a CD44high NK1.1pos (NK) stage (31,
instructive model is now widely accepted on 124). This sequence is characteristically ac-
the basis of the finding that although canon- companied by a massive cellular expansion oc-
ical Vα14-Jα18 rearrangements are rare and curring between the CD44low NK1.1neg stage
stochastic (114), once expressed (e.g., in TCR and the CD44high NK1.1neg stage (125). This
transgenic mice), an NKT TCR will induce expansion phase following positive selection
the full NKT cell lineage differentiation (115, and leading to the acquisition of a memory
116). phenotype is in line with the innate role of
NKT cells, which requires high copy number
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and effector/memory properties for prompt

Developmental Stages and effective responses, but it represents a
The production of CD1d-αGalCer tetramers key difference between the development of
specific for the canonical Vα14 TCRs (117– NKT cells and that of conventional T cells.
119) has transformed this area of study by Furthermore, during these stages a DN pop-
allowing the identification of developmen- ulation arises by downregulation of CD4 in
tal steps independently of the expression ∼30%–50% of the cells, as shown in cell
of NK1.1 (Figure 4). The first detectable transfer experiments (120), and by genetic
stages have a CD24high cortical pheno- fate mapping with ROSA26R reporter mice
type and include a CD4intermediate CD8intermediate crossed to CD4-cre deleter mice (123). DN
(double-positive, DPdull ) stage, followed by cells exhibit some functional differences with
a CD4high CD8neg stage. These developmen- CD4 cells, which are more pronounced in hu-
tal intermediates immediately follow posi- man than in mouse (126–128), and tend to
tive selection, as they express CD69 and are be more of the Th1 phenotype. The factors
not found in the CD1d-deficient thymus, determining this sublineage remain unclear,
but they are present at extremely low fre- as DN cells appear to share the same TCR
quencies (∼10−6 ) (120). The preselection DP, repertoire as the CD4 subset. A majority of
observed easily in Vα14-Jα18 TCRα-chain the CD44high NK1.1neg cells emigrate to pe-
transgenic mice (115), still escape tetramer ripheral tissues, where they stop proliferat-
detection in wild-type mice owing to the rar- ing and rapidly express NK1.1, a NK marker
ity of stochastic Vα14-Jα18 rearrangements available in the C57BL/6 background, fol-
and the low TCR level at this stage. Inves- lowed by other NK lineage receptors such as
tigators have attempted intrathymic transfer NKG2D, CD94/NKG2A, Ly49A, C/I, and
of purified DP cells to demonstrate the pres- G2 (31, 32, 124). Thymic emigration as-
ence of NKT cell precursors, but, given the says using intrathymic injection of fluorescein
size of the inoculum (107 DP cells), these ex- isothiocyanate have revealed that up to 5% of
periments could not formally rule out that recent thymic emigrants to the spleen, repre-
rare DN contaminants gave rise to the NKT senting 5 × 104 cells, are CD44high NK1.1neg
cell product (121). Interestingly, in mice lack- NKT cells and rapidly acquire NK1.1 to join
ing RORγt—a transcription factor induced in the nondividing long-lived NK1.1+ pool of
DP thymocytes that is essential for prolonged ∼5 × 105 cells (31, 32). Interestingly, a frac-
survival until distal Vα to Jα rearrange- tion of the CD44high NK1.1neg cells do not em-
ments (such as Vα14 to Jα18) can proceed— igrate and instead proceed to terminal matu-
NKT cell development was interrupted (122, ration (CD44high NK1.1pos ) inside the thymus,
123). where they become long-lived resident cells, • Biology of NKT Cells 309

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DN DP DP Cortical
RORγt thymocytes



CD4 CD24hi
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CD4 CD44lo
NK1.1– PKCθ
IL-4 Bcl-10

CD4 NK1.1–
DN IFN-γ T-bet

CD4 CD8 CD4 CD4 NK1.1+
Emigrant Resident


Figure 4
Thymic NKT cell development. NKT cell precursors diverge from mainstream thymocyte development
at the CD4+ CD8+ double-positive (DP) stage. Upon expression of their canonical TCRα chain, which
requires survival signals induced by RORγt, NKT cell precursors interact with endogenous agonist
ligands such as iGb3, presented by CD1d expressed on other DP thymocytes in the cortex. Accessory
signals provided through homotypic interactions between SLAM family members recruit SAP and Fyn
to activate the NF-κB cascade. DP precursors downregulate CD8 to produce CD4+ cells, and a subset
later downregulates CD4 to produce CD4− CD8− double-negative (DN) cells. Unlike mainstream
T cells, NKT cell precursors undergo several rounds of cell division and acquire a memory/effector
phenotype prior to thymic emigration. Acquisition of NK lineage receptors, including NK1.1, occurs
after emigration to peripheral tissues, except for a minor subset of thymic NKT cell residents. The
transcription factor T-bet is required for induction of the IL-15 receptor β chain and survival at the
late-memory and NK1.1 stages. EC, epithelial cell; DC, dendritic cell.

310 Bendelac · Savage · Teyton

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a peculiar fate of uncertain significance in the sion at the β locus, and transition to the DP
mouse thymus (32) that may be absent in the stage, where rearrangements are initiated at
human thymus (129). the TCRα locus. NKT cell precursors fol-
These developmental stages are associ- low the same pre-Tα path as mainstream
ated with sharply defined functional changes. T cells (120, 134). Therefore, the question
Thus, the CD44low NK1.1neg cells are ex- arises whether the biased usage of Vβ8, Vβ7,
clusive IL-4 producers upon TCR stimula- and Vβ2 in mouse (and Vβ11 in human) is
tion in vitro, whereas the CD44high NK1.1neg due to the inability of the Vα14-Jα18 TCRα
cells produce both IL-4 and IFN-γ and the chain to pair with the other Vβs or whether it
CD44high NK1.1pos cells produce more IFN-γ is due to positive or negative selection. Prema-
than IL-4 (31, 124). This is reflected faithfully ture expression of a Vα14-Jα18 TCRα trans-
in the spontaneous expression of high lev- gene at the DN3 stage created a population
els of GFP (green fluorescent protein) by the of thymocytes with a broad Vβ repertoire,
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CD44low NK1.1neg and CD44high NK1.1neg ruling out a Vβ pairing issue (135). Of these
cells of IL-4-GFP “4get” knockin mice, and transgenic cells, however, only those express-
in the expression of high levels of YFP (yellow ing the biased Vβ set responded to iGb3,
fluorescent protein) by the CD44high NK1.1pos whereas a broader set of Vβs responded to
cells of IFN-γ-YFP “Yeti” knockins, which αGalCer, demonstrating that the Vβ bias is
reflect open chromatin in the corresponding imparted by selection events. Furthermore,
cytokine loci (130). Vβ7 cells responded to the lowest concentra-
Because a panoply of NK receptors is ex- tions of iGb3, in agreement with several ob-
pressed with kinetics and frequencies similar servations that Vβ7+ NKT cells are relatively
to those of NK cells, components of a gen- diminished upon CD1d overexpression (con-
eral NK lineage program are likely activated. sistent with negative selection) and increased
Interestingly, however, the extent and pro- upon CD1d underexpression (consistent with
file of NK receptor expression vary in differ- decreased positive selection of the lower affin-
ent tissues, with thymic NKT cells express- ity Vβ8 and Vβ2) (62, 136, 137). Vβ7 cells
ing a repertoire similar to that of splenic NK were also preferentially expanded in a fetal
cells and spleen and liver NKT cells express- thymic organ culture system after exposure
ing these receptors at lower frequencies (131). to exogenous iGb3 (62). Because the Vβ7 >
Whether these differences reflect different Vβ8 > Vβ2 affinity hierarchy of these Vβs
stages of differentiation or an environmen- precisely reflects their respective degree of
tal influence on the acquisition or selection of enrichment during thymic selection, the Vβ
the NK receptor repertoire is not clear. Note repertoire of NKT cells appears to be shaped
that, despite their potential to regulate TCR mainly by positive selection, with little contri-
signaling thresholds to antigen (132), includ- bution from pairing bias or negative selection
ing natural ligand (133), the functions of NK in natural conditions. However, NK lineage
receptors remain to be elucidated in a physi- T cells are not inherently resistant to negative
ological context. selection, as they tend to disappear in condi-
tions of increased signaling (136, 138, 139).

Contribution of T Cell Receptor Vβ

Chains to Natural Ligand Cellular Interactions
Recognition In contrast with MHC class I molecules,
TCR Vβ-Dβ-Jβ rearrangements occur at the mouse and human CD1d are induced at
DN3 stage to produce a TCRβ chain that the DP stage and downregulated at the
pairs with the pre-Tα to form a receptor single-positive (SP) stage (82). This expres-
that induces cellular expansion, allelic exclu- sion pattern explains why cortical thymocytes • Biology of NKT Cells 311

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represent the thymic cell type, where CD1d gagement by CD1d-expressing cortical thy-
expression is necessary and sufficient for mocytes. This signaling is expected to dif-
NKT cell selection and lineage differentia- fer from that of conventional T cells for at
tion. Thus, NKT cells were absent in chimeric least two reasons. One is that the natural lig-
mice lacking CD1d expression in the DP com- and is an agonist that would normally induce
partment (140). Conversely, in pLck-CD1d negative selection in the mainstream lineage.
transgenic and chimeric mouse models where This is illustrated directly by the autoreac-
CD1d was exclusively expressed on corti- tive IL-2 response of NKT hybridomas to
cal thymocytes, NKT cells developed nearly DP thymocytes (18) and by the proliferative
normally and notably preserved their effec- and cytokine response of fresh NKT cells to
tor properties, with the exception of a rela- synthetic iGb3 (26). The other reason is that
tive decrease in NK receptor expression and the developing NKT cell precursors inter-
some hyperreactivity to TCR stimulation (86, act with cortical DP thymocytes rather than
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139). CD1d is also found on thymic CD11b+ with epithelial cells, implying that homotypic
macrophages, CD11c+ DCs, and epithelial rather than heterotypic cellular contacts are
cells (86), but this expression appeared to play involved and therefore recruit accessory re-
only an auxiliary role in NKT cell develop- ceptors or factors that elicit different signaling
ment, as shown by the normalization of NK pathways.
receptor expression and TCR hyperreactiv- In this context, the reports that Fyn knock-
ity upon crossing pLck-CD1d to Eα (MHC out (142, 143) and SLAM-associated protein
class II)-CD1d mice. Interestingly, in another (SAP) knockout (144–146) mice lacked NKT
Lck-CD1d transgenic model in which CD1d cells have attracted considerable attention be-
was expressed at a high level on peripheral cause the Src kinase FynT was recently shown
T cells, NKT cells appeared to be hypore- to signal downstream of the SLAM family
sponsive, and liver disease was observed (141). of homotypic interaction receptors through
Intrathymic transfer experiments and SAP (147–150). Several members of this fam-
thymic graft experiments further re- ily (151) are expressed on cortical thymocytes,
vealed that the acquisition of NK1.1 by reinforcing the hypothesis of homotypic in-
CD44high NK1.1neg NKT cells was decreased, teractions signaling through SAP and FynT
but not arrested, in the absence of CD1d during TCR recognition of CD1d ligands on
in the thymus or the periphery, although cortical thymocytes. Whether and which of
life span and effector functions were rela- these SLAM family members are involved are
tively preserved (32). These observations under investigation. In addition, the stages
suggest that interactions with CD1d ligands at which these interactions might influence
expressed by cell types other than DP occur NKT cell development and differentiation re-
throughout NKT cell development in the main to be defined. Notably, the report that
thymus and the periphery, consistent with a Vα14-Jα18 TCRα transgene corrected the
the autoreactivity of the Vα14 TCR, and, Fyn knockout–associated defect implied that
although not absolutely required, they this stage would precede TCRα expression
nevertheless promote terminal NKT cell (152), although interpretation of TCR trans-
differentiation. genic results should be careful given the de-
scription of transgenic lineage artifacts (115,
135). Indeed, more recent studies in our lab-
Molecular Interactions and Signaling oratory indicate that this correction is partial
The above studies imply that an understand- and due to the leaky phenotype of the Fyn
ing of the NKT cell lineage commitment re- knockout because the SAP knockout was not
volves around the signaling events imparted to reconstituted (K. Griewank and A. Bendelac,
NKT cell precursors during their TCR en- unpublished results).

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The emerging scenario, therefore, is that lating the decision between conventional and
homotypic interactions between SLAM fam- NKT cell–like lineages. Thus, conventional
ily members initiated in the cortex during CD8 T cells lacking these kinases upregu-
Vα14 TCR engagement by CD1d/iGb3- lated eomesodermin and the IL-15 receptor
expressing cortical thymocytes lead to FynT and turned into NKT cell–like cells that re-
signaling after SAP recruitment to the cy- quired ligand on bone marrow–derived rather
tosolic tyrosine motifs of SLAM family mem- than epithelial cells (163, 164). Interestingly,
bers (153). FynT signaling can activate the mice expressing MHC class II molecules on
canonical NF-κB pathway and may account, thymocytes through transgenic expression of
in conjunction with TCR signaling, for the the transcription factor CIITA selected an un-
well-established requirement of this pathway usual population of CD4 T cells resembling
in NKT cell development (Figure 4). In- NKT cells by their expression of a memory
deed, mice expressing a dominant-negative phenotype (165).
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IκBα transgene and those lacking NF- Additional NKT cell precursor-intrinsic
κBp50 exhibited developmental arrest at the factors regulate NKT cell development. For
CD44high NK1.1neg stage, which was partially example, mice lacking Runx1 (123) or Dock2
rescued by a Bcl-xL transgene, suggesting a (166) or mice overexpressing BATF, a ba-
survival role for NF-κB (154, 155). The pre- sic leucine zipper transcription factor and an
cise connections between TCR, FynT, and AP-1 inhibitor, exhibited severe defects early
NF-κB remain to be elucidated. PKCθ and in NKT cell development (167, 168).
Bcl-10 have been implicated in the signaling Although NKT cells interact with corti-
pathways of both FynT and the TCR leading cal thymocytes rather than epithelial cells for
to NF-κB activation (156), and their ablation TCR/ligand and SLAM family interactions,
impaired NKT cell development (157, 158), mice carrying defective components of the al-
although the NKT cell defects were relatively ternative NK-κB pathway, such as NIK or
modest. FynT has also been connected to Rel-B, in their thymic stroma exhibit severe
the Ras-GTPase-activating protein Ras-GAP and early disruption of NKT cell develop-
through the Dok1/2 adaptor proteins (149, ment (155, 169). Because these mutations also
159), suggesting that signals emanating from induce profound abnormalities of the thy-
SLAM family members may regulate signal- mus architecture, thymic lymphocyte emigra-
ing downstream of the TCR to avoid nega- tion, and thymic DCs, there may be multiple
tive selection through Ras while promoting causes of the NKT cell defects (170). Lym-
survival through NF-κB. photoxin α1β2 (expressed on thymocytes)
The molecular regulation of the NK pro- signaling through the lymphotoxin β receptor
gram activated between CD44high NK1.1neg (expressed on stromal cells) can activate this
and CD44high NK1.1pos cells remains enig- alternative pathway, but only modest NKT
matic. The transcription factor T-bet induces cell defects have been reported in the corre-
expression of the IL-2Rβ component of the sponding mutant mice (171–173).
IL-15 receptor, which is important for the Finally, GM-CSF was reported to control
survival of CD44high NK1.1neg and terminally the effector differentiation of NKT cells dur-
differentiated CD44high NK1.1pos cells (160– ing development by a mechanism that ren-
162). However, the range of functions of ders them competent for cytokine secretion
T-bet and its homolog eomesodermin in this (174).
developmental pathway, particularly with re-
spect to the induction of the NK differentia-
tion program, remains to be investigated. Re- NKT CELL FUNCTIONS
cent studies have suggested that Tec family NKT cells have been implicated in a
kinases Itk and Rlk play a central role in regu- broad array of disease conditions ranging • Biology of NKT Cells 313

ANRV306-IY25-12 ARI 11 February 2007 12:20

from transplant to tumors, various forms of characterized a cascade of activation events

autoimmunity, atherosclerosis, allergy, and following the exogenous administration of
infections. NKT ligands such as αGalCer (Figure 5).
The central feature is a reciprocal activation
of NKT cells and DCs, which is initiated
NKT Cell Activation by upon the presentation of αGalCer by rest-
Administration of Ligand In Vivo ing DCs to NKT cells, inducing NKT cells
The central concept underlying nearly all to upregulate CD40L and Th1 and Th2 cy-
NKT cell functions is the recognition by tokines and chemokines; CD40 cross-linking
the whole NKT cell population of endoge- induces DCs to upregulate CD40, B7.1 and
nous ligands such as iGb3 (autoreactivity) B7.2, and IL-12, which in turn enhances
or of microbial cell wall glycolipids such as NKT cell activation and cytokine produc-
α-glycuronylceramides. Several studies have tion (175, 176). Propagation of this reaction
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MΦ Liver sinusoid



NKT Vα14 CD1d:

IFN-γ Jα18 lipid

NK IL-4, IL-12



Figure 5
Cellular and molecular network activated by the NKT ligand αGalCer. DCs and perhaps also Kupffer
cells (macrophages) lining the liver sinusoids (where NKT cells accumulate) are at the center of a cellular
network of cross-activation, starting with NKT cell upregulation of CD40L, secretion of Th1 and Th2
cytokines and chemokines, and DC superactivation to prime adaptive CD4 and CD8 T cell responses.
NKT cells can provide help directly to B cells for antibody production and can also rapidly activate NK
cells. CXCR6/CXCL16 interactions provide essential survival signals for NKT cells. EC, endothelial cell.

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involves the activation of NK cell cytolysis proposed that the lipid with shorter sphin-
and IFN-γ production (177, 178) and, most gosin OCH failed to engage the TCR for a
importantly, the upregulation of DC costim- long enough period of time to induce IFN-γ.
ulatory properties and MHC class I– and On the other hand, plasmon resonance deter-
MHC class II–mediated antigen presentation, minations of TCR on and off rates, and even
particularly cross-priming, which serves as a crystal structures of the long (KRN7000) and
bridge to prime robust adaptive immune re- acyl shortened (PBS25, C8 acyl chain) ver-
sponses (179–181). Importantly, TLR signal- sion of αGalCer bound to CD1d have shown
ing is not involved in these responses. Thus, no significant differences (77). An alternative
αGalCer and related variants are being ac- hypothesis is based on the observation that
tively investigated for their ability to serve different NKT ligands preferentially reach
as vaccine adjuvants alone or in conjunction different cell types upon injection in vivo, sug-
with synergistic TLR ligands (182). In ad- gesting that increased Th1 responses may re-
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dition, the immunomodulatory properties of sult from the predominant uptake of lipid by
repeated injection of NKT ligands may be IL-12-secreting cell types such as DCs (77,
exploited to treat or prevent immunological 194). Perhaps of relevance to this issue is the
diseases (183). fact that all Th2 ligands described so far have
Mature NKT cells produce massive increased solubility in water owing to their
amounts of IFN-γ, but they are unique shorter lipid tail or the presence of insatura-
among lymphocytes for their ability to explo- tions. This property could modify their routes
sively release IL-4 (184), in addition to other of trafficking and uptake, favoring presenta-
key Th2 cytokines such as IL-13. The Th1 tion by non-IL-12-producing cells, such as
versus Th2 outcome of their activation is par- B cells. Finally, mucosal rather than systemic
tially understood. Systemic injection of the modes of administration may also modify the
original αGalCer compound induces an early Th1/Th2 output of NKT cells owing to a pre-
burst of IL-4 detected in the serum, followed existing bias in the cytokine environment.
by a more prolonged burst of IFN-γ by NKT
cells and transactivated NK cells, as well as of
IL-12 originating in part from DCs (185, Dual Reactivity to Self and Microbial
186). However, NKT cells also undergo a Ligands: A Paradigm for NKT Cell
rapid downregulation of their TCR, followed Activation and Function During
by massive apoptosis within 3–4 days of ac- Bacterial Infections
tivation, resulting in a long-lasting depletion Glycosphingolipids closely related to
until regeneration occurs in part from thymic αGalCer were reported in the cell wall
precursors (187–189). More sustained and ef- of Sphingomonas (53, 54), a prominent
ficient responses have been described upon in- Gram-negative, LPS-negative member of
jection of αGalCer-pulsed DCs, particularly an abundant class of bacteria on Earth,
with respect to the production of IFN-γ, re- α-proteobacteria (Figure 6). Sphingomonas
sulting in a superior adjuvant effect for the is a ubiquitous bacterium whose cell wall
priming of cytotoxic T lymphocytes (CTL) glycosphingolipids include the dominant
(190, 191). α-branched glucuronyl and galacturonyl
Interestingly, some variants of the original ceramides (GSL-1) and the less abundant di-
αGalCer KRN7000 have shown decreased (GSL-2), tri- (GSL-3), and tetra- (GSL-4)
Th1 compared to Th2 cytokine induction. glycosylated species shown in Figure 1.
These Th2 variants have shorter or insatu- Although these glycosphingolipids form
rated lipid chains (185, 192, 193). The mech- structures reminiscent of LPS (Figure 6),
anisms underlying these differences are de- their synthesis pathway and role in the
bated and may be diverse. Oki et al. (186) microbial cell wall are not well understood. • Biology of NKT Cells 315

ANRV306-IY25-12 ARI 11 February 2007 12:20

E. coli


Lipid A
Figure 6 Outer membrane
Outer membrane
of the cell walls of Membrane proteins
Sphingomonas and Peptidoglycan
Escherichia coli. The Inner membrane
inner leaflet of the
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outer membrane is
composed of
whereas the outer
leaflet is made of
LPS for E. coli. In Sphingomonas
the case of
between one and Glycosphingolipids
four carbohydrates
substitute for LPS.
Note the thin layer Outer membrane
of peptidoglycan
separating the Membrane proteins
inner and outer Peptidoglycan
membranes in both Inner membrane
cell walls. Cytoplasm

GSL-1 activates large proportions of mouse tively weak stimulants. High doses of Sph-
and human NKT cells (23–25, 55), but it is ingomonas induce a lethal toxic shock simi-
unclear at present whether the more complex lar to the one associated with Gram-negative,
GSL-2, -3, and -4 can be recognized by NKT LPS-positive bacteria. However, in the case of
cells or even whether they can be processed Sphingomonas, NKT cell–deficient mice are
efficiently into GSL-1 by host APCs. protected. These striking observations have
During infection, Sphingomonas is phago- led to the hypothesis that NKT cells and their
cytosed by macrophages and DCs and elic- canonical TCR specificity evolved to meet
its an activation cascade similar to exogenous the challenges of these Gram-negative, LPS-
αGalCer. NKT cell activation enhances mi- negative bacteria. Although Sphingomonas is
crobial clearance by 15- to 1000-fold within a promiscuous bacterium that can cause se-
the first 2–3 days of infection (23, 24). vere infection, particularly in immunocom-
Sphingomonas can also induce DC activation promised hosts, other more deadly mem-
through TLR-mediated signaling, but this bers of the class of α-proteobacteria may
direct effect is weak relative to the cross- have provided stronger evolutionary pressures
activation of DCs by NKT cells because on the NKT cell system. Particularly inter-
peptidoglycan and bacterial DNA are rela- esting is the case of Ehrlichia, a tick-borne

316 Bendelac · Savage · Teyton

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pathogen and member of the Rickettsiales that lysosome, and by blocking experiments with
is of widespread significance for mammals, the lectin Griffonia simplicifolia IB4, which
including wild and domesticated ruminants, recognizes the terminal sugar of the Galα1-
dogs, and humans from some regions of the 3Gal epitope of iGb3 bound to CD1d and
world such as Africa and East Asia. Ehrlichia blocks NKT cell activation (23). Strikingly,
muris activates NKT cells independently of NKT cell activation by Gram-negative, LPS-
iGb3, and its clearance was profoundly altered positive Salmonella is absolutely dependent
in CD1d- or Jα18-deficient animals (23). upon TLR signaling through the adaptors
Ehrlichia is a Gram-negative, LPS-negative MyD88 and Trif, and upon IL-12 release by
obligate intracellular bacterium, whose cell the APC, although the precise TLR combi-
wall composition has not been elucidated. nation and the corresponding microbial struc-
Interestingly, many other bacteria, particu- tures involved remain to be determined. Thus,
larly the Gram-negative, LPS-positive ones, the proposed scenario suggests that TLR sig-
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can activate NKT cells. However, rather than naling leading, but not limited, to IL-12 secre-
provide their own NKT ligands like Sphin- tion enhances the ability of DCs to stimulate
gomonas or Ehrlichia, these bacteria appear NKT cells through presentation of endoge-
to trigger autoreactive NKT cell responses nous ligands (Figure 7).
(23, 60). In the case of Salmonella, this is Whether TLR signaling induces an up-
suggested by the abrogation of NKT cell regulation of iGb3 or changes in the expres-
activation in the presence of DCs lacking sion of other factors such as, for example, NK
β-hexosaminidase B, the enzyme responsible receptor ligands is unclear. Contrary to an
for the generation of iGb3 from iGb4 in the early report (195), NKT cells do not usually

Direct microbial recognition Indirect microbial recognition

NKT cell
LPS-negative Bacterial Ag TLR4
bacteria iGb3


Bacterial Ag iGb3

Late endosome/lysosome

Figure 7
Dual recognition of self and microbial glycosphingolipids during microbial infections. On the left,
infection by Gram-negative, LPS-negative Sphingomonas induces direct activation of NKT cells through
recognition of microbial cell wall α-glycuronylceramide. On the right, infection by Gram-negative,
LPS-positive Salmonella activates TLR4 through LPS and induces IL-12, revealing constitutive
autoreactive recognition of iGb3 through the secretion of IFN-γ (indirect microbial recognition). • Biology of NKT Cells 317

ANRV306-IY25-12 ARI 11 February 2007 12:20

constitute the predominant cell type that pro- Parasitic Infections

duces IFN-γ in response to IL-12 in vivo
Shofield and colleagues (204) suggested that
(60, 196). This explains why they generally
the production of IgG antibodies to the
do not appear to be essential in fighting
malaria circumsporozoite antigen, a key com-
Gram-negative, LPS-positive bacteria. How-
ponent of protective immune responses in
ever, an impact on bacterial clearance has
humans, depended on NKT cell recogni-
been observed in the case of lung infection
tion of malarial glycosylphosphatidylinosi-
with Pseudomonas aeruginosa, where CD1d-
tol antigens in a mouse model. However,
deficient mice exhibited a ∼20-fold increased
additional experiments failed to detect a
bacterial count in the lung within 6–24 h
CD1d-dependent component to this antibody
postinoculation and an approximately three-
response, and glycosylphosphatidylinositols
fold decrease in MIP-2 and neutrophils in the
have not been identified as NKT cell anti-
bronchoalveolar lavage (197). This may not
gens in other reports (205, 206). In the con-
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be the case at other sites of infection (198).

text of helminth infection, DCs pulsed with
Variations have been noted as well in reports
Schistosoma mansoni eggs activated NKT cells
assessing the role of NKT cells versus NK
to secrete Th1 and Th2 cytokines in vitro in a
cells in LPS-induced toxic shock in vivo (199,
β-hexosaminidase-B-dependent but MyD88-
independent manner, suggesting recognition
of the self ligand iGb3 in the absence of TLR
Primary Biliary Cirrhosis and signaling (207).
An intriguing connection between primary
biliary cirrhosis (PBC), Sphingomonas, and Viral Infections
NKT cells has emerged recently. PBC is a Relatively modest defects in the clearance of
disease characterized by the presence of an- some viruses have been reported in CD1d-
timitochondrial antibodies, liver lymphocytic deficient mice infected with encephalomy-
infiltrates, and the chronic destruction of the ocarditis virus (208) or coxsackie B3 (209),
biliary epithelium, which leads to cirrhosis but these defects were not observed in Jα18-
(201). Interestingly, the autoantibodies recog- deficient mice, ruling out a specific role
nize an epitope of the mitochondrial PDC- of Vα14 NKT cells. Infections with lym-
E2 enzyme that is particularly well conserved phocytic choriomeningitis virus, mouse cy-
in Novosphingobium aromaticivorans, a strain tomegalovirus, vaccinia virus, and coronavirus
of Sphingomonas. Furthermore, PBC patients, were unaffected. Studies in humans have sug-
including those lacking antimitochondrial an- gested a profibrotic role of Vα24 NKT cells
tibodies, were specifically seropositive against in hepatitis C (85) and the accumulation of
Sphingomonas, which was detected by PCR in non-Vα24 CD1d-restricted T cells (210). Al-
stool samples of 25% of diseased or healthy though a specific role of Vα14 NKT cells
individuals, suggesting that PBC may be in- in HSV infection remains controversial (211,
duced by aberrant host reactivity to this bac- 212), recent studies have suggested that vi-
terium (202). PBC patients also showed an en- ral invasion may be associated with counter-
richment of Vα24 NKT cells in liver biopsies, measures against CD1d or NKT cells. For
but a depletion in blood (203). In light of the example, HSV-1 drastically and specifically
recent finding that Sphingomonas cell wall gly- impaired CD1d recycling from the lysosome
colipids specifically activate NKT cells, these to the plasma membrane, an essential pathway
studies suggest that NKT cells may play a key for glycolipid antigen presentation to NKT
role in the pathogeny of PBC by promoting cells (96). Kaposi sarcoma–associated herpes
aberrant responses to Sphingomonas. virus encodes two modulators of immune

318 Bendelac · Savage · Teyton

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recognition, MIR1 and MIR2, that downreg- Type I diabetes. The relative deficiency of
ulated CD1d in addition to other immunolog- NKT cells in NOD mice (36, 37), combined
ically relevant molecules such as MHC class with the notion that these cells represent a po-
I, CD86, and intracellular adhesion molecule tent source of Th2 cytokines, prompted the
(ICAM)-1 through ubiquitination of lysine original speculation of a causal relationship
residues in their cytoplasmic tail (95). The with diabetes. Early claims that humans with
lethal outcome of infections with Epstein- type I diabetes exhibited severe NKT cell de-
Barr virus in patients with X-linked lympho- fects and that their sera had less IL-4 than
proliferative (XLP) immunodeficiency syn- controls (213, 214) were not confirmed when
drome due to SAP mutations was hypothe- more specific methodologies became available
sized to result from the absence of NKT cells (38, 215). Researchers interpreted reports of
(144). Which of these effects or associations aggravated disease in CD1d-deficient NOD
reflect a specific viral evasion/immune defense mice (216, 217) as suggesting that, although
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strategy and the nature of the NKT ligands in- defective, the residual NKT cells in NOD
volved in these infectious conditions remain mice still suppressed autoimmunity. How-
to be determined. ever, independent studies in different colonies
of CD1d-deficient and Jα18-deficient mice
failed to support these claims (218), and par-
NKT Cells in Noninfectious tial reconstitution of NKT cells in NOD
Diseases mice carrying the B6 Nkt1 locus did not pro-
A role of NKT cells has been suggested in a tect against diabetes (34). Transgenic expres-
wide variety of disease conditions. At present, sion of the Vα14-Jα18 TCRα chain in NOD
however, many reports, lacking a detailed mice prevented diabetes, but this could be
mechanistic understanding, remain isolated explained by the reduced frequency of islet-
or are based merely on the analysis of NKT specific T cells and the general Th2 bias of
cell–deficient mice. Rather than compiling an these mice (219). Likewise, the suppression
exhaustive list of the published claims, this re- of diabetes by αGalCer multi-injection regi-
view provides a critical appraisal of selected mens could be the mere consequence of mas-
reports carrying important conceptual or clin- sive cytokine release (220, 221). More di-
ical implications. One frequently overlooked rect transfer experiments using diabetogenic
but recurrent methodological issue inherent T cells and NKT cells have suggested sup-
in the use of CD1d- or Jα18-deficient mice pressive or enhancing roles of NKT cells
is the extent to which gene-deficient mice in different experimental systems (222, 223).
are matched with littermate controls with re- Although other more circumstantial studies
spect to genetic background and environmen- have suggested a role of NKT cells in this
tal factors. This is particularly important in disease, it seems reasonable to conclude at this
studies of complex multigenetic diseases such point that there is no decisive evidence for a
as diabetes, lupus, cancer, or asthma. In ad- substantial or specific role of NKT cells in
dition, the injection of αGalCer as a gain- mouse or human type I diabetes.
of-function experiment should be interpreted
with caution because the massive release of cy-
tokines induced by this procedure is unlikely Lupus. Hyperreactive NKT cells were
to model chronic diseases. It may not be sur- shown to accumulate in aging NZB/W mice
prising, therefore, that some claims have be- (224) and suggested to help B cells produce
come controversial or will need to be rein- anti-DNA antibodies (225). However, studies
terpreted, complicating the task of drawing a of CD1d-deficient lupus-prone mice have
clear picture of the involvement of NKT cells not yielded concordant results (226–228),
in noninfectious diseases. and injections of αGalCer ameliorated or • Biology of NKT Cells 319

ANRV306-IY25-12 ARI 11 February 2007 12:20

aggravated disease, depending on the mouse suppress antitumor CTL responses. Because
strain (229). Jα18-deficient mice did not share the pheno-
type of CD1d-deficient mice, the study con-
Cancer. Similar to the general immune sup- cluded that other less well-known types of
pression of T cells commonly encountered in CD1d-restricted T cells might be involved
cancerous states, NKT cells were decreased or (236). As in the MCA-induced tumor trans-
functionally hyporeactive in cancer-bearing plants, these tumors did not express CD1d,
mice and humans (230, 231). One tumor shed yet CD1d expression by host cells, presumably
glycosphingolipids that could inhibit the stim- APCs, was required to observe the NKT cell
ulation of NKT cells in vitro (232). How- effects. In contrast, the growth of the CD1d-
ever, multiple mechanisms are likely to con- transfected RMA/S tumor cell line cells was
tribute to the deficiency of both T and NKT inhibited by Vα14 NKT cells (237). In con-
cells. In one report, the frequency of sarcomas clusion, the notion that mVα14 and hVα24
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six months after intramuscular injection of NKT cells regulate cancer rejection is based
the chemical carcinogen methylcholantrene largely on tumor transplant models, and the
(MCA) decreased two- to threefold in Jα18 relevance to natural clinical conditions re-
knockout NKT cell–deficient mice (233). mains to be determined.
This observation, which suggested that NKT
cells, similar to γδ T cells and NK cells, may Asthma. CD1d- and Jα18-deficient mice
be agents of immune surveillance against pri- were reported to exhibit decreased allergen-
mary cancers has remained isolated. induced airway hyperreactivity in the alum-
In a tumor transplant model, subcutaneous ovalbumin model of asthma, where mice are
injection of a fibrosarcoma tumor line derived intraperitoneally sensitized with ovalbumin
from MCA-inoculated Jα18-deficient mice mixed in alum and subsequently challenged
produced tumors that grew faster in Jα18- with ovalbumin inhalation (238, 239). How-
deficient compared with wild-type mice and ever, similar studies in another laboratory
were prevented by transfers of purified NKT have failed to observe differences between
cells into Jα18-deficient hosts (234). CD1d CD1d-deficient and wild-type mice (R. Lock-
expression and the presence of CD8 T cells sley, personal communication). In humans
in the host were required for tumor rejection, with persistent, moderate-to-severe asthma,
implying ligand recognition on host-derived Vα24 NKT cells dominated the bronchial
cells, presumably APCs, rather than on tu- Th2 infiltrate (240). The extent of this NKT
mor cells. The nature of the tumor-associated cell expansion has been disputed, however,
NKT ligands has not been identified. These perhaps reflecting differences in the cohorts
experiments also revealed a specialized func- of asthma patients examined or the methods
tion of liver DN—as compared with CD4— for identifying NKT cells (241).
NKT cells in this Th1-mediated response
In apparent contrast with this fibrosar- Atherosclerosis. CD1d deficiency de-
coma model, CD1d-deficient mice controlled creased the level of atherosclerosis in apoE-
the growth of otherwise relapsing subcuta- or LDL receptor–deficient mice, although
neous transplants of the 15-12RM tumor line, the effects observed were only mild and
suggesting that a natural CD1d-dependent transient in some studies (241, 242).
mechanism suppressed tumor rejection (235).
Further studies dissected a complex cellu- Other disease conditions. Additional ob-
lar network that involved IL-13-producing servations suggesting a suppressive role of
CD1d-restricted CD4 suppressors interact- NKT cells in some models of delayed-
ing with TGF-β-producing myeloid cells to type hypersensitivity (242, 243), in anterior

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chamber–associated immune deviation (244), forms innate-like functions (19). The pres-
and in burn injury (245) have been reported. ence of a more diverse population has been
In summary, contrasting with numerous suggested recently, more convincingly in hu-
reports suggesting a contribution of NKT mans, indicating that an adaptive population
cells in a range of noninfectious diseases, a of lipid-specific CD1d-restricted T cells may
convincing picture has not yet emerged as to be available (210, 247, 248). The biology of
the strength or consistency of the observed these cells remains largely unexplored, and fu-
effects, their mechanisms, or their relevance ture studies in this area would resolve a fasci-
to physiological or clinical conditions. Future nating and long-standing debate in the field
experiments are needed to define those dis- of T cell recognition. Indeed, glycolipids are
eases and conditions that are regulated specif- not easily mutated or modified, and although
ically by mVα14 or hVα24 NKT cells and to the potential theoretical combinations of car-
dissect the mechanisms involved. bohydrates are extremely diverse, the universe
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of microbial glycolipids is limited owing to en-

zyme specificity for both donor and acceptor
THE LARGER CD1 UNIVERSE substrates in glycolipid synthesis. Thus, the
Although T cells recognizing lipids pre- glycolipid-specific repertoire did not evolve
sented by other CD1 isotypes were the first under the same pressure that operated on the
discovered (44), their study now represents peptide-specific repertoire, where single mu-
only a small fraction of the current in- tations produce new T cell epitopes. How
vestigations on CD1-mediated antigen pre- diverse and specific this glycolipid-specific
sentation, which focus overwhelmingly on repertoire may be is an important question
the CD1d/NKT cell system. CD1d is the for future research because conserved glycol-
only representative in mouse and rat of a ipids may represent ideal, fixed targets for
larger family of β2-microglobulin-associated vaccine development. In addition, how cross-
MHC-like molecules that, in other mam- reactive the MHC- and CD1-restricted TCR
malian species, comprises CD1a, -b, and -c, repertoires are is a fundamental issue that
as well as CD1e (44). CD1 and MHC are en- remains to be investigated. Given that the
coded in different loci, but recent genomic groove of CD1 molecules is significantly nar-
studies in chicken suggest that they originated rower than that of MHC proteins and that at
from the same primordial MHC locus (246). least a proportion of the TCR repertoire ap-
CD1a, -b, and -c differ in their location in pears to be intrinsically MHC-restricted (249,
different endosomal compartments, in early 250), one would assume that the peptide-
recycling to late endosome and lysosome, and specific and glycolipid-specific TCR reper-
also in the architecture of their lipid-binding toire should be essentially non-cross-reactive,
grooves, which suggests that each is special- a prediction that remains to be tested.
ized to capture different lipids in different en-
dosomal compartments (44). Individual self
and microbial lipid-specific T cell clones have SUMMARY
been derived in vitro in humans, but relatively Recent studies have elucidated novel and
little is known about the T cell types and TCR striking aspects of NKT cell development
repertoires associated with CD1a, -b, and -c and of the cell and structural biology of
and about their function in the human system. lipid antigen processing and recognition. Key
With respect to CD1d, however, it is candidate antigens have been identified that
well established that the major population of provide a framework for understanding the
CD1d-restricted T cells in mouse is the NKT evolution and function of this innate-like lin-
cell population that expresses semi-invariant eage, particularly in microbial infections. Fu-
TCRs, predominantly Vα14-Jα18, and per- ture work will clarify the range and nature • Biology of NKT Cells 321

ANRV306-IY25-12 ARI 11 February 2007 12:20

of the most physiologically relevant ligands develop a mechanistic understanding of the

and the structural basis of their recognition broad and sometimes controversial array of
by the semi-invariant TCRs. These solid ad- diseases in which NKT cells are increasingly
vances in fundamental biology should help implicated.

We thank past and present members of our laboratories for their contributions to the un-
derstanding of NKT cell biology; Seth Scanlon and Omita Trivedi, for help with the figures;
and Richard Locksley, Diane Mathis, and Thomas Blankenstein for sharing unpublished re-
sults. Dirk Zajonc generated the structural representation in Figure 2. This work is supported
by the Howard Hughes Medical Institute (A.B.) and by a program project grant from the
National Institutes of Health (A.B., P.B.S., L.T.). No review on NKT cell biology can ade-
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by Universidad Nacional Autonoma de Mexico on 02/27/14. For personal use only.

quately describe every interesting paper, and we apologize to those investigators whose work
could not be cited because of space limitations.

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Annual Review of

Contents Volume 25, 2007

Annu. Rev. Immunol. 2007.25:297-336. Downloaded from
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Peter C. Doherty p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x
Challenged by Complexity: My Twentieth Century in Immunology
Peter C. Doherty p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1
The Impact of Glycosylation on the Biological Function and Structure
of Human Immunoglobulins
James N. Arnold, Mark R. Wormald, Robert B. Sim, Pauline M. Rudd,
and Raymond A. Dwek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 21
The Multiple Roles of Osteoclasts in Host Defense: Bone Remodeling
and Hematopoietic Stem Cell Mobilization
Orit Kollet, Ayelet Dar, and Tsvee Lapidot p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 51
Flying Under the Radar: The Immunobiology of Hepatitis C
Lynn B. Dustin and Charles M. Rice p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 71
Resolution Phase of Inflammation: Novel Endogenous
Anti-Inflammatory and Proresolving Lipid Mediators and Pathways
Charles N. Serhan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p101
Immunobiology of Allogeneic Hematopoietic Stem Cell
Lisbeth A. Welniak, Bruce R. Blazar, and William J. Murphy p p p p p p p p p p p p p p p p p p p p p p p139
Effector and Memory CTL Differentiation
Matthew A. Williams and Michael J. Bevan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p171
TSLP: An Epithelial Cell Cytokine that Regulates T Cell
Differentiation by Conditioning Dendritic Cell Maturation
Yong-Jun Liu, Vasilli Soumelis, Norihiko Watanabe, Tomoki Ito,
Yui-Hsi Wang, Rene de Waal Malefyt, Miyuki Omori, Baohua Zhou,
and Steven F. Ziegler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p193
Discovery and Biology of IL-23 and IL-27: Related but Functionally
Distinct Regulators of Inflammation
Robert A. Kastelein, Christopher A. Hunter, and Daniel J. Cua p p p p p p p p p p p p p p p p p p p p p p221

AR306-FM ARI 13 February 2007 11:22

Improving T Cell Therapy for Cancer

Ann M. Leen, Cliona M. Rooney, and Aaron E. Foster p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p243
Immunosuppressive Strategies that are Mediated by Tumor Cells
Gabriel A. Rabinovich, Dmitry Gabrilovich, and Eduardo M. Sotomayor p p p p p p p p p p p p267
The Biology of NKT Cells
Albert Bendelac, Paul B. Savage, and Luc Teyton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p297
Regulation of Cellular and Humoral Immune Responses by the SLAM
and SAP Families of Molecules
Cindy S. Ma, Kim E. Nichols, and Stuart G. Tangye p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p337
Mucosal Dendritic Cells
Annu. Rev. Immunol. 2007.25:297-336. Downloaded from
by Universidad Nacional Autonoma de Mexico on 02/27/14. For personal use only.

Akiko Iwasaki p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381

Immunologically Active Autoantigens: The Role of Toll-Like
Receptors in the Development of Chronic Inflammatory Disease
Ann Marshak-Rothstein and Ian R. Rifkin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p419
The Immunobiology of SARS
Jun Chen and Kanta Subbarao p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443
Nonreceptor Protein-Tyrosine Phosphatases in Immune Cell Signaling
Lily I. Pao, Karen Badour, Katherine A. Siminovitch, and Benjamin G. Neel p p p p p p p473
Fc Receptor-Like Molecules
Randall S. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p525
The Death Domain Superfamily in Intracellular Signaling of Apoptosis
and Inflammation
Hyun Ho Park, Yu-Chih Lo, Su-Chang Lin, Liwei Wang, Jin Kuk Yang,
and Hao Wu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p561
Cellular Responses to Viral Infection in Humans: Lessons from
Epstein-Barr Virus
Andrew D. Hislop, Graham S. Taylor, Delphine Sauce, and Alan B. Rickinson p p p p p p587
Structural Basis of Integrin Regulation and Signaling
Bing-Hao Luo, Christopher V. Carman, and Timothy A. Springer p p p p p p p p p p p p p p p p p p p619
Zoned Out: Functional Mapping of Stromal Signaling
Microenvironments in the Thymus
Howard T. Petrie and Juan Carlos Zúñiga-Pflücker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p649
T Cells as a Self-Referential, Sensory Organ
Mark M. Davis, Michelle Krogsgaard, Morgan Huse, Johannes Huppa,
Bjoern F. Lillemeier, and Qi-jing Li p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p681
The Host Defense of Drosophila melanogaster
Bruno Lemaitre and Jules Hoffmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p697

vi Contents
AR306-FM ARI 13 February 2007 11:22

Ontogeny of the Hematopoietic System

Ana Cumano and Isabelle Godin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p745
Chemokine:Receptor Structure, Interactions, and Antagonism
Samantha J. Allen, Susan E. Crown, and Tracy M. Handel p p p p p p p p p p p p p p p p p p p p p p p p p p787
IL-17 Family Cytokines and the Expanding Diversity of Effector
T Cell Lineages
Casey T. Weaver, Robin D. Hatton, Paul R. Mangan, and Laurie E. Harrington p p p821

Annu. Rev. Immunol. 2007.25:297-336. Downloaded from
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Cumulative Index of Contributing Authors, Volumes 15–25 p p p p p p p p p p p p p p p p p p p p p p p p853

Cumulative Index of Chapter Titles, Volumes 15–25 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p860


An online log of corrections to Annual Review of Immunology chapters (if any, 1997 to
the present) may be found at

Contents vii