4 cd1 nature

The CD1 family of MHC-class-I-like glycoproteins (CD1a, CD1b, CD1c, CD1d and CD1e) present both foreign and self lipids as cognate antigens to T cells. The CD1 proteins share sequence homology and overall domain structure with MHC class I molecules, being comprised of a heavy chain with three extracell-ular domains that are non-covalently associated with β2-microglobulin (β2m). On the basis of sequence analy-sis, the CD1 isoforms can be classified into three groups: group 1 comprises CD1a, CD1b and CD1c; group 2 comprises CD1d; and group 3 comprises CD1e. All mammals studied so far have been found to express CD1 molecules. Interestingly, however, humans express all CD1 isoforms (CD1a–CD1e), whereas muroid rodents express only CD1d. Group 1 CD1 molecules mainly present lipid antigens to clonally diverse T cells that mediate adaptive immunity to the vast range of micro-bial lipid antigens. By contrast, CD1d (group 2) mol-ecules present lipid antigens to natural killer T (NKT) cells, a subset of which, the invariant NKT (iNKT) cells, expresses an invariant T-cell receptor (TCR) α-chain, responds rapidly en masse following antigen recognition and is a potent effector of innate immunity.

CD1 proteins survey the endocytic pathway to intersect and bind lipid antigens. Several molecules involved in lipid metabolism have recently been shown to also func-tion in CD1-mediated lipid-antigen presentation, such as saposins, which mediate the loading of lipids onto CD1 molecules in lysosomes. In this Review, we describe the pathway of CD1 antigen presentation, including the delivery, processing, loading and presentation of lipid antigens to T cells. New insights now delineate the

strategies by which antigen-presenting cells (APCs) stimulate CD1-restricted T-cell responses and how these responses are linked to the types of lipid antigen (self versus foreign), the CD1 isoform (group 1 versus group 2) and the responding T-cell population (clonally specific versus population en masse). The mechanisms by which CD1-restricted T cells affect a broad range of immune responses can now be understood. For a detailed overview on the roles of NKT cells in disease, the reader is referred to other excellent reviews on this subject1,2.

Diversity of lipids presented by CD1 moleculesThe lipid antigens presented by CD1 molecules include a broad array of classes, ranging from foreign lipids that are unique to specific microorganisms to common mammalian self lipids.

Foreign lipid antigens. Many of the unique lipids found in Mycobacterium tuberculosis can be presented by CD1a, CD1b and CD1c to activate clonally diverse T cells. These include the abundant mycolates, such as the free fatty acid mycolic acid, and mycolates with esterified glycans, such as glucose monomycolate. Mycolic acid (BOX 1) was the first CD1-presented lipid antigen to be identified3. M. tuberculosis-derived lipoglycans (such as lipoarabino-mannan), which are composed of a phosphatidylinositol anchor, a macromolecular polysaccharide backbone and capping motifs, or their component phosphatidylinositol mannosides4, were found to activate CD1b-restricted T cells5. By contrast, Mycobacterium spp. lipids that are closely related to mannosyl β-1-phosphomycoketides are

Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 1 Jimmy Fund Way, Boston, Massachusetts 02115, USA.Correspondence to M.B.B. e-mail: [email protected]:10.1038/nri2191

Endocytic pathwayA trafficking pathway used by all cells for the internalization of molecules from the plasma membrane to lysosomes.

Capping motifsCarbohydrates that are attached to the branches of the arabinan domain in lipoarabinomannan (LAM). In the case of Man-LAM, which is found in Mycobacterium tuberculosis and other pathogenic species of mycobacteria, the carbohydrates are mannose groups. Ara-LAM is not capped and it is found in non-pathogenic, fast growing strains of mycobacteria.

CD1 antigen presentation: how it worksDuarte C. Barral and Michael B. Brenner

Abstract | The classic concept of self–non-self discrimination by the immune system focused on the recognition of fragments from proteins presented by classical MHC molecules. However, the discovery of MHC-class-I-like CD1 antigen-presentation molecules now explains how the immune system also recognizes the abundant and diverse universe of lipid-containing antigens. The CD1 molecules bind and present amphipathic lipid antigens for recognition by T-cell receptors. Here, we outline the recent advances in our understanding of how the processes of CD1 assembly, trafficking, lipid-antigen binding and T-cell activation are achieved and the new insights into how lipid antigens differentially elicit CD1-restricted innate and adaptive T-cell responses.

NATuRe RevIewS | immunology vOluMe 7 | DeCeMBeR 2007 | 929

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Nature Reviews | Immunology

Isoglobotrihexosylceramide: self

Didehydroxymycobactin: M. tuberculosis

a

b

c

Diacylated sulpholipid: M. tuberculosis

Keto mycolic acid: M. tuberculosis

Phosphatidylinositol: self

α-Galactosyldiacylglycerol: B. burgdorferi

α-Galactosylceramide: synthetic or marine sponge

α-Glucuronosylceramide: Sphingomonas spp.

Sulphatide: self

O

HN

O

O

O

NH

NH

O

N

O

OH

NH

O

O

O

O

O OH

O

HO

O OSO 3 H

OH OH

HO

O

HOOC

HO

O

– O P

O O

O

O

O O O

HO HO

OH

OH

OH

O

O

O

O

O OHO

HO

HO

OH

HN

O

O

OH

O

O

O OH

OH

O HO OH

OH HO

O OH

OH

HO

HN

O

O

OH

OOH

OH

HO

OH

HN

O OH

OH

O

OOH

OH

HO OH

O

HN

O

OH

O

O OH

OH

OSO3–

OH

HO

HO

HO

TrehaloseA disaccharide formed by two glucose units.

SiderophoresLow molecular-weight compounds that are secreted by numerous types of bacteria and that have a high affinity for iron and other metal ions. These molecules chelate metal ions and carry them into the cell through specific receptors. They are bacterial virulence factors.

SphingosineAn amino alcohol that can be linked to a fatty acid via the amino group to form the basic structure of sphingolipids.

Box 1 | Structures of microbial and self lipids presented by CD1 molecules

Mycobacterial lipids (see figure, part a), such as sulpholipids from Mycobacterium tuberculosis, have a sulphate group on a trehalose moiety, which is acylated by two to four very long chain branched fatty acids. The diacylated sulpholipid has been shown to be recognized by CD1b-restricted T cells8. Lipopeptides, such as didehydroxymyco-bactin, an intermediate in the biosynthesis of the mycobacterial iron scavenger mycobactin siderophores, can also be presented by CD1a molecules124. Mycobacterial mycolates contain α-alkyl branched β-hydroxy fatty acids in which the main meromycolate chain (C20–C90) may contain R-substitutions such as the keto function group that is shown3. Diacylglycerols, such as the α-galactosyldiacylglycerol from the spirochete Borrelia burgdorferi, and the common mammalian phosphoglycerides, such as phosphatidylinositol (part b), can be presented by CD1d to stimulate invariant natural killer T (iNKT) cells11,18. Glycosphingolipids (partc) are among the most studied lipids presented by CD1 molecules. α-galactosylceramide is a synthetic antigen mimic that, in contrast to mammalian glycosphingolipids, contains an α-linkage between the 1′ carbon of the sugar and the sphingosine base125. A similar α-linkage glycosphingolipid (α-glucuronosylceramide) found in α-proteobacteria activates iNKT cells9,10. By contrast, abundant mammalian β-linked ceramides, such as sulphatide from myelin, and isoglobo-series glycosphingolipids, such as isoglobotrihexosylceramide, can be presented by CD1 molecules19,23.

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Purified protein derivativeA protein from Mycobacterium tuberculosis that is used in the tuberculin sensitivity test, which determines previous interactions of the host with the bacterium. A positive tuberculin test is generally taken as an indication of previous exposure to M. tuberculosis.

α-Galactosylceramide(α-GalCer). A synthetic or marine-sponge-derived glycolipid containing an α-anomeric glycosidic linkage of the galactose residue to the sphingosine base. This lipid, and structurally related ones, potently activates CD1d-restricted natural killer T cells that express the semi-invariant Vα14–Jα18 T-cell receptor in mice (and the Vα24–Jα18 equivalent receptor in humans).

presented by CD1c to human T-cell clones6. Among the other unique mycobacterial lipids are lipopeptides and sulpholipids (BOX 1), which can be presented by CD1a and CD1b, respectively. Importantly, reactivity against several of these lipids has been observed with increased frequency in patients infected with M. tuberculosis or in healthy individuals who are positive for the purified protein derivative6–8, suggesting a role for these lipids in host defence against tuberculosis infection. Although all of the mycobacterial lipids noted above are recognized by a clonal population of CD1a-, CD1b- and CD1c-restricted T cells, so far, similar reactivity for individual iNKT cells has not been observed. Instead, different microorganism-derived lipids of several chemical classes have been shown to activate iNKT cells.

Studies of iNKT cells for many years focused on their activation by the synthetic mimic α-galactosylceramide (α-GalCer)(BOX 1). This raised the question of whether similar structures existed in microorganisms in nature. Although their occurrence among pathogens remains unclear, non-pathogenic, soil and water α-proteobacteria have now been found to contain iNKT-cell stimulating α-linked sphingolipids9,10 (BOX 1). Instead of α-linked sphingolipids, another class of lipids, the diacylglycerols (BOX 1) that are present in the spirochete Borrelia burg-dorferi (the aetiological agent of lyme disease in humans), were found to stimulate iNKT cells11. Further, the lipo-phosphoglycan of the protozoan parasite Leishmania donovani was found to bind CD1d and stimulate iNKT cells in vitro12. Importantly, iNKT-cell-deficient mice showed increased susceptibility to L. donovani

and B. burgdorferi infection11,12. So, microbial lipids that are distinct to those derived from M. tuberculosis and that are presented by CD1a, CD1b and CD1c can be presented by CD1d and can stimulate iNKT cells.

Mammalian self-lipid antigens. The first reported examples of group-1-CD1-restricted human T cells13 and CD1d-restricted mouse T cells14,15 were examples of self-reactive responses. It is widely assumed that iNKT cells recognize self lipids during selection in the thymus16. Yet, it is also clear that the same iNKT cells bearing self-lipid–CD1d-reactive TCRs also recognize foreign-lipid antigens in the periphery1. Similarly, group-1-CD1-restricted human T cells are weakly self reactive and display stronger reactivity to foreign-lipid antigens17. So, the combination of self- and foreign-antigen reactivity is likely to be an inherent feature of CD1-restricted T cells, as it is for MHC-restricted T cells.

In cell-free assays, it was shown that CD1d-restricted T cells depend on loading of the CD1 molecules with mammalian self lipids to activate NKT-cell hybri-domas18. These studies revealed that small mammalian phosphoglycerides, such as phosphatidylinositol (BOX 1), phosphatidylglycerol and phosphatidylethanolamine, could stimulate various NKT-cell populations. One fea-ture common to these self antigens was that they were weak agonists compared with the potent α-GalCer mimic. Several glycosphingolipids have also been shown to be presented by CD1 molecules (TABLE 1). Sulphatide, a major component of myelin (BOX 1), was found to be a promiscuous self-lipid antigen that could be presented by CD1a, CD1b and CD1c and activate clonally restricted human T cells19. Further, GM1 and related gangliosides were also found to be presented by CD1b, suggesting that self-lipid antigens may be important targets for autoimmune T cells in condi-tions such as multiple sclerosis20,21. Glycosphingolipids have also been reported to stimulate iNKT cells. The ganglioside GD3, which is markedly overexpressed in human melanomas, is a major target for ‘autoanti-bodies’. The injection of mice with human melanoma cells or GD3-loaded dendritic cells (DCs) also stimu-lates a CD1d-restricted NKT-cell response22. Recently, iGb3 (isoglobotrihexosylceramide), a member of the globo/isoglobo-series glycosphingolipids (BOX 1), was found to activate iNKT cells in vitro10,23. However, the sug-gestion that iGb3, as a single glycosphingolipid, is the critical self-lipid antigen controlling both iNKT-cell development in the thymus and activation in the periph-ery in vivo has been challenged by the failure to detect iGb3 in mouse or human thymi using sensitive high-performance liquid chromatography methods24 and by the finding that iGb3-synthase-deficient mice, which lack the entire family of isoglobo-series glycosphingo-lipids, have normal numbers of iNKT cells and iNKT-cell development25. So, it seems likely that there may be several self-lipid antigens that collectively provide a range of ligands for positive and negative selection of NKT cells in the thymus and the activation of NKT cells in the periphery.

Table 1 | CD1-restricted lipid antigens

Source Antigen CD1isoform Refs

Mycobacterium tuberculosis and other mycobacteria

Mycolic acids CD1b 3

Glucose monomycolate CD1b 132

Sulpholipid (diacylated sulphoglycolipid)

CD1b 8

Phosphatidylinositol mannosides CD1b, CD1d 5,133

Mannosylated lipoarabinomannan CD1b 5

Mannosyl-β1-phosphomycoketides CD1c 6,134

Didehydroxymycobactin CD1a 124

Sphingomonas spp. α-Glucuronosylceramide CD1d 9,10

Borrelia burgdorferi α-Galactosyldiacylglycerol CD1d 11

Leishmania donovani Lipophosphoglycan CD1d 12

Mammalian (self) Phosphatidylinositol CD1d 18

Phosphatidylglycerol CD1d 18

Phosphatidylethanolamine CD1d 18

GM1 CD1b 20,21

GD3 CD1d 22

Sulphatide CD1a, CD1b, CD1c

19

Isoglobotrihexosylceramide CD1d 10,23

Synthetic or marine sponge

α-Galactosylceramide CD1d 125

R E V I E W S




a b

c

α-GalCer

α2

α3

β2-microglobulin

α1 Phe70

Phe70

Cys12

Val12

A'

A' T'C'

F'

F'

HN

O

O

OH

OOH

OH

HO HO

OH

HO

O

O

O

OHHO

HOHO

C16 spacer lipid

α-GalCer variant

Glucose monomycolate (C60)

Lyme diseaseA disease caused by the bacterium Borrelia burgdorferi or other Borrelia spp. that is transmitted to humans via the bites of infected blacklegged ticks. Symptoms can include skin rash, fever, fatigue, headache, muscle pain, stiff neck and swelling of the knee and other large joints. Most cases can be successfully treated with antibiotics.

Globo/isoglobo-series glycosphingolipids One arm of the glycosphingo-lipid family, which is characterized by an α-linked galactose sugar in the third sugar position. Globotrihexo-sylceramide (Gb3) has an α1–4 linked galactose sugar, whereas isoglobotrihexosyl-ceramide (iGb3) has an α1–3 linked galactose sugar.

Structure of CD1 proteinsSimilar to MHC class I molecules, CD1 heavy chains consist of α1 and α2 domains that form the antigen-binding region, contained within two antiparallel α-helical structures that are situated on a β-pleated sheet (FIG. 1a). The α1 and α2 antigen-binding region is linked to an immunoglobulin-like α3 domain, which is attached to the membrane by a transmembrane seg-ment, followed by a short cytoplasmic tail. However, unlike MHC class I molecules, CD1 proteins bind alkyl chains in hydrophobic channels that reside beneath the surface of CD1 molecules, whereas the hydrophilic head groups of the lipid antigens protrude where the hydro-phobic channels open to the membrane distal surface of the CD1 molecule (FIG. 1a). These head moieties are stabilized by hydrogen bonds, which also contribute to the correct positioning of the lipid antigens. CD1 proteins have deeper and more voluminous antigen-binding compartments than MHC class I molecules. Moreover, the different architecture of the antigen-binding compartment of different CD1 isoforms allows them to bind distinct lipid antigens. The antigen- binding groove of mouse CD1d can be divided into two channels: the A′ channel and the F′ channel, classified by comparison with the pockets of the MHC class I peptide-binding groove26 (FIG. 1b). A narrow entrance into the groove is found at the junction between the

F′ and the A′ channels. There are similar channels in CD1a, with the A′ channel having a fixed length, in which a closed terminus limits the length of the alkyl chains that can be accommodated, like a ‘molecular ruler’, to approximately 18–23 carbon atoms27. Interestingly, the F′ channel of CD1a can bind either an alkyl chain or a peptide, which enables the molecule to bind and present antigens to T cells that have one or two alkyl chains, such as the lipopeptide didehydroxymycobac-tin and sulphatide, respectively27,28. By contrast, CD1b can accommodate longer alkyl chains and contains four channels: the A′, F′, C′ and T′ channels29 (FIG. 1c). In CD1b, the A′ and F′ channels are connected via the T′ tunnel, which forms a superchannel that can accom-modate multiple alkyl chains or a single alkyl chain of up to 60 carbons. Moreover, besides the surface region where the A′ and F′ channels converge, CD1b has an extra portal where the C′ channel contacts the molecular surface. This allows egress of the alkyl chain under the α2 helix of CD1b such that chains longer than 16 carbon atoms might extend out of this chan-nel. Together, these features enable the CD1b groove to accommodate very long fatty acid chains and, poten-tially, lipids with three alkyl chains29.

Recently, the crystal structure of the TCR–α-GalCer–CD1d ternary complex was elucidated30. As predicted, the TCR of the NKT cell contacts the protruding head

Figure 1 | CrystalstructuresofCD1bandmouseCD1dloadedwithlipids.a | A ribbon diagram of the mouse CD1d crystal structure loaded with a synthetic variant of α-galactosylceramide (α-GalCer) that contains a shorter fatty acid chain (the α-GalCer variant is shown in stick representation with the carbon backbone in yellow and oxygen atoms in red)130. A C16 spacer lipid was found to fill the empty space in the A′ channel of CD1d, which would be occupied by the longer fatty acid of the non-variant α-GalCer. b | Surface representation of the mouse CD1d antigen-binding groove loaded with the same lipids as in a. The A′ and F′ hydrophobic channels are shown, as well as two amino-acid residues that form the A′ pole (Cys12 and Phe70) and divert the channel containing the lipid alkyl chain around them. The short fatty acid chain of the lipid inserts into the A′ channel, whereas the sphingosine chain is inserted into the F’ channel. c | Surface representation of CD1b loaded with the C60 species of glucose monomycolate47. The long fatty acid chain sequentially traverses the A′, T′ and F′ superchannel and the α-branched chain inserts into the C′ channel. The diagrams were generated using CCP4MG software131.

R E V I E W S



AP2

AP3

CD1 (heavy chain)

MHC class I

MHC class II

Calnexin Calreticulin

β2m

Clathrin-coated pit

b

d

c

e

f

Trans-Golgi network

Golgi

ER

Nucleus a

MHC class II compartment or lysosome

Endocytic recycling compartment


Ii

ERp57

Sorting endosome

Plasma membrane

Peptide

Self lipid Signal sequencesShort peptide sequences involved in the post-translational targeting of proteins. In particular, the endoplasmic reticulum (ER) signal sequence is recognized by the signal-recognition peptide after synthesis of the signal and the protein is co-translationally inserted into the ER lumen. The signal sequence is normally removed by a signal peptidase.

Secretory pathway A trafficking pathway from the endoplasmic reticulum to the plasma membrane that is taken by newly synthesized molecules that are destined to be secreted.

group of α-GalCer and the CD1 α-helices. Interestingly, the footprint of the NKT-cell TCR on the surface of the α-GalCer–CD1d complex is substantially different from the footprint of MHC-class-I-restricted TCRs, being parallel to the long axis of the CD1d binding groove and positioned in the extreme end of CD1d, above the F′ pocket; and with the invariant TCR α-chain regions CDR1 (complementarity-determining region 1) and CDR3 dominating the recognition of the antigen.

CD1 trafficking in the secretory pathwayAssembly of CD1 molecules. Newly synthesized CD1 molecules have signal sequences for translocation into the lumen of the endoplasmic reticulum (eR). Following synthesis, they rapidly become glycosylated by the addition of N-linked oligosaccharides. This enables the newly synthesized heavy chains to bind the eR chaper-ones calnexin and calreticulin, which have specificity for monoglucosylated immature glycans31–33 (FIG. 2a). Similar

to MHC class I molecules, CD1d also associates with the thiol oxidoreductase eRp57, which is involved in the formation of disulphide bonds33–35 (FIG. 2a). In general, CD1 proteins depend on the association with β2m for their exit from the eR and for trafficking through the secretory pathway to the cell surface31,36,37, although CD1d can also be expressed at the cell surface independently of β2m and might still be functional38–41.

Loading of endogenous lipids onto CD1 in the ER. Studies in which natural ligands have been eluted from CD1 have revealed that CD1b and CD1d associate with endogenous lipids, including phosphatidylinositol and glycosylphosphatidylinositols42–44. The use of native mass spectrometry to study lipid–CD1 complexes revealed that CD1b was associated with phosphati-dylcholine45. Another study that used a recombinant form of CD1d containing a KDel eR-retention signal showed that phosphatidylinositol associated with the

Figure 2 |intracellulartraffickingofCD1molecules.a | CD1 heavy chains are assembled in the endoplasmic reticulum (ER), where they bind the chaperones calnexin, calreticulin and ERp57. They also bind β2-microglobulin (β2m) non-covalently in the ER. b | CD1 molecules then follow the secretory route through the Golgi apparatus to the plasma membrane. MHC class I and II molecules also assemble in the ER and follow a similar route, with MHC class II molecules (in complex with invariant chain (Ii)) being diverted from the trans-Golgi network to the endosomes. c | CD1 molecules are internalized in clathrin-coated pits via the interaction of the adaptor complex AP2 with tyrosine-based sorting motifs present in the cytoplasmic tails of CD1. From the sorting endosome, CD1 molecules can follow two main routes. d | CD1 molecules such as CD1a and CD1c can follow the slow recycling pathway, back to the plasma membrane, through the endocytic recycling compartment. e | CD1 molecules such as CD1b and mouse CD1d can traffic to late endosomal and lysosomal compartments via the interaction of AP3 also with tyrosine-based motifs contained in the cytoplasmic tails of these CD1 molecules. f |CD1 and MHC class II molecules recycle from lysosomal compartments to the plasma membrane. During their trafficking, CD1 molecules are thought to be loaded with a lipid molecule.

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ChylomicronsLarge lipoprotein particles primarily composed of triglycerides, secreted by the intestine into the lymphatic system and degraded by lipoprotein lipase.

Endoglycosidase-H resistanceEndoglycosidase H cleaves high-mannose oligosaccharides from N-linked glycoproteins. The acquisition of resistance to this enzyme is related to the processing of the high mannose into complex oligosaccharides in the medial Golgi apparatus and therefore indicates progression through the secretory pathway.

Invariant chain(Ii). A non-polymorphic molecule that associates with MHC class II proteins. By occupying the antigen-binding cleft, the invariant chain stabilizes newly synthesized MHC class II molecules in the endoplasmic reticulum and directs the mature molecules to compartments in which binding with antigenic peptides occurs.

Surface plasmon resonanceA technique used to measure molecular interactions by observing how much of an input molecule (for example, a protein) is bound to a chip immobilized with another molecule. The amount of bound input is directly proportional to the change in the light reflected off the immobilized chip, which is specifically measured. This technique can be used to calculate single-molecule affinities, as well as binding on and off rates.

Slow recycling pathwayOne arm of the early recycling pathway that involves trafficking through early endosomes and the endocytic recycling compartment.

recombinant CD1d, which supports the idea that self lipids are loaded into CD1 molecules during their assembly in the eR43,44. Such eR-loaded self lipids might be antigenic or alternatively they might function as chaperones to facilitate the assembly and to stabilize CD1 molecules in the eR. Consistent with this, no CD1b crystal structures have been obtained without a lipid or other molecule filling the entire antigen-binding groove of the molecule, suggesting that the hydrophobic groove is stable only when occupied by a bound lipid45–47.

Role for MTP in CD1 antigen presentation. Micro-somal triglyceride transfer protein (MTP; also known as MTTP) is an eR-resident lipid-transfer protein that is required for the proper assembly and secretion of apolipoprotein-B-containing lipoproteins, namely very low-density lipoproteins (vlDls) by the liver and chylomicrons by the intestine48,49. Interestingly, the inhi-bition of MTP in APCs causes a defect in the presenta-tion of lipid antigens by CD1d, and mice lacking MTP are also deficient in CD1d antigen presentation50,51. Although the precise mechanism of action of MTP has not been delineated, MTP was shown to transfer a phospholipid (phosphatidylethanolamine) to mouse CD1d in vitro51, and therefore could have a role in the loading of CD1d with endogenous lipids that are important both for NKT-cell selection in the thymus and antigen presentation by APCs in the periphery. unexpectedly, MTP deficiency was also shown to inhibit the recycling of mouse CD1d from lysosomes to the cell surface, suggesting possible effects of MTP that are distal from its location in the eR52.

CD1 trafficking from the ER to the plasma membrane. Studies of the time-course of acquisition of endoglycosidase-H resistance of CD1b and the appearance of CD1b at the cell surface suggest that, following assembly in the eR, this isoform follows the secretory pathway through the Golgi directly to the plasma membrane53 (FIG. 2b). CD1d has also been observed to associate with MHC class II molecules and the invariant chain (Ii), which can direct CD1 complexes with these proteins from the trans-Golgi network to endosomal compartments without first reach-ing the plasma membrane54,55. However, the functional significance of this alternative CD1 trafficking pathway is not known.

CD1 trafficking in the endocytic pathwayCD1 internalization from the plasma membrane. The internalization of CD1 from the plasma membrane is essential for its ability to sample antigens in the endo-cytic system. After trafficking to the plasma membrane, CD1 molecules appear to follow a dominant, pre-sumably constitutive, pathway of internalization into endosomes (FIG. 2c). This clathrin-dependent pathway is common to proteins that contain well character-ized tyrosine-based sorting motifs of the YXXφ type (where Y is a tyrosine, X is any amino acid and φ is a bulky hydrophobic residue), which bind the adaptor protein complex 2 (AP2) and allow sorting of cargo

proteins into clathrin-coated pits56. using surface plasmon resonance, the cytoplasmic tails of CD1b, CD1c and mouse CD1d were shown to bind AP2 or one of its subunits, and immunoelectron microscopy studies of CD1b and CD1c showed that these molecules promi-nently localize to clathrin-coated pits and vesicles53,57–59. Accordingly, a marked reduction in CD1b and CD1d internalization, and consequent accumulation on the cell surface, occurred when the respective cytoplasmic tails of these molecules were deleted, demonstrating the importance of these domains for internalization of the proteins from the plasma membrane53,54,57,59–61. In contrast to the other isoforms, CD1a does not contain any sorting motifs in its cytoplasmic tail, and yet it is also internalized from the plasma membrane into endosomal compartments by an unknown mechanism.

CD1 trafficking through the endocytic pathway. After internalization into the early or sorting endosomes, the different CD1 isoforms follow different trafficking path-ways. CD1a and CD1c, but not CD1b, co-localize with a dominant-negative form of the small GTPase ARF6 (ARF6-T27N) in the endocytic recycling compartment, indicating that CD1a and CD1c follow the slow recycling pathway back to the plasma membrane58,62 (FIG. 2d). Indeed, CD1a was shown to co-localize with RAB11, another marker for the endocytic recycling compartment, and to recycle back to the plasma membrane from an intra-cellular pool63.

Some of the tyrosine-based sorting motifs, such as those in CD1b and mouse CD1d molecules, can bind both AP2, which mediates CD1 internalization from the plasma membrane, and AP3, which diverts these CD1 molecules from the early recycling path-way to late endosomes and lysosomes59,64–66 (FIG. 2e). Accordingly, AP3-deficient cells have increased cell-surface expression of CD1b, with an accumulation of molecules in the early recycling pathway and a near absence in lysosomes64. The functional importance of AP3-dependent CD1b trafficking is highlighted by the defects displayed by the AP3-deficient cells in the CD1b-restricted presentation of a microbial-lipid antigen, such as glucose monomycolate64. In the case of mouse CD1d, AP3-deficient mice show a similar defect in CD1d localization to lysosomes and CD1d-mediated antigen presentation, which is reflected in a significant reduction in the number of NKT cells in these mice65,66. Interestingly, unlike CD1b and mouse CD1d, the tyrosine-based motifs of CD1c and CD1d (human) are not capable of binding AP3, as assessed using yeast two-hybrid assays64. As a result, AP3-deficient human cells do not display any defects in the presentation of CD1c- mediated lipid antigens. The importance of CD1 traf-ficking through the endocytic pathway for lipid loading is highlighted by studies that examined the function of CD1 molecules that do not internalize properly from the plasma membrane owing to mutations in the cyto-plasmic tail. Indeed, mutant mouse CD1d and CD1b molecules that lack the cytoplasmic tail are defective in the presentation of lipid antigens to iNKT cells and T cells, respectively60,61,67.

R E V I E W S




PinocytosisA type of endocytosis in which fluid is taken up by cells.

C-type lectinsReceptor proteins that bind carbohydrates in a calcium-dependent manner. The binding activity of C-type lectins is based on the structure of the carbohydrate-recognition domain, which is highly conserved between members of this family.

Langerhans cellsA type of dendritic cell that is resident in the epidermal layer of the skin.

So, once internalized from the plasma membrane, CD1a molecules traffic mainly through the endocytic recycling compartment, whereas CD1b and mouse CD1d molecules traffic mainly through late endosomes and lysosomes. These distinct trafficking routes taken by the different CD1 isoforms presumably allow each isoform to survey distinct intracellular compartments for lipid antigens. Of the CD1 isoforms, CD1c appears to have the most promiscuous localization, as it can be found in both early and late endocytic compartments58.

CD1 recycling from endosomes to the plasma membrane. The sequence of antigen loading onto CD1 molecules probably involves exchanging self lipids that are bound to CD1 during assembly in the eR or delivery to the plasma membrane with other self or foreign lipids that are encountered within endosomes. Once this has occurred, CD1 molecules must return to the plasma membrane for presentation of the antigens to T cells. For CD1a molecules, return to the plasma membrane can readily be accounted for because of their extensive localization in the endocytic recycling compartment. like other cell-surface molecules that are internalized into the endocytic recycling compartment from the plasma membrane, after a period of retention, they correspondingly recycle back to the plasma membrane. In recent years, it has become well recognized that recycling from late endosomes and lysosomes back to the plasma membrane is also a functionally significant trafficking route for molecules that are not degraded in these compartments, such as MHC class II molecules68,69 (FIG. 2f). Although the route followed by CD1 molecules from lysosomes to the plasma membrane has not been clearly delineated, this pathway is the target for immune evasion by herpes simplex virus 1 (HSv1), as it was shown that this virus can inhibit the recycling of CD1d to the plasma mem-brane, with consequent accumulation of the protein in lysosomes70.

CD1 trafficking in mature DCs. The recycling of molecules from lysosomes to the plasma membrane is influenced by the maturation of DCs. Strikingly, MHC class II molecules are rapidly delivered from lysosomes to the plasma membrane within hours of receiving DC maturation signals71. This is one of the several changes DCs undergo when exposed to maturation stimuli, which also include decreased pinocytosis and increased half-life of MHC class II molecules on the cell surface71–74. By contrast, it is not clear whether the recycling of CD1 molecules shows similar changes in response to DC maturation. Indeed, cell-surface expression of CD1a, CD1b, CD1c and CD1d only shows minor changes on DC maturation, and the presentation of glucose monomycolate is not affected by DC maturation, whereas the presentation of an MHC-class-II-presented peptide is markedly enhanced in mature DCs74. Another difference between CD1 and MHC class II molecules is the continued active internalization of CD1b after DC maturation, whereas the internalization of MHC class II molecules is strongly reduced in mature DCs72,75. Therefore, MHC class II

and CD1 trafficking are differentially regulated during DC maturation.

Lipid traffickingAs described, CD1 molecules can survey different cell-ular compartments by virtue of differential endosomal trafficking. Collectively, CD1 molecules ensure a broad survey of endocytic compartments for endogenous and exogenous lipid antigens, which are ultimately presented to T cells. Therefore, CD1 molecules must intersect rele-vant lipid antigens in endocytic compartments for lipid loading to occur.

Delivery of lipid antigens to APCs. Cells continuously take up lipids for their metabolic needs. Most of these are transported in the blood in complex with apoli-poproteins, forming lipid-transport particles76. One mechanism of uptake of lipid-transport particles is the lDl receptor (lDlR)-dependent internalization of the vlDl particles into endosomes. The lDlR binds apolipoprotein e (apoe) on vlDl particles and deliv-ers it to the endocytic system77 (FIG. 3b). The low pH in endosomes facilitates the release of bound apolipo-proteins from the receptor. The lDlR is then recycled to the plasma membrane, whereas the lipoproteins are delivered to late endosomes and lysosomes for degradation78. Interestingly, apoe can bind exogenous CD1 lipid antigens and dramatically enhances the CD1d-dependent presentation of a lipid antigen that requires lysosomal processing79. It has also been shown that APCs secrete large amounts of apoe, suggesting a mechanism for how APCs sample the extracellular milieu for lipid antigens by secreting and taking up apoe-bound lipids from their local environment79.

Other families of receptors may also be involved in the uptake of lipid antigens by APCs. Scavenger recep-tors, including SR-A, SR-BI, lOX1 (lectin-type oxidized lDl receptor 1; also known as OlR1) and CD36, bind modified forms of lDl, such as acetylated lDl and oxidized lDl, and also apoptotic cells80, and may there-fore be involved in the uptake of exogenous lipids from infected cells (FIG. 3b).

C-type lectins bind carbohydrate moieties via their carbohydrate-recognition domains (CRDs) in a cal-cium-dependent manner81 (FIG. 3b). Most C-type lectins bind mannosylated moieties, which are present in several CD1 lipid antigens. Indeed, lipoarabinoman-nan has been shown to be internalized and delivered to late endosomal and lysosomal compartments by the macrophage mannose receptor82. Another C-type lectin, langerin, is expressed by Langerhans cells and localizes to Birbeck granules, an endosomal recycling compartment in which CD1a is also found83. The failure of APCs to present CD1a-dependent lipid antigens when langerin is blocked by antibodies suggests that langerin might be involved in the uptake of glycolipid antigens84.

Finally, the uptake of particulate material or the pathogens themselves by phagocytosis is also an impor-tant route of delivery of exogenous and microbial lipid antigens into the endocytic system, where CD1 isoforms can bind them (FIG. 3b).

R E V I E W S



Clathrin-coated pit

Clathrin

b

c

ER

Nucleus

a

LDLR

ApoE

?

Self lipid

CD1MTP

Pathogen

C-type lectin Scavenger receptor

MHC class II compartment or lysosome

Phagocytosis

CD1e

Saposin B Microbial lipid


Plasma membrane

Intracellular trafficking of lipids. Our understand-ing of intracellular lipid trafficking is very incomplete. Nevertheless, it is now appreciated that lipids are distrib-uted differently among compartments and, consequently, different compartments of the endocytic pathway have distinct lipid compositions. For example, early endo-somes have been shown to be enriched in PtdIns3P (phosphatidylinositol-3-phosphate) and cholesterol, whereas late endosomes are enriched in PtdIns(3,5)P2 (phosphatidylinositol-3,5-bisphosphate) and bis(mono-acylglycero)phosphate (also known as lysobisphosphatidic acid)85,86. Furthermore, it has been shown that some lipids traffic intracellularly according to their biophysical prop-erties. lipid analogues that contain long and saturated lipid tails traffic to late endocytic compartments, whereas lipids with short or unsaturated tails, and the same head group, follow the endocytic recycling pathway87. Indeed, the long-chain version of the CD1b-restricted antigen glucose monomycolate (containing 80 carbons) prefer-entially accumulates in late endocytic compartments, when compared with a shorter version (containing 32 carbons)88. The stereochemistry of the lipid has also been shown to influence lipid internalization and trafficking89. Given that lipids are sorted into different compartments and traffic according to their biophysical properties, it has been proposed that CD1 trafficking evolved to sample the compartments to which the lipid antigens traffic90.

Lipid exchange in the endocytic pathwaySeveral lines of evidence indicate that microbial and self antigens may be predominantly loaded onto CD1 molecules in the late endosomal or lysosomal compart-ments. This would explain why the steady state distribu-tion of many CD1 molecules (such as CD1b and mouse CD1d) is mainly localized to these compartments. The many examples of defects in presentation that occur when CD1 molecules fail to be internalized from the plasma membrane and/or delivered to late endosomes and lysosomes are consistent with the acquisition of lipid antigens in endosomal compartments. Some self lipids are loaded onto CD1 molecules in the eR, which sug-gests that the nascent CD1 molecules internalized from the plasma membrane contain bound lipids that can be exchanged for others in the endocytic compartments, such as late endosomes and lysosomes. Given that the lipids in endocytic compartments are probably inserted into endosomal membranes, these lipid antigens must be transferred from the internal or limiting membrane of an endosome into the CD1 antigen-binding groove. This process is facilitated by accessory molecules that have been recently identified.

Saposins. Saposins are membrane-perturbing sphingo-lipid activator proteins that were previously charac-terized for their role in the lysosomal degradation of

Figure 3 | lipidloadingandexchangeintheendocyticpathway.a | Self lipids (shown in orange) from the endoplasmic reticulum (ER) are loaded onto CD1 molecules. In the case of CD1d, this process is facilitated by a poorly characterized mechanism that involves microsomal triglyceride transfer protein (MTP). b | Four possible mechanisms for the uptake of foreign lipid antigens are shown: clathrin-dependent internalization of apolipoprotein E (apoE)–lipid complexes bound to the low-density lipoprotein receptor (LDLR); phagocytosis of particulate material or whole pathogens; C-type lectins, which can bind mannose residues on glycolipids; and internalization through scavenger receptors, which can bind modified forms of LDL and apoptotic cells. c | The exchange of endogenous lipids, loaded in the ER or the secretory pathway, by foreign lipids or different endogenous lipid antigens (in blue), takes place in endocytic compartments, such as lysosomes. Several accessory molecules, such as saposins and CD1e, have been implicated in the loading of lipids in these compartments. In the case of saposin B, the protein probably binds lipids, extracts them from membranes and transfers them onto CD1d molecules.

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Class-II-associated invariant chain peptide(CLIP). A fragment of invariant chain that occupies the MHC class II antigen-binding groove and prevents loading of antigenic peptides.

Metachromatic leukodystrophyAn inherited lipid storage disorder that affects the growth and/or development of the myelin sheet. Although several cell types can be affected, the pathology is essentially associated with the nervous system. This disease has infantile, juvenile and adult forms.

Gaucher’s diseaseA heterogeneous genetic lipid storage disorder. The most common form, type 1, is characterized by anaemia, low platelet counts, enlarged liver and spleen and skeletal disorders and may involve lung and kidney impairment.

glycosphingolipids. Although not enzymatically active, deficiencies in these glycoproteins lead to sphingolipid-storage disorders by failing to activate the enzymatic deg-radation of glycosphingolipids by glycosidic enzymes91 (BOX 2). Saposins are generated from a single precursor protein, prosaposin, that yields saposins A–D upon its endosomal proteolytic cleavage91. Prosaposin-deficient fibroblasts were found to have impaired presentation of CD1b- and mouse CD1d-restricted exogenous antigens in vitro92,93. Interestingly, the CD1b presentation defect could be rescued with the addition of saposin C but not other saposins92. Such selectivity for saposin–CD1 combinations is further supported by the finding that presentation of α-GalCer by CD1d is preferentially restored by saposin B94. In addition, prosaposin- deficient mice exhibit defects in CD1d-mediated antigen presentation to NKT cells, as well as in the thymic devel-opment of NKT cells95. Saposins are known to mobilize sphingolipids from donor membranes and transfer them to acceptor membranes (FIG. 3c). Indeed, saposin B was shown to transfer GM1, phosphatidylinositol and phosphatidylcholine between membranes96,97. The exact mechanism by which saposins mediate the transfer of lipid antigens onto CD1 is not known, but it was found by co-immunoprecipitation that saposin C and, to a lesser extent, saposin D interact directly with CD1b and that saposin A and lipid-loaded CD1d can form a complex, as analysed by western blotting after native isoelectric focusing92,95. These studies suggest that saposins may directly bind lipid antigens, extract them from endosomal membranes and then transfer the lipids while bound to CD1. However, this mechanism would require that saposins be capable of binding every lipid that CD1 molecules present. Although this is conceiv-able, it is likely that some saposins have a more general role as membrane disruptors that facilitate access to lipids or destabilize lipid topologies in membranes, rendering them more available for loading onto CD1 molecules without directly transferring them (BOX 2). Other molecules might also be involved in the loading

of lipid antigens onto CD1, in lysosomes, as recently shown for Niemann-Pick type C2 protein98.

CD1e. CD1e is the only CD1 isoform that is not expressed on the cell surface of DCs99,100. In immature DCs, CD1e is mainly localized in the Golgi apparatus, whereas in mature DCs it is nearly exclusively detected in late endo-somes and lysosomes, where it is cleaved into a functional soluble form99–101. It has been suggested that CD1e could be involved in antigen processing as it was found that it facilitates the α-mannosidase-dependent processing of PIM6 into PIM2 (REF. 101).

In the case of peptide loading onto MHC class II mol-ecules, an accessory protein, HlA-DM (known as H-2M in mice) facilitates the displacement of class-II-associated invariant chain peptide (ClIP) from the peptide-binding groove to allow new peptides to bind to MHC class II molecules102,103. So far, no specific mechanism for displac-ing the eR-bound lipids to favour loading of other lipids onto CD1 molecules has been identified. Future studies will be needed to fully understand the specific molecules and topologies of lipid-antigen loading onto CD1 mol-ecules. Interestingly, it is clear that some lipids may load onto CD1 molecules on the cell surface88,104 or in early endosomes, where saposins have not been detected.

Functions of CD1-restricted T cellsCharacteristics of group-1-CD1-restricted T cells. Group-1-CD1-restricted T cells specific for microbial-lipid anti-gens have highly diverse TCR α- and β-chains, which are indistinguishable from comparable peptide-specific TCRs105. CD1a-, CD1b- and CD1c-restricted T cells were initially identified among the CD4–CD8– double-negative T-cell subset but have now been recognized to be even more common in the CD4+ and CD8+ αβ TCR T-cell pool3,7,106–109. Most microbe-specific group-1-CD1-restricted T cells appear to be T helper 1 (TH1)-like cells7,107–109. The clonal heterogeneity of group-1-CD1-restricted T cells and their expansion in infection suggests that they provide adaptive immunity to microbial-lipid

Box 2 | Saposins

The saposins (saposin A to saposin D) are small, non-enzymatic proteins that localize to lysosomes and facilitate the hydrolysis of different glycosphingolipids in these compartments. For example, saposin B ‘activates’ the degradation of sulphatide by arylsulphatase A, and saposin C promotes the degradation of glucosylceramide by glucosylceramide-β-glucosidase. Consequently, defects in these saposins lead to the accumulation of the referred substrates in lysosomes and result in a variant form of metachromatic leukodystrophy and in a juvenile variant of Gaucher’s disease, respectively. Saposin C and saposin D bind and destabilize phospholipid membranes in a pH-dependent manner126. The neutralization of the highly negatively charged surface of saposin C, by protonation of acidic residues upon a decrease in endosomal pH, is thought to facilitate the interaction with membranes, as the negatively charged surface might hinder the insertion into the apolar interior of membranes or cause electrostatic repulsion from the negatively charged groups of the membrane127. This destabilizes the membrane bilayer architecture and may make glycolipid substrates more accessible to hydrolases. By contrast, saposin B binds poorly to phospholipid membranes and does not destabilize them significantly, even at low pH126. Saposin B forms a dimer in solution and contains a large hydrophobic cavity where lipids can bind128. Indeed, a phospholipid was shown to co-purify and crystallize with saposin B and could be modelled in the dimer structure, in which the acyl chains are buried inside the hydrophobic cavity of saposin B, whereas the hydrophilic head group is exposed to the solvent in a manner similar to lipid-loaded CD1 molecules. So, saposin B can extract and transfer sphingolipids and phospholipids between membranes and does so more efficiently at low pH97,129. Therefore, this saposin is considered to be a lipid ‘solubilizer’ that forms a complex with the lipid after extracting it from the membrane, whereas saposin C is considered to be a membrane disruptor, increasing exposure of lipid head groups to enzymes. Both of these mechanisms could facilitate the transfer of lipids from lysosomal membranes to CD1 molecules.

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Toll-like receptor(TLR). A family of pattern-recognition receptors that recognize conserved molecules from pathogens, such as lipopolysaccharide or endotoxin, initiating innate immune responses.

SpirochetesPhylum of flagellated helical-shaped, Gram-negative bacteria.

antigens, analogous to the adaptive immunity of MHC-restricted T cells against microbial-protein antigens (TABLE 2).

Innate-like function of iNKT cells. CD1d-restricted iNKT cells comprise a large pool of up to several percent of all T cells that respond rapidly and display the fea-tures of innate immune cells (TABLE 2). Resting NKT cells have a memory or partially activated phenotype and respond rapidly following TCR stimulation to produce cytokines, such as interferon-γ (IFNγ) and interleukin-4 (Il-4), and to become cytotoxic T lymphocytes (CTls). Although all iNKT cells use nearly the same TCR α-chain sequence, they include both CD4+ and CD4– subsets that are distinct in terms of various func-tions. For example, human CD8+ and double-negative iNKT cells are more likely to be cytotoxic and produce TH1-type cytokines, whereas CD4+ iNKT cells are more likely to produce both TH1-type and TH2-type cytokines after stimulation110,111, and these subsets also have some-what different patterns of chemokine and chemokine receptor expression112,113. Moreover, the CD4– subset of NKT cells has the main capacity for tumour rejection in several mouse models114.

Once activated, iNKT cells rapidly stimulate DCs, macrophages and NK cells and recruit neutrophils to expand the immediate innate immune response and have an impact on the subsequent adaptive T-cell and B-cell responses. There is a striking reciprocal stimulation axis between DCs and NKT cells, such that activation of iNKT cells by DCs, using either α-GalCer or CD1-restricted self reactivity, results in upregulation of CD40 ligand expression and IFNγ secretion by the NKT cells, which in turn stimulates DC maturation115–117. The cycle is self-amplifying as Il-12 production by the DCs in this context further activates the iNKT cells118. The secretion of IFNγ by iNKT cells activates macrophages to enhance their capacity for killing intracellular microorganisms119,120. Therefore, NKT cells contribute to the innate phase of immunity to microorganisms through their role at the centre of a broad immune response (TABLE 2). In fact, the full innate immune response to the Toll-like receptor (TlR) agonist lipopolysaccharide is blunted in NKT-cell-deficient mice121. Although iNKT cells are mainly associated with

the innate phase of the immune response, it is also clear that their early activation can provide help to B cells, influencing antibody production122, as well as enhancing MHC-restricted T-cell responses to peptide antigens123.

Activation of CD1-restricted T cells by microorganisms. The strategy for the activation of group-1-CD1-restricted T cells by microbial antigens appears to be similar to that used for the recognition of microbial peptides presented by classical MHC molecules. A clonally restricted T-cell response occurs that is rigorously determined by recog-nition of the lipid-antigen–CD1 complex by the TCR (FIG. 4a). As discussed earlier, microbial lipids presented as cognate antigens that activate iNKT cells have also been recently identified. Sphingolipids in α-proteobacte-ria, diacylglycerols in spirochetes and lipophosphoglycans in Leishmania spp. parasites stimulate iNKT cells and are recognized directly by the invariant vα14–Jα18 (mouse) or vα24–Jα18 (human) TCRs (FIG. 4b). Currently, α-GalCer-loaded CD1d tetramers are considered the gold standard for identifying iNKT cells. By compari-son, tetramers of CD1d loaded with Sphingomonas spp. α-glucuronosylceramide and B. burgdorferi α-galactosyl-diacylglycerol stain 25% and 12% of iNKT cells, respec-tively9–11. These subpopulations of iNKT cells are activated en masse by such antigens.

So far, no cognate lipid antigens that are recognized by the iNKT-cell TCR have been found in the main Gram-negative and Gram-positive bacterial pathogens that are prominent in human disease, and viruses do not encode enzymatic machinery for lipid synthesis. In the search for a mechanism by which a wider range of microorganisms might be able to stimulate iNKT cells, even in the absence of specific cognate foreign lipid antigens, an alternative strategy was found that depends on cytokines (such as Il-12) in addition to TCR stimulation by CD1d-presented self antigens118 (FIG. 4c). Indeed, the stimulation by an array of Gram-negative bacteria (such as Salmonella typhimu-rium) or Gram-positive bacteria (such as Staphylococcus aureus) cultured with myeloid DCs could be blocked by either CD1d-specific or Il-12-specific monoclonal anti-bodies in vitro and in vivo. So, TlR-stimulated cytokines from APCs can drive iNKT cells interacting with the same or nearby APC, enabling them to respond to virtually any

Table 2 | Characteristics of CD1-restricted T cells

group-1-CD1-restrictedTcells

CD1d-restrictedinKTcells CD1d-restricteddiversenKTcells

Antigens Microbial and self lipids Microbial and self lipids Unknown

T-cellpopulation Clonally diverse Canonical TCRα but polyclonal Clonally diverse

TCR TCRα: diverse; TCRβ: diverse TCRα: invariant Vα14 or Vα24 and Jα18; TCRβ: limited Vβ repertoire with diverse CDR3

TCRα: diverse; TCRβ:diverse

Precursorfrequency

One per thousands, unique specificity for single antigen

<1% of T cells in humans; 2–50% of T cells in mice; pool of cells that responds en masse to a single antigen

Unknown

memmory Yes No Unknown

immunity Adaptive, slow Innate-like, rapid (hours to few days) Unknown

CDR3, complementarity-determining region 3; iNKT cell, invariant natural killer T cell; TCR, T-cell receptor.

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Microorganism

Clonally unique T cells

TCR

CD1a, CD1b or CD1c

Plasmamembrane

Microbial-lipidantigenLysosome

Phagosome

CD1d

iNKT cells

Vα14(Vα24)–Jα18 TCR

CD1dIL-12TLR

Self-lipid antigen

Cognate microbial-antigenactivation of T cells restricted by CD1a, CD1b or CD1c

a Cognate microbial-antigenactivation of CD1d-restricted iNKT cells

b Cytokine-driven self-antigen activation of CD1d-restricted iNKT cells

c

Saposin

Lipid

Antigen-presenting cell

microorganism that can stimulate the APC accordingly118. That this cytokine-dependent mechanism is depend-ent on TlR engagement of the APC was confirmed in studies showing that S. typhimurium-exposed wild-type bone-marrow-derived DCs, but not cells deficient in the TlR-adaptor proteins MyD88 (myeloid differen-tiation primary-response gene 88) or TRIF (Toll/Il-1- receptor (TIR)-domain-containing adaptor protein inducing IFNβ), were able to stimulate iNKT cells in vitro10. So, there are two major mechanisms for the activation of iNKT cells, either via cognate microbial antigens (for those organisms that have them) or via self antigens and APC-derived cytokines (for organisms with potent TlR agonists)118,121 (FIG. 4).

Concluding remarksThe structure of CD1 molecules enables the hydrocarbon chains of lipids of a wide variety of types to embed them-selves in the hydrophobic channels of the CD1 antigen-binding groove, below the surface of the molecule. The TCR then recognizes the surface-exposed polar moieties of the lipids along with CD1 surface determinants, in a manner similar to that of peptide recognition by MHC molecules. To survey the intracellular compartments of APCs for lipid antigens, CD1 molecules traffic from the eR to the cell surface and then back into the cell, where

they circulate through various endocytic compartments before returning to the plasma membrane bearing lipid antigens. CD1 molecules first bind lipid antigens in the eR and then may exchange these for other self- or microbial-lipid antigens that are encountered along the endocytic pathway. The eR loading may involve MTP, and eR-loaded lipids may be exchanged in lysosomes by saposins. Foreign lipid antigens may be delivered to APCs in phagocytosed microorganisms or in lipoprotein particles. lipid-antigen–CD1 complexes expressed on the cell surface then can activate both adaptive and innate-like lymphocyte responses. Group-1-CD1-restricted T cells appear to function as clonally diverse adaptive immune cells that expand following infection. By contrast, CD1d-restricted iNKT cells are innate-like lymphocytes that respond within hours after activation as a pool of charged cells that are potent sources of cytokines and elicit broad responses. Recent studies of lipid antigens provide insight into this surprising range of T-cell responses, which are closely linked to the distinct CD1 isoforms and the invari-ant and diverse T-cell responses they elicit. even more remarkably, the ability of iNKT cells to be activated by self antigens and APC-derived cytokines in the absence of cognate foreign antigen recognition appears an entirely unexpected mechanism of T-cell response that has no parallel among MHC-restricted responses.

Figure 4 | StrategiesforactivationmechanismsofCD1-restrictedTcells.a | Activation of CD1a-, CD1b- and CD1c-restricted T cells. Distinct microbial lipid antigens presented by CD1a, CD1b and CD1c are recognized by low precursor frequency T cells bearing individual diverse αβ T-cell receptors (TCRs). b | Cognate microbial-antigen activation of invariant natural killer T (iNKT) cells. Certain bacteria contain lipid antigens recognized by the Vα14–Jα18 (mouse) or Vα24–Jα18 (human) invariant TCR that stimulate a large pool of iNKT cells en masse. c | Cytokine-driven self-antigen activation of iNKT cells. Even in the absence of a cognate microbial lipid antigen for the iNKT-cell TCR, most microorganisms can activate iNKT cells by stimulating antigen-presenting cells to produce interleukin-12 (IL-12), which in combination with the self-lipid antigens presented by CD1d can stimulate potent iNKT-cell responses. This allows rapid activation of a large pool of iNKT cells without direct microbial lipid antigen recognition by the TCR.

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AcknowledgementsWe thank R. Grenha and R. Tatituri for valuable help in the preparation of the original figures, M. Brigl, L. Léon, R. Tatituri and D. B. Moody for critical reading of the manuscript and members of our laboratory for helpful discussions. D.C.B. is the recipient of a postdoctoral fellowship from the Arthritis Foundation and M.B.B. is supported by grants of the National Institutes of Health, USA. We apologize to those whose work could not be included due to space limitations.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneCD1a | CD1b | CD1c | CD1d | CD1e | MTP | prosaposin

FURTHER INFORMATIONMichael B. Brenner’s homepage: http://www.hms.harvard.edu/dms/immunology/fac/Brenner.html

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