The Biology of NKT Cells

Transcription

1 Annu. Rev. Immunol : First published online as a Review in Advance on December 6, 2006 The Annual Review of Immunology is online at immunol.annualreviews.org This article s doi: /annurev.immunol Copyright c 2007 by Annual Reviews. All rights reserved /07/ $20.00 The Biology of NKT Cells Albert Bendelac, 1 Paul B. Savage, 2 and Luc Teyton 3 1 Howard Hughes Medical Institute, Committee on Immunology and Department of Pathology University of Chicago, Chicago, Illinois 60637; abendela@bsd.uchicago.edu 2 Department of Chemistry, Brigham Young University, Provo, Utah 84602; paul savage@byu.edu 3 Department of Immunology, Scripps Research Institute, La Jolla, California 92037; lteyton@scripps.edu Key Words natural killer T cell, lymphocyte development, innate immunity, α-proteobacteria, Sphingomonas, Ehrlichia, Salmonella, glycolipid, CD1d, antigen presentation Abstract Recognized more than a decade ago, NKT cells differentiate from mainstream thymic precursors through instructive signals emanating during TCR engagement by CD1d-expressing cortical thymocytes. Their semi-invariant αβ TCRs recognize isoglobotrihexosylceramide, 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 immunopathological 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 recognition. 297

2 Natural killer T (NKT) cell: a T cell expressing a CD1d-restricted, lipid-specific T cell receptor combining a canonical Vα14-Jα18 α chain with a variable Vβ8, -7, or -2 β chain in mouse or Vα24-Jα18/Vβ11 in human CD1: a family of MHC-like molecules that specialize in presenting lipid antigens to T lymphocytes α-glycuronylceramides: glycolipids that substitute for LPS in the cell wall of Gram-negative, LPS-negative bacteria such as Sphingomonas INTRDUCTIN Several lines of research led to the identification of NKT cells as a separate lineage of T lymphocytes. The first sightings included (a) the identification of a canonical Vα14- Jα18 ( Jα18 was previously known as Jα281 or Jα15) rearrangement in a set of hybridomas derived from mouse KLH (keyhole limpet hemocyanin)-specific suppressor T cells (1 3), and later in cdna extracted from lymphoid organs of unimmunized mice (4, 5); (b) the identification of a subset of mouse CD4 8 double-negative (DN) T cells with avβ8 usage bias (6, 7); and (c) the identification of a recurrent Vα24-Jα18 rearrangement in human DN peripheral blood lymphocytes (8, 9). These observations were pieced together when a subset of CD4 and DN IL-4-producing thymocytes co-expressing NK lineage receptors was independently identified and shown to express a biased set of Vβ8, Vβ7, and Vβ2 T cell receptor (TCR) β chains (10 13) combined with a canonical Vα14-Jα18 in mouse (14) and with the homologous Vα24-Jα18/Vβ11 pair in human (14, 15). The finding that the mouse and human NKT cells were autoreactive to cells expressing CD1d (15 18), a member of the CD1 family of MHC-like molecules, completed the initial characterization of this lineage and raised modern questions relating to their development, specificity, and function. These issues have been treated in more than 1500 reports over the past 10 years, more than 300 of which were published in the past year alone. We attempt to organize a critical understanding of the general biology of NKT cells, mainly of the predominant mvα14 and hvα24 subsets, on the basis of recent fundamental advances and newly emerging concepts. wing to space limitations, it is not possible to exhaustively review or mention all the studies, many of which suggest new roles of NKT cells in various diseases and remain relatively preliminary or isolated. We focus on bacterial infections where the role of NKT cells is well established and examine a selection of autoimmune, allergic, and tumor conditions of broad clinical interest, where the function of NKT cells remains speculative or controversial. DEFINITIN NKT cells are narrowly defined as a T cell lineage expressing NK lineage receptors, including NK1.1 in the C57BL/6 background, in addition to semi-invariant CD1d-restricted αβ TCRs. More than 80% of these TCRs are Vα14-Jα18/Vβ8, Vβ7, and Vβ2 in mouse (or Vα24-Jα18/Vβ11 in human), with the remaining representing a collection of rare but recurrent Vα3.2-Jα9/Vβ8, Vα8/Vβ8, and other TCRs (19, 20). Whereas both the Vα14 and the non-vα14 NKT cells exhibit autoreactivity to CD1d-expressing cells, particularly thymocytes, their antigen specificities do not overlap. Thus, mvα14 and hvα24 NKT cells, irrespective of their Vβ-Dβ-Jβ chain usage, recognize a marine sponge derived α-galactosylceramide (αgalcer) (21, 22) and closely related microbial α-glycuronylceramides (23 25), as well as the self antigen isoglobotrihexosylceramide (igb3) (26). In contrast, the self and foreign antigens recognized by non-vα14 NKT cells remain to be identified. A striking, generic difference between Vα14 and non-vα14 NKT cells is that the natural Vα14 NKT ligands, including igb3, require endosomal trafficking of CD1d and intact lysosomal functions for presentation at the cell surface, whereas the non-vα14 ligands are normally presented by a tail-truncated CD1d, which is defective in endosomal trafficking and likely presents antigens loaded in the secretory pathway or at the cell surface (27). These CD1d-restricted NKT cells should be distinguished from CD1d-restricted T cells that express noninvariant TCRs and from a variety of other non- CD1d-restricted T cells that express NK lineage receptors (28, 29). Although some studies have recently implicated non-vα14 CD1drestricted T cells in various diseases, this 298 Bendelac Savage Teyton

3 review focuses mainly on the canonical mvα14 and hvα24 NKT cells. SPECIES AND TISSUE DISTRIBUTIN Vα14 NKT cells have been well characterized in mouse, where they represent 0.5% of the T cell population in the blood and peripheral lymph nodes, 2.5% of T cells in the spleen, mesenteric, and pancreatic lymph nodes, and up to 30% of T cells in the liver. Although their precise distribution within the lymphoid organs is still unknown, they reside within the liver sinusoids, which they appear to patrol. Their expression of CXCR6 matches the expression of CXCL16 on the endothelial cells lining the sinusoids and appears to be important for survival rather than for migration (30). NKT cell frequency in the whole thymus is 0.5%, but they represent up to 5% of the recent thymic emigrants found in the spleen (31, 32). Although the tissue distribution is less well studied in humans, Vα24 NKT cells appear to be 10 times less frequent in all these locations. However, high and low NKT cell expressors exist in mice and in humans, and NKT cell frequency appears to be a stable phenotype under the genetic control of at least two recessive loci in mouse (33, 34). Low Vα14 NKT cell expressors in mice include ND and SJL (35 37). The range of frequencies found in human blood varies by up to 100-fold between individuals but is under strict genetic control, as shown by identical twin studies (38). Similar frequencies have been found in nonhuman primates (39). Vα14 NKT cells are present in rats (40, 41), and, based on genomic and functional studies of CD1d, they may be absent in cows (42). NKT LIGANDS Although disputed initially (43), there is now a general consensus that CD1d, like other CD1 family members, evolved to present lipids to T cells (44). However, the nature and the source of the various lipids that bind naturally to CD1d remain poorly elucidated. Early studies of CD1d immunoprecipitates obtained from cell detergent lysates suggested a predominance of phospholipids particularly glycosylphosphatidylinositols, an anchor for various surface proteins, and phosphatidylinositols (45, 46). However, because these early studies used detergents that could potentially displace natural lipids bound to CD1d, or soluble forms of CD1d that did not traffic through the endosome and might have acquired irrelevant lipids from membrane compartments or culture medium, their interpretation is uncertain. Future studies of CD1d molecules engineered to express an enzymatic cleavage site at the membraneproximal portion of their extracellular domain constitute an attractive approach to reexamining this fundamental issue. Despite a lack of direct biochemical studies of CD1-bound lipids, combinations of genetic, cell biological, and chemical approaches have nevertheless uncovered some key NKT ligands discussed below. Marine Sponge αgalcer The first NKT ligand emerged from studies initiated at Kirin Pharmaceuticals to identify natural anticancer medicines. Extracts from Agelas mauritianus, a marine sponge collected in the kinawan sea, prolonged survival of mice bearing B16 melanoma (47). The structure of the active principle was identified as an α-branched galactosylceramide and slightly modified for optimal efficacy to produce a compound termed KRN7000, also commonly referred to as αgalcer (Figure 1) (48). The lipid nature of this compound, its strong effect on liver metastasis, and its activation of dendritic cells (DCs) independent of MHC class I or class II (49) led to the identification of Vα14 NKT cells as their target (21). As a surrogate ligand of very high activity in vitro and in vivo, in the picomolar range αgalcer has been used broadly in Biology of NKT Cells 299

4 a αgalcer (KRN7000) C6" C2' H C1" HN C2C3 C4 b GSL-1 HC H H HN GSL-2 GSL-3 GSL-4 c igb3 H H H H H H H H H H H H NH 2 HC H H H H H H H HN NH 2 HC NH 2 HC H H HN HN 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. 300 Bendelac Savage Teyton

5 various functional assays and to generate the first CD1d tetramers specific for mouse and human NKT cells. The affinity of interaction between CD1d-αGalCer and mouse TCRs is one of the highest ever recorded for natural TCR/ligand pairs with a Kd 100 nm, owing to a slow off rate, for several Vα14-Jα18/Vβ8 combinations examined (50, 51) and may be significantly lower in the human system ( 7 μm) (52). Although the expression of this ligand in marine sponges could not be linked with any physiologically relevant function, the striking properties of αgalcer have provided early support for the hypothesis that the conserved TCRs of NKT cells evolved to recognize conserved lipids. More than 95% of cloned mouse and human NKT cells recognize αgalcer, irrespective of their variable CDR3 β sequence, and the mouse CD1dαGalCer tetramers stain human and nonhuman primate NKT cells as well (22, 39), attesting to the high degree of conservation of this recognition system. Microbial Ligands The lack of physiological relevance of αgalcer should be revisited with the recent discovery that closely related structures that substitute for lipopolysaccharide (LPS) are found in the cell wall of Sphingomonas, a Gram-negative, LPS-negative member of the class of α-proteobacteria (53, 54). These glycosphingolipids are responsible for the strong stimulation of NKT cells and their role in clearing infection (23 25, 55). The most abundant glycosphingolipids have only one sugar, galacturonyl or glucuronyl, α-anomerically branched to the ceramide backbone (Figure 1, GSL-1). Thus, they differ from the stimulating αgalcer or αglccer mainly by the carboxyl group in C6, a position permissive to NKT cell recognition (56, 57). ther more complex but less abundant glycosphingolipids include GSL-2, -3, and -4 (Figure 1). Because in general it is known that extracts from A. mauritianus have different properties depending on seaα-prtebacteria α-proteobacteria constitute one of the most ubiquitous classes of Gram-negative bacteria on Earth. They exhibit a wide range of lifestyles, from free-living to obligate intracellular pathogens, and are found in marine and soil environments. bligate intracellular organisms include the Rickettsiales, with lethal tick-borne pathogens such as Rickettsia and Ehrlichia, agents of the ancient plague epidemic typhus, Rocky Mountain spotted fever, and other severe febrile and typhus-like syndromes. Whereas some of the Rickettsiae express LPS, the Ehrlichiae lack the genes required for LPS and peptidoglycan synthesis, and the composition of its cell wall is mysterious. Mitochondria represent the ultimate example of α-proteobacteria that have established an obligate relationship with eukaryotic hosts. Bartonella and Brucella (an LPS expressor) belong to a group phylogenetically related to the Rickettsiales. Sphingomonas is a ubiquitous bacterium found in marine (e.g., sponges and corals) and terrestrial environments that is actively studied by industrial microbiologists because of its ability to degrade xenobiotic aromatic compounds. Its 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 infections, particularly in immunocompromised individuals. Intriguingly, on the basis of the presence of a specific antibody response in patients sera, it has been implicated in the etiopathogeny of primary biliary cirrhosis, a chronic autoimmune disease targeting intrahepatic bile ducts. son and location and because these sponges are often colonized by α-proteobacterial symbionts, particularly by Sphingomonas (58), the marine sponge αgalcer may in fact have originated from bacterial symbionts. Self Ligand igb3 Although the discovery of bacterial NKT ligands provides a fascinating new perspective on the evolutionarily relevant functions of NKT cells, considerable attention has also focused on self ligands. Indeed, mouse and human NKT cells exhibit conspicuous low-level autoreactivity to various CD1d-expressing cell types (15, 17, 59). This autoreactivity and Biology of NKT Cells 301

6 the presence of IL-12, triggered by Tolllike receptor (TLR) signaling, are required for the commonly observed IFN-γ secretion by NKT cells during immune responses against Gram-negative, LPS-positive bacteria (23, 60). Autoreactivity may also underlie the thymic development of NKT cells (18), which includes an expansion phase after positive selection (31) and the acquisition of a memory phenotype independent of microbial exposure or TLR signaling (61). Recent findings demonstrate that the glycosphingolipid igb3 (Figure 1), both natural and synthetic, could activate a majority of mouse Vα14 and human Vα24 NKT cells, irrespective of their Vβ chain, upon presentation by DCs or plastic-bound CD1d/iGb3 preformed complexes (26, 62, 63). igb3 appears to be a weaker agonist than αgalcer, requiring 30- to 100-fold higher concentrations to achieve the same level of stimulation. This may explain the failure to stain NKT cells using CD1d/iGb3 tetramers. However, solubility issues and more stringent requirements for professional antigenpresenting cells (APCs) may contribute to its lower apparent activity, and the affinity of CD1d/iGb3-TCR interactions remains to be measured directly, particularly to dissect the contribution of on and off rates. Different lines of experiments suggest that igb3 is an important physiological NKT ligand. β-hexosaminidase-b-deficient mice, which lack the ability to degrade igb4 into igb3 in the lysosome, exhibited a 95% decrease in thymic NKT cell production, and β-hexosaminidase-b-deficient thymocytes could not stimulate autoreactive Vα14 NKT cell hybridomas (26). Notably, unlike other mutations of enzymes or transporters involved in lipid metabolism and associated with lipid storage, the defect in β-hexosaminidase-b-deficient cells appeared to be specific in that β-hexosaminidase-bdeficient bone marrow derived DCs normally presented several complex derivatives of αgalcer that required lysosomal processing prior to NKT cell recognition, but lost their ability to process and present igb4 the precursor to igb3 or GalNAcβ1,4GalαCer, both of which require removal of the outer, β-branched hexosamine for NKT cell recognition. In addition, the Griffonia simplicifolia isolectin B4 (IB4) specific for the terminal Galα1,3Gal blocked CD1d-mediated presentation of both exogenous igb3 and endogenous ligand (natural autoreactivity), but not αgalcer. These studies suggest that igb3 is an important physiological ligand of NKT cells. Additional findings reviewed below suggest that igb3 may also be the natural ligand activating NKT cells during Gram-negative, LPS-positive infections. These results are therefore consistent with the requirement for endosomal trafficking of CD1d (27, 64) and the role of lysosomal saposins functioning as glycosphingolipid exchange proteins in the presentation of the NKT ligand in vivo (65, 66). It should be noted, however, that the presence of igb3 among CD1d-bound lipids remains to be demonstrated and that igb3 itself has not yet been directly identified in human or mouse tissue, a task complicated by the rarity of igb3 and the dominance of the regioisomer Gb3. Furthermore, other than the enzymatic pathways of synthesis and degradation, little is known about the general biology of igb3, its subcellular location, or its function. ther NKT Ligands α-galactosyldiacylglycerols expressed by Gram-negative LPS-negative Borrelia burgdorferi, the agent of Lyme disease, resemble α-galactosylceramide and could directly stimulate NKT cells (67). However, recognition of intact or heat-killed bacteria could not be demonstrated, and only one isolated report has suggested defective bacterial clearance in vivo (68). Purified phosphatidylinositolmannoside PIM4, a mycobacterial membrane phospholipid, was reported to elicit IFN-γ but not IL-4 production from a fraction of mouse and human NKT cells, and PIM4-loaded CD1d tetramers showed weak staining of a fraction 302 Bendelac Savage Teyton

7 of NKT cells (69). However, CD1d-deficient mice did not reveal defects in mycobacterial clearance (70), and a synthetic PIM4 failed to stimulate NKT cells (67). Because multiple components of the mycobacterial cell wall are strong activators of TLR expressed by APCs, contaminating lipids associated with the PIM4 preparation may cause indirect stimulation of NKT cells through presentation of their endogenous ligand and amplification of IFN-γ production by TLR-induced IL-12 (see Dual Reactivity to Self and Microbial Ligands: A Paradigm for NKT Cell Activation and Function During Bacterial Infections). Purified phospholipids originally extracted from tumors, such as phosphatidylinositol, phosphatidylethanolamine, and phosphatidylglycerol, weakly stimulated some Vα14 and non-vα14 NKT hybridomas when loaded onto recombinant CD1d, but there is little support at present for their physiological importance because neither the tumor nor the synthetic lipids could expand or activate fresh NKT cells in vivo or in vitro (71). Another report suggested the presence of CD1drestricted phosphatidylethanolamine-specific αβ and γδ T cells in the blood of patients with pollen allergies, although few clones expressed the canonical Vα24 TCR (72, 73). Human melanomas overexpress the ganglioside GD3, and, on the basis of CD1d/ GD3 tetramer staining, immunization with the human melanoma SK-MEL-28 was reported to expand a very small subset of Vα14 NKT cells in mice in vivo (74). These studies, however, did not demonstrate a role for NKT cells in rejection of GD3-overexpressing tumors. Another common glycosphingolipid, β-galactosylceramide, was shown to induce downregulation of NKT cell numbers and TCR surface level in whole spleens examined in vivo and in vitro (75). These effects were relatively modest even at high concentrations of lipids, and a direct stimulation or expansion of cloned NKT cells could not be observed. Because mice lacking β-galactosylceramide (76) also did not exhibit NKT cell defects, the physiological relevance of these observations remains intriguing. In summary, despite some exciting breakthroughs, this difficult and essential area of study is somewhat controversial and remains a work in progress. wing to an array of criteria, including stimulation or staining by recombinant CD1d complexed with synthetic ligands, lack of TLR signaling requirement, stimulation of proliferation and cytokine secretion by large populations of fresh NKT cells in mouse and human, and genetic or functional indications of relevance in vivo during physiological processes and diseases, igb3 and microbial α-glycuronylceramides represent the most compelling NKT ligands identified so far. Their identification considerably reinforces the view that NKT cells and their canonical mvα14-jα18/hvα24-jα18 TCRs evolved to recognize conserved ligands and to perform innate-like rather than adaptive functions. The significance of other reported individual specificities without functional correlates remains uncertain. STRUCTURAL BILGY F GLYCLIPID RECGNITIN Recent reports of the crystal structure of several CD1d/lipid complexes have far-reaching implications. The lipid-binding pocket of CD1d is particularly well adapted to bind self and microbial glycosphingolipids, with the acyl chain in the A hydrophobic pocket and the sphingosin chain in the F hydrophobic channel (77 79). For αgalcer and the closely similar α-glycuronylceramides, the α1 helix Arg79 and Asp80 establish hydrogen bonds with the hydroxyl groups of the sphingosine. The α2 helix Asp153 stabilizes the galactose through hydrogen bonds with the 2 and 3 hydroxyl group, solidly anchoring the protruding sugar in a position parallel to the plan of the α helices and explaining the exquisite stimulatory properties of several hydroxyl groups (Figure 2). Because α-anomeric glycosylceramides do not exist in Biology of NKT Cells 303

8 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 C 8 acyl chain in the A pocket and with the C 18 sphingosine in the F pocket. Note the deeply buried spacer C 16 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 signature of microbial invasion. Notably, CD1d produced in fly cells included a spacer lipid present at the bottom of the A pocket, which preempted the loading of full-length mammalian glycosphingolipid and explained why in general short lipids have proven easier to load onto CD1d in the absence of lipid transfer proteins. However, lipids with long and short (C 8 ) acyl chain produced identical conformations when complexed with CD1d, and they bound the TCR with similar on and off rates (77, 80). CD1d-iGb3 complexes have not yet been reported, but modeling suggests that the β-linked sugar should emerge orthogonal to the plan of the α helices (77), which raises the general issue of how the TCR will recognize two radically different structures and, in particular, accommodate the three protruding sugars. Intriguing insights have come from a report that the human Vα24/Vβ11 TCR displays an unusual cavity between the CDR3 α and β loops (81), suggesting an unusual mode of recognition of the trisaccharide within this TCR cavity. Future crystallographic studies of CD1d-iGb3 and ternary complexes with the TCR should clarify these fundamental issues and illuminate novel aspects of carbohydrate recognition by immune receptors. CELL BILGY F LIPID PRESENTATIN BY CD1d CD1d is prominently and constitutively expressed by APCs such as DCs, macrophages, and B cells (82, 83), particularly marginal zone B cells (82), with relatively modest changes associated with TLR activation and inflammatory cytokines (84). CD1d is also strikingly expressed on cortical thymocytes, where it is essential for NKT cell development (18), and on Kupffer cells and endothelial cells lining liver sinusoids, where the highest frequencies of NKT cells are found in mice (30). Hepatocytes express CD1d constitutively in 304 Bendelac Savage Teyton

9 mouse and upon disease induction in human, for example, in the context of hepatitis C (85). CD1d expression in the liver is not required, however, for NKT cell homing (86), and neither is CXCR6 expression by NKT cells, although CXCR6/CXCL16 interactions are essential for survival in this organ (30). CD1d is upregulated on microglial cells during inflammation (87). Similar to the MHC class II system, most other solid tissue cells and non-antigen-presenting hematopoietic cells express low or undetectable levels of CD1d. Trafficking of CD1d The intracellular trafficking of CD1d has been studied thoroughly (Figure 3). Biosynthesis of the heavy chain associated with β2-microglobulin involves the endoplasmic reticulum chaperones calnexin and calreticulin and the thiol oxidoreductase ERp57 (88). It is logical to assume that endogenous lipids in the endoplasmic reticulum would fill the groove of CD1d, and one study suggested the presence of phosphatidylinositol (45), with the caveat that contamination by membrane phospholipids could not be formally excluded. CD1d rapidly reaches the plasma membrane within 30 min after biosynthesis and undergoes extensive internalization and recycling between the plasma membrane and endosomal/lysosomal compartments in a manner dependent upon a tyrosine motif encoded in the CD1d cytoplasmic tail (89 91). The tyrosine motif in the cytoplasmic tail primarily binds adaptor protein (AP)-2 and AP-3 in mouse (92, 93), where the bulk of CD1d accumulates in the lysosome, and AP-2 in humans, where CD1d tends to reside in the late endosome (94). Additional but largely redundant contributions by the invariant chain or invariant chain/mhc class II complexes that bind weakly to CD1d have been documented in mouse and human (89, 90). The CD1d intracytoplasmic tail also expresses a lysine targeted for ubiquitination by the MIR proteins of the Kaposi sarcoma associated herpes virus, causing downregulation from the cell surface without degradation (95). Interestingly, another herpes virus, herpes simplex virus-1 (HSV-1), induces CD1d downregulation from the cell surface, but the mechanism appears to be distinct, involving lysosomal retention through impaired recycling to the plasma membrane (96). Intersection of CD1d and Lipids in Late Endosome and Lysosome Tail-truncated CD1d molecules fail to access the late endosome and lysosome, causing a profound disruption of CD1d-mediated antigen presentation in vitro in cell lines and in vivo in knockin mice. Particularly affected are the presentation of the NKT endogenous ligand (27) and, consequently, the thymic generation of Vα14 NKT cells (64). The presentation of diglycosylated αgalcer variants requiring processing prior to NKT cell recognition, an important tool for research (56), or of igb4, which requires processing into igb3 prior to recognition, is also abolished (26). However, other lipids that do not require processing still exhibit variable requirements for the late endosome and lysosome trafficking of CD1d, either partial in the case αgalcer (three- to fivefold shift in dose response) or substantial in the case of igb3 (>10-fold shift). Recent studies of lipid uptake, trafficking, and loading have begun to shed some light on these observations. Lipid Uptake and Trafficking Lipids in the circulating blood or in culture medium are bound to lipoproteins, and a dominant role for VLDL in the serum and its receptor, the LDL receptor, at the cell surface has been proposed for the clathrin-mediated uptake of some lipids into endosomal compartments (Figure 3) (97). ther extracellular lipids can be captured by the mannose receptor langerin (98, 99) or can insert themselves directly in the outer leaflet of the Biology of NKT Cells 305

10 igb3 Vα14 TCR Exogenous lipid Vα14 TCR Exogenous lipid VLDL βhexb AP-2/AP-3 CD1d β2-m MTP? Figure 3 igb3 Golgi igb4 ER Saposins Late endosome/lysosome Phagosome LDLR 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 through clathrin-dependent or -independent pathways (100). Glycosphingolipids tagged with a fluorochrome, BDIPY, on the acyl chain reached the late endosome and were rapidly sorted to the endoplasmic reticulum and the Golgi. In contrast, a prodan-conjugated (on carbohydrate C6 ) αgalcer accumulated selectively in the lysosome (102). These 306 Bendelac Savage Teyton

11 pathways overlap only partially with those governing the trafficking of endogenous glycosphingolipids, which are synthesized in the lumenal part of the Golgi and thought to reach the plasma membrane first, then the endosome, through clathrin-dependent and -independent endocytosis until they are degraded in the lysosome (103). How exogenously administered or endogenous intracellular lipids choose between these pathways and the consequence for antigen presentation are questions that are just beginning to be addressed and may depend on intrinsic properties such as length or insaturation of alkyl chains (104), composition of the polar head, and solubility in aqueous environments, as well as extrinsic variations in the mode of administration such as use of detergents, liposomes, or lipid-protein complexes. The development of new methodologies, genetic manipulation, and reagents will be required to address these essential questions. In addition, recognition of microbial lipids in the context of infection most likely involves different pathways because the uptake of bacteria is governed by different sets of cell surface receptors and the release of cell wall lipids would occur through degradation of the microorganism in the lysosome before processing and loading onto CD1d. Lipid Exchange Proteins Although an intrinsic, ph-dependent mechanism appears to favor the acquisition of some lipids by CD1 proteins, perhaps through a conformational change (105, 106), lipid exchange now appears to be regulated by specialized lipid transfer proteins. By using various detergents, early studies of lipid binding to CD1 molecules tacitly dealt with the fact that in general lipids are insoluble in water, forming micelles that cannot transfer monomeric lipids onto CD1. These detergents, however, also tended to dislodge lipids bound to CD1, as shown directly in the crystal structure of CD1b complexed with phosphatidylinositol, where two molecules of detergent cohabited with the lipid in the groove (107). In contrast, during biological processes, membrane lipids are extracted and transported by lipid exchange proteins (108). Prosaposin is a protein precursor to four individual saposins, A, B, C, and D, released by proteolytic cleavage in the lysosome. Prosaposin-deficient mice provided the first genetic link between NKT cells and lipid metabolism, as they lacked NKT cells and exhibited greatly impaired ability to present various endogenous and exogenous NKT ligands (65, 66). In cell-free assays, recombinant saposins readily mediated lipid exchange between liposomes and CD1d in a nonenzymatic process requiring equimolar concentrations of CD1d and saposins (65). Although they exhibited some overlap in lipid specificity, individual saposins differed in their ability to load particular lipids. More detailed studies of the effects of these and other lipid exchange proteins such as NPC2 and the GM2 activator are required to understand their function individually or cooperatively at different phases of lipid processing and loading. In addition, the structural basis of the lipid exchange mechanism and its relative specificity for lipid subsets remain to be elucidated. Another lipid transfer protein expressed in the endoplasmic reticulum, microsomal triglyceride transfer protein (MTP), assists in the folding of apolipoprotein B by loading lipids during biosynthesis. Coprecipitation of MTP with CD1d suggested that MTP might play a similar role for CD1 molecules (109). Indeed, genetic or drug-induced inhibition of MTP was associated with defects in lipid antigen presentation (109, 110). MTP was suggested to transfer phosphatidylethanolamine onto CD1d in a cell-free assay, but the efficiency of this process remains to be established, and cell biological studies are required in vivo to fully understand the role of MTP in CD1d-mediated lipid presentation. CD1e is a member of the human CD1 family that is not expressed at the plasma membrane but is instead found as a cleaved soluble protein in the lysosome. Recent experiments Biology of NKT Cells 307

12 have shown that CD1e could assist the enzymatic degradation of phosphatidylinositolmannoside, suggesting that this protein may have diverged from other CD1 molecules to perform ancillary functions rather than to carry out direct antigen presentation (111). Membrane Transporters NPC1 is a complex membrane multispan protein present in the late endosome that is mutated in Niemann-Pick type C1 disease and associated with a lipid storage phenotype similar to NPC2, a soluble lipid transfer protein present in the lysosome. NPC1-mutant mice exhibited broad defects of NKT cell development and CD1d-mediated lipid presentation, which could be attributed in part to an arrest of lipid transport from late endosome to lysosome (102). The precise function of NPC1 remains unknown, and it is unclear how this putative flippase translocating lipid between leaflets of the membrane bilayer could induce general alterations of lipid trafficking. ther Glycosidases and Lipid Storage Diseases Mutations of several proteins involved in glycosphingolipid degradation or transport are accompanied by lipid storage within distended lysosomal vesicles, the impact of which depends on the enzyme, the cell type, the mouse strain, and the age at which cells are examined (100, 101). This lipid accumulation may disrupt rate-limiting steps of lipid metabolism and indirectly alter CD1- mediated lipid antigen presentation through defective lipid trafficking or lipid competition for loading CD1d. For example, while NPC1-mutant cells showed a block in lipid transport from late endosome to lysosome, this block could be partially reversed by inhibitors of glycosphingolipid synthesis such as N-butyldeoxygalactonojirimycin, presumably through alleviation of the lipid overload (102). Bone marrow derived DCs from mice lacking β-hexosaminidase B, α-galactosidase A, or galactosylceramidase did not show much alteration of general lipid functions because they conserved their ability to process several complex diglycosylated derivatives of αgalcer for presentation to NKT cells (26, 56, 65), although a divergent report was recently published (101). In contrast, β-galactosidase-deficient cells exhibited more general defects than expected from the specificity of the mutated enzyme ( J. Mattner and A. Bendelac, unpublished data, and Reference 101). Cathepsins Paradoxically, studies of cathepsin-mutant mice led to the first reports of defects in NKT cell development and CD1d-mediated lipid antigen presentation. This is particularly well established for cathepsin L because mutant thymocytes, but not DCs (perhaps owing to the redundancy of other cathepsins), failed to stimulate Vα14 NKT hybridomas in vitro and consequently failed to select NKT cells in vivo (112). Although its target remains to be identified, cathepsin L may be directly or indirectly required for thymocytes to process prosaposin into saposins. NKT CELL DEVELPMENT Based on their canonical TCR receptors and antigenic specificities, their unusual expression of NK lineage markers, their peculiar tissue distribution, and their functional properties independent of environmental exposure to microbes, NKT cells constitute a separate lineage. Two models that explained the basis of the NKT cell lineage were initially opposed. ne model suggested that NKT cells originated from precursors committed prior to TCR expression (committed precursor model), whereas the other model proposed that the lineage was instructed after TCR expression and interaction with NKT ligands (TCR instructive model). The first model was based on a report suggesting the presence 308 Bendelac Savage Teyton

13 of cells expressing the canonical Vα14 TCR at day 9.5 of gestation (113), well before a thymus was formed, but these data have not been reproduced with the new, more specific CD1d tetramer reagents. Instead, the TCR instructive model is now widely accepted on the basis of the finding that although canonical Vα14-Jα18 rearrangements are rare and stochastic (114), once expressed (e.g., in TCR transgenic mice), an NKT TCR will induce the full NKT cell lineage differentiation (115, 116). Developmental Stages The production of CD1d-αGalCer tetramers specific for the canonical Vα14 TCRs ( ) has transformed this area of study by allowing the identification of developmental steps independently of the expression of NK1.1 (Figure 4). The first detectable stages have a CD24 high cortical phenotype and include a CD4 intermediate CD8 intermediate (double-positive, DP dull ) stage, followed by a CD4 high CD8 neg stage. These developmental intermediates immediately follow positive selection, as they express CD69 and are not found in the CD1d-deficient thymus, but they are present at extremely low frequencies ( 10 6 ) (120). The preselection DP, observed easily in Vα14-Jα18 TCRα-chain transgenic mice (115), still escape tetramer detection in wild-type mice owing to the rarity of stochastic Vα14-Jα18 rearrangements and the low TCR level at this stage. Investigators have attempted intrathymic transfer of purified DP cells to demonstrate the presence of NKT cell precursors, but, given the size of the inoculum (10 7 DP cells), these experiments could not formally rule out that rare DN contaminants gave rise to the NKT cell product (121). Interestingly, in mice lacking RRγt a transcription factor induced in DP thymocytes that is essential for prolonged survival until distal Vα to Jα rearrangements (such as Vα14 to Jα18) can proceed NKT cell development was interrupted (122, 123). As cells progress to the mature CD24 low stage, three more stages are described: first a CD44 low NK1.1 neg stage (naive), then a CD44 high NK1.1 neg (memory) stage, and finally a CD44 high NK1.1 pos (NK) stage (31, 124). This sequence is characteristically accompanied by a massive cellular expansion occurring between the CD44 low NK1.1 neg stage and the CD44 high NK1.1 neg stage (125). This expansion phase following positive selection and leading to the acquisition of a memory phenotype is in line with the innate role of NKT cells, which requires high copy number and effector/memory properties for prompt and effective responses, but it represents a key difference between the development of NKT cells and that of conventional T cells. Furthermore, during these stages a DN population arises by downregulation of CD4 in 30% 50% of the cells, as shown in cell transfer experiments (120), and by genetic fate mapping with RSA26R reporter mice crossed to CD4-cre deleter mice (123). DN cells exhibit some functional differences with CD4 cells, which are more pronounced in human than in mouse ( ), and tend to be more of the Th1 phenotype. The factors determining this sublineage remain unclear, as DN cells appear to share the same TCR repertoire as the CD4 subset. A majority of the CD44 high NK1.1 neg cells emigrate to peripheral tissues, where they stop proliferating and rapidly express NK1.1, a NK marker available in the C57BL/6 background, followed by other NK lineage receptors such as NKG2D, CD94/NKG2A, Ly49A, C/I, and G2 (31, 32, 124). Thymic emigration assays using intrathymic injection of fluorescein isothiocyanate have revealed that up to 5% of recent thymic emigrants to the spleen, representing cells, are CD44 high NK1.1 neg NKT cells and rapidly acquire NK1.1 to join the nondividing long-lived NK1.1 + pool of cells (31, 32). Interestingly, a fraction of the CD44 high NK1.1 neg cells do not emigrate and instead proceed to terminal maturation (CD44 high NK1.1 pos ) inside the thymus, where they become long-lived resident cells, Biology of NKT Cells 309

14 DN DN Vα14-Jα18 RRγt DP TCR/iGb3/CD1d DP Cortical thymocytes SLAM/SLAM? SAP/Fyn Bcl-xL DC EC CD4 CD8 CD4 CD4 CD4 DN CD4 DN Emigrant CD24 hi CD69 hi CD24 lo CD44 lo NK1.1 IL-4 CD4 CD24 lo CD44 hi NK1.1 IL-4 IFN-γ MHC I MHC II CD1d TCR CD4 DN Resident PKCθ Bcl-10 NF-κB T-bet IL-15Rβ CD44 hi NK1.1 + IL-4 IFN-γ 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 RRγt, 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

15 a peculiar fate of uncertain significance in the mouse thymus (32) that may be absent in the human thymus (129). These developmental stages are associated with sharply defined functional changes. Thus, the CD44 low NK1.1 neg cells are exclusive IL-4 producers upon TCR stimulation in vitro, whereas the CD44 high NK1.1 neg cells produce both IL-4 and IFN-γ and the CD44 high NK1.1 pos cells produce more IFN-γ than IL-4 (31, 124). This is reflected faithfully in the spontaneous expression of high levels of GFP (green fluorescent protein) by the CD44 low NK1.1 neg and CD44 high NK1.1 neg cells of IL-4-GFP 4get knockin mice, and in the expression of high levels of YFP (yellow fluorescent protein) by the CD44 high NK1.1 pos cells of IFN-γ-YFP Yeti knockins, which reflect open chromatin in the corresponding cytokine loci (130). Because a panoply of NK receptors is expressed with kinetics and frequencies similar to those of NK cells, components of a general NK lineage program are likely activated. Interestingly, however, the extent and profile of NK receptor expression vary in different tissues, with thymic NKT cells expressing a repertoire similar to that of splenic NK cells and spleen and liver NKT cells expressing these receptors at lower frequencies (131). Whether these differences reflect different stages of differentiation or an environmental influence on the acquisition or selection of the NK receptor repertoire is not clear. Note that, despite their potential to regulate TCR signaling thresholds to antigen (132), including natural ligand (133), the functions of NK receptors remain to be elucidated in a physiological context. Contribution of T Cell Receptor Vβ Chains to Natural Ligand Recognition TCR Vβ-Dβ-Jβ rearrangements occur at the DN3 stage to produce a TCRβ chain that pairs with the pre-tα to form a receptor that induces cellular expansion, allelic exclusion at the β locus, and transition to the DP stage, where rearrangements are initiated at the TCRα locus. NKT cell precursors follow the same pre-tα path as mainstream T cells (120, 134). Therefore, the question arises whether the biased usage of Vβ8, Vβ7, and Vβ2 in mouse (and Vβ11 in human) is due to the inability of the Vα14-Jα18 TCRα chain to pair with the other Vβs or whether it is due to positive or negative selection. Premature expression of a Vα14-Jα18 TCRα transgene at the DN3 stage created a population of thymocytes with a broad Vβ repertoire, ruling out a Vβ pairing issue (135). f these transgenic cells, however, only those expressing the biased Vβ set responded to igb3, whereas a broader set of Vβs responded to αgalcer, demonstrating that the Vβ bias is imparted by selection events. Furthermore, Vβ7 cells responded to the lowest concentrations of igb3, in agreement with several observations that Vβ7 + NKT cells are relatively diminished upon CD1d overexpression (consistent with negative selection) and increased upon CD1d underexpression (consistent with decreased positive selection of the lower affinity Vβ8 and Vβ2) (62, 136, 137). Vβ7 cells were also preferentially expanded in a fetal thymic organ culture system after exposure to exogenous igb3 (62). Because the Vβ7 > Vβ8 > Vβ2 affinity hierarchy of these Vβs precisely reflects their respective degree of enrichment during thymic selection, the Vβ repertoire of NKT cells appears to be shaped mainly by positive selection, with little contribution from pairing bias or negative selection in natural conditions. However, NK lineage T cells are not inherently resistant to negative selection, as they tend to disappear in conditions of increased signaling (136, 138, 139). Cellular Interactions In contrast with MHC class I molecules, mouse and human CD1d are induced at the DP stage and downregulated at the single-positive (SP) stage (82). This expression pattern explains why cortical thymocytes Biology of NKT Cells 311

16 represent the thymic cell type, where CD1d expression is necessary and sufficient for NKT cell selection and lineage differentiation. Thus, NKT cells were absent in chimeric mice lacking CD1d expression in the DP compartment (140). Conversely, in plck-cd1d transgenic and chimeric mouse models where CD1d was exclusively expressed on cortical thymocytes, NKT cells developed nearly normally and notably preserved their effector properties, with the exception of a relative decrease in NK receptor expression and some hyperreactivity to TCR stimulation (86, 139). CD1d is also found on thymic CD11b + macrophages, CD11c + DCs, and epithelial cells (86), but this expression appeared to play only an auxiliary role in NKT cell development, as shown by the normalization of NK receptor expression and TCR hyperreactivity upon crossing plck-cd1d to Eα (MHC class II)-CD1d mice. Interestingly, in another Lck-CD1d transgenic model in which CD1d was expressed at a high level on peripheral T cells, NKT cells appeared to be hyporesponsive, and liver disease was observed (141). Intrathymic transfer experiments and thymic graft experiments further revealed that the acquisition of NK1.1 by CD44 high NK1.1 neg NKT cells was decreased, but not arrested, in the absence of CD1d in the thymus or the periphery, although life span and effector functions were relatively preserved (32). These observations suggest that interactions with CD1d ligands expressed by cell types other than DP occur throughout NKT cell development in the thymus and the periphery, consistent with the autoreactivity of the Vα14 TCR, and, although not absolutely required, they nevertheless promote terminal NKT cell differentiation. Molecular Interactions and Signaling The above studies imply that an understanding of the NKT cell lineage commitment revolves around the signaling events imparted to NKT cell precursors during their TCR engagement by CD1d-expressing cortical thymocytes. This signaling is expected to differ from that of conventional T cells for at least two reasons. ne is that the natural ligand is an agonist that would normally induce negative selection in the mainstream lineage. This is illustrated directly by the autoreactive IL-2 response of NKT hybridomas to DP thymocytes (18) and by the proliferative and cytokine response of fresh NKT cells to synthetic igb3 (26). The other reason is that the developing NKT cell precursors interact with cortical DP thymocytes rather than with epithelial cells, implying that homotypic rather than heterotypic cellular contacts are involved and therefore recruit accessory receptors or factors that elicit different signaling pathways. In this context, the reports that Fyn knockout (142, 143) and SLAM-associated protein (SAP) knockout ( ) mice lacked NKT cells have attracted considerable attention because the Src kinase FynT was recently shown to signal downstream of the SLAM family of homotypic interaction receptors through SAP ( ). Several members of this family (151) are expressed on cortical thymocytes, reinforcing the hypothesis of homotypic interactions signaling through SAP and FynT during TCR recognition of CD1d ligands on cortical thymocytes. Whether and which of these SLAM family members are involved are under investigation. In addition, the stages at which these interactions might influence NKT cell development and differentiation remain to be defined. Notably, the report that a Vα14-Jα18 TCRα transgene corrected the Fyn knockout associated defect implied that this stage would precede TCRα expression (152), although interpretation of TCR transgenic results should be careful given the description of transgenic lineage artifacts (115, 135). Indeed, more recent studies in our laboratory indicate that this correction is partial and due to the leaky phenotype of the Fyn knockout because the SAP knockout was not reconstituted (K. Griewank and A. Bendelac, unpublished results). 312 Bendelac Savage Teyton

17 The emerging scenario, therefore, is that homotypic interactions between SLAM family members initiated in the cortex during Vα14 TCR engagement by CD1d/iGb3- expressing cortical thymocytes lead to FynT signaling after SAP recruitment to the cytosolic tyrosine motifs of SLAM family members (153). FynT signaling can activate the canonical NF-κB pathway and may account, in conjunction with TCR signaling, for the well-established requirement of this pathway in NKT cell development (Figure 4). Indeed, mice expressing a dominant-negative IκBα transgene and those lacking NFκBp50 exhibited developmental arrest at the CD44 high NK1.1 neg stage, which was partially rescued by a Bcl-xL transgene, suggesting a survival role for NF-κB (154, 155). The precise connections between TCR, FynT, and NF-κB remain to be elucidated. PKCθ and Bcl-10 have been implicated in the signaling pathways of both FynT and the TCR leading to NF-κB activation (156), and their ablation impaired NKT cell development (157, 158), although the NKT cell defects were relatively modest. FynT has also been connected to the Ras-GTPase-activating protein Ras-GAP through the Dok1/2 adaptor proteins (149, 159), suggesting that signals emanating from SLAM family members may regulate signaling downstream of the TCR to avoid negative selection through Ras while promoting survival through NF-κB. The molecular regulation of the NK program activated between CD44 high NK1.1 neg and CD44 high NK1.1 pos cells remains enigmatic. The transcription factor T-bet induces expression of the IL-2Rβ component of the IL-15 receptor, which is important for the survival of CD44 high NK1.1 neg and terminally differentiated CD44 high NK1.1 pos cells ( ). However, the range of functions of T-bet and its homolog eomesodermin in this developmental pathway, particularly with respect to the induction of the NK differentiation program, remains to be investigated. Recent studies have suggested that Tec family kinases Itk and Rlk play a central role in regulating the decision between conventional and NKT cell like lineages. Thus, conventional CD8 T cells lacking these kinases upregulated eomesodermin and the IL-15 receptor and turned into NKT cell like cells that required ligand on bone marrow derived rather than epithelial cells (163, 164). Interestingly, mice expressing MHC class II molecules on thymocytes through transgenic expression of the transcription factor CIITA selected an unusual population of CD4 T cells resembling NKT cells by their expression of a memory phenotype (165). Additional NKT cell precursor-intrinsic factors regulate NKT cell development. For example, mice lacking Runx1 (123) or Dock2 (166) or mice overexpressing BATF, a basic leucine zipper transcription factor and an AP-1 inhibitor, exhibited severe defects early in NKT cell development (167, 168). Although NKT cells interact with cortical thymocytes rather than epithelial cells for TCR/ligand and SLAM family interactions, mice carrying defective components of the alternative NK-κB pathway, such as NIK or Rel-B, in their thymic stroma exhibit severe and early disruption of NKT cell development (155, 169). Because these mutations also induce profound abnormalities of the thymus architecture, thymic lymphocyte emigration, and thymic DCs, there may be multiple causes of the NKT cell defects (170). Lymphotoxin α1β2 (expressed on thymocytes) signaling through the lymphotoxin β receptor (expressed on stromal cells) can activate this alternative pathway, but only modest NKT cell defects have been reported in the corresponding mutant mice ( ). Finally, GM-CSF was reported to control the effector differentiation of NKT cells during development by a mechanism that renders them competent for cytokine secretion (174). NKT CELL FUNCTINS NKT cells have been implicated in a broad array of disease conditions ranging Biology of NKT Cells 313

18 from transplant to tumors, various forms of autoimmunity, atherosclerosis, allergy, and infections. NKT Cell Activation by Administration of Ligand In Vivo The central concept underlying nearly all NKT cell functions is the recognition by the whole NKT cell population of endogenous ligands such as igb3 (autoreactivity) or of microbial cell wall glycolipids such as α-glycuronylceramides. Several studies have NK CXCL16 IFN-γ CXCR6 B NKT characterized a cascade of activation events following the exogenous administration of NKT ligands such as αgalcer (Figure 5). The central feature is a reciprocal activation of NKT cells and DCs, which is initiated upon the presentation of αgalcer by resting DCs to NKT cells, inducing NKT cells to upregulate CD40L and Th1 and Th2 cytokines and chemokines; CD40 cross-linking induces DCs to upregulate CD40, B7.1 and B7.2, and IL-12, which in turn enhances NKT cell activation and cytokine production (175, 176). Propagation of this reaction IL-4, IL-13 MΦ CD40L Vα14 Jα18 CD40 IL-12 CD1d: lipid EC IFN-γ DC Liver sinusoid CD8 killer CD4 helper 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. 314 Bendelac Savage Teyton

19 involves the activation of NK cell cytolysis and IFN-γ production (177, 178) and, most importantly, the upregulation of DC costimulatory properties and MHC class I and MHC class II mediated antigen presentation, particularly cross-priming, which serves as a bridge to prime robust adaptive immune responses ( ). Importantly, TLR signaling is not involved in these responses. Thus, αgalcer and related variants are being actively investigated for their ability to serve as vaccine adjuvants alone or in conjunction with synergistic TLR ligands (182). In addition, the immunomodulatory properties of repeated injection of NKT ligands may be exploited to treat or prevent immunological diseases (183). Mature NKT cells produce massive amounts of IFN-γ, but they are unique among lymphocytes for their ability to explosively release IL-4 (184), in addition to other key Th2 cytokines such as IL-13. The Th1 versus Th2 outcome of their activation is partially understood. Systemic injection of the original αgalcer compound induces an early burst of IL-4 detected in the serum, followed 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, 186). However, NKT cells also undergo a rapid downregulation of their TCR, followed by massive apoptosis within 3 4 days of activation, resulting in a long-lasting depletion until regeneration occurs in part from thymic precursors ( ). More sustained and efficient responses have been described upon injection of αgalcer-pulsed DCs, particularly with respect to the production of IFN-γ, resulting in a superior adjuvant effect for the priming of cytotoxic T lymphocytes (CTL) (190, 191). Interestingly, some variants of the original αgalcer KRN7000 have shown decreased Th1 compared to Th2 cytokine induction. These Th2 variants have shorter or insaturated lipid chains (185, 192, 193). The mechanisms underlying these differences are debated and may be diverse. ki et al. (186) proposed that the lipid with shorter sphingosin CH failed to engage the TCR for a long enough period of time to induce IFN-γ. n the other hand, plasmon resonance determinations of TCR on and off rates, and even crystal structures of the long (KRN7000) and acyl shortened (PBS25, C 8 acyl chain) version of αgalcer bound to CD1d have shown no significant differences (77). An alternative hypothesis is based on the observation that different NKT ligands preferentially reach different cell types upon injection in vivo, suggesting that increased Th1 responses may result from the predominant uptake of lipid by IL-12-secreting cell types such as DCs (77, 194). Perhaps of relevance to this issue is the fact that all Th2 ligands described so far have increased solubility in water owing to their shorter lipid tail or the presence of insaturations. This property could modify their routes of trafficking and uptake, favoring presentation by non-il-12-producing cells, such as B cells. Finally, mucosal rather than systemic modes of administration may also modify the Th1/Th2 output of NKT cells owing to a preexisting bias in the cytokine environment. Dual Reactivity to Self and Microbial Ligands: A Paradigm for NKT Cell Activation and Function During Bacterial Infections Glycosphingolipids closely related to αgalcer were reported in the cell wall of Sphingomonas (53, 54), a prominent Gram-negative, LPS-negative member of an abundant class of bacteria on Earth, α-proteobacteria (Figure 6). Sphingomonas is a ubiquitous bacterium whose cell wall glycosphingolipids include the dominant α-branched glucuronyl and galacturonyl ceramides (GSL-1) and the less abundant di- (GSL-2), tri- (GSL-3), and tetra- (GSL-4) glycosylated species shown in Figure 1. Although these glycosphingolipids form structures reminiscent of LPS (Figure 6), their synthesis pathway and role in the microbial cell wall are not well understood. Biology of NKT Cells 315

20 E. coli LPS Porin Lipid A Figure 6 uter membrane of the cell walls of Sphingomonas and Escherichia coli. The inner leaflet of the outer membrane is composed of phospholipids, whereas the outer leaflet is made of LPS for E. coli. In the case of Sphingomonas, glycosphingolipids containing between one and four carbohydrates substitute for LPS. Note the thin layer of peptidoglycan separating the inner and outer membranes in both cell walls. Cytoplasm Sphingomonas Cytoplasm uter membrane Membrane proteins Peptidoglycan Inner membrane Glycosphingolipids uter membrane Membrane proteins Peptidoglycan Inner membrane GSL-1 activates large proportions of mouse and human NKT cells (23 25, 55), but it is unclear at present whether the more complex GSL-2, -3, and -4 can be recognized by NKT cells or even whether they can be processed efficiently into GSL-1 by host APCs. During infection, Sphingomonas is phagocytosed by macrophages and DCs and elicits an activation cascade similar to exogenous αgalcer. NKT cell activation enhances microbial clearance by 15- to 1000-fold within the first 2 3 days of infection (23, 24). Sphingomonas can also induce DC activation through TLR-mediated signaling, but this direct effect is weak relative to the crossactivation of DCs by NKT cells because peptidoglycan and bacterial DNA are relatively weak stimulants. High doses of Sphingomonas induce a lethal toxic shock similar to the one associated with Gram-negative, LPS-positive bacteria. However, in the case of Sphingomonas, NKT cell deficient mice are protected. These striking observations have led to the hypothesis that NKT cells and their canonical TCR specificity evolved to meet the challenges of these Gram-negative, LPSnegative bacteria. Although Sphingomonas is a promiscuous bacterium that can cause severe infection, particularly in immunocompromised hosts, other more deadly members of the class of α-proteobacteria may have provided stronger evolutionary pressures on the NKT cell system. Particularly interesting is the case of Ehrlichia, a tick-borne 316 Bendelac Savage Teyton

21 pathogen and member of the Rickettsiales that is of widespread significance for mammals, including wild and domesticated ruminants, dogs, and humans from some regions of the world such as Africa and East Asia. Ehrlichia muris activates NKT cells independently of igb3, and its clearance was profoundly altered in CD1d- or Jα18-deficient animals (23). Ehrlichia is a Gram-negative, LPS-negative obligate intracellular bacterium, whose cell wall composition has not been elucidated. Interestingly, many other bacteria, particularly the Gram-negative, LPS-positive ones, can activate NKT cells. However, rather than provide their own NKT ligands like Sphingomonas or Ehrlichia, these bacteria appear to trigger autoreactive NKT cell responses (23, 60). In the case of Salmonella, this is suggested by the abrogation of NKT cell activation in the presence of DCs lacking β-hexosaminidase B, the enzyme responsible for the generation of igb3 from igb4 in the Direct microbial recognition Gram-negative, LPS-negative bacteria lysosome, and by blocking experiments with the lectin Griffonia simplicifolia IB4, which recognizes the terminal sugar of the Galα1-3Gal epitope of igb3 bound to CD1d and blocks NKT cell activation (23). Strikingly, NKT cell activation by Gram-negative, LPSpositive Salmonella is absolutely dependent upon TLR signaling through the adaptors MyD88 and Trif, and upon IL-12 release by the APC, although the precise TLR combination and the corresponding microbial structures involved remain to be determined. Thus, the proposed scenario suggests that TLR signaling leading, but not limited, to IL-12 secretion enhances the ability of DCs to stimulate NKT cells through presentation of endogenous ligands (Figure 7). Whether TLR signaling induces an upregulation of igb3 or changes in the expression of other factors such as, for example, NK receptor ligands is unclear. Contrary to an early report (195), NKT cells do not usually NKT cell Bacterial Ag igb3 IL-12p40 TLR4 Indirect microbial recognition LPS Gram-negative bacteria? Bacterial Ag igb3 Figure 7 Late endosome/lysosome Dual recognition of self and microbial glycosphingolipids during microbial infections. n the left, infection by Gram-negative, LPS-negative Sphingomonas induces direct activation of NKT cells through recognition of microbial cell wall α-glycuronylceramide. n 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

22 constitute the predominant cell type that produces IFN-γ in response to IL-12 in vivo (60, 196). This explains why they generally do not appear to be essential in fighting Gram-negative, LPS-positive bacteria. However, an impact on bacterial clearance has been observed in the case of lung infection with Pseudomonas aeruginosa, where CD1ddeficient mice exhibited a 20-fold increased bacterial count in the lung within 6 24 h postinoculation and an approximately threefold decrease in MIP-2 and neutrophils in the bronchoalveolar lavage (197). This may not be the case at other sites of infection (198). Variations have been noted as well in reports assessing the role of NKT cells versus NK cells in LPS-induced toxic shock in vivo (199, 200). Primary Biliary Cirrhosis and Sphingomonas An intriguing connection between primary biliary cirrhosis (PBC), Sphingomonas, and NKT cells has emerged recently. PBC is a disease characterized by the presence of antimitochondrial antibodies, liver lymphocytic infiltrates, and the chronic destruction of the biliary epithelium, which leads to cirrhosis (201). Interestingly, the autoantibodies recognize an epitope of the mitochondrial PDC- E2 enzyme that is particularly well conserved in Novosphingobium aromaticivorans, a strain of Sphingomonas. Furthermore, PBC patients, including those lacking antimitochondrial antibodies, were specifically seropositive against Sphingomonas, which was detected by PCR in stool samples of 25% of diseased or healthy individuals, suggesting that PBC may be induced by aberrant host reactivity to this bacterium (202). PBC patients also showed an enrichment of Vα24 NKT cells in liver biopsies, but a depletion in blood (203). In light of the recent finding that Sphingomonas cell wall glycolipids specifically activate NKT cells, these studies suggest that NKT cells may play a key role in the pathogeny of PBC by promoting aberrant responses to Sphingomonas. Parasitic Infections Shofield and colleagues (204) suggested that the production of IgG antibodies to the malaria circumsporozoite antigen, a key component of protective immune responses in humans, depended on NKT cell recognition of malarial glycosylphosphatidylinositol antigens in a mouse model. However, additional experiments failed to detect a CD1d-dependent component to this antibody response, and glycosylphosphatidylinositols have not been identified as NKT cell antigens in other reports (205, 206). In the context of helminth infection, DCs pulsed with Schistosoma mansoni eggs activated NKT cells to secrete Th1 and Th2 cytokines in vitro in a β-hexosaminidase-b-dependent but MyD88- independent manner, suggesting recognition of the self ligand igb3 in the absence of TLR signaling (207). Viral Infections Relatively modest defects in the clearance of some viruses have been reported in CD1ddeficient mice infected with encephalomyocarditis virus (208) or coxsackie B3 (209), but these defects were not observed in Jα18- deficient mice, ruling out a specific role of Vα14 NKT cells. Infections with lymphocytic choriomeningitis virus, mouse cytomegalovirus, vaccinia virus, and coronavirus were unaffected. Studies in humans have suggested a profibrotic role of Vα24 NKT cells in hepatitis C (85) and the accumulation of non-vα24 CD1d-restricted T cells (210). Although a specific role of Vα14 NKT cells in HSV infection remains controversial (211, 212), recent studies have suggested that viral invasion may be associated with countermeasures against CD1d or NKT cells. For example, HSV-1 drastically and specifically impaired CD1d recycling from the lysosome to the plasma membrane, an essential pathway for glycolipid antigen presentation to NKT cells (96). Kaposi sarcoma associated herpes virus encodes two modulators of immune 318 Bendelac Savage Teyton

23 recognition, MIR1 and MIR2, that downregulated CD1d in addition to other immunologically relevant molecules such as MHC class I, CD86, and intracellular adhesion molecule (ICAM)-1 through ubiquitination of lysine residues in their cytoplasmic tail (95). The lethal outcome of infections with Epstein- Barr virus in patients with X-linked lymphoproliferative (XLP) immunodeficiency syndrome due to SAP mutations was hypothesized to result from the absence of NKT cells (144). Which of these effects or associations reflect a specific viral evasion/immune defense strategy and the nature of the NKT ligands involved in these infectious conditions remain to be determined. NKT Cells in Noninfectious Diseases A role of NKT cells has been suggested in a wide variety of disease conditions. At present, however, many reports, lacking a detailed mechanistic understanding, remain isolated or are based merely on the analysis of NKT cell deficient mice. Rather than compiling an exhaustive list of the published claims, this review provides a critical appraisal of selected reports carrying important conceptual or clinical implications. ne frequently overlooked but recurrent methodological issue inherent in the use of CD1d- or Jα18-deficient mice is the extent to which gene-deficient mice are matched with littermate controls with respect to genetic background and environmental factors. This is particularly important in studies of complex multigenetic diseases such as diabetes, lupus, cancer, or asthma. In addition, the injection of αgalcer as a gainof-function experiment should be interpreted with caution because the massive release of cytokines induced by this procedure is unlikely to model chronic diseases. It may not be surprising, therefore, that some claims have become controversial or will need to be reinterpreted, complicating the task of drawing a clear picture of the involvement of NKT cells in noninfectious diseases. Type I diabetes. The relative deficiency of NKT cells in ND mice (36, 37), combined with the notion that these cells represent a potent source of Th2 cytokines, prompted the original speculation of a causal relationship with diabetes. Early claims that humans with type I diabetes exhibited severe NKT cell defects and that their sera had less IL-4 than controls (213, 214) were not confirmed when more specific methodologies became available (38, 215). Researchers interpreted reports of aggravated disease in CD1d-deficient ND mice (216, 217) as suggesting that, although defective, the residual NKT cells in ND mice still suppressed autoimmunity. However, independent studies in different colonies of CD1d-deficient and Jα18-deficient mice failed to support these claims (218), and partial reconstitution of NKT cells in ND mice carrying the B6 Nkt1 locus did not protect against diabetes (34). Transgenic expression of the Vα14-Jα18 TCRα chain in ND mice prevented diabetes, but this could be explained by the reduced frequency of isletspecific T cells and the general Th2 bias of these mice (219). Likewise, the suppression of diabetes by αgalcer multi-injection regimens could be the mere consequence of massive cytokine release (220, 221). More direct transfer experiments using diabetogenic T cells and NKT cells have suggested suppressive or enhancing roles of NKT cells in different experimental systems (222, 223). Although other more circumstantial studies have suggested a role of NKT cells in this disease, it seems reasonable to conclude at this point that there is no decisive evidence for a substantial or specific role of NKT cells in mouse or human type I diabetes. Lupus. Hyperreactive NKT cells were shown to accumulate in aging NZB/W mice (224) and suggested to help B cells produce anti-dna antibodies (225). However, studies of CD1d-deficient lupus-prone mice have not yielded concordant results ( ), and injections of αgalcer ameliorated or Biology of NKT Cells 319

24 aggravated disease, depending on the mouse strain (229). Cancer. Similar to the general immune suppression of T cells commonly encountered in cancerous states, NKT cells were decreased or functionally hyporeactive in cancer-bearing mice and humans (230, 231). ne tumor shed glycosphingolipids that could inhibit the stimulation of NKT cells in vitro (232). However, multiple mechanisms are likely to contribute to the deficiency of both T and NKT cells. In one report, the frequency of sarcomas six months after intramuscular injection of the chemical carcinogen methylcholantrene (MCA) decreased two- to threefold in Jα18 knockout NKT cell deficient mice (233). This observation, which suggested that NKT cells, similar to γδ T cells and NK cells, may be agents of immune surveillance against primary cancers has remained isolated. In a tumor transplant model, subcutaneous injection of a fibrosarcoma tumor line derived from MCA-inoculated Jα18-deficient mice produced tumors that grew faster in Jα18- deficient compared with wild-type mice and were prevented by transfers of purified NKT cells into Jα18-deficient hosts (234). CD1d expression and the presence of CD8 T cells in the host were required for tumor rejection, implying ligand recognition on host-derived cells, presumably APCs, rather than on tumor cells. The nature of the tumor-associated NKT ligands has not been identified. These experiments also revealed a specialized function of liver DN as compared with CD4 NKT cells in this Th1-mediated response (128). In apparent contrast with this fibrosarcoma model, CD1d-deficient mice controlled the growth of otherwise relapsing subcutaneous transplants of the 15-12RM tumor line, suggesting that a natural CD1d-dependent mechanism suppressed tumor rejection (235). Further studies dissected a complex cellular network that involved IL-13-producing CD1d-restricted CD4 suppressors interacting with TGF-β-producing myeloid cells to suppress antitumor CTL responses. Because Jα18-deficient mice did not share the phenotype of CD1d-deficient mice, the study concluded that other less well-known types of CD1d-restricted T cells might be involved (236). As in the MCA-induced tumor transplants, these tumors did not express CD1d, yet CD1d expression by host cells, presumably APCs, was required to observe the NKT cell effects. In contrast, the growth of the CD1dtransfected RMA/S tumor cell line cells was inhibited by Vα14 NKT cells (237). In conclusion, the notion that mvα14 and hvα24 NKT cells regulate cancer rejection is based largely on tumor transplant models, and the relevance to natural clinical conditions remains to be determined. Asthma. CD1d- and Jα18-deficient mice were reported to exhibit decreased allergeninduced airway hyperreactivity in the alumovalbumin model of asthma, where mice are intraperitoneally sensitized with ovalbumin mixed in alum and subsequently challenged with ovalbumin inhalation (238, 239). However, similar studies in another laboratory have failed to observe differences between CD1d-deficient and wild-type mice (R. Locksley, personal communication). In humans with persistent, moderate-to-severe asthma, Vα24 NKT cells dominated the bronchial Th2 infiltrate (240). The extent of this NKT cell expansion has been disputed, however, perhaps reflecting differences in the cohorts of asthma patients examined or the methods for identifying NKT cells (241). Atherosclerosis. CD1d deficiency decreased the level of atherosclerosis in apoeor LDL receptor deficient mice, although the effects observed were only mild and transient in some studies (241, 242). ther disease conditions. Additional observations suggesting a suppressive role of NKT cells in some models of delayedtype hypersensitivity (242, 243), in anterior 320 Bendelac Savage Teyton

25 chamber associated immune deviation (244), and in burn injury (245) have been reported. In summary, contrasting with numerous reports suggesting a contribution of NKT cells in a range of noninfectious diseases, a convincing picture has not yet emerged as to the strength or consistency of the observed effects, their mechanisms, or their relevance to physiological or clinical conditions. Future experiments are needed to define those diseases and conditions that are regulated specifically by mvα14 or hvα24 NKT cells and to dissect the mechanisms involved. THE LARGER CD1 UNIVERSE Although T cells recognizing lipids presented by other CD1 isotypes were the first discovered (44), their study now represents only a small fraction of the current investigations on CD1-mediated antigen presentation, which focus overwhelmingly on the CD1d/NKT cell system. CD1d is the only representative in mouse and rat of a larger family of β2-microglobulin-associated MHC-like molecules that, in other mammalian species, comprises CD1a, -b, and -c, as well as CD1e (44). CD1 and MHC are encoded in different loci, but recent genomic studies in chicken suggest that they originated from the same primordial MHC locus (246). CD1a, -b, and -c differ in their location in different endosomal compartments, in early recycling to late endosome and lysosome, and also in the architecture of their lipid-binding grooves, which suggests that each is specialized to capture different lipids in different endosomal compartments (44). Individual self and microbial lipid-specific T cell clones have been derived in vitro in humans, but relatively little is known about the T cell types and TCR repertoires associated with CD1a, -b, and -c and about their function in the human system. With respect to CD1d, however, it is well established that the major population of CD1d-restricted T cells in mouse is the NKT cell population that expresses semi-invariant TCRs, predominantly Vα14-Jα18, and performs innate-like functions (19). The presence of a more diverse population has been suggested recently, more convincingly in humans, indicating that an adaptive population of lipid-specific CD1d-restricted T cells may be available (210, 247, 248). The biology of these cells remains largely unexplored, and future studies in this area would resolve a fascinating and long-standing debate in the field of T cell recognition. Indeed, glycolipids are not easily mutated or modified, and although the potential theoretical combinations of carbohydrates are extremely diverse, the universe of microbial glycolipids is limited owing to enzyme specificity for both donor and acceptor substrates in glycolipid synthesis. Thus, the glycolipid-specific repertoire did not evolve under the same pressure that operated on the peptide-specific repertoire, where single mutations produce new T cell epitopes. How diverse and specific this glycolipid-specific repertoire may be is an important question for future research because conserved glycolipids may represent ideal, fixed targets for vaccine development. In addition, how crossreactive the MHC- and CD1-restricted TCR repertoires are is a fundamental issue that remains to be investigated. Given that the groove of CD1 molecules is significantly narrower than that of MHC proteins and that at least a proportion of the TCR repertoire appears to be intrinsically MHC-restricted (249, 250), one would assume that the peptidespecific and glycolipid-specific TCR repertoire should be essentially non-cross-reactive, a prediction that remains to be tested. SUMMARY Recent studies have elucidated novel and striking aspects of NKT cell development and of the cell and structural biology of lipid antigen processing and recognition. Key candidate antigens have been identified that provide a framework for understanding the evolution and function of this innate-like lineage, particularly in microbial infections. Future work will clarify the range and nature Biology of NKT Cells 321

26 of the most physiologically relevant ligands and the structural basis of their recognition by the semi-invariant TCRs. These solid advances in fundamental biology should help develop a mechanistic understanding of the broad and sometimes controversial array of diseases in which NKT cells are increasingly implicated. ACKNWLEDGMENTS We thank past and present members of our laboratories for their contributions to the understanding of NKT cell biology; Seth Scanlon and mita Trivedi, for help with the figures; and Richard Locksley, Diane Mathis, and Thomas Blankenstein for sharing unpublished results. 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 adequately describe every interesting paper, and we apologize to those investigators whose work could not be cited because of space limitations. LITERATURE CITED 1. Sumida T, Takei I, Taniguchi M Activation of acceptor-suppressor hybridoma with antigen specific suppressor T cell factor of two-chain type: requirement of the antigenand the I-J-restricting specificity. J. Immunol. 133: Sumida T, Taniguchi M Novel mechanisms of specific suppression of antihapten antibody response mediated by monoclonal antihapten antibody. J. Immunol. 134: Imai K, Kanno M, Kimoto H, Shigemoto T, Yamamoto S, Taniguchi M Sequence and expression of transcripts of the T-cell antigen receptor α-chain gene in a functional, antigen-specific suppressor-t cell hybridoma. Proc. Natl. Acad. Sci. USA 83: Koseki H, Imai K, Nakayama F, Sado T, Moriwaki K, Taniguchi M Homogeneous junctional sequence of the V14 + T cell antigen receptor a chain expanded in unprimed mice. Proc. Natl. Acad. Sci. USA 87: Koseki H, Asano H, Inaba T, Miyashita N, Moriwaki K, et al Dominant expression of a distinctive V14 + T cell antigen receptor a chain in mice. Proc. Natl. Acad. Sci. USA 88: Budd RC, Miescher GC, Howe RC, Lees RK, Bron C, Macdonald HR Developmentally regulated expression of T cell receptor beta chain variable domains in immature thymocytes. J. Exp. Med. 166: Fowlkes BJ, Kruisbeek AM, Ton-That H, Weston MA, Coligan JE, et al A novel population of T-cell receptor ab-bearing thymocytes which predominantly expresses a single Vβ8 gene family. Nature 329: Porcelli S, Yockey CE, Brenner MB, Balk SP Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4 8 α/β T cells demonstrates preferential use of several Vβ genes and an invariant TCR α chain. J. Exp. Med. 178: Dellabona P, Padovan E, Casorati G, Brockhaus M, Lanzavecchia A An invariant Vα24-JαQ/Vβ11 T cell receptor is expressed in all individuals by clonally expanded CD4 8 T cells. J. Exp. Med. 180: Bendelac A, Schwartz RH CD4 + and CD8 + T cells acquire specific lymphokine secretion potentials during thymic maturation. Nature 353: Bendelac Savage Teyton

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41 Contents Annual Review of Immunology Volume 25, 2007 Frontispiece Peter C. Doherty x Challenged by Complexity: My Twentieth Century in Immunology Peter C. Doherty 1 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 21 The Multiple Roles of steoclasts in Host Defense: Bone Remodeling and Hematopoietic Stem Cell Mobilization rit Kollet, Ayelet Dar, and Tsvee Lapidot 51 Flying Under the Radar: The Immunobiology of Hepatitis C Lynn B. Dustin and Charles M. Rice 71 Resolution Phase of Inflammation: Novel Endogenous Anti-Inflammatory and Proresolving Lipid Mediators and Pathways Charles N. Serhan 101 Immunobiology of Allogeneic Hematopoietic Stem Cell Transplantation Lisbeth A. Welniak, Bruce R. Blazar, and William J. Murphy 139 Effector and Memory CTL Differentiation Matthew A. Williams and Michael J. Bevan 171 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 mori, Baohua Zhou, and Steven F. Ziegler 193 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 221 v