[Frontiers in Bioscience S3, 236-251, January 1, 2011]

Natural killer T cells in health and disease

Lan Wu, Luc Van Kaer

Department of Microbiology and Immunology, Room A-5301, Medical Center North, 1161 21st Avenue South, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2363, USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Development and maintenance of NKT cells
4. NKT cell activation
5. NKT cell effector functions
6. NKT cell functions
7. NKT cell-based vaccine adjuvants
8. NKT cell-based immunotherapies
9. Summary and perspective
10. Acknowledgements
11. References

1. ABSTRACT

Natural killer T (NKT) cells are a subset of T lymphocytes that share surface markers and functional characteristics with both conventional T lymphocytes and natural killer cells. Most NKT cells express a semi-invariant T cell receptor that reacts with glycolipid antigens presented by the major histocompatibility complex class I-related protein CD1d on the surface of antigen-presenting cells. NKT cells become activated during a variety of infections and inflammatory conditions, rapidly producing large amounts of immunomodulatory cytokines. NKT cells can influence the activation state and functional properties of multiple other cell types in the immune system and, thus, modulate immune responses against infectious agents, autoantigens, tumors, tissue grafts and allergens. One attractive aspect of NKT cells is that their immunomodulatory activities can be readily harnessed with cognate glycolipid antigens, such as the marine sponge-derived glycosphingolipid alpha-galactosylceramide. These properties of NKT cells are being exploited for therapeutic intervention to prevent or treat cancer, infections, and autoimmune and inflammatory diseases.

2. INTRODUCTION

Cells of the immune system are typically divided into cells that belong to the innate or the adaptive arms of the immune system. Cells of the innate immune system express receptors that recognize molecular patterns shared among many different microbes whereas cells of the adaptive immune system express receptors with exquisite specificity for foreign antigens. Another distinction between cells of the innate and adaptive immune systems is that the former respond in a similar manner to each repeated exposure to the same microbe, whereas the latter respond more vigorously and faster to repeated exposure to the same antigen. Natural killer T (NKT) cells are a subset of immune cells that share characteristics of both the innate and adaptive arms of the immune system (for general reviews on NKT cells see (1-5)). Like conventional T lymphocytes, NKT cells express a T cell receptor (TCR), which is generated by somatic DNA rearrangement. However, whereas the TCR repertoire of conventional T cells is highly diverse, most NKT cells, commonly referred to as invariant or type I NKT cells, express a semi-invariant TCR (6). Murine invariant NKT cells express Valpha14-Jalpha18 chains paired with Vbeta8.2, -7, or -2, and human invariant NKT cells express homologous Valpha24-Jalpha18 chains paired with Vbeta11. This semi-invariant TCR reacts with glycolipid antigens presented by the major histocompatibility complex (MHC) class I-related protein CD1d (see Figure 1A). A substantial fraction of NKT cells in mice and humans expresses the co-receptor CD4, most of the remaining NKT cells lack CD4 and CD8 expression, and a small subset of NKT cells in humans but not mice express CD8. NKT cells also constitutively express a variety of markers, including CD25, CD44, CD69 and CD122 that are typical of T cells with an activated or memory phenotype. In addition to these T cell markers, NKT cells express receptors such as NK1.1 in mice or CD161 in humans that are characteristic of the natural killer (NK) cell lineage, which belongs to the innate arm of the immune system. Consistent with their activated phenotype, TCR engagement of NKT cells results in rapid elicitation of effector functions, including cytokine secretion and cytotoxicity. A hallmark of these cells is their capacity to rapidly produce a mixture of cytokines, including interleukin (IL)-4 and interferon (IFN)-gamma, which are the signature cytokines produced by T helper type 1 (Th1) cells and Th2 cells, respectively. NKT cells are found in substantial numbers in thymus, spleen, liver and bone marrow, but only low numbers are present in peripheral blood and lymph nodes. The size of the NKT cell population in humans is much lower than that in mice and shows high variability among different individuals.

In addition to type I NKT cells, a subset of NKT cells lacks expression of the invariant TCRalpha chain, and instead expresses a more diverse set of TCRs (6). These cells are referred to as type II NKT cells or variant NKT cells. Like type I NKT cells, type II NKT cells recognize glycolipid antigens presented by CD1d molecules and have potent immunomodulatory activities (7). Additional T cell subsets with surface receptors and functions that are similar to NKT cells have been identified, and these cells are usually referred to as NKT-like cells (6). This review will focus on type I NKT cells, which we will simply call NKT cells.

3. DEVELOPMENT AND MAINTENANCE OF NKT CELLS

Like conventional T cells, NKT cells develop in the thymus (8). Generation of the invariant TCR by VDJ recombination is a stochastic event. Acquisition of the NKT cell phenotype appears to be driven by interaction of the invariant TCR with CD1d. Mice that are deficient in CD1d expression have a complete block in NKT cell development (9-11). Positive selection of NKT cells requires interaction of the invariant TCR on immature NKT cells expressing both CD4 and CD8 with CD1d expressed by cortical thymocytes themselves (12-14). This step in NKT cell development depends on co-stimulatory interactions between immature NKT cells and CD1d-expressing cortical thymocytes mediated by signaling lymphocyte activation molecules (SLAM) (15-17). SLAM molecules engage in homotypic interactions resulting in activation of the SLAM adaptor protein (SAP), which signals through the Src tyrosine kinase Fyn. Consequently, SAP-deficient mice, and patients with X-linked lymphoproliferative disease due to SAP-deficiency, lack NKT cells but contain normal numbers of conventional T cells (18-20). This step in NKT cell development is controlled by the transcription factor c-Myb, which regulates CD1d expression, the half-life of double-positive thymocytes, and the expression of SLAM and SAP (21). The interaction between the invariant TCR of NKT cells likely involves endogenous glycolipid antigens that drive positive selection of NKT cells. It has been suggested that isoglobotrihexosylceramide (iGb3), a lysosomal glycosphingolipid that weakly activates mature NKT cells, can perform this function (22). However, not all evidence supports this possibility and it is likely that multiple glycolipids are involved.

There is also evidence that NKT cells undergo negative selection in order to avoid overt activation in the periphery (8). During early stages of their development NKT cells express the transcription factor Nur77 (23), which is associated with negative selection in conventional T cells. Treatment of fetal thymus organ cultures with the NKT cell agonist alpha-galactosylceramide (alpha-GalCer; see Figure 1B for the structure of alpha-GalCer) resulted in the loss of NKT cells, suggesting negative selection (24, 25). Further, overexpression of CD1d on dendritic cells, which play a critical role in intrathymic negative selection, resulted in reduced numbers of NKT cells and reduced responsiveness of these cells to TCR engagement (24). Conversely, selective expression of CD1d on thymocytes resulted in NKT cells that were hyperresponsive to stimulation with alpha-GalCer (26). However, in another transgenic mouse where CD1d was selectively expressed on thymocytes and peripheral T cells, the selected NKT cells were hyporesponsive to alpha-GalCer stimulation but developed liver pathology in the absence of exogenous manipulation (27).

Following positive selection, NKT cells undergo a series of differentiation steps that result in the mature NKT cell pool. Our understanding of this process is largely based on studies in mice (8). Early in their maturation, positively selected NKT cells downregulate CD8, and a subset of these cells also downregulate CD4. Silencing of CD8 in mouse NKT cells appears to be driven by the transcription factor Th-POK (Th, poxviruses and Zinc finger, and Kruppel family) (28). These immature NKT cells undergo extensive proliferation and are potent IL-4 producers, but make little IFN-gamma. Most immature NKT cells leave the thymus at this stage, in a process that is controlled by the lymphotoxin-beta receptor and the sphingosine 1-phosphate receptor. Next, immature NKT cells, both in the thymus and the periphery, acquire NK cell receptors such as NK1.1 (CD161) and increase their capacity to produce IFN-gamma. In mice a substantial fraction of mature CD4-negative and CD8-negative NKT cells remain in the thymus as long-term resident cells.

The maturation of NKT cells is guided by a variety of cytokines, cell surface molecules, signal transducers, transcription factors and other regulatory factors, which have been reviewed elsewhere (8). A key step in the maturation of NKT cells is their acquisition of innate effector functions. The transcription factor promyelocytic leukemia zinc finger (PLZF) has been implicated in this process (29). PLZF is expressed by NKT cells and a few other T cell subsets with innate effector functions but not by conventional T lymphocytes. PLZF-deficient mice had sharply reduced numbers of NKT cells and the remaining cells had lost their characteristic memory phenotype and accumulated in lymph nodes (30, 31). Conversely, transgenic expression of PLZF in thymocytes resulted in the development of MHC class II-restricted, CD4-expressing T cells with rapid effector functions (31). Another key step in the maturation of NKT cells is the acquisition of cytokine secretion capability. These cells constitutively express cytokine gene transcripts but require intrathymic signaling mediated by granulocyte monocyte colony-stimulating factor (GM-CSF) in order to become competent for cytokine secretion (32).

The homeostasis and maintenance of NKT cells in the periphery is regulated by the cytokine IL-15 and to a lesser extent IL-7 (33, 34), which is similar to NK and memory CD8 T cells. In contrast to conventional T cells, which require interaction with their MHC ligands for maintenance, NKT cell homeostasis does not require CD1d expression (26, 33, 34). Recruitment and maintenance of NKT cells in the liver further involves the chemokine receptor CXCR6 (35, 36), expression of integrin LFA-1 (37, 38), and the transcription factor Id2 (39). Finally, a recent study further showed that the composition of commensal microbiota in the gut can impact the abundance of NKT cells in mice (40). Specifically, germfree mice had a moderate reduction of these cells as compared with mice maintained under specific pathogen-free conditions, and the precise composition of commensal microorganisms impacted NKT cell abundance as well.

4. NKT CELL ACTIVATION

Several endogenous and exogenous glycolipids that can activate NKT cells have been identified (41). The self-glycolipid iGb3 can activate NKT cells but its role in NKT cell development and functions is unclear (22). Some NKT cell hybridomas reacted with phospholipids, including phosphatidylinositol and phosphatidylethanolamine (42). Further, a substantial portion of human NKT cell clones was shown to react with lyso-phospholipids (43), which serve as lipid messengers during normal physiological responses. A subset of NKT cells can also respond to ganglioside GD3 (44), which is highly expressed in tumors of neuroectodermal origin. NKT cells also react with multiple glycolipids of microbial origin (41). A small fraction of NKT cells reacts with a tetramannosylated form of phosphatidylinositol called PIM4, derived from Mycobacterium bovis BCG (45). Similarly, a substantial portion of mouse and human NKT cells was able to react with diacylglycerol-based glycolipids derived from Borrelia burgdorferi (46), the etiological agent of Lyme disease. It has also been reported that a subset of NKT cells can recognize lipophosphoglycan from the protozoan parasite Leishmania donovani (47) and it has been suggested that a lipopeptidophosphoglycan in the membrane of Entamoeba histolytica (48) can activate NKT cells as well. Alpha-glycuronosylceramides, which are found in bacteria of the Novosphingobium (formerly called Sphingomonas) and Ehrlichia genera, can potently activate both mouse and human NKT cells (49-51). This included alpha-galacturonosylceramide and alpha-glucuronosylceramide from Novosphingobium species. These reagents bear profound structural similarity with alpha-GalCer, which had been previously identified as a potent NKT cell agonist present in extracts derived from the marine sponge Agelas mauritianus (52). It is now believed that alpha-GalCer is actually derived from Novosphingobium bacteria that colonized the marine sponge. An optimized synthetic version of alpha-GalCer, called KRN7000, has been employed by many laboratories to study the functions and therapeutic properties of NKT cells (53). Many synthetic variants of KRN7000 and other NKT cell ligands have also been studied. Collectively, these studies have revealed a remarkable capacity of NKT cells to react with a wide range of lipid structures.

The structural basis for the capacity of the invariant TCR of NKT cells to react with a wide array of glycolipid and lipid antigens has been investigated (54, 55). Structural studies of human and mouse CD1d have revealed that CD1d contains a hydrophobic antigen-binding groove, with two pockets that can accommodate lipid tails (56). Structures of CD1d with alpha-GalCer confirmed the binding of lipid tails in the CD1d pockets, with the polar head group protruding out of the CD1d binding groove (57). Structures of CD1d with a version of alpha-GalCer with a shortened acyl chain (58), a Novosphingobium-derived glycolipid (59) and the putative endogenous antigen iGb3 (60) revealed the presence of a spacer lipid, providing part of the explanation for the capacity of CD1d to bind a wide variety of glycolipid and lipid antigens. The crystal structures of the canonical human and mouse NKT cell receptor bound with CD1d and alpha-GalCer revealed that the NKT cell receptor binds parallel to the long axis of CD1d, rather than the diagonal type of binding that has been observed for TCRs of conventional T cells (61, 62). Interactions of the NKT cell receptor with CD1d and glycolipid were dominated by the TCRalpha chain, which is surprising because the alpha chain is invariant. These observations have been confirmed and extended by extensive mutational analysis of the NKT cell receptor (61, 63-66). These mutational analyses have further indicated that the TCRbeta chain can influence the fine-specificity of the NKT cell receptor (66).

The mechanisms by which glycolipid antigens are processed and presented to NKT cells have also been investigated (67). Following its synthesis in the endoplasmic reticulum (ER), CD1d molecules are loaded with ER-resident phospholipids that largely function as chaperones to stabilize CD1d complexes and facilitate their transport to the cell surface. Loading of CD1d with these phospholipids is assisted by the lipid transport protein microsomal triglyceride transfer protein (MTP) (68). Surface displayed CD1d is subsequently internalized and routed to endosomal-lysosomal compartments where the phospholipids are released from CD1d. Lipids might be delivered to these compartments following engagement of pattern recognition receptors on antigen-presenting cells with microbial molecular patterns (67), or following internalization of apolipoprotein E-containing lipid complexes via lipoprotein receptors (69). A recent study further showed that loading of alpha-GalCer on CD1d is regulated by the enzyme fatty acid amide hydrolase (70). After delivery to endosomal-lysosomal compartments some complex lipids might require further processing by resident lipases and glycosidases (71). Loading of glycolipids on CD1d is assisted by lipid transfer proteins such as the saposins, the GM2-activator protein and the Niemann-Pick C2 protein (72-74). Several pathogens, including Kaposi sarcoma-associated herpes virus, HIV-1, herpes simplex virus-1, vesicular stomatitis virus, vaccinia virus and Chlamydia trachomatis have evolved mechanisms to interfere with the CD1d antigen processing pathway and, thus, impair glycolipid antigen presentation to NKT cells (75, 76).

In addition to the direct activation of NKT cells by cognate glycolipid antigens, NKT cells can become activated indirectly (77) (see Figure 2). This has been studied most extensively for Gram-negative bacteria that contain lipopolysaccharide (LPS) in their cell wall. NKT cells became activated when dendritic cells were cultured with Salmonella bacteria and this could be blocked by antibodies against IL-12 or CD1d (78). This activation also required signaling through toll-like receptors (TLR). Consistent with this finding, LPS was able to substitute for the bacteria in activating NKT cells. Similar findings have been observed for other bacteria and other types of microorganisms (79). In some cases, activation of antigen-presenting cells by TLR ligands was shown to modify lipid biosynthetic pathways, the composition of CD1d-bound lipids or possibly expression of NKT cell ligands (80-82). In the case of the Gram-positive bacterium Listeria monocytogenes, it was shown that production of IFN-beta induced upregulation of CD1d in infected antigen-presenting cells (83). Collectively, these studies have led to the general concept that activation of antigen-presenting cells via TLR signaling results in the production of cytokines (i.e., IL-12, IL-18 and/or type I interferons) by the antigen-presenting cells that can synergize with interactions of the invariant TCR with CD1d-lipid complexes for NKT cell activation (79). Of note, CD1d expression on the antigen-presenting cells was not always required for NKT cell activation (84). This pathway of NKT cell activation might be sufficiently conserved to permit activation of these cells during other situations, such as during the inflammatory responses that are associated with many pathological conditions.

5. NKT CELL EFFECTOR FUNCTIONS

The effector functions of NKT cells have been explored most extensively when these cells were stimulated with alpha-GalCer (53). A hallmark of NKT cells is their capacity to rapidly produce a variety of cytokines following TCR stimulation. This includes typical Th1 cytokines such as IFN-gamma and tumor necrosis factor (TNF) and typical Th2 cytokines such as IL-4, IL-10 and IL-13, but also IL-2, transforming growth factor (TGF)-beta and several chemokines. Further, a subset of NKT cells produces the Th17 cytokine IL-17 (85). NKT cells produce cytokines early after their activation, after which cytokine production ceases. In mice, IL-4 production peaks around 6 hrs and IFN-gamma production peaks around 24 hrs after in vivo alpha-GalCer treatment. NKT cells activated in this manner are also capable of substantial expansion in multiple organs, after which NKT cell numbers gradually decline to reach pre-injection levels around 2 weeks after the initial alpha-GalCer treatment (86, 87). These alpha-GalCer-experienced NKT cells are then resistant to restimulation with alpha-GalCer, as they have adopted an anergic phenotype, which lasts for up to 2 months (88, 89).

The level of activation and the profile of cytokines produced by NKT cells can be profoundly modulated by the context in which these cells become activated (53, 90-92). For example, NKT cell activation can be modulated by engagement of co-stimulatory molecules on these cells such as CD28, CD40L, OX40, and programmed death (PD)-1. Likewise, distinct NKT cell agonists can skew the cytokine profile of NKT cells. While KRN7000, the prototypical alpha-GalCer, induces a mixed Th1/Th2 cytokine profile in NKT cells, some structural variants such as OCH (93) and C20:2 (94) promote a Th2-biased cytokine profile, whereas some other structural variants such as alpha-C-GalCer (95) and alpha-carba-GalCer (96) promote a Th1-biased cytokine production profile. These properties of NKT cells have been exploited in the development of vaccine adjuvants and immunotherapies (see below).

NKT cells are capable of substantial crosstalk with many other cell types from the innate and adaptive immune system (91, 92). In particular, activated NKT cells are potent stimulators of dendritic cell functions, a property that has received substantial interest for the development of NKT cell-based adjuvants (97). Activated NKT cells also promote the activation of natural killer (NK) cells, macrophages, neutrophils, and conventional B and T cells. In many cases, NKT cells also influence the quality of an immune response by regulating the differentiation of naïve CD4 T cells into Th cell effectors. These complex interactions of NKT cells with other cell types in the immune system are influenced by the precise nature of the stimulus and form the basis for the capacity of these cells to modulate a variety of immune responses.

6. NKT CELL FUNCTIONS

NKT cells can contribute to protective immune responses against a wide variety of pathogens, including bacteria, viruses, fungi, protozoa and parasites (79, 98). Some of these microorganisms, including Borrelia burgdorferi (99) and species of the Novosphingobium and Ehrlichia genera (50), contain glycolipid or lipid antigens that can directly activate NKT cells. However, most microorganisms likely activate NKT cells in an indirect manner, using the pathway described above and depicted in Figure 2. NKT cells can also contribute to disease pathogenesis during infection. For example, although these cells contribute to protective immunity against infection with a low dose of Novosphingobium capsulata, at high dose inoculation, they can induce a potent cytokine storm that leads to a lethal sepsis (50). These cells also play a pathogenic role following intranasal infection with Chlamydia muridarum, but surprisingly, were protective for infection by the related organism Chlamydia pneumoniae (100). Some studies further showed that the outcome of infection can be influenced by the genetic background of the animals employed. For example, NKT cells protected BALB/c mice against disease induced by respiratory syncytial virus infection, but exacerbated disease in mice of the C57BL/6 background (101). Similarly, NKT cells protected BALB/c mice against cerebral malaria but exacerbated disease in C57BL/6 mice (102). Interestingly, a recent study showed that NKT cells can regulate colonization of the intestine of mice with commensal and pathogenic bacteria (103).

NKT cells have been implicated in natural immunity against tumors (104). This has been demonstrated in experimental models involving transplantable tumors, tumors induced by the chemical methylcholanthrene, and tumors that develop in mice heterozygous for the p53 tumor suppressor gene. This natural immunity against tumors appears to be due to production of IFN-gamma to activate NK cells and CD8 T cells, and by inducing IL-12 production in dendritic cells. Consistent with this protective role of NKT cells against tumors in mice, infiltration of tumors with NKT cells appears to be positively correlated with survival of cancer patients.

NKT cells contribute to a variety of autoimmune and inflammatory conditions (105, 106). In most mouse models of type 1 diabetes, multiple sclerosis and systemic lupus erythematosus, NKT cells play a protective role. Although NKT cell-deficient animals do not develop any obvious disease pathologies, aged Jalpha18-deficient mice developed signs of kidney disease and antibodies directed against double-stranded DNA (107), which are characteristic of lupus-like disease. A recent study (108) has provided important insight into the potential mechanisms by which NKT cells can modulate lupus development. These investigators showed that apoptotic cells, which are often increased in lupus, can activate NKT cells, resulting in suppression of autoreactive, CD1d-expressing B cells and lupus development in mice (108). NKT cells can also promote autoimmunity in some cases. In models of collagen-induced and antibody-mediated arthritis NKT cells played a pathogenic role (109). Similarly, these cells were important for the development of primary biliary cirrhosis in transgenic mice overexpressing a dominant-negative form of the TGF-beta receptor, and in mice infected with Novosphingobium aromaticivorans (110-112). NKT cells also play a pathogenic role in mouse models for multiple inflammatory diseases and hypersensitivities, including allergic airway hyperresponsiveness and asthma (113), atherosclerosis (114-116), contact hypersensitivity (117), rejection of tissue transplants (118), colitis induced by the hapten oxazolone (119), liver disease induced by the T cell mitogen concanavalin A (120), ischemia-reperfusion injury in multiple organs (121) and during sickle cell disease (122), and sepsis syndrome induced by LPS (84). Consistent with the proposed role of NKT cells in autoimmunity and inflammation, mice or patients with autoimmune or inflammatory diseases often have alterations in the abundance and cytokine production profile of NKT cells. However, it is unclear whether these alterations in NKT cells contribute to disease development or if they are secondary to the disease process.

The hypothesis that NKT cells play an important role in immune tolerance is supported by studies with experimental models of tolerance induction. This was first demonstrated in a model of tolerance in the eye, termed anterior chamber-induced immune deviation (123). Tolerance induced by injection of protein antigens in the eye was dependent on NKT cells in the spleen (124). A similar tolerogenic role of NKT cells has been demonstrated in transplant tolerance induced by blocking antibodies directed against CD4 or co-stimulatory molecules (125), immune suppression induced by ultraviolet exposure (126) or following burn wounds (127), and tolerance to the fetus (128). NKT cells in the host also play a protective role in protecting the host from graft-vs-host disease (GVHD) following bone marrow transplantation (129). Importantly, the latter activities of NKT cells did not impair immune responses against tumors in the host (i.e., graft-vs-leukemia responses).

7. NKT CELL-BASED VACCINE ADJUVANTS

The immune potentiating properties of NKT cells have been exploited for the development of vaccine adjuvants (130, 131). These properties of NKT cells are based on their capacity to stimulate the maturation of dendritic cells, which, in turn, promote potent cell-mediated immune responses. In vivo injection of alpha-GalCer into mice led to phenotypic and functional maturation of dendritic cells, resulting in the priming of CD4 and CD8 effector T cells. Dendritic cells potently upregulated a variety of co-stimulatory molecules, including CD80, CD86 and CD70, and secreted copious amounts of cytokines such as IL-12 and TNF-alpha. These activities of NKT cells required CD40-CD40L interactions between the dendritic cells and NKT cells. Additional studies further showed that NKT cells can also promote antibody responses. Mechanistic studies suggested that activation of NKT cells by alpha-GalCer enhances antibody responses to co-administered T cell-dependent antigens by inducing persistent plasma cell responses (132). Because CD1d expression on the B cells was required, it is likely that direct interactions between B cells and NKT cells were critical for these activities of alpha-GalCer on antibody responses. Thus, alpha-GalCer has strong adjuvant activities that promote both cell-mediated and humoral immune responses.

The adjuvant activities of NKT cells have been tested in vaccines against several pathogens and tumors (130, 131, 133, 134). This was first demonstrated for malaria vaccines, using irradiated sporozoites or recombinant viruses expressing a malaria antigen (135). These adjuvant properties of alpha-GalCer required IFN-gamma production by NKT cells. Alpha-GalCer was also effective as a mucosal adjuvant in vaccine formulations directed against influenza virus, in which case mucosal IgA antibodies were produced. Similarly, alpha-GalCer promoted the efficacy of an HIV-1 DNA vaccine, promoting a 10-fold increase in humoral immune responses. Multiple strategies have been employed to develop tumor vaccines. Some investigators have employed tumors expressing neoantigens such as ovalbumin, in which case mice were immunized with ovalbumin antigens and treated with alpha-GalCer and then challenged with the ovalbumin-expressing tumor cells. Another strategy employed alpha-GalCer treatment at the time of immunization with clinically relevant peptide epitopes, such as the HLA-A2-restricted peptide derived from the NY-ESO-1 antigen. In another approach alpha-GalCer was co-administered with irradiated tumor cells, in which case NKT cell activation promoted the maturation of dendritic cells that captured and presented tumor antigens to CD4 and CD8 T cells. In a variation of this approach, CD1d-expressing tumor cells were pulsed with alpha-GalCer and then used to immunize mice. In the latter case, immunity to the tumors was long-lived, persisting for 6-12 months (136).

8. NKT CELL-BASED IMMUNOTHERAPIES

Alpha-GalCer was originally discovered during a search for reagents derived from a marine sponge with potent anti-metastatic activities in mice (137). These original findings paved the way for numerous studies investigating the anti-tumor activities of alpha-GalCer and its structural variants (104, 138). Tumor models employed included transplantable tumors, tumors induced by the chemical carcinogen methylcholanthrene and spontaneous tumors in Her2/neu transgenic mice and in p53-deficient mice. Mechanistic studies revealed that CD1d expression on the tumors was not required for the anti-metastatic activities of alpha-GalCer, and provided evidence for a critical role of IFN-gamma, produced by NKT cells themselves, NK cells and cytotoxic T lymphocytes. Consistent with the critical role of IFN-gamma, the alpha-GalCer analogue alpha-C-GalCer demonstrated superior anti-metastatic activities (95). Similarly, delivery of the glycolipid by dendritic cells provided improved therapeutic activities (139). The anti-metastatic activities of alpha-GalCer have also been tested in cancer patients (133). Treatment modalities employed included repeated injection with free alpha-GalCer or alpha-GalCer-loaded dendritic cells, and treatment with NKT cells expanded in vitro with alpha-GalCer. These clinical trials revealed that it is very challenging to induce potent NKT cell responses in humans, in part because humans have low numbers of NKT cells as compared with mice. Another challenge is that NKT cells in cancer patients are often dysfunctional. One trial, which involved repeated treatment of patients with non-small lung cancer using alpha-GalCer-stimulated peripheral blood mononuclear cells, has shown some promising clinical benefit to patients (140).

The capacity of alpha-GalCer to impact infection has been investigated for a wide variety of microorganisms (79, 98). In most cases, treatment was associated with pathogen clearance, in a manner that depended on the capacity of alpha-GalCer to activate dendritic cells and other innate immune cells. Consistent with this notion, alpha-C-GalCer was more effective that alpha-GalCer in clearing malaria parasites from mice infected with sporozoites (95). Because alpha-GalCer treatment was typically only effective during a narrow time window of a few days before and after infection, this strategy will likely have limited benefit for treatment of human infectious diseases. Clinical trials with patients chronically infected with hepatitis B virus (141) or hepatitis C virus (142) have found no clinical benefit of alpha-GalCer treatment.

Alpha-GalCer and its structural analogues have been tested in a variety of autoimmune diseases, usually employing repeated injections (105, 143). Alpha-GalCer has proven efficacious for prevention of diabetes in non-obese diabetic mice (144-147), multiple sclerosis-like disease in multiple mouse models (93, 148-150), some induced and genetic models of systemic lupus erythematosus (151, 152), collagen-induced rheumatoid arthritis (153, 154), and experimental models of autoimmune myasthenia gravis (155), autoimmune thyroiditis (156), and ocular autoimmunity (157). However, in some cases, NKT cell activation exacerbated disease, which was observed in an adoptive transfer model for type 1 diabetes in C57BL/6 mice (158), some studies with multiple sclerosis (159) or lupus models (152, 160), and antibody-mediated arthritis (161). The outcome of NKT cell activation on the disease process was often influenced by the glycolipid dose, timing of treatment, genetic background of the animals and precise experimental model tested. Generally, disease prevention was associated with a shift in the immune response towards Th2 immunity and/or suppression of Th1 and/or Th17 cell responses. In many cases cytokines such as IL-4 and IL-10 appeared to be critical for disease protection, but in some models IFN-gamma appeared to play a protective role as well. Consistent with the idea that immune deviation plays a role, the alpha-GalCer analogues OCH and C20:2 demonstrated superior disease protection in some models. In addition to general deviation of the immune response, multiple other mechanisms have been implicated in the capacity of activated NKT cells to suppress autoimmunity, including the induction of tolerogenic dendritic cells and regulatory T cells, induction of anergy in pathogenic T cells, direct effects on B cells, or general immune suppression. It is likely that NKT cell activation by distinct NKT cell agonists impacts distinct autoimmune diseases in a different manner and that mechanisms involved are diverse. For example, a single injection of either alpha-GalCer or alpha-C-GalCer was able to protect DBA/1 mice against collagen-induced arthritis (153). In the case of alpha-GalCer the cytokine IL-10 appeared to play a critical role, whereas the effects of alpha-C-GalCer on disease amelioration were associated with a general immune suppression. Similarly, alpha-GalCer, OCH and alpha-C-GalCer all protected mice against ocular autoimmunity, but alpha-C-GalCer was most effective, in a manner that was associated with suppression of both Th1 and Th17 cell responses (157). The diverse and often unpredictable outcomes of NKT cell activation on autoimmunity in mouse models will complicate translation of these preclinical studies to the clinical setting.

A few studies have investigated the capacity of NKT cell activation to modulate GVHD responses. In a model of allogeneic bone marrow transplantation where recipients were nonlethally irradiated, alpha-GalCer treatment of the recipient mice reduced GVHD by stimulating host NKT cells and inducing Th2 deviation in donor T cells (162, 163). However, in a model of allogeneic stem cell transplantation where recipient animals received a fully myeloablative dose of irradiation and using delayed glycolipid administration, alpha-GalCer exacerbated GVHD, in a manner involving donor NK and T cell activation (164). Nevertheless, using the latter model, the Th2-deviating alpha-GalCer analogue C20:2 potently inhibited GVHD.

In a model of inflammatory bowel disease induced by dextran sodium sulphate, which elicits inflammation by permitting access of intestinal microorganisms to the subepithelial space in the gut, alpha-GalCer and OCH were able to confer disease protection (165, 166). In the case of OCH, even a single treatment was effective, in a manner that correlated with a reduction in IFN- expression and an increase in IL-4 expression in the mucosal tissue (165).

The effects of NKT cell activation in models of atherosclerosis have also been investigated (167). Alpha-GalCer exacerbated spontaneous atherosclerosis in apolipoprotein (apo) E-deficient mice (114-116). However, in a model where atherosclerosis was induced by Western-type diet feeding and collar placement around the carotid arteries, alpha-GalCer treatment had no effect on atherosclerosis in apoE-deficient mice, but led to a reduction in plaque size in low density lipoprotein receptor-deficient mice (168). The investigators of the latter studies proposed that these differences between the two types of knockout animals might be due to the critical role of apoE in delivering glycolipids to NKT cells.

In allergic reactions and asthma, NKT cell activation during the sensitization phase exacerbated disease (169). Conversely, a CD1d-dependent antagonist of NKT cells, di-palmitoyl-phosphatidyl ethanolamine polyethylene glycol, prevented development of allergen-induced airway hyperreactivity in mice (170).

9. SUMMARY AND PERSPECTIVE

NKT cells are regulatory T cells that bridge the innate and adaptive immune system. NKT cells can directly respond to glycolipid antigens that bind with CD1d molecules, and these cells can also become activated indirectly in response to TLR agonists and other inflammatory mediators such as pro-inflammatory cytokines. Activated NKT cells are capable of rapidly producing copious amounts of cytokines that can activate cells of the innate and adaptive immune systems and influence the character of the ensuing immune response. Consequently, NKT cells have been implicated in a variety of immune responses and diseases, including infections, cancer, tissue graft rejection, allergies, autoimmunity and inflammatory diseases. Numerous glycolipid antigens that can differentially induce distinct effector functions of NKT cells have been identified, providing potent tools for the development of vaccine adjuvants and immunotherapies for a variety of diseases. Nevertheless, a number of challenges for applying these tools in the clinical setting remain. Thus far, NKT cell activation during therapy of cancer or infectious disease in humans has had limited success. More will need to be learned about the behavior of NKT cells and how their therapeutic properties can be best exploited. With the proper tools it might be possible to target specific subsets of NKT cells with unique and distinct effector functions. In addition, because humans have lower numbers of NKT cells than mice, it will be critical to develop potent techniques to elicit the biological functions of these cells in patients. In this context, methods to introduce the invariant TCR of human NKT cells into stem cells followed by programming towards the T cell lineage has been considered (171). Finally, combination therapies are being tested in animal models. Collectively, these studies hold much promise for the clinical application of NKT cell-based immunotherapies.

10. ACKNOWLEDGEMENTS

We thank colleagues and members of our laboratory for helpful discussions. Work of the authors was supported by grants from the National Institutes of Health and a discovery grant from the Diabetes Research and Training Center at Vanderbilt University School of Medicine.

11. REFERENCES

1. M. Taniguchi, M. Harada, S. Kojo, T. Nakayama and H. Wakao: The regulatory role of Va14 NKT cells in innate and acquired immune response. Annu. Rev. Immunol., 21, 483-513 (2003)
;doi:10.1146/annurev.immunol.21.120601.141057

2. M. Brigl and M. B. Brenner: CD1: antigen presentation and T cell function. Annu. Rev. Immunol., 22, 817-890 (2004)
;doi:10.1146/annurev.immunol.22.012703.104608

3. M. Kronenberg: Toward an understanding of NKT cell biology: progress and paradoxes. Annu. Rev. Immunol., 26, 877-900 (2005)
;doi:10.1146/annurev.immunol.23.021704.115742

4. A. Bendelac, P. B. Savage and L. Teyton: The biology of NKT cells. Annu. Rev. Immunol., 25, 297-336 (2007)
;doi:10.1146/annurev.immunol.25.022106.141711

5. L. Van Kaer: NKT cells: T lymphocytes with innate effector functions. Curr. Opin. Immunol., 19(3), 354-364 (2007)
;doi:10.1016/j.coi.2007.03.001

6. D. I. Godfrey, H. R. MacDonald, M. Kronenberg, M. J. Smyth and L. Van Kaer: NKT cells: what's in a name? Nat. Rev. Immunol., 4(3), 231-237 (2004)
;doi:10.1038/nri1309

7. P. Arrenberg, R. Halder and V. Kumar: Cross-regulation between distinct natural killer T cell subsets influences immune response to self and foreign antigens. J. Cell. Physiol., 218(2), 246-250 (2009)
;doi:10.1002/jcp.21597

8. D. I. Godfrey, S. Stankovic and A. G. Baxter: Raising the NKT cell family. Nat. Immunol., 11(3), 197-206 (2010)
;doi:10.1038/ni.1841

9. S. K. Mendiratta, W. D. Martin, S. Hong, A. Boesteanu, S. Joyce and L. Van Kaer: CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity, 6(4), 469-477 (1997)
;doi:10.1016/S1074-7613(00)80290-3

10. Y. H. Chen, N. M. Chiu, M. Mandal, N. Wang and C. R. Wang: Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity, 6(4), 459-467 (1997)
;doi:10.1016/S1074-7613(00)80289-7

11. S. T. Smiley, M. H. Kaplan and M. J. Grusby: Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells. Science, 275(5302), 977-979 (1997)
;doi:10.1126/science.275.5302.977

12. A. Bendelac: Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J. Exp. Med., 182(6), 2091-2096 (1995)
;doi:10.1084/jem.182.6.2091

13. J. S. Bezbradica, T. Hill, A. K. Stanic, L. Van Kaer and S. Joyce: Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+ stage of thymic ontogeny. Proc. Natl. Acad. Sci. USA, 102(8), 5114-5119 (2005)
;doi:10.1073/pnas.0408449102

14. T. Egawa, G. Eberl, I. Taniuchi, K. Benlagha, F. Geissmann, L. Hennighausen, A. Bendelac and D. R. Littman: Genetic evidence supporting selection of the Va14i NKT cell lineage from double-positive thymocyte precursors. Immunity, 22(6), 705-716 (2005)
;doi:10.1016/j.immuni.2005.03.011

15. C. Borowski and A. Bendelac: Signaling for NKT cell development: the SAP-FynT connection. J. Exp. Med., 201(6), 833-836 (2005)
;doi:10.1084/jem.20050339

16. C. S. Ma, K. E. Nichols and S. G. Tangye: Regulation of cellular and humoral immune responses by the SLAM and SAP families of molecules. Annu. Rev. Immunol., 25, 337-379 (2007)
;doi:10.1146/annurev.immunol.25.022106.141651

17. K. Griewank, C. Borowski, S. Rietdijk, N. Wang, A. Julien, D. G. Wei, A. A. Mamchak, C. Terhorst and A. Bendelac: Homotypic interactions mediated by Slamf1 and Slamf6 receptors control NKT cell lineage development. Immunity, 27(5), 751-762 (2007)
;doi:10.1016/j.immuni.2007.08.020

18. B. Chung, A. Aoukaty, J. Dutz, C. Terhorst and R. Tan: Signaling lymphocytic activation molecule-associated protein controls NKT cell functions. J. Immunol., 174(6), 3153-3157 (2005)

19. K. E. Nichols, J. Hom, S. Y. Gong, A. Ganguly, C. S. Ma, J. L. Cannons, S. G. Tangye, P. L. Schwartzberg, G. A. Koretzky and P. L. Stein: Regulation of NKT cell development by SAP, the protein defective in XLP. Nat. Med., 11, 340-345 (2005)
;doi:10.1038/nm1189

20. B. Pasquier, L. Yin, M. C. Fondaneche, F. Relouzat, C. Bloch-Queyrat, N. Lambert, A. Fischer, G. de Saint-Basile and S. Latour: Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product. J. Exp. Med., 201(5), 695-701 (2005)
;doi:10.1084/jem.20042432

21. T. Hu, A. Simmons, J. Yuan, T. P. Bender and J. Alberola-Ila: The transcription factor c-Myb primes CD4+CD8+ immature thymocytes for selection into the iNKT lineage. Nat. Immunol., 11(5), 435-441 (2010)
;doi:10.1038/ni.1865

22. D. Zhou, J. Mattner, C. Cantu, N. Schrantz, N. Yin, Y. Gao, Y. Sagiv, K. Hudspeth, Y. Wu, T. Yamashita, S. Teneberg, D. Wang, R. Proia, S. B. Levery, P. B. Savage, L. Teyton and A. Bendelac: Lysosomal glycosphingolipid recognition by NKT cells. Science, 306(5702), 1786-1789 (2004)
;doi:10.1126/science.1103440

23. A. K. Stanic, J. S. Bezbradica, J. J. Park, N. Matsuki, A. L. Mora, L. Van Kaer, M. R. Boothby and S. Joyce: NF-kappa B controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes. J. Immunol., 172(4), 2265-2273 (2004)

24. T. Chun, M. J. Page, L. Gapin, J. L. Matsuda, H. Xu, H. Nguyen, H. S. Kang, A. K. Stanic, S. Joyce, W. A. Koltun, M. J. Chorney, M. Kronenberg and C. R. Wang: CD1d-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells. J. Exp. Med., 197(7), 907-918 (2003)
;doi:10.1084/jem.20021366

25. D. G. Pellicci, A. P. Uldrich, K. Kyparissoudis, N. Y. Crowe, A. G. Brooks, K. J. Hammond, S. Sidobre, M. Kronenberg, M. J. Smyth and D. I. Godfrey: Intrathymic NKT cell development is blocked by the presence of alpha-galactosylceramide. Eur. J. Immunol., 33(7), 1816-1823 (2003)
;doi:10.1002/eji.200323894

26. D. G. Wei, H. Lee, S. H. Park, L. Beaudoin, L. Teyton, A. Lehuen and A. Bendelac: Expansion and long-range differentiation of the NKT cell lineage in mice expressing CD1d exclusively on cortical thymocytes. J. Exp. Med., 202(2), 239-248 (2005)
;doi:10.1084/jem.20050413

27. M. I. Zimmer, A. Colmone, K. Felio, H. Xu, A. Ma and C. R. Wang: A cell-type specific CD1d expression program modulates invariant NKT cell development and function. J. Immunol., 176(3), 1421-1430 (2006)

28. I. Engel, K. Hammond, B. A. Sullivan, X. He, I. Taniuchi, D. Kappes and M. Kronenberg: Co-receptor choice by V alpha14i NKT cells is driven by Th-POK expression rather than avoidance of CD8-mediated negative selection. J. Exp. Med., 207(5), 1015-1029 (2010)
;doi:10.1084/jem.20090557

29. L. Gapin: The making of NKT cells. Nat. Immunol., 9(9), 1009-1011 (2008)
;doi:10.1038/ni0908-1009

30. D. Kovalovsky, O. U. Uche, S. Eladad, R. M. Hobbs, W. Yi, E. Alonzo, K. Chua, M. Eidson, H. J. Kim, J. S. Im, P. P. Pandolfi and D. B. Sant'Angelo: The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions. Nat. Immunol., 9(9), 1055-1064 (2008)
;doi:10.1038/ni.1641

31. A. K. Savage, M. G. Constantinides, J. Han, D. Picard, E. Martin, B. Li, O. Lantz and A. Bendelac: The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity, 29(3), 391-403 (2008)
;doi:10.1016/j.immuni.2008.07.011

32. J. S. Bezbradica, L. E. Gordy, A. K. Stanic, S. Dragovic, T. Hill, D. Hawiger, D. Unutmaz, L. Van Kaer and S. Joyce: Granulocyte-macrophage colony-stimulating factor regulates effector differentiation of invariant natural killer T cells during thymic ontogeny. Immunity, 25, 487-497 (2006)
;doi:10.1016/j.immuni.2006.06.017

33. T. Ranson, C. A. Vosshenrich, E. Corcuff, O. Richard, V. Laloux, A. Lehuen and J. P. Di Santo: IL-15 availability conditions homeostasis of peripheral natural killer T cells. Proc. Natl. Acad. Sci. USA, 100(5), 2663-2668 (2003)
;doi:10.1073/pnas.0535482100

34. J. L. Matsuda, L. Gapin, S. Sidobre, W. C. Kieper, J. T. Tan, C. D. Surh and M. Kronenberg: Homeostasis of Va14i NKT cells. Nat. Immunol., 3, 966-974 (2002)
;doi:10.1038/ni837

35. F. Geissmann, T. O. Cameron, S. Sidobre, N. Manlongat, M. Kronenberg, M. J. Briskin, M. L. Dustin and D. R. Littman: Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids. PloS Biol., 3(4), 650-661 (2005)
;doi:10.1371/journal.pbio.0030113

36. E. Germanov, L. Veinotte, R. Cullen, E. Chamberlain, E. C. Butcher and B. Johnston: Critical role for the chemokine receptor CXCR6 in homeostasis and activation of CD1d-restricted NKT cells. J. Immunol., 181(1), 81-91 (2008)

37. T. Ohteki, C. Maki, S. Koyasu, T. W. Mak and P. S. Ohashi: Cutting edge: LFA-1 is required for liver NK1.1+TCR alpha beta+ cell development: evidence that liver NK1.1+TCR alpha beta+ cells originate from multiple pathways. J. Immunol., 162(7), 3753-3756 (1999)

38. M. Emoto, H. W. Mittrucker, R. Schmits, T. W. Mak and S. H. Kaufmann: Critical role of leukocyte function-associated antigen-1 in liver accumulation of CD4+NKT cells. J. Immunol., 162(9), 5094-5098 (1999)

39. L. A. Monticelli, Y. Yang, J. Knell, L. M. D'Cruz, M. A. Cannarile, I. Engel, M. Kronenberg and A. W. Goldrath: Transcriptional regulator Id2 controls survival of hepatic NKT cells. Proc. Natl. Acad. Sci. USA, 106(46), 19461-19466 (2009)
;doi:10.1073/pnas.0908249106

40. B. Wei, G. Wingender, D. Fujiwara, D. Y. Chen, M. McPherson, S. Brewer, J. Borneman, M. Kronenberg and J. Braun: Commensal microbiota and CD8+ T cells shape the formation of invariant NKT cells. J. Immunol., 184(3), 1218-1226 (2010)
;doi:10.4049/jimmunol.0902620

41. M. M. Venkataswamy and S. A. Porcelli: Lipid and glycolipid antigens of CD1d-restricted natural killer T cells. Semin. Immunol., 22(2), 68-78 (2010)
;doi:10.1016/j.smim.2009.10.003

42. J. E. Gumperz, C. Roy, A. Makowska, D. Lum, M. Sugita, T. Podrebarac, Y. Koezuka, S. A. Porcelli, S. Cardell, M. B. Brenner and S. M. Behar: Murine CD1d-restricted T cell recognition of cellular lipids. Immunity, 12(2), 211-221 (2000)
;doi:10.1016/S1074-7613(00)80174-0

43. L. M. Fox, D. G. Cox, J. L. Lockridge, X. Wang, X. Chen, L. Scharf, D. L. Trott, R. M. Ndonye, N. Veerapen, G. S. Besra, A. R. Howell, M. E. Cook, E. J. Adams, W. H. Hildebrand and J. E. Gumperz: Recognition of lyso-phospholipids by human natural killer T lymphocytes. PLoS Biol., 7(10), e1000228 (2009)
;doi:10.1371/journal.pbio.1000228

44. D. Y. Wu, N. H. Segal, S. Sidobre, M. Kronenberg and P. B. Chapman: Cross-presentation of disialoganglioside GD3 to natural killer T cells. J. Exp. Med., 198(1), 173-181 (2003)
;doi:10.1084/jem.20030446

45. K. Fischer, E. Scotet, M. Niemeyer, H. Koebernick, J. Zerrahn, S. Maillet, R. Hurwitz, M. Kursar, M. Bonneville, S. H. E. Kaufmann and U. E. Schaible: Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc. Natl. Acad. Sci. USA, 101(29), 10685-10690 (2004)
;doi:10.1073/pnas.0403787101

46. Y. Kinjo, E. Tupin, D. Wu, M. Fujio, R. Garcia-Navarro, M. Benhnia, D. M. Zajonc, G. Ben-Menachem, G. D. Ainge, G. F. Painter, A. Khurana, K. Hoebe, S. M. Behar, B. Buetler, I. A. Wilson, M. Tsuji, T. J. Sellati, C. H. Wong and M. Kronenberg: Natural killer T cells recognize diacylglycerol antigens from pathogenic bacteria. Nat. Immunol., 7(9), 978-986 (2006)
;doi:10.1038/ni1380

47. J. L. Amprey, J. S. Im, S. J. Turco, H. W. Murray, P. A. Illarionov, G. S. Besra, S. A. Porcelli and G. F. Spath: A subset of liver NK T cells is activated during Leishmania donovani infection by CD1d-bound lipophosphoglycan. J. Exp. Med., 200(7), 895-904 (2004)
;doi:10.1084/jem.20040704

48. H. Lotter, N. Gonzalez-Roldan, B. Lindner, F. Winau, A. Isibasi, M. Moreno-Lafont, A. J. Ulmer, O. Holst, E. Tannich and T. Jacobs: Natural killer T cells activated by a lipopeptidophosphoglycan from Entamoeba histolytica are critically important to control amebic liver abscess. PLoS Pathog., 5(5), e1000434 (2009)
;doi:10.1371/journal.ppat.1000434

49. Y. Kinjo, D. Y. Wu, G. Kim, G.-W. Xing, M. A. Poles, D. D. Ho, M. Tsuji, K. Kawahara, C.-H. Wong and M. Kronenberg: Recognition of bacterial glycosphingolipids by natural killer T cells. Nature, 434(7032), 520-525 (2005)
;doi:10.1038/nature03407

50. J. Mattner, K. L. DeBord, N. Ismail, R. D. Goff, C. I. Cantu, D. Zhou, P. Saint-Mezard, V. Wang, Y. Gao, N. Yin, K. Hoebe, O. Schneewind, D. Walker, B. Buetler, L. Teyton, P. B. Savage and A. Bendelac: Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature, 434(7032), 525-529 (2005)
;doi:10.1038/nature03408

51. V. Sriram, W. Du, J. Gervay-Hague and R. R. Brutkiewicz: Cell wall glycosphingolipids of Sphingomonas paucimobilis are CD1d-specific ligand for NKT cells. Eur. J. Immunol., 35(6), 1692-1701 (2005)
;doi:10.1002/eji.200526157

52. T. Kawano, J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, H. Koseki and M. Taniguchi: CD1d-restricted and TCR-mediated activation of Va14 NKT cells by glycosylceramides. Science, 278(5343), 1626-1629 (1997)
;doi:10.1126/science.278.5343.1626

53. V. V. Parekh, M. T. Wilson and L. Van Kaer: iNKT-cell responses to glycolipids. Crit. Rev. Immunol., 25(3), 183-213 (2005)
;doi:10.1615/CritRevImmunol.v25.i3.20

54. D. I. Godfrey, D. G. Pellicci, O. Patel, L. Kjer-Nielsen, J. McCluskey and J. Rossjohn: Antigen recognition by CD1d-restricted NKT T cell receptors. Semin. Immunol., 22(2), 61-67 (2010)
;doi:10.1016/j.smim.2009.10.004

55. W. C. Florence, R. K. Bhat and S. Joyce: CD1d-restricted glycolipid antigens: presentation principles, recognition logic and functional consequences. Expert. Rev. Mol. Med., 10, e20 (2008)
;doi:10.1017/S1462399408000732

56. Z. Zeng, A. R. Castano, B. W. Segelke, E. A. Stura, P. A. Peterson and I. A. Wilson: Crystal structure of mouse CD1: An MHC-like fold with a large hydrophobic binding groove. Science, 277(5324), 339-345 (1997)
;doi:10.1126/science.277.5324.339

57. M. Koch, V. S. Stronge, D. Shepherd, S. D. Gadola, B. Mathew, G. Ritter, A. R. Fersht, G. S. Besra, R. R. Schmidt, E. Y. Jones and V. Cerundolo: The crystal structure of human CD1d with and without a-galactosylceramide. Nat. Immunol., 6(8), 819-826 (2005)
;doi:10.1038/ni1225

58. D. M. Zajonc, C. r. Cantu, J. Mattner, D. Zhou, P. B. Savage, A. Bendelac, I. A. Wilson and L. Teyton: Structure and function of a potent agonist for the semi-invariant natural killer T cell receptor. Nat. Immunol., 6(8), 810-818 (2005)
;doi:10.1038/ni1224

59. D. Wu, D. M. Zajonc, M. Fujio, B. A. Sullivan, Y. Kinjo, M. Kronenberg, I. A. Wilson and C. H. Wong: Design of natural killer T cell activators: structure and function of a microbial glycosphingolipid bound to mouse CD1d. Proc. Natl. Acad. Sci. USA, 103(11), 3972-3977 (2006)
;doi:10.1073/pnas.0600285103

60. D. M. Zajonc, P. B. Savage, A. Bendelac, I. A. Wilson and L. Teyton: Crystal structures of mouse CD1d-iGb3 complex and its cognate Valpha14 T cell receptor suggest a model for dual recognition of foreign and self glycolipids. J. Mol. Biol., 377(4), 1104-1116 (2008)
;doi:10.1016/j.jmb.2008.01.061

61. D. G. Pellicci, O. Patel, L. Kjer-Nielsen, S. S. Pang, L. C. Sullivan, K. Kyparissoudis, A. G. Brooks, H. H. Reid, S. Gras, I. S. Lucet, R. Koh, M. J. Smyth, T. Mallevaey, J. L. Matsuda, L. Gapin, J. McCluskey, D. I. Godfrey and J. Rossjohn: Differential recognition of CD1d-alpha-galactosyl ceramide by the V beta 8.2 and V beta 7 semi-invariant NKT T cell receptors. Immunity, 31(1), 47-59 (2009)
;doi:10.1016/j.immuni.2009.04.018

62. N. A. Borg, K. S. Wun, L. Kjer-Nielsen, M. C. Wilce, D. G. Pellicci, R. Koh, G. S. Besra, M. Bharadwaj, D. I. Godfrey, J. McCluskey and J. Rossjohn: CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor. Nature, 448(7149), 44-49 (2007)
;doi:10.1038/nature05907

63. J. P. Scott-Browne, J. L. Matsuda, T. Mallevaey, J. White, N. A. Borg, J. McCluskey, J. Rossjohn, J. Kappler, P. Marrack and L. Gapin: Germline-encoded recognition of diverse glycolipids by natural killer T cells. Nat. Immunol., 8, 1105-1113 (2007)
;doi:10.1038/ni1510

64. K. S. Wun, N. A. Borg, L. Kjer-Nielsen, T. Beddoe, R. Koh, S. K. Richardson, M. Thakur, A. R. Howell, J. P. Scott-Browne, L. Gapin, D. I. Godfrey, J. McCluskey and J. Rossjohn: A minimal binding footprint on CD1d-glycolipid is a basis for selection of the unique human NKT TCR. J. Exp. Med., 205(4), 939-949 (2008)
;doi:10.1084/jem.20072141

65. T. Mallevaey, J. P. Scott-Browne, J. L. Matsuda, M. H. Young, D. G. Pellicci, O. Patel, M. Thakur, L. Kjer-Nielsen, S. K. Richardson, V. Cerundolo, A. R. Howell, J. McCluskey, D. I. Godfrey, J. Rossjohn, P. Marrack and L. Gapin: T cell receptor CDR2 beta and CDR3 beta loops collaborate functionally to shape the iNKT cell repertoire. Immunity, 31(1), 60-71 (2009)
;doi:10.1016/j.immuni.2009.05.010

66. W. C. Florence, R. K. Bhat and S. Joyce: CD1d-restricted glycolipid antigens: presentation principles, recognition logic and functional consequences. Expert Rev. Mol. Med., 10, e20 (2008)
;doi:10.1017/S1462399408000732

67. M. Salio, J. D. Silk and V. Cerundolo: Recent advances in processing and presentation of CD1 bound lipid antigens. Curr. Opin. Immunol., 22(1), 81-88 (2010)
;doi:10.1016/j.coi.2009.12.008

68. S. Brozovic, T. Nagaishi, M. Yoshida, S. Betz, A. Salas, D. Chen, A. Kaser, J. Glickman, T. Kuo, A. Little, J. Morrison, N. Corazza, J. Y. Kim, S. P. Colgan, S. G. Young, M. Exley and R. S. Blumberg: CD1d function is regulated by microsomal triglyceride transfer protein. Nat. Med., 10, 535-539 (2004)
;doi:10.1038/nm1043

69. P. van den Elzen, S. Garg, L. Leon, M. Brigl, E. A. Leadbetter, J. E. Gumperz, C. C. Dascher, T. Y. Cheng, F. M. Sacks, P. A. Illarionov, G. S. Besra, S. C. Kent, D. B. Moody and M. B. Brenner: Apolipoprotein-mediated pathways of lipid antigen presentation. Nature, 437(7060), 906-910 (2005)
;doi:10.1038/nature04001

70. S. Freigang, V. Zadorozhny, M. K. McKinney, P. Krebs, R. Herro, J. Pawlak, L. Kain, N. Schrantz, K. Masuda, Y. Liu, P. B. Savage, A. Bendelac, B. F. Cravatt and L. Teyton: Fatty acid amide hydrolase shapes NKT cell responses by influencing the serum transport of lipid antigen in mice. J. Clin. Invest., 20(6), 1873-1884 (2010)

71. T. I. Prigozy, O. Naidenko, P. Qasba, D. Elewaut, L. Brossay, A. Khurana, T. Natori, Y. Koezuka, A. Kulkarni and M. Kronenberg: Glycolipid antigen processing for presentation by CD1d molecules. Science, 291(5504), 664-667 (2001)
;doi:10.1126/science.291.5504.664

72. W. Yuan, X. Qi, P. Tsang, S. J. Kang, P. A. Illarionov, G. S. Besra, J. Gumperz and P. Cresswell: Saposin B is the dominant saposin that facilitates lipid binding to human CD1d molecules. Proc. Natl. Acad. Sci. USA, 104(13), 5551-5556 (2007)
;doi:10.1073/pnas.0700617104

73. N. Schrantz, Y. Sagiv, Y. Liu, P. B. Savage, A. Bendelac and L. Teyton: The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells. J. Exp. Med., 204(4), 841-852 (2007)
;doi:10.1084/jem.20061562

74. D. Zhou, C. r. Cantu, Y. Sagiv, N. Schrantz, A. B. Kukarni, X. Qi, D. J. Mahuran, C. R. Morales, G. A. Grabowski, K. Benlagha, P. B. Savage, A. Bendelac and L. Teyton: Editing of CD1d-bound lipid antigens by endosomal lipid transfer proteins. Science, 303, 523-527 (2004)
;doi:10.1126/science.1092009

75. L. Van Kaer and S. Joyce: Viral evasion of antigen presentation: not just for peptides anymore. Nat. Immunol., 7, 795-797 (2006)
;doi:10.1038/ni0806-795

76. K. Kawana, A. J. Quayle, M. Ficarra, J. A. Ibana, L. Shen, Y. Kawana, H. Yang, L. Marrero, S. Yavagal, S. J. Greene, Y. X. Zhang, R. B. Pyles, R. S. Blumberg and D. J. Schust: CD1d degradation in Chlamydia trachomatis-infected epithelial cells is the result of both cellular and chlamydial proteasomal activity. J. Biol. Chem., 282(10), 7368-7375 (2007)
;doi:10.1074/jbc.M610754200

77. L. Van Kaer and S. Joyce: Innate immunity: NKT cells in the spotlight. Curr. Biol., 15(11), R429-R431 (2005)
;doi:10.1016/j.cub.2005.05.032

78. M. Brigl, L. Bry, S. C. Kent, J. E. Gumperz and M. B. Brenner: Mechanism of CD1d-restricted natural killer T cell activation during microbial infection. Nat. Immunol., 4(12), 1230-1237 (2003)
;doi:10.1038/ni1002

79. E. Tupin, T. Kinjo and M. Kronenberg: The unique role of natural killer T cells in the response to microorganisms. Nat. Rev. Microbiol., 5(6), 405-417 (2007)
;doi:10.1038/nrmicro1657

80. M. Salio, A. O. Speak, D. Shepherd, P. Polzella, P. A. Illarionov, N. Veerapen, G. S. Besra, F. M. Platt and V. Cerundolo: Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation. Proc. Natl. Acad. Sci. USA, 104(51), 20490-20495 (2007)
;doi:10.1073/pnas.0710145104

81. C. Paget, T. Mallevaey, A. O. Speak, D. Torres, J. Fontaine, K. C. Sheehan, M. Capron, B. Ryffel, C. Faveeuw, M. Leite de Moraes, F. Platt and F. Trottein: Activation of invariant NKT cells by toll-like receptor 9-stimulated dendritic cells requires type I interferon and charged glycosphingolipids. Immunity, 27(4), 597-609 (2007)
;doi:10.1016/j.immuni.2007.08.017

82. K. Muindi, M. Cernadas, G. F. Watts, L. Royle, D. C. Neville, R. A. Dwek, G. S. Besra, P. M. Rudd, T. D. Butters and M. B. Brenner: Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphingolipids presented by CD1d. Proc. Natl. Acad. Sci. USA, 107(7), 3052-3057 (2010)
;doi:10.1073/pnas.0915056107

83. G. Raghuraman, Y. Geng and C.-R. Wang: IFN-b-mediated up-regulation of CD1d in bacteria-infected APCs. J. Immunol., 177, 7841-7848 (2006)

84. N. A. Nagarajan and M. Kronenberg: Invariant NKT cells amplify the innate immune response to lipopolysaccharide. J. Immunol., 178, 2706-2713 (2007)

85. M. L. Michel, A. C. Keller, C. Paget, M. Fujio, F. Trottein, P. B. Savage, C. H. Wong, E. Schneider, M. Dy and M. C. Leite-de-Moraes: Identification of an IL-17-producing NK1.1(neg) iNKT cell population involved in airway neutrophilia. J. Exp. Med., 204(5), 995-1001 (2007)
;doi:10.1084/jem.20061551

86. M. T. Wilson, C. Johansson, D. Olivares-Villagomez, A. K. Singh, A. K. Stanic, C. R. Wang, S. Joyce, M. J. Wick and L. Van Kaer: The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion. Proc. Natl. Acad. Sci. USA, 100(19), 10913-10918 (2003)
;doi:10.1073/pnas.1833166100

87. N. Y. Crowe, A. P. Uldrich, K. Kyparissoudis, K. J. L. Hammond, K. Hayakawa, S. Sidobre, R. Keating, M. Kronenberg, M. J. Smyth and D. I. Godfrey: Glycolipid antigen drives rapid expansion and sustained cytokine production by NKT cells. J. Immunol., 171(8), 4020-4027 (2003)

88. V. V. Parekh, M. T. Wilson, D. Olivares-Villagomez, A. K. Singh, L. Wu, C.-R. Wang, S. Joyce and L. Van Kaer: Glycolipid antigen induces long-term natural killer T cell anergy in mice. J. Clin. Invest., 115(9), 2572-2583 (2005)
;doi:10.1172/JCI24762

89. A. P. Ulrich, N. Y. Crowe, K. Kyparissoudis, D. G. Pellicci, Y. Zhan, A. M. Lew, P. Bouillet, A. Strasser, M. J. Smyth and D. I. Godfrey: NKT cell stimulation with glycolipid antigen in vivo: costimulation-dependent expansion, bim-dependent contraction, and hyporesponsiveness to further antigenic challenge. J. Immunol., 175(5), 3092-3101 (2005)

90. V. V. Parekh, A. K. Singh, M. T. Wilson, D. Olivares-Villagomez, J. S. Bezbradica, H. Inazawa, H. Ehara, T. Sakai, I. Serizawa, L. Wu, C. R. Wang, S. Joyce and L. Van Kaer: Quantitative and qualitative differences in the in vivo response of NKT cells to distinct a- and b-anomeric glycolipids. J. Immunol., 173(6), 3693-3706 (2004)

91. V. V. Parekh, S. Lalani and L. Van Kaer: The in vivo response of invariant natural killer T cells to glycolipid antigens. Int. Rev. Immunol., 26(1-2), 31-48 (2006)

92. J. L. Matsuda, T. Mallevaey, J. Scott-Browne and L. Gapin: CD1d-restricted iNKT cells, the 'Swiss-Army knife' of the immune system. Curr. Opin. Immunol., 20(3), 358-368 (2008)
;doi:10.1016/j.coi.2008.03.018

93. K. Miyamoto, S. Miyake and T. Yamamura: A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells. Nature, 413(6855), 531-534 (2001)
;doi:10.1038/35097097

94. K. O. Yu, J. S. Im, A. Molano, Y. Dutronc, P. A. Illarionov, C. Forestier, N. Fujiwara, I. Arias, S. Miyake, T. Yamamura, Y. T. Chang, G. S. Besra and S. A. Porcelli: Modulation of CD1d-restricted NKT cell responses by using N-acyl variants of a-galactosylceramides. Proc. Natl. Acad. Sci. USA, 102(9), 3383-3388 (2005)
;doi:10.1073/pnas.0407488102

95. J. Schmieg, G. Yang, R. W. Franck and M. Tsuji: Superior protection against malaria and melanoma metastases by a C-glycoside analogue of the natural killer T cell ligand a-galactosylceramide. J. Exp. Med., 198(11), 1631-1641 (2003)
;doi:10.1084/jem.20031192

96. T. Tashiro, E. Sekine-Kondo, T. Shigeura, R. Nakagawa, S. Inoue, M. Omori-Miyake, T. Chiba, N. Hongo, S. Fujii, K. Shimizu, Y. Yoshiga, T. Sumida, K. Mori, H. Watarai and M. Taniguchi: Induction of Th1-biased cytokine production by alpha-carba-GalCer, a neoglycolipid ligand for NKT cells. Int. Immunol., 22(4), 319-328 (2010)
;doi:10.1093/intimm/dxq012

97. S. Fujii, K. Shimizu, C. Smith, L. Bonifaz and R. M. Steinman: Activation of natural killer T cells by a-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein. J. Exp. Med., 198(2), 267-279 (2003)
;doi:10.1084/jem.20030324

98. S. M. Behar and S. A. Porcelli: CD1-restricted T cells in host defense to infectious diseases. Curr. Top. Microbiol. Immunol., 314, 215-250 (2007)
;doi:10.1007/978-3-540-69511-0_9

99. E. Tupin, M. R. Benhnia, Y. Kinjo, R. Patsey, C. J. Lena, M. C. Haller, M. J. Caimano, M. Imamura, C. H. Wong, S. Crotty, J. D. Radolf, T. J. Sellati and M. Kronenberg: NKT cells prevent chronic joint inflammation after infection with Borrelia burgdorferi. Proc. Natl. Acad. Sci. USA, 105(50), 19863-19868 (2008)
;doi:10.1073/pnas.0810519105

100. A. G. Joyee, H. Qiu, S. Wang, Y. Fan, L. Bilenki and X. Yang: Distinct NKT cell subsets are induced by different Chlamydia species leading to differential adaptive immunity and host resistance to the infections. J. Immunol., 178(2), 1048-1058 (2007)

101. T. R. Johnson, S. Hong, L. Van Kaer, Y. Koezuka and B. S. Graham: NK T cells contribute to expansion of CD8+ T cells and amplification of antiviral immune responses to respiratory syncytial virus. J. Virol., 76(9), 4294-4303 (2002)
;doi:10.1128/JVI.76.9.4294-4303.2002

102. D. S. Hansen, M. A. Siomos, L. Buckingham, A. A. Scalzo and L. Schofield: Regulation of murine cerebral malaria pathogenesis by CD1d-restricted NKT cells and the natural killer complex. Immunity, 18(3), 391-402 (2003)
;doi:10.1016/S1074-7613(03)00052-9

103. E. E. Nieuwenhuis, T. Matsumoto, D. Lindenbergh, R. Willemsen, A. Kaser, Y. Simons-Oosterhuis, S. Brugman, K. Yamaguchi, H. Ishikawa, Y. Aiba, Y. Koga, J. N. Samsom, K. Oshima, M. Kikuchi, J. C. Escher, M. Hattori, A. B. Onderdonk and R. S. Blumberg: Cd1d-dependent regulation of bacterial colonization in the intestine of mice. J. Clin. Invest., 119(5), 1241-1250 (2009)
;doi:10.1172/JCI36509

104. M. Terabe and J. A. Berzofsky: The role of NKT cells in tumor immunity. Adv. Cancer. Res., 101, 277-348 (2008)
;doi:10.1016/S0065-230X(08)00408-9

105. L. Wu and L. Van Kaer: Natural killer T cells and autoimmune disease. Curr. Mol. Med., 9(1), 4-14 (2009)
;doi:10.2174/156652409787314534

106. C. L. Gabriel, L. Wu, V. V. Parekh and L. Van Kaer: Invariant natural killer T cell-based therapy of autoimmune diseases. Curr. Immunol. Rev., 6, 88-101 (2010)
;doi:10.2174/157339510791111682

107. G. Sireci, D. Russo, F. Dieli, S. A. Porcelli, M. Taniguchi, M. P. La Manna, D. Di Liberto, F. Scarpa and A. Salerno: Immunoregulatory role of Jalpha281 T cells in aged mice developing lupus-like nephritis. Eur. J. Immunol., 37(2), 425-433 (2007)
;doi:10.1002/eji.200636695

108. F. Wermeling, S. M. Lind, E. D. Jordo, S. L. Cardell and M. C. Karlsson: Invariant NKT cells limit activation of autoreactive CD1d-positive B cells. J. Exp. Med., 207(5), 943-952 (2010)
;doi:10.1084/jem.20091314

109. K. Coppieters, P. Dewint, K. Van Beneden, P. Jacques, S. Seeuws, G. Verbruggen, D. Deforce and D. Elewaut: NKT cells: manipulable managers of joint inflammation. Rheumatology, 46(4), 565-571 (2007)
;doi:10.1093/rheumatology/kel437

110. Y. H. Chuang, Z. X. Lian, G. X. Yang, S. A. Shu, Y. Moritoki, W. M. Ridgway, A. A. Ansari, M. Kronenberg, R. A. Flavell, B. Gao and M. E. Gershwin: Natural killer T cells exacerbate liver injury in a transforming growth factor beta receptor II dominant-negative mouse model of primary biliary cirrhosis. Hepatology, 47(2), 571-580 (2008)
;doi:10.1002/hep.22052

111. J. Mattner, P. B. Savage, P. Leung, S. S. Oertelt, V. Wang, O. Trivedi, S. T. Scanlon, K. Pendem, L. Teyton, J. Hart, W. M. Ridgway, L. S. Wicker, M. E. Gershwin and A. Bendelac: Liver autoimmunity triggered by NKT cells. Cell Host Microbe, 3, 304-315 (2008)
;doi:10.1016/j.chom.2008.03.009

112. S. Joyce and L. Van Kaer: Invariant natural killer T cells trigger adaptive lymphocytes to churn up bile. Cell Host Microbe, 3, 275-277 (2008)
;doi:10.1016/j.chom.2008.04.007

113. O. Akbari, P. Stock, E. Meyer, M. Kronenberg, S. Sidobre, T. Nakayama, M. Taniguchi, M. J. Grusby, R. H. DeKruyff and D. T. Umetsu: Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity. Nat. Med., 9, 582-588 (2003)
;doi:10.1038/nm851

114. E. Tupin, A. Nicoletti, R. Elhage, M. Rudling, H. G. Ljunggren, G. K. Hansson and G. P. Berne: CD1d-dependent activation of NKT cells aggravates atherosclerosis. J. Exp. Med., 199(3), 417-422 (2004)
;doi:10.1084/jem.20030997

115. A. S. Major, M. T. Wilson, J. L. McCaleb, Y. Ru Su, A. K. Stanic, S. Joyce, L. Van Kaer, S. Fazio and M. F. Linton: Quantitative and qualitative differences in proatherogenic NKT cells in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol., 24(12), 2351-2357 (2004)
;doi:10.1161/01.ATV.0000147112.84168.87

116. Y. Nakai, K. Iwabuchi, S. Fujii, N. Ishimori, N. Dashtsoodol, K. Watano, T. Mishima, C. Iwabuchi, S. Tanaka, J. S. Bezbradica, T. Nakayama, M. Taniguchi, S. Miyake, T. Yamamura, A. Kitabatake, S. Joyce, L. Van Kaer and K. Onoe: Natural killer T cells accelerate atherogenesis in mice. Blood, 104(7), 2051-2059 (2004)
;doi:10.1182/blood-2003-10-3485

117. R. A. Campos, M. Szczepanik, A. Itakura, M. Akahira-Azuma, S. Sidobre, M. Kronenberg and P. W. Askenase: Cutaneous immunization rapidly activates liver invariant Va14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact hypersensitivity. J. Exp. Med., 198(12), 1785-1796 (2003)
;doi:10.1084/jem.20021562

118. M. I. Zimmer, H. P. Nguyen, B. Wang, H. Xu, A. Colmone, K. Felio, H. J. Choi, P. Zhou, M. L. Alegre and C. R. Wang: Polymorphisms in CD1d affect antigen presentation and the activation of CD1d-restricted T cells. Proc. Natl. Acad. Sci. USA, 106(6), 1909-1914 (2009)
;doi:10.1073/pnas.0808476106

119. F. Heller, I. J. Fuss, E. E. Nieuwenhuis, R. S. Blumberg and W. Strober: Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity, 17, 629-638 (2002)
;doi:10.1016/S1074-7613(02)00453-3

120. K. Takeda, Y. Hayakawa, L. Van Kaer, H. Matsuda, H. Yagita and K. Okumura: Critical contribution of liver natural killer T cells to a murine model of hepatitis. Proc. Natl. Acad. Sci. USA, 97(10), 5498-5503 (2000)
;doi:10.1073/pnas.040566697

121. C. M. Lappas, Y. J. Day, M. A. Marshall, V. H. Engelhard and J. Linden: Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury by inhibiting CD1d-dependent NKT cell activation. J Exp Med, 203(12), 2639-2648 (2006)
;doi:10.1084/jem.20061097

122. K. L. Wallace, M. A. Marshall, S. I. Ramos, J. A. Lannigan, J. J. Field, R. M. Strieter and J. Linden: NKT cells mediate pulmonary inflammation and dysfunction in murine sickle cell disease through production of IFN-gamma and CXCR3 chemokines. Blood, 114(3), 667-676 (2009)
;doi:10.1182/blood-2009-02-205492

123. K. H. Sonoda, M. Exley, S. Snapper, S. P. Balk and J. Stein-Streilein: CD1-reactive natural killer T cells are required for development of systemic tolerance through an immune-privileged site. J. Exp. Med., 190(9), 1215-1226 (1999)
;doi:10.1084/jem.190.9.1215

124. J. Y. Niederkorn: Role of NKT cells in anterior chamber-associated immune deviation. Expert. Rev. Clin. Immunol., 5(2), 137-144 (2009)
;doi:10.1586/1744666X.5.2.137

125. K. I. Seino, K. Fukao, K. Muramoto, K. Yanagisawa, Y. Takada, S. Kakuta, Y. Iwakura, L. Van Kaer, K. Takeda, T. Nakayama, M. Taniguchi, H. Bashuda, H. Yagita and K. Okumura: Requirement for natural killer T (NKT) cells in the induction of allograft tolerance. Proc. Natl. Acad. Sci. USA, 98(5), 2577-2581 (2001)
;doi:10.1073/pnas.041608298

126. A. M. Moodycliffe, D. Nghiem, G. Clydesdale and S. E. Ullrich: Immune suppression and skin cancer development: regulation by NKT cells. Nat. Immunol., 1(6), 521-525 (2000)
;doi:10.1038/82782

127. D. E. Faunce, R. L. Gamelli, M. A. Choudhry and E. J. Kovacs: A role for CD1d-restricted NKT cells in injury-associated T cell suppression. J Leuk Biol, 73(6), 747-755 (2003)
;doi:10.1189/jlb.1102540

128. K. Ito, M. Karasawa, T. Kawano, T. Akasaka, H. Koseki, Y. Akutsu, E. Kondo, S. Sekiya, K. Sekikawa, M. Harada, M. Yamashita, T. Nakayama and M. Taniguchi: Involvement of decidual Va14 NKT cells in abortion. Proc. Natl. Acad. Sci. USA, 97(2), 740-744 (2000)
;doi:10.1073/pnas.97.2.740

129. A. B. Pillai, T. I. George, S. Dutt, P. Teo and S. Strober: Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation. J. Immunol., 178(10), 6242-6251 (2007)

130. S. Kim, S. Lalani, V. V. Parekh, L. Wu and L. Van Kaer: Glycolipid ligands of invariant natural killer T cells as vaccine adjuvants. Expert Rev. Vaccines, 7(10), 1519-1532 (2008)
;doi:10.1586/14760584.7.10.1519

131. V. Cerundolo, J. D. Silk, S. H. Masri and M. Salio: Harnessing invariant NKT cells in vaccination strategies. Nat. Rev. Immunol., 9(1), 28-38 (2009)
;doi:10.1038/nri2451

132. T. S. Devera, H. B. Shah, G. A. Lang and M. L. Lang: Glycolipid-activated NKT cells support the induction of persistent plasma cell responses and antibody titers. Eur. J. Immunol., 38(4), 1001-1011 (2008)
;doi:10.1002/eji.200738000

133. S. Fujii: Exploiting dendritic cells and natural killer T cells in immunotherapy against malignancies. Trends Immunol., 29(5), 242-249 (2008)
;doi:10.1016/j.it.2008.02.002

134. X. Li, M. Fujio, M. Imamura, D. Wu, S. Vasan, C. H. Wong, D. D. Ho and M. Tsuji: Design of a potent CD1d-binding NKT cell ligand as a vaccine adjuvant. Proc Natl Acad Sci USA, in press

135. G. Gonzalez-Aseguinolaza, L. Van Kaer, C. C. Bergmann, J. M. Wilson, J. Schmieg, M. Kronenberg, T. Nakayama, M. Taniguchi, Y. Koezuka and M. Tsuji: Natural killer T cell ligand a-galactosylceramide enhances protective immunity induced by malaria vaccines. J. Exp. Med., 195(5), 617-624 (2002)
;doi:10.1084/jem.20011889

136. K. Shimizu, Y. Kurosawa, M. Taniguchi, R. M. Steinman and S. Fujii: Cross-presentation of glycolipid from tumor cells loaded with alpha-galactosylceramide leads to potent and long-lived T cell mediated immunity via dendritic cells. J. Exp. Med., 204(11), 2641-2653 (2007)
;doi:10.1084/jem.20070458

137. T. Natori, Y. Koezuka and T. Higa: Agelasphins, novel a-galactosylceramides from the marine sponge Agelas mauritianus. Tetrahedron Lett., 34(35), 5591-5592 (1993)
;doi:10.1016/S0040-4039(00)73889-5

138. S. Motohashi and T. Nakayama: Clinical applications of natural killer T cell-based immunotherapy for cancer. Cancer Sci., 99(4), 638-645 (2008)
;doi:10.1111/j.1349-7006.2008.00730.x

139. S. Fujii, K. Shimizu, M. Kronenberg and R. M. Steinman: Prolonged IFN-g-producing NKT response induced with a-galactosylceramide-loaded DCs. Nat. Immunol., 3(9), 867-874 (2002)
;doi:10.1038/ni827

140. S. Motohashi, K. Nagato, N. Kunii, H. Yamamoto, K. Yamasaki, K. Okita, H. Hanaoka, N. Shimizu, M. Suzuki, I. Yoshino, M. Taniguchi, T. Fujisawa and T. Nakayama: A phase I-II study of alpha-galactosylceramide-pulsed IL-2/GM-CSF-cultured peripheral blood mononuclear cells in patients with advanced and recurrent non-small cell lung cancer. J. Immunol., 182(4), 2492-2501 (2009)
;doi:10.4049/jimmunol.0800126

141. A. M. Woltman, M. J. Ter Borg, R. S. Binda, D. Sprengers, B. M. von Blomberg, R. J. Scheper, K. Hayashi, N. Nishi, A. Boonstra, R. van der Molen and H. L. Janssen: Alpha-galactosylceramide in chronic hepatitis B infection: results from a randomized placebo-controlled Phase I/II trial. Antivir. Ther., 14(6), 809-818 (2009)
;doi:10.3851/IMP1295

142. B. J. Veldt, H. J. van der Vliet, B. M. von Blomberg, H. van Vlierberghe, G. Gerken, N. Nishi, K. Hayashi, R. J. Scheper, R. J. de Knegt, A. J. van den Eertwegh, H. L. Janssen and C. M. van Nieuwkerk: Randomized placebo controlled phase I/II trial of alpha-galactosylceramide for the treatment of chronic hepatitis C. J. Hepatol., 47(3), 356-365 (2007)
;doi:10.1016/j.jhep.2007.04.018

143. L. Van Kaer: a-Galactosylceramide therapy for autoimmune diseases: prospects and obstacles. Nat. Rev. Immunol., 5(1), 31-42 (2005)
;doi:10.1038/nri1531

144. S. Sharif, G. A. Arreaza, P. Zucker, Q. S. Mi, J. Sondhi, O. V. Naidenko, M. Kronenberg, Y. Koezuka, T. L. Delovitch, J. M. Gombert, M. Leite-De-Moraes, C. Gouarin, R. Zhu, A. Hameg, T. Nakayama, M. Taniguchi, F. Lepault, A. Lehuen, J. F. Bach and A. Herbelin: Activation of natural killer T cells by a-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes. Nat. Med., 7(9), 1057-1062 (2001)
;doi:10.1038/nm0901-1057

145. Y. N. Naumov, K. S. Bahjat, R. Gausling, R. Abraham, M. A. Exley, Y. Koezuka, S. B. Balk, J. L. Strominger, M. Clare-Salzer and S. B. Wilson: Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets. Proc. Natl. Acad. Sci. USA, 98(24), 13838-13843 (2001)
;doi:10.1073/pnas.251531798

146. B. Wang, Y. B. Geng and C. R. Wang: CD1-restricted NK T cells protect nonobese diabetic mice from developing diabetes. J. Exp. Med., 194(3), 313-320 (2001)
;doi:10.1084/jem.194.3.313

147. S. Hong, M. T. Wilson, I. Serizawa, L. Wu, N. Singh, O. V. Naidenko, T. Miura, T. Haba, D. C. Scherer, J. Wei, M. Kronenberg, Y. Koezuka and L. Van Kaer: The natural killer T-cell ligand a-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice. Nat. Med., 7(9), 1052-1056 (2001)
;doi:10.1038/nm0901-1052

148. A. Jahng, I. Maricic, C. Aguilera, S. Cardell, R. C. Halder and V. Kumar: Prevention of autoimmunity by targeting a distinct, noninvariant CD1d-reactive T cell population reactive to sulfatide. J. Exp. Med., 199(7), 947-957 (2004)
;doi:10.1084/jem.20031389

149. R. Furlan, A. Bergami, D. Cantarella, E. Brambilla, M. Taniguchi, P. Dellabona, G. Casorati and G. Martino: Activation of invariant NKT cells by a-GalCer administration protects mice from MOG35-55 induced EAE: critical roles for administration route and IFN-g. Eur. J. Immunol., 33(7), 1830-1838 (2003)
;doi:10.1002/eji.200323885

150. A. K. Singh, M. T. Wilson, S. Hong, D. Olivares-Villagomez, C. Du, A. K. Stanic, S. Joyce, S. Sriram, Y. Koezuka and L. Van Kaer: Natural killer T cell activation protects mice against experimental autoimmune encephalomyelitis. J. Exp. Med., 194(12), 1801-1811 (2001)
;doi:10.1084/jem.194.12.1801

151. J.-Q. Yang, V. Saxena, H. Xu, L. Van Kaer, C. R. Wang and R. R. Singh: Repeated a-galactosylceramide administration results in expansion of Va14 NKT cells and alleviates inflammatory dermatitis in MRL-lpr/lpr mice. J. Immunol., 171(8), 4439-4446 (2003)

152. A. K. Singh, J.-Q. Yang, V. V. Parekh, J. Wei, C.-R. Wang, S. Joyce, R. R. Singh and L. Van Kaer: The natural killer T cell ligand a-galactosylceramide prevents or promotes pristane-induced lupus in mice. Eur. J. Immunol., 35(4), 1143-1154 (2005)
;doi:10.1002/eji.200425861

153. K. Coppieters, K. Van Beneden, P. Jacques, P. Dewint, A. Vervloet, B. Vander Cruyssen, S. Van Calenbergh, G. Chen, R. W. Franck, G. Verbruggen, D. Deforce, P. Matthys, M. Tsuji, P. Rottiers and D. Elewaut: A single early activation of invariant NK T cells confers long-term protection against collagen-induced arthritis in a ligand-specific manner. J. Immunol., 179(4), 2300-2309 (2007)

154. A. Miellot, R. Zhu, S. Diem, M. C. Boissier, A. Herbelin and N. Bessis: Activation of invariant NK T cells protects against experimental rheumatoid arthritis by an IL-10-dependent pathway. Eur. J. Immunol., 35(12), 3704-3713 (2005)
;doi:10.1002/eji.200535235

155. R. Liu, A. La Cava, X.-F. Bai, Y.-h. Jee, M. Price, D. I. Campagnolo, P. Christadoss, T. L. Vollmer, L. Van Kaer and F.-D. Shi: Cooperation of invariant NKT cells and CD4+CD25+ regulatory T cells in the prevention of autoimmune myasthenia. J. Immunol., 175(12), 7898-7904 (2005)

156. Y. Nagayama, K. Watanabe, M. Niwa, S. M. McLachlan and B. Rapoport: Schistosoma mansoni and a-galactosylceramide: prophylactic effect of Th1 immune suppression in a mouse model of Graves' hyperthyroidism. J. Immunol., 173(3), 2167-2173 (2004)

157. R. S. Grajewski, A. M. Hansen, R. K. Agarwal, M. Kronenberg, S. Sidobre, S. B. Su, P. B. Silver, M. Tsuji, R. W. Franck, A. P. Lawton, C. C. Chan and R. R. Caspi: Activation of invariant NKT cells ameliorates experimental ocular autoimmunity by a mechanism involving innate IFN-gamma production and dampening of the adaptive Th1 and Th17 responses. J. Immunol., 181(7), 4791-4797 (2008)

158. J. P. Driver, F. Scheuplein, Y. G. Chen, A. E. Grier, S. B. Wilson and D. V. Serreze: Invariant natural killer T-cell control of type 1 diabetes: a dendritic cell genetic decision of a silver bullet or Russian roulette. Diabetes, 59(2), 423-432 (2010)
;doi:10.2337/db09-1116

159. A. W. Jahng, I. Maricic, B. Pedersen, N. Burdin, O. Naidenko, M. Kronenberg, Y. Koezuka and V. Kumar: Activation of natural killer T cells potentiates or prevents experimental autoimmune encephalomyelitis. J. Exp. Med., 194(12), 1789-1799 (2001)
;doi:10.1084/jem.194.12.1789

160. D. Zeng, Y. Liu, S. Sidobre, M. Kronenberg and S. Strober: Activation of natural killer T cells in NZB/W mice induces Th1-type immune responses exacerbating lupus. J. Clin. Invest., 112(8), 1211-1222 (2003)

161. H. Y. Kim, H. J. Kim, H. S. Min, S. Kim, W. S. Park, S. H. Park and D. H. Chung: NKT cells promote antibody-induced joint inflammation by suppressing transforming growth factor beta1 production. J. Exp. Med., 201(1), 41-47 (2005)
;doi:10.1084/jem.20041400

162. D. Hashimoto, S. Asakura, S. Miyake, T. Yamamura, L. Van Kaer, C. Liu, M. Tanimoto and T. Teshima: Stimulation of host NKT cells by synthetic glycolipid regulates acute graft-versus-host disease by inducing Th2 polarization of donor T cells. J. Immunol., 174(1), 551-556 (2005)

163. K. Haraguchi, T. Takahashi, A. Matsumoto, T. Asai, Y. Kanda, M. Kurokawa, S. Ogawa, H. Oda, M. Taniguchi, H. Hirai and S. Chiba: Host-residual invariant NK T cells attenuate graft-versus-host immunity. J. Immunol., 175(2), 1320-1328 (2005)

164. R. D. Kuns, E. S. Morris, K. P. Macdonald, K. A. Markey, H. M. Morris, N. C. Raffelt, T. Banovic, A. L. Don, V. Rowe, A. C. Burman, A. D. Clouston, C. Farah, G. S. Besra, P. A. Illarionov, M. J. Smyth, S. A. Porcelli and G. R. Hill: Invariant natural killer T cell-natural killer cell interactions dictate transplantation outcome after alpha-galactosylceramide administration. Blood, 113(23), 5999-6010 (2009)
;doi:10.1182/blood-2008-10-183335

165. Y. Ueno, S. Tanaka, M. Sumii, S. Miyake, S. Tazuma, M. Taniguchi, T. Yamamura and K. Chayama: Single dose of OCH improves mucosal T helper type 1/T helper type 2 cytokine balance and prevents experimental colitis in the presence of Va14 natural killer T cells in mice. Infl. Bow. Dis., 11(1), 35-41 (2005)
;doi:10.1097/00054725-200501000-00005

166. L. J. Saubermann, P. Beck, Y. P. De Jong, R. S. Pitman, M. S. Ryan, H. S. Kim, M. Exley, S. Snapper, S. P. Balk, S. J. Hagen, O. Kanauchi, K. Motoki, T. Sakai, C. Terhorst, Y. Koezuka, D. K. Podolsky and R. S. Blumberg: Activation of natural killer T cells by a-galactosylceramide in the presence of CD1d provides protection against colitis in mice. Gastroenterol., 119(1), 119-128 (2000)
;doi:10.1053/gast.2000.9114

167. N. A. Braun, R. Covarrubias and A. S. Major: Natural killer T cells and atherosclerosis: form and function meet pathogenesis. J. Innate Immun., 2(4), 316-324 (2010)
;doi:10.1159/000296915

168. G. H. van Puijvelde, E. J. van Wanrooij, A. D. Hauer, P. de Vos, T. J. van Berkel and J. Kuiper: Effect of natural killer T cell activation on the initiation of atherosclerosis. Thromb. Haemost., 102(2), 223-230 (2009)

169. E. H. Meyer, R. H. DeKruyff and D. T. Umetsu: iNKT cells in allergic disease. Curr. Top. Microbiol. Immunol., 314, 269-291 (2007)
;doi:10.1007/978-3-540-69511-0_11

170. V. Lombardi, P. Stock, A. K. Singh, J. Kerzerho, W. Yang, B. A. Sullivan, X. Li, T. Shiratsuchi, N. E. Hnatiuk, A. R. Howell, K. O. Yu, S. A. Porcelli, M. Tsuji, M. Kronenberg, S. B. Wilson and O. Akbari: A CD1d-dependent antagonist inhibits the activation of invariant NKT cells and prevents development of allergen-induced airway hyperreactivity. J. Immunol., 184(4), 2107-2115 (2010)
;doi:10.4049/jimmunol.0901208

171. H. Wakao: NKT cells: from totipotency to regenerative medicine. Arch. Immunol. Ther. Exp., 57(2), 117-128 (2009)
;doi:10.1007/s00005-009-0009-x

Key Words: Natural killer T cells, Innate Immunity, Glycolipid Antigens, CD1d, Immunotherapy, Immunomodulation, Vaccine Adjuvants, Review

Send Correspondence to: Luc Van Kaer, Department of Microbiology and Immunology, Room A-5301, Medical Center North, 1161 21st Avenue South, Vanderbilt University School of Medicine, Nashville, TN 37232-2363, USA, Tel: 1-615-343-2707, Fax: 1-615-343-2972, E-mail:luc.van.kaer@vanderbilt.edu