Invariant T-cell Receptor Research Articles

Structure-Activity Relationship Studies of Novel Glycosphingolipids That Stimulate Natural Killer T-Cells

KRN7000, an anticancer drug candidate developed by Kirin Brewery Co. in 1995, is an α-galactosyl ceramide. It is a ligand making a complex with CD1d protein, and it stimulates invariant natural killer T (NKT) cells, which are one of the lineages of immunocytes. NKT cells activated by recognition of the CD1d/KRN7000 complex with its invariant T-cell receptor (TCR) can induce both protective and regulatory immune responses. To determine the recognition and activation mechanisms of NKT cells and to develop drug candidates more effective than KRN7000, a large number of analogs of KRN7000 have been synthesized. Some of them show potent bioactivities and have the potential of being utilized as therapeutic agents. In this review, structure-activity relationship studies of novel glycolipids which stimulate NKT cells efficiently are summarized.

Dendritic Cell Internalization of α-Galactosylceramide from CD8 T Cells Induces Potent Antitumor CD8 T-cell Responses

Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.

Endogenous Ligands Selectively Stimulate Highly Avid Autoreactive Human Invariant Natural Killer T Cells with Distinctive T-Cell Receptor Vβ11 CDR3 Sequence Motifs

Abstract 999Human CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of innate T-cells that carry an invariant T-cell receptor (TCR) Vα24 chain paired with a TCR Vβ11 chain. They constitutively express a variety of activation and memory markers and possess the capacity to rapidly produce a variety of cytokines including IFN-γ and IL-4 upon TCR engagement. As an immune response launches, iNKT cells can induce innate immune responses and serve as a bridge between innate and adaptive immunity. In addition, it has also been shown that iNKT cells themselves have anti-tumor and anti-infectious effects in mice. However, to translate these findings and develop iNKT cell-mediated immunotherapy, iNKT cells must recognize target cells while sparing normal cells. Autoreactivity is primarily dictated by 1) the specificity and avidity of the TCR on iNKT cells; 2) the expression level of CD1d on target cells; and 3) the density and stability of endogenous ligand(s) presented by CD1d on target cells.In this study, we sought to determine the TCR Vβ11 CDR3 sequence motifs that distinguish high avidity and low avidity iNKT cells. We developed an artificial antigen-presenting cell (aAPC) to expand iNKT cells by transducing K562 with CD1d, CD80, and CD83. Using this CD1d+aAPC loaded with or without α-galactosylceramide (αGC), we expanded CD1d-restricted iNKT cells by stimulating primary CD3+ T cells expressing canonical iNKT TCR Vα24 chain. Expanded iNKT cells were stained with αGC-loaded CD1d tetramers and were found to express TCR Vβ11 chain in conjunction with transgenic canonical TCR Vα24 chain. Sequence analysis of cloned Vβ11 CDR3 revealed that the clonality of iNKT cells generated using unloaded aAPC was significantly lower than that of iNKT cells generated using loaded aAPC. Surprisingly, when cotransfected with canonical TCR Vα24 chain, some TCR Vβ11 chains isolated from iNKT cells generated using unloaded aAPC were stained with unloaded CD1d tetramers produced in HEK293 cells. This result suggests that these reconstituted iNKT TCR recognized endogenous ligand(s) derived from HEK293 cells in the context of CD1d. A comprehensive analysis of the structural avidity demonstrated that these TCR Vβ11 chains reconstituted TCR with significantly higher structural avidity than those cloned from NKT cells generated using loaded aAPC. However, this significant difference was only observed when the structural avidity was measured using unloaded tetramers but not αGC-loaded tetramers. A univariate analysis found that structural avidity was significantly higher when 1) Vβ11 CDR3 used J2-5; 2) Vβ11 CDR3 consisted of exactly 23 amino acids; and 3) Vβ11 CDR3 encoded 3 or more acidic amino acids (asparagic and glutamic acids). A multivariate analysis confirmed that all three variables were independent predictors of higher structural avidity. Furthermore, each variable is sufficient to increase the structural avidity of an iNKT TCR with low structural avidity. Intriguingly, unloaded mouse CD1d tetramers produced in HEK293 cells also stained a human iNKT TCR with high structural avidity reconstituted on both human and mouse T cells. This result suggests that the human iNKT TCR with high structural avidity possessed a cross-species reactivity to mouse CD1d in conjunction with endogenous ligand(s) derived from HEK293 cells.We next studied the autoreactivity of cloned human iNKT TCR with high structural avidity. A human T cell line, Jurkat, reconstituted with high avidity iNKT TCR, was able to recognize cells expressing CD1d endogenously (itself, SUP-T1, and primary human monocytes) and ectopically (K562, C1R, Hela). Furthermore, mouse T cells expressing human iNKT TCR with high structural avidity recognized mouse cell lines, B16, EL4, and 58, all endogenously expressing CD1d. Finally, human primary T cells transduced with the iNKT TCR with high structural avidity were autoreactive, secreting both IFN-γ and IL-4 in response to CD1d+ target cells.Using our CD1d+ aAPC-based system, we successfully isolated human iNKT cells with high structural avidity and identified the TCR Vβ11 CDR3 sequence motifs that dictate the structural avidity of iNKT cells. To develop clinically effective iNKT cell-mediated immunotherapy without unwanted autoimmunity, it will be critically important to define the range of iNKT TCR avidity that enables reactivity to pathologic but not normal cells. Disclosures:No relevant conflicts of interest to declare.

INKT Cell Frequency in Peripheral Blood of Caucasian Children and Adolescent: The Absolute iNKT Cell Count is Stable from Birth to Adulthood

Human invariant natural killer T cells (iNKT cells) are a unique population of T cells that express a semi-invariantly rearranged T cell receptor (TCR) and are involved in a variety of immunoregulatory processes. We assessed the frequency of peripheral blood iNKT cells in 64 healthy Caucasian children from 7 months to 18 years of age and five cord blood samples by flow cytometry. iNKT cells were measured as CD3(+) cells co-expressing TCRVα24 and TCRVβ11 and using the monoclonal antibody 6B11, which recognizes specifically their invariant TCR rearrangement. The absolute number of iNKT cells ranged from 86 to 10,499 (CD3(+) /TCRVα24(+) / TCRVβ11(+)) and 233 to 11,167 (CD3(+) /6B11(+)) iNKT cells per millilitre of blood. This range is stable from birth to adulthood. The relative iNKT cell count was found to be 0.003-0.71% (CD3(+) /TCRVα24/TCRVβ11) and 0.019-0.776% (CD3/6B11) of peripheral blood T cells and shows only a slight increase with age.

CD1d-Independent Activation of Invariant Natural Killer T Cells by Staphylococcal Enterotoxin B through Major Histocompatibility Complex Class II/T Cell Receptor Interaction Results in Acute Lung Injury

There are two important mechanisms of activation of invariant natural killer T cells (iNKT cells) by microbes: direct activation of the invariant T-cell receptor (TCR) by microbial glycolipids presented by CD1d and indirect activation, mediated by the responses of antigen-presenting cells to microbes. In this study, we provide evidence for a novel CD1d-independent direct activation of iNKT cells involving a microbial protein superantigen presented in the context of major histocompatibility complex class II (MHC-II), which plays a critical role in pathogenesis, thereby redefining the role of iNKT cells. Intranasal exposure to staphylococcal enterotoxin B (SEB) in C57BL/6 wild-type mice caused acute lung injury (ALI) characterized by vascular leak, cytokine storm, and infiltration of mononuclear cells in the lungs. In contrast, the vascular leak and inflammation were decreased by ~50% in NKT cell-deficient Jα18(-/-) and CD1d(-/-) mice following SEB exposure, which was reversed following adoptive transfer of iNKT cells into CD1d(-/-) mice. In vitro, SEB could directly stimulate iNKT cells in a CD1d-independent manner via MHC-II/TCR interaction, specifically involving Vβ8. These studies not only demonstrate that iNKT cells can be activated directly by a bacterial protein superantigen independent of CD1d but also indicate that in addition to the conventional T cells, iNKT cells play a critical role in SEB-mediated ALI.

Cord Blood Vα24-Vβ11+ Natural Killer T Cells Display a Th2-Chemokine Receptor Profile and Cytokine Responses

BackgroundThe fetal immune system is characterized by a Th2 bias but it is unclear how the Th2 predominance is established. Natural killer T (NKT) cells are a rare subset of T cells with immune regulatory functions and are already activated in utero. To test the hypothesis that NKT cells are part of the regulatory network that sets the fetal Th2 predominance, percentages of Vα24+Vβ11+ NKT cells expressing Th1/Th2-related chemokine receptors (CKR) were assessed in cord blood. Furthermore, IL-4 and IFN-γ secreting NKT cells were quantified within the single CKR+ subsets.ResultsCord blood NKT cells expressed the Th2-related CCR4 and CCR8 at significantly higher frequencies compared to peripheral blood NKT cells from adults, while CXCR3+ and CCR5+ cord blood NKT cells (Th1-related) were present at lower percentages. Within CD4negCD8neg (DN) NKT cells, the frequency of IL-4 producing NKT cells was significantly higher in cord blood, while frequencies of IFN-γ secreting DN NKT cells tended to be lower. A further subanalysis showed that the higher percentage of IL-4 secreting DN NKT cells was restricted to CCR3+, CCR4+, CCR5+, CCR6+, CCR7+, CCR8+ and CXCR4+ DN subsets in cord blood. This resulted in significantly decreased IFN-γ /IL-4 ratios of CCR3+, CCR6+ and CCR8+ cord blood DN NKT cells. Sequencing of VA24AJ18 T cell receptor (TCR) transcripts in sorted cord blood Vα24Vβ11 cells confirmed the invariant TCR alpha-chain ruling out the possibility that these cells represent an unusual subset of conventional T cells.ConclusionsDespite the heterogeneity of cord blood NKT cells, we observed a clear Th2-bias at the phenotypic and functional level which was mainly found in the DN subset. Therefore, we speculate that NKT cells are important for the initiation and control of the fetal Th2 environment which is needed to maintain tolerance towards self-antigens as well as non-inherited maternal antigens.

Comparison of Invariant NKT Cells with Conventional T Cells by Using Gene Set Enrichment Analysis (GSEA)

BackgroundInvariant Natural killer T (iNKT) cells, a distinct subset of CD1d-restricted T cells with invariant Vαβ TCR, functionally bridge innate and adaptive immunity. While iNKT cells share features with conventional T cells in some functional aspects, they simultaneously produce large amount of Th1 and Th2 cytokines upon T-cell receptor (TCR) ligation. However, gene expression pattern in two types of cells has not been well characterized.Methodswe performed comparative microarray analyses of gene expression in murine iNKT cells and conventional CD4+CD25-γδTCR- T cells by using Gene Set Enrichment Analysis (GSEA) method.ResultsHere, we describe profound differences in gene expression pattern between iNKT cells and conventional CD4+CD25-γδTCR- T cells.ConclusionOur results provide new insights into the functional competence of iNKT cells and a better understanding of their various roles during immune responses.

MEK Kinase 1 signaling regulates Natural Killer T cells (50.38)

Abstract MEK Kinase 1 (MEKK1) is of critical importance in controlling the thymus-dependent immune response by regulating Mitogen-Activated Protein Kinase (MAPK) activation from many receptors. Natural Killer T (NKT) cells are a distinct subset of lymphocytes that are characterized by the cell surface expression of a single invariant T-cell receptor (TCR) and Natural Killer (NK) cell markers. The activation of NKT cells is initiated by the engagement of the NKT receptor with glycolipid antigens presented by the molecule CD1d. Careful examination of Mekk1 kinase-deficient mice (Mekk1ΔKD) reveals multiple defects in NKT cells. Mekk1ΔKD NKT cells have deficient: MAPK signal transduction, proliferative expansion, apoptosis, and aberrant cytokine production. Mekk1ΔKD mice display elevated liver damage in a ConA-induced acute hepatitis liver injury model. My results demonstrate for the first time that MEKK1 is a critical MAPK Kinase kinase (MAP3K) signalling component for the regulation of NKT cells.

Natural killer T cells are important in the pathogenesis of asthma: The many pathways to asthma

The pathogenesis of bronchial asthma, a complex trait associated with a number of environmental factors (eg, allergens, infection, air pollution, exercise, and obesity), involves multiple cell types and several distinct cellular and molecular pathways. These pathways include adaptive and innate immunity and involve T(H)2 cells, mast cells, basophils, eosinophils, neutrophils, airway epithelial cells, and subsets of a newly described cell type called natural killer T (NKT) cells. A role for subsets of NKT cells in asthma has been suggested by extensive studies in animal models of asthma induced with allergen, viral infection, ozone exposure, or bacterial components, suggesting that NKT cells function in concert with T(H)2 cells or independently of adaptive immunity in causing airway hyperreactivity. The clinical relevance of NKT cells in human asthma is supported by the observation that NKT cells are present in the lungs of some patients with asthma, particularly patients with severe, poorly controlled asthma, although additional research is required to more precisely define the specific role of NKT cells in human asthma. These studies of NKT cells greatly expand our understanding of possible mechanisms that drive the development of asthma, particularly in the case of asthma associated with neutrophils, viral infection, and air pollution.

Transcriptional regulator Id2 controls survival of hepatic NKT cells

Natural killer T cells expressing an invariant T-cell receptor (iNKT) regulate activation of both innate and adaptive immunity in many contexts. iNKT cells accumulate in the liver and rapidly produce prodigious amounts of numerous cytokines upon activation, impacting the immune response to viral infection, immunosurveillance for malignant cells, and liver regeneration. However, little is known about the factors controlling iNKT homeostasis, survival and hepatic localization. Here, we report that the absence of the transcriptional regulator Id2 resulted in a severe, intrinsic defect in the accumulation of hepatic iNKT cells. Id2-deficient iNKT cells showed increased cell death in the liver, although migration and functional activity were not impaired in comparison to Id2-expressing iNKT cells. Id2-deficient iNKT cells exhibited diminished expression of CXCR6, a critical determinant of iNKT cell accumulation in the liver, and of the anti-apoptotic molecules bcl-2 and bcl-X(L), compared to Id2-sufficient iNKT cells. Furthermore, survival and accumulation of iNKT cells lacking Id2 expression was rescued by deficiency in bim, a key pro-apoptotic molecule. Thus, Id2 was necessary to establish a hepatic iNKT cell population, defining a role for Id2 and implicating the Id targets, E protein transcription factors, in the regulation of iNKT cell homeostasis.

Research Highlights: Immunotherapy

Research Highlights: Immunotherapy

Human invariant natural killer T cells: implications for immunotherapy

Human invariant natural killer T cells are a unique lymphocyte population that have an invariant T-cell receptor and recognize glycolipids instead of peptides in the restriction of CD1d molecules. These natural killer T cells play important roles in anti-tumor immunity, transplantation immunity, allergy, autoimmunity and microbial immunity. Since human natural killer T cells show high-level biological activity such as cytokine production, an anti-tumor effect and regulatory T-cell control, they may be a useful tool in immune-cell therapy. In this review, we summarize the immune responses mediated by human natural killer T cells, especially in tumor and transplantation immunity, and discuss their potential in clinical applications.

Natural killer T cell-mediated immunotherapy for malignant diseases

Human invariant Valpha24 Natural Killer T (NKT) cells are unique lymphocyte subsets, characterized by an invariant T-cell receptor Valpha24 chain paired with Vbeta11. Recent findings have highlighted the role of NKT cells in tumor immunity. Valpha24 NKT cells are activated by a specific glycolipid ligand, alpha-Galactosylceramide and rapidly produce high levels of cytokines upon stimulation, thereby modulating other immune cells such as NK cells antigen-specific CD4+ and CD8+ T cells and dendritic cells. Abnormalities in the numbers and functions of Valpha24 NKT cells have been observed in patients with various malignant diseases. Therefore, therapeutic strategies have recently focused on the reconstitution of an adequate number of functionally sufficient Valpha24 NKT cells which is thought to be logical and reasonable for cancer treatment. The quantitative alteration and functional impairment of circulating Valpha24 NKT cells are herein reviewed in various cancer-bearing patients and the progress to date in the clinical applications of NKT cell-based tumor immunotherapy is summarized.

Contrasting roles for Vα14+natural killer T cells in a viral model for multiple sclerosis

Most natural killer (NK) T cells express an invariant Valpha14 T-cell receptor. To explore the contribution of NKT cells in an animal model for multiple sclerosis, Theiler's murine encephalomyelitis virus (TMEV) infection, TMEV-infected mice were treated with Valpha14 antibody. Treatment during the early stage of infection delayed the onset of demyelinating disease with higher interleukin-4 production, whereas administration during the late stage or weekly resulted in more severe demyelination with enhanced virus persistence. The effect of in vivo depletion of NKT cells differed depending on the stage of infection, suggesting contrasting roles for NKT cells over the disease course.

Invariant Natural Killer T Cell-Based Immunotherapy for Cancer

Human Valpha24 invariant natural killer T (iNKT) cells are a distinct lymphocyte population, characterized by an invariant T-cell receptor Valpha24 chain paired mainly with Valpha11. Valpha24 iNKT cells are activated by a glycolipid ligand - alpha-galactosylceramide - and produce a large amount of Th1 and Th2 cytokines, thereby modulating the function of other cells. iNKT cells have the capability to control a wide variety of immune responses, including antitumor immunity. Abnormalities in the number and function of Valpha24 iNKT cells have been observed in patients with malignant diseases accompanied with a poor clinical outcome. Therefore, therapeutic strategies that focused on the restoration of Valpha24 iNKT cell population and function would be a reasonable rationale for the treatment of cancer. In this article, the progress to date in the clinical studies of iNKT cell-based immunotherapy is briefly reviewed and the role of Valpha24 iNKT cells in cancer immunotherapy is highlighted.

Prolongation of Cardiac Allograft Survival by Rapamycin and the Invariant Natural Killer T Cell Glycolipid Agonist OCH

Invariant natural killer T (iNKT) cells are glycolipid-responsive cells with potent immunomodulatory properties. Although iNKT cells have been implicated in cardiac allograft tolerance, whether in vivo triggering of iNKT cells with Th2-promoting glycolipids offers a therapeutic benefit in heart transplantation remains unexplored. C3H (H-2k) hearts were transplanted into C57BL/6 (H-2b) mice. The recipients were left untreated or received the Th2-promoting iNKT cell agonist OCH, the antirejection agent rapamycin, or both. Allografts were recovered on postoperative day 8 or at endpoint, stained with hematoxylin-eosin, and analyzed for intragraft transcript levels of effector cytokines and iNKT cells' invariant T-cell receptor segment Valpha14-Jalpha18. The presence of circulating alloantibodies was assessed in recipients' sera at similar time points. A second fully mismatched cardiac allograft model (BALB/c-to-C57BL/6) was used to further validate the efficacy of our treatment regimens. Combination immunotherapy with OCH and rapamycin significantly enhanced C3H allograft survival and led to nearly normal graft histology with minimal vascular changes and mononuclear cell infiltration, and an almost normal IgG1:IgG2a ratio in recipients' sera. These were accompanied by elevated intragraft mRNA levels of interleukin (IL)-4, and to a lesser extent IL-10 and IL-13, and high transcript levels of Valpha14-Jalpha18 T-cell receptor gene segment. Furthermore, when used alone or together with rapamycin, OCH delayed allograft rejection in our BALB/c-to-C57BL/6 model. In vivo administration of OCH may deviate alloimmune responses towards a Th2 phenotype and prolong allograft survival. Select iNKT cell glycolipid agonists can therefore be used in monotherapy or combination immunotherapy of transplant rejection.

CD1d-restricted glycolipid antigens: presentation principles, recognition logic and functional consequences

Invariant natural killer T (iNKT) cells are innate lymphocytes whose functions are regulated by self and foreign glycolipid antigens presented by the antigen-presenting molecule CD1d. Activation of iNKT cells in vivo results in rapid release of copious amounts of effector cytokines and chemokines with which they regulate innate and adaptive immune responses to pathogens, certain types of cancers and self-antigens. The nature of CD1d-restricted antigens, the manner in which they are recognised and the unique effector functions of iNKT cells suggest an innate immunoregulatory role for this subset of T cells. Their ability to respond fast and our ability to steer iNKT cell cytokine response to altered lipid antigens make them an important target for vaccine design and immunotherapies against autoimmune diseases. This review summarises our current understanding of CD1d-restricted antigen presentation, the recognition of such antigens by an invariant T-cell receptor on iNKT cells, and the functional consequences of these interactions.

Type I NKT cells protect (and type II NKT cells suppress) the host's innate antitumor immune response to a B-cell lymphoma

AbstractNatural killer T (NKT) cells are a T-cell subpopulation known to possess immunoregulatory functions and recognize CD1d molecules. The majority of NKT cells express an invariant T-cell receptor (TCR) α chain rearrangement (Vα14Jα18 in mice; Vα24Jα18 in humans) and are called type I NKT cells; all other NKT cells are type II. In the current study, we have analyzed the roles for these NKT-cell subsets in the host’s innate antitumor response against a murine B-cell lymphoma model in vivo. In tumor-bearing mice, we found that type I NKT cells conferred protection in a CD1d-dependent manner, whereas type II NKT cells exhibited inhibitory activity. Pro- and anti-inflammatory cytokines secreted by splenocytes from tumor-bearing mice correlated with tumor progression. Myeloid cells (CD11b+Gr1+) were present in large numbers at the tumor site and in the spleen of tumor-bearing type I NKT–deficient mice, suggesting that antitumor immunosurveillance was inhibited by CD11b+Gr1+ cells. Overall, these data suggest that there are distinct roles for NKT-cell subsets in response to a B-cell lymphoma in vivo, pointing to potential novel targets to be exploited in immunotherapeutic approaches against blood cancers.

Clinical applications of natural killer T cell–based immunotherapy for cancer

Human invariant V alpha 24 natural killer T (NKT) cells are a novel, distinct lymphocyte population, characterized by an invariant T-cell receptor V alpha 24 chain paired with V beta 11. V alpha 24 NKT cells are activated by a specific glicolipid ligand, alpha-GalCer, and rapidly produce a large amount of Th1 and Th2 cytokines, thereby modulating other immune cells such as antigen-specific CD4 and CD8 T cells, NK cells, and dendritic cells. Recent studies have shown that NKT cells play pivotal regulatory roles in many immune responses, including antitumor immunity. We herein review the quantitative alteration and functional deterioration of circulating V alpha 24 NKT cells in various cancer-bearing patients. We also summarize the recent progress in the clinical studies of NKT cell-based tumor immunotherapy. Novel immunological results including the increased peripheral blood V alpha 24 NKT cells and IFN-producing cells after the immunotherapy were revealed. The details of the safety profile and the antitumor responses were also disclosed. Although the objective clinical responses still remain unclear, some encouraging results have emerged. Therefore, NKT cell-based immunotherapy may potentially be an effective strategy for the treatment of cancer patients.

Chapter 8 The Role of NKT Cells in Tumor Immunity

NKT cells are a relatively newly recognized member of the immune community, with profound effects on the rest of the immune system despite their small numbers. They are true T cells with a T cell receptor (TCR), but unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a nonclassical MHC molecule. As members of both the innate and adaptive immune systems, they bridge the gap between these, and respond rapidly to set the tone for subsequent immune responses. They fill a unique niche in providing the immune system a cellular arm to recognize lipid antigens. They play both effector and regulatory roles in infectious and autoimmune diseases. Furthermore, subsets of NKT cells can play distinct and sometimes opposing roles. In cancer, type I NKT cells, defined by their invariant TCR using Valpha14Jalpha18 in mice and Valpha24Jalpha18 in humans, are mostly protective, by producing interferon-gamma to activate NK and CD8(+) T cells and by activating dendritic cells to make IL-12. In contrast, type II NKT cells, characterized by more diverse TCRs recognizing lipids presented by CD1d, primarily inhibit tumor immunity. Moreover, type I and type II NKT cells counter-regulate each other, forming a new immunoregulatory axis. Because NKT cells respond rapidly, the balance along this axis can greatly influence other immune responses that follow. Therefore, learning to manipulate the balance along the NKT regulatory axis may be critical to devising successful immunotherapies for cancer.