Natural killer T (NKT) lymphocytes express an invariant T cell antigen receptor (TCR) encoded by the Valpha14 and Jalpha281 gene segments. A glycosylceramide-containing alpha-anomeric sugar with a longer fatty acyl chain (C26) and sphingosine base (C18) was identified as a ligand for this TCR. Glycosylceramide-mediated proliferative responses of Valpha14 NKT cells were abrogated by treatment with chloroquine-concanamycin A or by monoclonal antibodies against CD1d/Vbeta8, CD40/CD40L, or B7/CTLA-4/CD28, but not by interference with the function of a transporter-associated protein. Thus, this lymphocyte shares distinct recognition systems with either T or NK cells.

A major group of natural killer (NK) T cells express an invariant Vα14+ T cell receptor (TCR) specific for the lipoglycan α-galactosylceramide (α-GalCer), which is presented by CD1d. These cells may have an important immune regulatory function, but an understanding of their biology has been hampered by the lack of suitable reagents for tracking them in vivo. Here we show that tetramers of mouse CD1d loaded with α-GalCer are a sensitive and highly specific reagent for identifying Vα14+ NK T cells. Using these tetramers, we find that α-GalCer–specific T lymphocytes are more widely distributed than was previously appreciated, with populations of largely NK1.1− but tetramer-binding T cells present in the lymph nodes and the intestine. Injection of α-GalCer leads to the production of both interferon γ and interleukin 4 by nearly all NK T cells in the liver and the majority of the spleen within 2 h. These cells mostly disappear by 5 h, and they do not reappear after 1 wk. Curiously, tetramer-positive thymocytes do not rapidly synthesize cytokines, nor do they undergo decreases in cell number after lipid antigen stimulation, although they express equivalent TCR levels. In summary, the data presented here demonstrate that α-GalCer–specific NK T cells undergo a unique and highly compartmentalized response to antigenic stimulation.

Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice may be favored by immune dysregulation leading to the hyporesponsiveness of regulatory T cells and activation of effector T-helper type 1 (Th1) cells. The immunoregulatory activity of natural killer T (NKT) cells is well documented, and both interleukin (IL)-4 and IL-10 secreted by NKT cells have important roles in mediating this activity. NKT cells are less frequent and display deficient IL-4 responses in both NOD mice and individuals at risk for T1D (ref. 8), and this deficiency may lead to T1D (refs. 1,6-9). Thus, given that NKT cells respond to the alpha-galactosylceramide (alpha-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines, we reasoned that activation of NKT cells by alpha-GalCer might prevent the onset and/or recurrence of T1D. Here we show that alpha-GalCer treatment, even when initiated after the onset of insulitis, protects female NOD mice from T1D and prolongs the survival of pancreatic islets transplanted into newly diabetic NOD mice. In addition, when administered after the onset of insulitis, alpha-GalCer and IL-7 displayed synergistic effects, possibly via the ability of IL-7 to render NKT cells fully responsive to alpha-GalCer. Protection from T1D by alpha-GalCer was associated with the suppression of both T- and B-cell autoimmunity to islet beta cells and with a polarized Th2-like response in spleen and pancreas of these mice. These findings raise the possibility that alpha-GalCer treatment might be used therapeutically to prevent the onset and recurrence of human T1D.

Diabetes in non-obese diabetic (NOD) mice is mediated by pathogenic T-helper type 1 (Th1) cells that arise because of a deficiency in regulatory or suppressor T cells. V alpha 14-J alpha 15 natural killer T (NKT) cells recognize lipid antigens presented by the major histocompatibility complex class I-like protein CD1d (refs. 3,4). We have previously shown that in vivo activation of V alpha 14 NKT cells by alpha-galactosylceramide (alpha-GalCer) and CD1d potentiates Th2-mediated adaptive immune responses. Here we show that alpha-GalCer prevents development of diabetes in wild-type but not CD1d-deficient NOD mice. Disease prevention correlated with the ability of alpha-GalCer to suppress interferon-gamma but not interleukin-4 production by NKT cells, to increase serum immunoglobulin E levels, and to promote the generation of islet autoantigen-specific Th2 cells. Because alpha-GalCer recognition by NKT cells is conserved among mice and humans, these findings indicate that alpha-GalCer might be useful for therapeutic intervention in human diseases characterized by Th1-mediated pathology such as Type 1 diabetes.

We have previously reported that hepatitis B virus (HBV)–specific CD8+ cytotoxic T lymphocytes and CD4+ helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting interferon (IFN)-γ when they recognize viral antigen. To determine whether an activated innate immune system can also inhibit HBV replication, in this study we activated natural killer T (NKT) cells in the liver of HBV transgenic mice by a single injection of α-galactosylceramide (α-GalCer), a glycolipid antigen presented to Vα14+NK1.1+ T cells by the nonclassical major histocompatibility complex class I–like molecule CD1d. Within 24 h of α-GalCer injection, IFN-γ and IFN-α/β were detected in the liver of HBV transgenic mice and HBV replication was abolished. Both of these events were temporally associated with the rapid disappearance of NKT cells from the liver, presumably reflecting activation-induced cell death, and by the recruitment of activated NK cells into the organ. In addition, prior antibody-mediated depletion of CD4+ and CD8+ T cells from the mice did not diminish the ability of α-GalCer to trigger the disappearance of HBV from the liver, indicating that conventional T cells were not downstream mediators of this effect. Finally, the antiviral effect of α-GalCer was inhibited in mice that are genetically deficient for either IFN-γ or the IFN-α/β receptor, indicating that most of the antiviral activity of α-GalCer is mediated by these cytokines. Based on these results, we conclude that α-GalCer inhibits HBV replication by directly activating NKT cells and by secondarily activating NK cells to secrete antiviral cytokines in the liver. In view of these findings, we suggest that, if activated, the innate immune response, like the adaptive immune response, has the potential to control viral replication during natural HBV infection. In addition, the data suggest that therapeutic activation of NKT cells may represent a new strategy for the treatment of chronic HBV infection.

Natural killer (NK) T cells are a lymphocyte subset with a distinct surface phenotype, an invariant T cell receptor (TCR), and reactivity to CD1. Here we show that mouse NK T cells can recognize human CD1d as well as mouse CD1, and human NK T cells also recognize both CD1 homologues. The unprecedented degree of conservation of this T cell recognition system suggests that it is fundamentally important. Mouse or human CD1 molecules can present the glycolipid α-galactosylceramide (α-GalCer) to NK T cells from either species. Human T cells, preselected for invariant Vα24 TCR expression, uniformly recognize α-GalCer presented by either human CD1d or mouse CD1. In addition, culture of human peripheral blood cells with α-GalCer led to the dramatic expansion of NK T cells with an invariant (Vα24+) TCR and the release of large amounts of cytokines. Because invariant Vα14+ and Vα24+ NK T cells have been implicated both in the control of autoimmune disease and the response to tumors, our data suggest that α-GalCer could be a useful agent for modulating human immune responses by activation of the highly conserved NK T cell subset.

NKT cells are associated with immunological control of autoimmune disease and cancer and can recognize cell surface mCD1d without addition of exogenous antigens. Cellular antigens presented by mCD1d have not been identified, although NKT cells can recognize a synthetic glycolipid, alpha-GalCer. Here we show that after addition of a lipid extract from a tumor cell line, plate-bound mCD1d molecules stimulated an NKT cell hybridoma. This hybridoma also responded strongly to three purified phospholipids, but failed to recognize alpha-GalCer. Seven of sixteen other mCD1d restricted hybridomas also showed a response to certain purified phospholipids. These findings suggest NKT cells can recognize cellular antigens distinct from alpha-GalCer and identify phospholipids as potential self-antigens presented by mCD1d.

We have recently identified ␣-galactosylceramide (␣-GalCer) as a specific ligand for an invariant V␣14͞ V␤8.2 T cell receptor exclusively expressed on the majority of V␣14 NKT cells, a novel subset of lymphocytes. Here, we report that ␣-GalCer selectively activates V␣14 NKT cells resulting in prevention of tumor metastasis. The effector mechanisms of the ligand-activated V␣14 NKT cells seem to be mediated by natural killer (NK)-like nonspecific cytotoxicity. Indeed, the cytotoxic index obtained by ␣-GalCeractivated V␣14 NKT cells was reduced by the addition of cold target tumor cells or by treatment with concanamycin A, which inhibits activation and secretion of perforin, but not by mAbs against molecules involved in the NKT cell recognition and conventional cytotoxicity, such as CD1d, V␤8, NK1.1, Ly49C, Fas, or Fas ligand. These results suggest that the ligand-activated V␣14 NKT cells kill tumor cells directly through a CD1d͞V␣14 T cell receptor-independent, NK-like mechanism.A novel lymphoid lineage, V␣14 natural killer (NK) T cells, distinct from other lymphoid cells including T cells, B cells, and NK cells, is characterized by the early development at day 9.5 of gestation before thymus formation (1) and also by coexpression of the NK receptor and a single, invariant T cell receptor (TCR) encoded by V␣14 and J␣281 gene segments (2-4) in association with a highly skewed set of V␤s, mainly V␤8.2 (5-13). Moreover, the generation of V␣14 NKT cells is exclusively dependent on the expression of the invariant V␣14 TCR as evidenced by the fact that V␣14 NKT cells do not develop in the invariant V␣14 TCR-deficient mice (14) and that the forced expression of the invariant V␣14 TCR leads exclusively to V␣14 NKT cell generation and blocks conventional T cell development (15, 16).Although physiological functions of V␣14 NKT cells remain to be elucidated, the extensive analysis has shown that V␣14 NKT cells are able to mediate allograft bone marrow rejection (17), control autoimmune disease development (18,19), and produce large amounts of both interleukin 4 (IL-4) and interferon ␥ (16,20,21). These findings suggest that V␣14-NKT cells play complicated roles in regulating immune responses more than simply as IL-4 or interferon ␥ providers for Th2 or Th1 cell development, respectively, so far reported (22,23). In addition, we have demonstrated recently that V␣14 NKT cells are a primary target of IL-12 and exert a major effector function in IL-12-mediated tumor rejection (14).A ligand for the invariant V␣14͞V␤8.2 TCR exclusively expressed on V␣14 NKT cells has been identified recently to be ␣-galactosylceramide (␣-GalCer) (16). In addition, ␣-GalCer is presented by a monomorphic non-majorhistocompatibility gene complex (non-MHC) class Ib molecule, CD1d, expressed on dendritic cells (DC), and the ligand͞CD1 complex selectively stimulates to proliferate V␣14 NKT cells but not other lymphocytes only if costimulatory signals generated by CD40͞CD40 ligand and B7͞ CD28 interactions are provided (16). The results suggest th...