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 lymphocyte subpopulation, the Valpha14 natural killer T (NKT) cells, expresses both NK1.1 and a single invariant T cell receptor encoded by the Valpha14 and Jalpha281 gene segments. Mice with a deletion of the Jalpha281 gene segment were found to exclusively lack this subpopulation. The Valpha14 NKT cell-deficient mice could no longer mediate the interleukin-12 (IL-12)-induced rejection of tumors. Although the antitumor effect of IL-12 was thought to be mediated through natural killer cells and T cells, Valpha14 NKT cells were found to be an essential target of IL-12, and they mediated their cytotoxicity by an NK-like effector mechanism after activation with IL-12.

The natural killer T (NKT) cell ligand α-galactosylceramide (α-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12–mediated antitumor activities. Because of these similarities between the activities of α-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by α-GalCer. We first established, using purified subsets of various lymphocyte populations, that α-GalCer selectively activates NKT cells for production of interferon (IFN)-γ. Production of IFN-γ by NKT cells in response to α-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, α-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1−/− or Vα14−/− mice. This effect of α-GalCer required the production of IFN-γ by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of α-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-γ production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by α-GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer.

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset–depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1+ T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3− NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I–deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jα281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or α-galactosylceramide.

The administration of concanavalin A (Con A) induces a rapid severe injury of hepatocytes in mice. Although the Con A–induced hepatitis is considered to be an experimental model of human autoimmune hepatitis, the precise cellular and molecular mechanisms that induce hepatocyte injury remain unclear. Here, we demonstrate that Vα14 NKT cells are required and sufficient for induction of this hepatitis. Moreover, interleukin (IL)-4 produced by Con A–activated Vα14 NKT cells is found to play a crucial role in disease development by augmenting the cytotoxic activity of Vα14 NKT cells in an autocrine fashion. Indeed, short-term treatment with IL-4 induces an increase in the expression of granzyme B and Fas ligand (L) in Vα14 NKT cells. Moreover, Vα14 NKT cells from either perforin knock-out mice or FasL-mutant gld/gld mice fail to induce hepatitis, and hence perforin–granzyme B and FasL appear to be effector molecules in Con A–induced Vα14 NKT cell–mediated hepatocyte injury.

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...

In vivo administration of NKT cell ligand, alpha-galactosylceramide (alpha-GalCer), caused the activation of NKT cells to induce a strong NK activity and cytokine production by CD1d-restricted mechanisms. Surprisingly, we also found that alpha-GalCer induced the activation of immunoregulatory cells involved in acquired immunity. Specifically, in vivo administration of alpha-GalCer resulted in the induction of the early activation marker CD69 on CD4(+) T cells, CD8(+) T cells and B cells in addition to macrophages and NKT cells. However, no significant induction of CD69 was observed on cells from CD1d- or V(alpha)14 NKT-deficient mice, indicating an essential role for the interaction between NKT cells and CD1d-expressing dendritic cells (DC) in the activation of acquired immunity in response to alpha-GalCer. Indeed, in vivo injection of alpha-GalCer resulted not only in the activation of NKT cells but also in the generation of CD69(+)CD8(+) T cells possessing both cytotoxic T lymphocyte (CTL) activity and IFN-gamma-producing ability. Tumor-specific CTL generation was also accelerated by alpha-GalCer. The critical role of CD40-CD40 ligand (CD40L)-mediated NKT-DC interaction during the development of CD69(+)CD8(+) CTL by alpha-GalCer was demonstrated by blocking experiments using anti-CD40L mAb. These findings provide direct evidence for a critical role of CD1d-restricted NKT cells and DC in bridging innate and acquired immunity.

The immunological mechanisms that regulate abortion are largely unknown. Here, we found that a distinct subset of lymphocytes, V␣14 NKT cells expressing an invariant antigen receptor encoded by V␣14͞J␣281 and V␤7 segments, accumulated in the decidua during pregnancy and provoked abortion upon stimulation with ␣-galactosylceramide (␣-GalCer), a specific ligand for V␣14 NKT cells. The ␣-GalCer-mediated abortion was not observed in V␣14 NKT-, IFN-␥-, tumor necrosis factor ␣-, or perforin-knock-out mice and appeared to be due to the degeneration of embryonic trophoblasts mediated by the activated V␣14 NKT cells whose perforin-dependent killing and production of IFN-␥ and tumor necrosis factor ␣ were essential. The possible role of the decidual V␣14 NKT cells in the pathogenesis of abortion is discussed.