Invariant NKT (iNKT) cells bridge innate and acquired immunity and play an important role in both protective and regulatory responses. The nature of the response is dictated by the initial cytokine environment: interaction with IL-10-producing cells induces negative regulatory T(h)2/regulatory T cell-type iNKT cells, while that with IL-12-producing cells results in pro-inflammatory T(h)1-type responses. Particularly, in the anti-tumor response, iNKT cells mediate adjuvant activity by their production of IFN-gamma, which in turn activates both innate and acquired immune systems. Thus, upon activation of iNKT cells, both MHC(-) and MHC(+) tumor cells can be efficiently eliminated. On the basis of these mechanisms, iNKT cell-targeted adjuvant cell therapies have been developed and have shown great promise in initial clinical trials on cancer patients.

NKT cells are characterized by their production of both T(h)1 and T(h)2 cytokines immediately after stimulation with alpha-galactosylceramide (alpha-GalCer), which is composed of alpha-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of alpha-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized alpha-carba-GalCer, which has an alpha-linked carba-galactosyl moiety; here, the 5a'-oxygen atom of the D-galactopyranose ring of alpha-GalCer is replaced by a methylene group. The alpha-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-gamma compared with alpha-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The alpha-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was alpha-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens.

Establishment of a system with efficient generation of natural killer T (NKT) cells from embryonic stem (ES) cells would enable us to identify the cells with NKT- IntroductionNatural killer T (NKT) cells are characterized by their expression of an invariant receptor encoded by V␣14-J␣18 in mice 1 and by V␣24-J␣18 in humans. 2,3 Because the invariant receptor is used only by NKT cells and not by conventional T cells, NKT cells are a distinct lineage from conventional T cells. NKT cells produce both T helper 1 (Th1) and Th2 cytokines, mediating strong adjuvant activity through their Th1 cytokine production essential for protective responses against tumors 4-6 and pathogens 7-9 and also protecting autoimmune disease development through their Th2 cytokine production. 10,11 Despite the importance of this cell type in the immune system, the identity of NKT progenitor cells and their subsequent development remain poorly understood. In previous reports, cells with NKT-cell potential were detected in the CD4CD8 double-positive (DP) thymocyte population, 12,13 indicating that NKT cells are branched off from conventional ␣␤T-cell precursors at the DP stage in the thymus. [12][13][14] It has also been shown that the early immature CD4 NKT cells produce only interleukin-4 (IL-4), but their potential to produce interferon ␥ (IFN-␥) is acquired at a later developmental stage. 15,16 However, it is still possible that NKT cells might be derived from a precursor population distinct from that of conventional ␣␤T cells, because it has previously been shown that NKT precursor cells express on their cell surface granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), 17 which is known to be a unique marker for myeloid cell lineages but not for lymphoid cells. In addition, because the invariant V␣14 receptor is used only by NKT cells and not by conventional T cells, NKT cells may be a distinct lineage from conventional ␣␤T cells. 18 Embryonic stem (ES) cells are a powerful model system in which to study in vitro lymphocyte differentiation, addressing the questions on the cells with NKT precursor potential and the ability of NKT cells to produce both Th1 and Th2 cytokines during their development. For example, embryoid bodies generated from ES cells contain CD34 ϩ cells that develop into lymphocytes when cocultured with OP9 stromal cells plus appropriate cytokines. 19 However, in most cases, ES cells generate B and NK cells, but not T or NKT cells on OP9 coculture. 20,21 To overcome these problems, OP9 stromal cells transduced with Notch ligand delta-like1 (OP9/Dll-1) are used for the directed differentiation of ES cells to T-cell lineages. Induction of Notch signals directs stem cells to differentiate into immature DP T cells and inhibits B-cell development, indicating that Notch signaling is required as a proximal event in T-cell commitment from progenitors. 22,23 Another approach is to use cloned ES (NKT-ES) cells established by nuclear transfer of a cell with a rearranged T-cell receptor (TCR) gene. 24,25 Interestingly...

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