In addition, repeated injections of ␣-GalCer or the related glycolipid OCH to apolipoprotein E knockout (apoE ؊/؊ ) mice during the early phase of atherosclerosis significantly enlarged the lesion areas compared with mice injected with vehicle control. However, administering ␣-GalCer to apoE ؊/؊ mice with established lesions did not significantly increase the lesion area but considerably decreased the collagen content. Atherosclerosis development in either AD-fed WT or apoE ؊/؊ mice was associated with the presence of V␣14J␣18 transcripts in the atherosclerotic arterial walls, indicating that NKT cells were recruited to these lesions. Thioglycolate-elicited macrophages pulsed with oxidized low-density lipoproteins expressed enhanced CD1d levels and induced NKT cells to produce interferon-␥, a potentially proatherogenic T-helper 1 (T H 1) cytokine. Collectively, we conclude that NKT cells are proatherogenic in mice. IntroductionAtherosclerosis is an inflammatory vascular disease that involves components of the innate and acquired immune systems. [1][2][3] Several studies have suggested that lymphocytes, which are detected in atherosclerotic lesions in humans and mice, 4,5 play a proatherogenic role. [6][7][8] Recently, the role of distinct lymphocyte subsets in the development of atherosclerosis has been evaluated. For example, emerging evidence indicates that T-helper 1 (T H 1) cells are proatherogenic, 9 whereas T H 2 cells are antiatherogenic. 10,11 These observations are further supported by the finding that T H 1 cytokines (eg,) are important in the progression of atherosclerosis [12][13][14][15] and that, among T H 2 cytokines, IL-10 is antiatherogenic. 16 On the other hand, recent studies have suggested that B cells play a protective role in atherogenesis. 17,18 Natural killer T (NKT) cells are a unique subset of lymphocytes that have surface markers and functions of T cells and NK cells. [19][20][21][22][23] Several characteristics of NKT cells suggest that they may play a role in the atherogenic process. Most NKT cells express an invariant V␣14J␣18 T-cell receptor (TCR)-V␣ chain paired with a restricted set of TCR-V␤ chains. These classical NKT cells recognize lipid antigens presented by the major histocompatibility complex (MHC) class 1-like molecule CD1d, produce copious amounts of IFN-␥ and IL-4 on activation, 22 and constitutively express Fas-ligand. 23 Moreover, NKT cells play a protective role in several autoimmune diseases, infections, and tumor progression/ metastasis. 20 Protective effects of NKT cells and their ligands in autoimmunity are largely attributed to their capacity to promote T H 2 immune responses. 24,25 However, in some situations, NKT cells can contribute to the development of T H 1 immune responses as well. 26 Therefore, it was difficult to predict whether NKT cells would play a proatherogenic or an antiatherogenic role. 2 To date, few studies have investigated the role of CD1d and CD1d-dependent T cells in atherogenesis. CD1d-expressing cells are present in human atherosclerotic...

This protocol describes methods to identify, purify and culture CD1d restricted invariant natural killer T (iNKT) cells from mouse tissue or human blood samples. The methods for identification and purification of iNKT cells are based on the interaction between iNKT cell receptor and its ligand. The iNKT cell receptor is composed of the invariant V alpha 14 J alpha 18/V beta 8.2 in mice or V alpha 24 J alpha 18/V beta 11 in humans and is expressed only on iNKT cells but not on conventional T cells. The iNKT cell antigen receptor in both species recognizes alpha-galactosylceramide (alpha-GalCer) presented by the MHC class I-like CD1d. Thus, alpha-GalCer-loaded CD1d dimer can be used for analysis and purification by fluorescence-activated cell sorting (FACS). Isolation of 1 x 10(6) purified iNKT cells from mouse thymus, spleen or liver requires 5-6 mice and takes 1-2 h for mononuclear cell preparation from mouse tissues, 1.5 h for enrichment by magnetic beads and 4 h for detection and purification of the iNKT cells by FACS. In the case of isolation of human peripheral blood mononuclear cells (PBMCs) from whole blood, it takes 2 h and requires 5 ml of blood to obtain 5 x 10(6) PBMCs, which contain 500-25,000 iNKT cells.

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.

Although invariant V14 natural killer T cells (NKT cells) are thought to be generated from CD4CD8 double-positive (DP) thymocytes, the developmental origin of CD4CD8 double-negative (DN) NKT cells still remains unresolved. Here we provide definitive genetic evidence obtained, through studies of mice with DP-stage-specific ablation of expression of the gene encoding the recombinase component RAG-2 (Rag2) and by a fate-mapping approach, that supports the proposal of the existence of an alternative developmental pathway through which a fraction of DN NKT cells with strong T-helper-type-1 (T1)-biased and cytotoxic characteristics develop from late DN-stage thymocytes, bypassing the DP stage. These findings provide new insight into understanding of the development of NKT cells and propose a role for timing of expression of the invariant T cell antigen receptor in determining the functional properties of NKT cells.

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...

Natural Killer T (NKT) cells are unique lymphocytes characterized by their expression of a single invariant antigen receptor encoded by Vα14Jα18 in mice and Vα24Jα18 in humans, which recognizes glycolipid antigens in association with the monomorphic CD1d molecule. NKT cells mediate adjuvant activity to activate both CD8T cells to kill MHC-positive tumor cells and NK cells to eliminate MHC-negative tumor at the same time in patients, resulting in the complete eradication of tumors without relapse. Therefore, the NKT cell-targeted therapy can be applied to any type of tumor and also to anyone individual, regardless of HLA type.Phase IIa clinical trials on advanced lung cancers and head and neck tumors have been completed and showed significantly prolonged median survival times with only the primary treatment. Another potential treatment option for the future is to use induced pluripotent stem cell (iPS)-derived NKT cells, which induced adjuvant effects on anti-tumor responses, inhibiting in vivo tumor growth in a mouse model.

Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells.

Current tumor therapies, including immunotherapies, focus on passive eradication or at least reduction of the tumor mass. However, cancer patients quite often suffer from tumor relapse or metastasis after such treatments. To overcome these problems, we have developed a natural killer T (NKT) cell-targeted immunotherapy focusing on active engagement of the patient’s immune system, but not directly targeting the tumor cells themselves. NKT cells express an invariant antigen receptor α chain encoded by Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans and recognize glycolipid ligand in conjunction with a monomorphic CD1d molecule. The NKT cells play a pivotal role in the orchestration of antitumor immune responses by mediating adjuvant effects that activate various antitumor effector cells of both innate and adaptive immune systems and also aid in establishing a long-term memory response. Here, we established NKT cell-targeted therapy using a newly discovered NKT cell glycolipid ligand, RK, which has a stronger capacity to stimulate both human and mouse NKT cells compared to previous NKT cell ligand. Moreover, RK mediates strong adjuvant effects in activating various effector cell types and establishes long-term memory responses, resulting in the continuous attack on the tumor that confers long-lasting and potent antitumor effects. Since the NKT cell ligand presented by the monomorphic CD1d can be used for all humans irrespective of HLA types, and also because NKT cell-targeted therapy does not directly target tumor cells, this therapy can potentially be applied to all cancer patients and any tumor types.