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Gnetin-C is a naturally occurring stilbene derived from the seeds of Gnetum gnemon L. , an edible plant native to Southeast Asia that is called melinjo. Although the biological properties and safety of G. gnemon extract, which contains nearly 3% Gnetin-C, have been confirmed in various human studies, whether or not pure Gnetin-C is safe for humans is unclear at present. We conducted a randomized, double-blind, placebo-controlled trial. Healthy subjects were randomly divided into two groups. The interventional group ( n = 6) was given Gnetin-C, and the control group ( n = 6) was provided a placebo, for 14 days. Lipid profiles, biomarkers of oxidative stress and circulating blood cells were assessed before and after the intervention. All subjects completed the study, with no side effects reported across the study duration. Gnetin-C supplementation demonstrated a statistically significant increase in the absolute number of circulating natural killer (NK) cells expressing the activating receptors NKG2D and NKp46. NK cells derived from subjects who received Gnetin-C for two weeks showed higher cytotoxicity against K562 target cells than those before receiving Gnetin-C. In addition, Gnetin-C also resulted in a significant decrease in the absolute neutrophil count in the blood compared with the placebo. Furthermore, Gnetin-C significantly reduced the levels of uric acid, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total adiponectin, and high-molecular-weight adiponectin. These data indicate that Gnetin-C has biological effects of enhancing the NK activity on circulating human immune cells. The immunomodulatory effects are consistent with a putative improvement in cancer immunosurveillance via the upregulation of the NKG2D receptor. The study was registered with UMIN-CTR, number 000030364, on 12 December 2017.

Natural killer (NK) cells are innate lymphocytes that exhibit many features of adaptive immunity, including clonal proliferation and long-lived memory. Here we demonstrate that the BTB-ZF transcription factor Zbtb32 (also known as ROG, FAZF, TZFP and PLZP) was essential for the proliferative burst and protective capacity of virus-specific NK cells. Signals from proinflammatory cytokines were both necessary and sufficient to induce high expression of Zbtb32 in NK cells. Zbtb32 facilitated NK cell proliferation during infection by antagonizing the anti-proliferative factor Blimp-1 (Prdm1). Our data support a model in which Zbtb32 acts as a cellular 'hub' through which proinflammatory signals instruct a 'proliferation-permissive' state in NK cells, thereby allowing their prolific expansion in response to viral infection.

To develop more effective vaccines and strategies to regulate chronic inflammatory diseases, it is important to understand the mechanisms of immunological memory. Factors regulating memory CD4(+) T helper (Th)-cell pool size and function remain unclear, however. We show that activation of type I invariant natural killer T (iNKT) cells with glycolipid ligands and activation of type II natural killer T (NKT) cells with the endogenous ligand sulfatide induced dramatic proliferation and expansion of memory, but not naïve, CD4 T cells. NKT cell-induced proliferation of memory Th1 and Th2 cells was dependent largely on the production of IL-2, with Th2-cell proliferation also affected by loss of IL-4. Type II NKT cells were also required for efficient maintenance of memory CD4 T cells in vivo. Activation of iNKT cells resulted in up-regulation of IFN-γ expression by memory Th2 cells. These IFN-γ-producing memory Th2 cells showed a decreased capability to induce Th2 cytokines and eosinophilic airway inflammation. Thus, activated NKT cells directly regulate memory CD4 T-cell pool size and function via the production of cytokines in vivo.

Rationale: Chronic inflammation in the myocardium is involved in the development of left ventricular (LV) remodeling and failure after myocardial infarction (MI). Invariant natural killer T (iNKT) cells have been shown to produce inflammatory cytokines and orchestrate tissue inflammation. However, no previous studies have determined the pathophysiological role of iNKT cells in post-MI LV remodeling., Objective: The purpose of this study was to examine whether the activation of iNKT cells might affect the development of LV remodeling and failure., Methods and Results: After creation of MI, mice received the injection of either α-galactosylceramide (αGC; n=27), the activator of iNKT cells, or phosphate-buffered saline (n=31) 1 and 4 days after surgery, and were followed during 28 days. Survival rate was significantly higher in MI+αGC than MI+PBS (59% versus 32%, P<0.05). LV cavity dilatation and dysfunction were significantly attenuated in MI+αGC, despite comparable infarct size, accompanied by a decrease in myocyte hypertrophy, interstitial fibrosis, and apoptosis. The infiltration of iNKT cells were increased during early phase in noninfarcted LV from MI and αGC further enhanced them. It also enhanced LV interleukin (IL)-10 gene expression at 7 days, which persisted until 28 days. AntienIL-10 receptor antibody abrogated these protective effects of αGC on MI remodeling. The administration of αGC into iNKT cell-deficient Jα18(-/-) mice had no such effects, suggesting that αGC was a specific activator of iNKT cells., Conclusions: iNKT cells play a protective role against post-MI LV remodeling and failure through the enhanced expression of cardioprotective cytokines such as IL-10.

Schnurri (Shn)-2 is a large zinc finger-containing protein implicated in cell growth, signal transduction and lymphocyte development. Here, we report that Shn-2-deficient (Shn-2(-/-)) mice develop CD3-positive lymphoma spontaneously. In Shn-2(-/-) mice, we observed decreased cytotoxicity of natural killer (NK) cells accompanied by decreased expression of perforin and granzyme-B. In addition, phosphorylation of signal transducer and activator of transcription (STAT) 5 was reduced in Shn-2(-/-) NK cells, while phosphorylation of STAT3 and protein expression of nuclear factor-κB p65 subunit were enhanced in Shn-2(-/-) NK cells. Moreover, cell-surface expression of activation molecules such as CD27, CD69 and CD122 were decreased on Shn-2(-/-) NK cells. Thus, Shn-2 is considered to play an important role in the activation and function of NK cells and the development of T cell lymphoma in vivo.

The progression of obesity is accompanied by a chronic inflammatory process that involves both innate and acquired immunity. Natural killer T (NKT) cells recognize lipid antigens and are also distributed in adipose tissue. To examine the involvement of NKT cells in the development of obesity, C57BL/6 mice (wild type; WT), and two NKT-cell-deficient strains, Jα18(-/-) mice that lack the type I subset and CD1d(-/-) mice that lack both the type I and II subsets, were fed a high fat diet (HFD). CD1d(-/-) mice gained the least body weight with the least weight in perigonadal and brown adipose tissue as well as in the liver, compared to WT or Jα18(-/-) mice fed an HFD. Histologically, CD1d(-/-) mice had significantly smaller adipocytes and developed significantly milder hepatosteatosis than WT or Jα18(-/-) mice. The number of NK1.1(+)TCRβ(+) cells in adipose tissue increased when WT mice were fed an HFD and were mostly invariant Vα14Jα18-negative. CD11b(+) macrophages (Mφ) were another major subset of cells in adipose tissue infiltrates, and they were divided into F4/80(high) and F4/80(low) cells. The F4/80(low)-Mφ subset in adipose tissue was increased in CD1d(-/-) mice, and this population likely played an anti-inflammatory role. Glucose intolerance and insulin resistance in CD1d(-/-) mice were not aggravated as in WT or Jα18(-/-) mice fed an HFD, likely due to a lower grade of inflammation and adiposity. Collectively, our findings provide evidence that type II NKT cells initiate inflammation in the liver and adipose tissue and exacerbate the course of obesity that leads to insulin resistance.

Functionally polarized helper T cells (Th cells) play crucial roles in the induction of tumor immunity. There is considerable knowledge about the contributions of IFN-producing Th1 cells that supports the role of cytotoxic cluster of differentiation (CD8) T cells and natural killer (NK) cells, but much less is known about how IL-4-producing Th2 cells contribute to tumor immunity. In this study, we investigated the cellular and molecular mechanisms employed by memory Th2 cells in sustaining tumor immunity by using a mouse model system wherein ovalbumin (OVA) is used as a specific tumor antigen. In this model, we found that OVA-specific memory Th2 cells exerted potent and long-lasting antitumor effects against NK-sensitive OVA-expressing tumor cells, wherein antitumor effects were mediated by NK cells. Specifically, NK cell cytotoxic activity and expression of perforin and granzyme B were dramatically enhanced by the activation of memory Th2 cells. Interleukin 4 (IL-4) produced by memory Th2 cells in vivo was critical for the antitumor effects of the NK cells, which IL-4 directly stimulated to induce their perforin- and granzyme-B-dependent cytotoxic activity. Our findings show that memory Th2 cells can induce potent antitumor immunity through IL-4-induced activation of NK cells, suggesting potential applications in cellular therapy for cancer patients., (©2011 AACR.)

T helper 2 (Th2) cells play crucial roles in the development of allergic asthma, while various distinct cell populations also contribute to the pathogenesis of the disease. Invariant natural killer T (iNKT) cells produce large amounts of cytokines such as IL-4 and IFNγ upon stimulation with a ligand, α-galactosylceramide, and regulate various immune responses. Recently, a critical role of iNKT cells in the mouse model of asthma and also in asthma patients has been reported, while some contradictory results have also been described. Here, we summarize the experimental results in mouse and human systems, and discuss the current understanding of the role of NKT cells in the pathogenesis of asthma, including a possible mechanism by which iNKT cells are activated in asthma patients., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

The aim of this clinical trial was to investigate the feasibility of intra-arterial infusion of in vitro-expanded Valpha24 natural killer T (NKT) cells combined with submucosal injection of alpha-galactosylceramide (KRN7000; alphaGalCer)-pulsed antigen-presenting cells (APC). A phase I clinical study was carried out in patients with head and neck squamous cell carcinoma (HNSCC). Patients with locally recurrent HNSCC refractory to standard therapy were eligible. Eight patients received super-selective transcatheter intra-arterial infusion of activated Valpha24 NKT cells into tumor-feeding arteries and nasal submucosal injections of alphaGalCer-pulsed APC twice with a 1-week interval. Valpha24 NKT cell-specific immune responses, safety, and antitumor effects were evaluated. The number of Valpha24 NKT cells and interferon-gamma-producing cells in peripheral blood mononuclear cells increased in seven out of eight patients enrolled. Grade 3 toxicity with a pharyngocutaneous fistula related to local tumor reduction was observed in one patient and mild adverse events with grade 1-2 symptoms occurred in seven patients. Regarding the clinical responses, three cases exhibited a partial but significant response, four were classified as stable disease, and one patient continued to develop progressive disease. The use of the intra-arterial infusion of activated Valpha24 NKT cells and the submucosal injection of alphaGalCer-pulsed APC has been shown to induce significant antitumor immunity and had beneficial clinical effects in the management of advanced HNSCC. The use of such therapeutic modalities may be helpful in the management of tumors and therefore needs to be explored in further detail. The clinical trial registration number was UMIN000000722.

In the present study, we examined the roles of natural killer T (NKT) cells in host defence against Legionella pneumophila in a mouse model. The survival rate of NKT cell-deficient Jalpha281 knock-out (KO) mice was significantly higher than that of wild-type mice. There was no bacterial overgrowth in the lungs, but Jalpha281 KO mice showed enhanced pulmonary clearance at a later stage of infection, compared with their wild-type counterparts. The severity of lung injury in L. pneumophila-infected Jalpha281 KO mice was less, as indicated by lung permeability measurements, such as lung weight and bronchoalveolar lavage fluid albumin concentration. Recruitment of inflammatory cells in the lungs was approximately twofold greater in Jalpha281 KO mice on day 3. Interestingly, higher values of interleukin (IL)-1beta and IL-18, and increased caspase-1 activity were noted in the lungs of Jalpha281 KO mice from an early time point (6 h). Exogenous alpha-galactosylceramide, a ligand of NKT cells, induced IL-12 and gamma interferon at 6 h, but suppressed IL-1beta at later time points in wild-type, whereas no effects were evident in Jalpha281 KO mice, as expected. Systemic administration of heat-killed L. pneumophila, but not Escherichia coli LPS, reproduced exaggerated production of IL-1beta in the lungs of Jalpha281 KO mice. These results demonstrate that NKT cells play a role in host defence against L. pneumophila, which is characterized by enhanced lung injury and decreased accumulation of inflammatory cells in the lungs. The regulation of IL-1beta, IL-18 and caspase-1 may be associated with the modulating effect of host responses by NKT cells.

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.

Wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a variety of mammalian cell types in vitro, but does not replicate in these cells. We investigated the effects of AcMNPV in the induction of the immune response and tumor metastasis in mice. After intravenous injection, AcMNPV was taken up by the liver and spleen, and preferentially infected dendritic cells (DCs) and B cells in the spleen; costimulatory molecules CD40, CD80, and CD86 were upregulated in the DCs. The hepatic mononuclear cells (MNCs) in these animals were highly cytotoxic to natural killer (NK)-sensitive YAC-1 and B16 melanoma cells, but not to NK-resistant EL4 cells. Intravenous injection of AcMNPV-induced NK cell proliferation in the liver and spleen, and enhanced antitumor immunity in mice with B16 liver metastases. Furthermore, such treatment increased the survival of C57BL/6, J alpha 281 (-/-), and interferon (IFN)-gamma (-/-) mice that were previously injected with B16 tumor cells. AcMNPV injection did not enhance the survival of NK cell-depleted mice. Moreover, one AcMNPV treatment effectively prolonged survival in a B16 liver metastasis model, and was equivalent to five treatments with recombinant interleukin-12 (IL-12) protein. These findings suggest that AcMNPV efficiently stimulates NK cell-mediated antitumor immunity.

Background: Chronic sinusitis (CS) with asthma generally exhibits a high degree of sinus tissue eosinophilia and recurrence often occurs even after surgical therapy. However, the cause has not yet been fully clarified., Aims of the Study: To elucidate the pathogenesis of this refractory disease, we examined the infiltration of natural killer T (NKT) and type 1 helper T (Th1)/type 2 helper T (Th2) cells, and the cytokine expression in the sinus mucosa., Methods: Sinus mucosal specimens were obtained surgically from 16 CS patients with nasal polyps. The NKT cells, Th1/Th2 cells and the expression of IL-4, IL-5, IL-13 and IFN-gamma were examined by a polymerase chain reaction or flow cytometry. Nasal mucosal specimens from six other patients with allergic rhinitis (AR) were examined in a similar manner., Results: The NKT cells were detected to varying degrees in the sinus mucosa from asthmatic CS patients, but neither in the nonasthmatics nor in the nasal mucosa from the patients with AR. The Th2 cells and Th2 cytokines were expressed at significantly higher levels in the sinus mucosa from the CS patients with asthma in comparison to those without asthma. However, the Th1 cell infiltration and IFN-gamma expression were not different between these groups., Conclusion: Natural killer T cells may, therefore, play important roles in the enhanced Th2 cytokine expression and increased infiltration of Th2 cells and eosinophils observed in the sinus mucosa from asthmatic CS patients through MHC-independent mechanisms.

The liver contains significant numbers of invariant natural killer T (iNKT) cells, which have an invariant T cell receptor-alpha chain and are activated in a CD1d-restricted manner. We examined the role of iNKT cells in the spontaneous tolerance of the major histocompatibility antigen complex-mismatched liver allograft model using Jalpha18 knockout mice that lack iNKT cells. Liver allografts lacking iNKT cells manifested not only infiltration but also hemorrhage and necrosis with significant reduction of graft survival and much less induction of tolerance compared with wild type (WT) liver allograft. In addition, allografts lacking iNKT cells grafted into iNKT-deficient recipients result in more severe inflammation than when grafted into WT recipients, while there was no significant difference with respect to induction of tolerance and graft survival. These results demonstrated that iNKT cells, especially donor-residual iNKT cells, constitute immune regulatory cells that play an important role in induction of allograft tolerance.

Valpha14 natural killer T (NKT) cells produce large amounts of both IL-4 and IFN-gamma upon stimulation with a ligand, alpha-galactosylceramide (alpha-GalCer), and play a crucial role in various immune responses, including allergic reactions. Interestingly, Valpha14 NKT cells are not essential for the induction of specific IgE response but they instead tend to induce suppression of specific IgE upon alpha-GalCer activation in vivo. The suppression in the IgE production is not detected either in Valpha14 NKT cell-deficient mice or in IFN-gamma-deficient mice. Therefore, activated Valpha14 NKT cells are able to exert a potent suppressive activity on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In an OVA-induced asthma model, alpha-GalCer administration inhibited airway inflammation and airway hyperreactivity by IFN-gamma from activated Valpha14 NKT cells, thus suggesting the negative regulation of Th2-responses by the activated Valpha14 NKT cells.

Previously, we demonstrated that Valpha14+ NKT cells and IFN-gamma are important upstream components in neutrophil-mediated host defense against infection with Streptococcus pneumoniae. In the present study, we extended these findings by elucidating the role of IFN-gamma in this Valpha14+ NKT cell-promoted process. Administration of recombinant IFN-gamma to Jalpha18KO mice prolonged the shortened survival, promoted the attenuated clearance of bacteria and improved the reduced accumulation of neutrophils and synthesis of MIP-2 and TNF-alpha in the lungs, in comparison to wild-type (WT) mice. In addition, intravenous transfer of liver mononuclear cells (LMNC) from WT mice into Jalpha18KO mice resulted in complete recovery of the depleted responses listed above, whereas such effects were not detected when LMNC were obtained from IFN-gammaKO or Jalpha18KO mice. Activation of Valpha14+ NKT cells by alpha-galactosylceramide (alpha-GalCer) significantly enhanced the clearance of bacteria, accumulation of neutrophils and synthesis of MIP-2 and TNF-alpha in the infected lungs; this effect was significantly inhibited by a neutralizing anti-IFN-gamma antibody. Finally, in a flow cytometric analysis, TNF-alpha synthesis was detected largely by CD11b(bright+) cells in the infected lungs. Our results demonstrated that IFN-gamma plays an important role in the neutrophil-mediated host protective responses against pneumococcal infection promoted by Valpha14+ NKT cells.

Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Valpha14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bepsilon cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2-modifying factor, resulting in Bepsilon cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell-dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.

Purpose: Human Valpha24 natural killer T (Valpha24 NKT) cells bearing an invariant Valpha24JalphaQ antigen receptor are activated by a glicolipid ligand alpha-galactosylceramide (alphaGalCer; KRN7000) in a CD1d-dependent manner. The human Valpha24 NKT cells activated with alphaGalCer and interleukin-2 have been shown to produce large amounts of cytokines, such as IFN-gamma, and also exerting a potent killing activity against various tumor cell lines. We did a phase I study with autologous activated Valpha24 NKT cell therapy., Experimental Design: Patients with advanced or recurrent non-small cell lung cancer received i.v. injections of activated Valpha24 NKT cells (level 1: 1 x 10(7)/m2 and level 2: 5 x 10(7)/m2) to test the safety, feasibility, and clinical response of this therapeutic strategy. Immunomonitoring was also done in all cases., Results: Six patients were enrolled in this study. No severe adverse events were observed during this study in any patients. After the first and second injection of activated Valpha24 NKT cells, an increased number of peripheral blood Valpha24 NKT cells was observed in two of three cases receiving a level 2 dose of activated Valpha24 NKT cells. The number of IFN-gamma-producing cells in peripheral blood mononuclear cells increased after the administration of activated Valpha24 NKT cells in all three cases receiving the level 2 dose. No patient was found to meet the criteria for either a partial or a complete response., Conclusions: The clinical trial with activated Valpha24 NKT cell administration was well tolerated and carried out safely with minor adverse events even in patients with advanced diseases.

CD1d-restricted NKT cells are reported to play a critical role in the host defense to pulmonary infection with Pseudomonas aeruginosa. However, the contribution of a major subset expressing a Valpha14-Jalpha18 gene segment remains unclear. In the present study, we re-evaluated the role of NKT cells in the neutrophilic inflammatory responses and host defense to this infection using mice genetically lacking Jalpha18 or CD1d (Jalpha18KO or CD1dKO mice). These mice cleared the bacteria in lungs at a comparable level to wild-type (WT) mice. There was no significant difference in the local neutrophilic responses, as shown by neutrophil counts and synthesis of MIP-2 and TNF-alpha, in either KO mice from those in WT mice. Administration of alpha-galactosylceramide, a specific activator of Valpha14+ NKT cells, failed to promote the bacterial clearance and neutrophilic responses, although the same treatment increased the synthesis of IFN-gamma, suggesting the involvement of this cytokine downstream of NKT cells. In agreement against this notion, these responses were not further enhanced by administration of recombinant IFN-gamma in the infected Jalpha18KO mice. Our data indicate that NKT cells play a limited role in the development of neutrophilic inflammatory responses and host defense to pulmonary infection with P. aeruginosa.

A unique lymphocyte population, CD1d-restricted NKT cells, has been revealed to be a key player in both the innate and acquired immune responses, including antitumor effects. Recent studies revealed that at least two subsets of CD1d-restricted NKT cells exist: type I, having invariant Valpha14 receptor; and type II, having heterogeneous non-Valpha14 receptor. The specific glycolipid ligand, alpha-GalCer, effectively stimulates mouse and human type I NKT cells. The activation of type I NKT cells substantially influences function of other various cell types, particularly DC, NK cells, CD4 Th1 cells, and CD8 cytotoxic T cells, all contributing to the antitumor immune responses. Recent studies also indicated that, unlike type I NKT cells, type II NKT cells have a potential to repress antitumor immune responses. In this review, we summarize the characteristics of the antitumor immune responses mediated by both mouse and human CD1d-restricted NKT cells and discuss their potential in clinical applications against cancer.

Mouse CD1d-restricted Valpha14 NKT cells are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster myoblast city, is critical for lymphocyte migration and regulates T cell responsiveness through immunological synapse formation, yet its role in Valpha14 NKT cells remains unknown. We found that DOCK2 deficiency causes marked reduction of Valpha14 NKT cells in the thymus, liver, and spleen. When alpha-galactosylceramide (alpha-GalCer), a ligand for Valpha14 NKT cells, was administrated, cytokine production was scarcely detected in DOCK2-deficient mice, suggesting that DOCK2 deficiency primarily affects generation of Valpha14 NKT cells. Supporting this idea, staining with CD1d/alpha-GalCer tetramers revealed that CD44- NK1.1- Valpha14 NKT cell precursors are severely reduced in the thymuses of DOCK2-deficient mice. In addition, studies using bone marrow chimeras indicated that development of Valpha14 NKT cells requires DOCK2 expression in T cell precursors, but not in APCs. These results indicate that DOCK2 is required for positive selection of Valpha14 NKT cells in a cell-autonomous manner, thereby suggesting that avidity-based selection also governs development of this unique subset of lymphocytes in the thymus.

Asthma is an inflammatory lung disease, in which conventional CD4+ T cells producing IL-4/IL-13 appear to play an obligatory pathogenic role. Here we show, in a mouse model of asthma, that activation of pulmonary IL-4/IL-13 producing invariant TCR+ CD1d-restricted natural killer T (NKT) cells is sufficient for the development of airway hyperreactivity (AHR), a cardinal feature of asthma, in the absence of conventional CD4+ T cells and adaptive immunity. Respiratory administration of glycolipid antigens that specifically activate NKT cells (alpha-GalactosylCeramide and a Sphingomonas bacterial glycolipid) rapidly induced AHR and inflammation typically associated with protein allergen administration. Naïve MHC class II-deficient mice, which lack conventional CD4+ T but have NKT cells, showed exaggerated baseline AHR and, when challenged with alpha-GalactosylCeramide, demonstrated even greater AHR. These studies demonstrate an expanded role for NKT cells, in which NKT cells not only produce cytokines that influence adaptive immunity but also function as critical effector cells that can induce AHR. These results suggest that NKT cells responding to glycolipid antigens, as well as conventional CD4+ T cells responding to peptide antigens, may be synergistic in the induction of AHR, although in some cases, each may independently induce AHR.

A role of natural killer T (NKT) cells in transplant rejection remains unknown. Here, we determined whether NKT cells participate in rejection of islet allografts, using NKT cell-deficient mice. Survival of islet allografts in streptozotocin-induced diabetic CD1d(-/-) mice or Valpha14 NKT cell(-/-) mice was significantly prolonged without immunosuppression when grafted into the liver, but not beneath the kidney capsule, compared with wild-type mice. Acceptance of intrahepatic islet allografts was achieved in CD1d(-/-) mice by a subtherapeutic dose of rapamycin, which was abrogated in conjunction with the transfer of hepatic mononuclear cells from wild-type, but not from CD1d(-/-), mice at islet transplantation. The second islet grafts from a donor-specific, but not from a third-party, strain in CD1d(-/-) mice bearing functional islet allografts were accepted without immunosuppression at 120 days after the initial transplantation. These findings demonstrate that NKT cells play a significant role in rejection of islet allografts in the liver of mice, but that NKT cells are not essential for induction of donor-specific unresponsiveness in this model. The current study indicates that NKT cells might be considered as a target for intervention to prevent islet allograft rejection when the liver is the site of transplantation.

Human invariant Valpha24+ natural killer T (NKT) cells display potent antitumor activity upon stimulation. Activation of endogenous Valpha24+ NKT cells would be one strategy for the treatment of cancer patients. For example, dendritic cells (DCs) loaded with a glycolipid NKT cell ligand, alpha-galactosylceramide (alphaGalCer, KRN7000), are a possible tool for the activation and expansion of functional Valpha24+ NKT cells in vivo. In this report, we demonstrate that the levels of expansion and the ability to produce IFN-gamma of Valpha24+ NKT cells induced by alphaGalCer-loaded whole PBMCs cultured with IL-2 and GM-CSF (IL-2/GM-CSF-cultured PBMCs) were superior to those of cells induced by monocyte-derived CD11c+ DCs (moDCs) developed with IL-4 and GM-CSF. Interestingly, CD11c+ cells in the IL-2/GM-CSF-cultured PBMCs showed a mature phenotype without further stimulation and exerted potent stimulatory activity on Valpha24+ NKT cells to enable them to produce IFN-gamma preferentially at an extent equivalent to mature moDCs induced by stimulation with LPS or a cytokine cocktail. Cocultivation with CD11c- cells in the IL-2/GM-CSF-cultured PBMCs induced maturation of moDCs. In particular, CD11c-CD3+ T cells appeared to play important roles in DC maturation. In addition, TNF-alpha was preferentially produced by CD11c-CD3+ T cells in IL-2/GM-CSF-cultured PBMCs and was involved in the maturation of moDCs. Thus, the maturation of DCs induced by CD11c- T cells through TNF-alpha production appears to result in the efficient expansion and activation of Valpha24+ NKT cells to produce IFN-gamma preferentially.

Valpha14 NKT cells exhibit various immune regulatory properties in vivo, but their precise mechanisms remain to be solved. In this study, we demonstrate the mechanisms of generation of regulatory dendritic cells (DCs) by stimulation of Valpha14 NKT cells in vivo. After repeated injection of alpha-galactosylceramide (alpha-GalCer) into mice, splenic DCs acquired properties of regulatory DCs in IL-10-dependent fashion, such as nonmatured phenotypes and increased IL-10 but reduced IL-12 production. The unique cytokine profile in these DCs appears to be regulated by ERK1/2 and IkappaB(NS). These DCs also showed an ability to suppress the development of experimental allergic encephalomyelitis by generating IL-10-producing regulatory CD4 T cells in vivo. These findings contribute to explaining how Valpha14 NKT cells regulate the immune responses in vivo.

A unique lymphocyte population, Valpha14 NKT cells, has recently been revealed to be a key player in the immune responses against tumors. Activation of Valpha14 NKT cells affects various cell types, particularly dendritic cells (DCs), NK cells, CD4 Th1 cells, and CD8 cytotoxic T cells in the innate and acquired immune systems, eventually resulting in the enhanced activation of NKT cell-mediated cellular cascade in the anti-tumor responses. The specific ligand, alpha-galactosylceramide (alpha-GalCer), effectively stimulates mouse and human NKT cells, making NKT cells an ideal target for the development of cancer immunotherapy. Clinical trials using alpha-GalCer have actually started in several centers in the world. In this review, we summarize the Valpha14 NKT cell-mediated cellular cascade in the anti-tumor response in mice and discuss potential clinical applications of alpha-GalCer-pulsed DC therapy.

Purpose: Human Valpha24 natural killer T (NKT) cells bearing an invariant Valpha24JalphaQ antigen receptor, the counterpart of murine Valpha14 NKT cells, are activated by a specific ligand, alpha-galactosylceramide (alphaGalCer, KRN7000), in a CD1d-dependent manner. I.v. administration of alphaGalCer-pulsed dendritic cells (DC) induces significant activation and expansion of Valpha14 NKT cells in the lung and resulting potent antitumor activities in mouse tumor metastatic models. We did a phase I dose escalation study with alphaGalCer-pulsed DCs in lung cancer patients., Experimental Design: Patients with advanced non-small cell lung cancer or recurrent lung cancer received i.v. injections of alphaGalCer-pulsed DCs (level 1: 5 x 10(7)/m(2); level 2: 2.5 x 10(8)/m(2); and level 3: 1 x 10(9)/m(2)) to test the safety, feasibility, and clinical response. Immunomonitoring was also done in all completed cases., Results: Eleven patients were enrolled in this study. No severe adverse events were observed during this study in any patient. After the first and second injection of alphaGalCer-pulsed DCs, dramatic increase in peripheral blood Valpha24 NKT cells was observed in one case and significant responses were seen in two cases receiving the level 3 dose. No patient was found to meet the criteria for partial or complete responses, whereas two cases in the level 3 group remained unchanged for more than a year with good quality of life., Conclusions: In this clinical trial, alphaGalCer-pulsed DC administration was well tolerated and could be safely done even in patients with advanced disease.

Natural killer type-1 (NK1)/NK2 terminology was proposed as an analogy to T helper type-1 (Th1)/Th2 subsets of CD4 T cells. In addition to well recognized IFN-gamma-producing NK1 cells, recent accumulating evidence suggests the presence and the regulatory function of NK2 cells, which produce type-2 cytokines, such as IL-5 and IL-13. Most studies on the production of cytokines by NK1/NK2 subsets have been performed in the human system. Recently, a mouse in vitro NK1/NK2 cell differentiation system was established. A linear 2-0-1 model has been proposed for the differentiation of NK1/NK2 cell subsets, but the branching NK1/NK2 cell differentiation model, comparable to Thl/Th2 cell differentiation, remains a likely explanation. In this review, we summarize the evidence indicating the unique features of NK1/NK2 cells and discuss the above two NK cell differentiation models. In addition, we point to interesting similarities in the expression of transcription factors that regulate cytokine production in NK cells and CD8 T cells.

NK cells differentiate into either NK1 or NK2 cells that produce IFN-gamma or IL-5 and IL-13, respectively. Little is known, however, about the molecular mechanisms that control NK1 and NK2 cell differentiation. To address these questions, we established an in vitro mouse NK1/NK2 cell differentiation culture system. For NK1/NK2 cell differentiation, initial stimulation with PMA and ionomycin was required. The in vitro differentiated NK2 cells produced IL-5 and IL-13, but the levels were 20 times lower than those of Th2 or T cytotoxic (Tc)2 cells. No detectable IL-4 was produced. Freshly prepared NK cells express IL-2Rbeta, IL-2RgammaC, and IL-4Ralpha. After stimulation with PMA and ionomycin, NK cells expressed IL-2Ralpha. NK1 cells displayed higher cytotoxic activity against Yac-1 target cells. The levels of GATA3 protein in developing NK2 cells were approximately one-sixth of those in Th2 cells. Both NK1 and NK2 cells expressed large amounts of repressor of GATA, the levels of which were equivalent to CD8 Tc1 and Tc2 cells and significantly higher than those in Th2 cells. The levels of histone hyperacetylation of the IL-4 and IL-13 gene loci in NK2 cells were very low and equivalent to those in naive CD4 T cells. The production of IL-5 and IL-13 in NK2 cells was found to be STAT6 dependent. Thus, similar to Th2 cells, NK2 cell development is dependent on STAT6, and the low level expression of GATA3 and the high level expression of repressor of GATA may influence the unique type 2 cytokine production profiles of NK2 cells.

We have investigated the potential role of CD1d-restricted natural killer T (NKT) cells in the development of atherosclerosis in mice. When fed an atherogenic diet (AD), NKT cell-deficient CD1d(-/-) mice had significantly smaller atherosclerotic lesions than AD-fed C57BL/6 (wild-type [WT]) mice. A significant reduction in atherosclerotic lesions was also demonstrated in AD-fed, low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice reconstituted with CD1d(-/-) bone marrow cells compared with the lesions observed in Ldlr(-/-)mice reconstituted with WT marrow cells. In addition, repeated injections of alpha-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 alpha-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 Valpha14Jalpha18 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-gamma, a potentially proatherogenic T-helper 1 (TH1) cytokine. Collectively, we conclude that NKT cells are proatherogenic in mice.

Both osteopontin (OPN) and natural killer T (NKT) cells play a role in the development of immunological disorders. We examined a functional link between OPN and NKT cells. Concanavalin A (Con A)-induced hepatitis is a well-characterized murine model of T cell-mediated liver diseases. Here, we show that NKT cells secrete OPN, which augments NKT cell activation and triggers neutrophil infiltration and activation. Thus, OPN- and NKT cell-deficient mice were refractory to Con A-induced hepatitis. In addition, a neutralizing antibody specific for a cryptic epitope of OPN, exposed by thrombin cleavage, ameliorated hepatitis. These findings identify NKT cell-derived OPN as a novel target for the treatment of inflammatory liver diseases.

Natural killer (NK) cells play a pivotal role in the immune reaction during the bone marrow allograft rejection. Little is known, however, about the molecular mechanisms underlying the NK cell-mediated allograft recognition and rejection. In this report, we assessed the role of a recently identified NK receptor, killer cell lectinlike receptor 1 (KLRE-1), by generating knock-out mice. KLRE-1-deficient mice were born at an expected frequency and showed no aberrant phenotype on growth and lymphoid development. Nevertheless, KLRE-1-deficient cells showed a severely compromised allogeneic cytotoxic activity compared with the wild-type cells. Furthermore, allogeneic bone marrow transfer culminated in colony formation in the spleen of KLRE-1-deficient mice, whereas no colony formation was observed in wild-type recipient mice. These results demonstrate that KLRE-1 is a receptor mediating recognition and rejection of allogeneic target cells in the host immune system.

Sarcoidosis is a systemic disorder associated with granuloma characterized by an abnormal T(h)1-type cytokine production and accumulation of T(h)1 CD4 T cells in the granuloma lesions, suggesting an importance of T(h)1 responses in sarcoidosis. However, the pathogenesis of sarcoidosis remains to be solved. Here, we investigated the nature of V(alpha)24 NKT cells with immunoregulatory functions in sarcoidosis. Patients with non-remitting sarcoidosis displayed a decrease in the number of V(alpha)24 NKT cells in peripheral blood, but an accumulation of these cells in granulomatous lesions. When stimulated with the specific glycolipid ligand, alpha-galactosylceramide, peripheral blood V(alpha)24 NKT cells from patients with non-remitting disease produced significantly less IFN-gamma than those from healthy volunteers, but normal levels of IL-4. The reduced IFN-gamma production was observed only in V(alpha)24 NKT cells and not conventional CD4 T cells, but was normal in patients with remitting disease, suggesting that non-remitting sarcoidosis involves an insufficient IFN-gamma production of V(alpha)24 NKT cells which is well correlated with disease activity. Thus, these results suggest that V(alpha)24 NKT cells play a crucial role in the disease status of sarcoidosis.

NKT cells expressing the invariant Valpha14 antigen receptor constitute a novel lymphocyte subpopulation with immunoregulatory functions. Stimulation via their invariant Valpha14 receptor with anti-CD3 or a ligand, alpha-galactosylceramide (alpha-GalCer), triggers activation of Valpha14 NKT cells, resulting in a rapid cytokine production such as IFN-gamma and IL-4. Soon after their receptor activation, Valpha14 NKT cells disappeared as judged by staining with CD1d tetramer loaded with alpha-GalCer (alpha-GalCer/CD1d tetramer), which has been believed to be due to apoptotic cell death. Here we show that such a disappearance was largely attributed to down-regulation of the Valpha14 receptor. In fact, Valpha14 NKT cells were relatively resistant to apoptosis compared to the conventional T cells as evidenced by less staining with Annexin-V, a limited DNA fragmentation, and their preferential expression of anti-apoptotic genes such as NAIP and MyD118. Furthermore, they did not become tolerant, and maintained their proliferative capacity and cytokine production even after their receptor down-regulation. These as yet unrecognized facets of Valpha14 NKT cells are discussed in relation to their regulatory functions.

Natural killer (NK) cells mediate bone marrow allograft rejection. However, the molecular mechanisms underlying such a rejection remain elusive. In previous analyses, it has been shown that NK cells recognize allogeneic target cells through Ly-49s and CD94/NKG2 heterodimers. Here, we describe identification and characterization of a novel murine NK receptor, NKG2I, belonging to the NKG2 family. NKG2I, which was composed of 226 amino acids, showed approximately 40% homology to the murine NKG2D and CD94 in the C-type lectin domain. Flow cytometric analysis with anti-NKG2I monoclonal antibody (mAb) revealed that expression of NKG2I was largely confined to NK and NKT cells, but was not seen in T cells. Furthermore, anti-NKG2I mAb inhibited NK cell-mediated cytotoxicity, whereas cross-linking of NKG2I enhanced interleukin 2- and interleukin 12-dependent interferon-gamma production. Similarly, the injection of anti-NKG2I mAb before the allogeneic bone marrow transfer in vivo impinged on the function of NKG2I, resulting in the enhanced colony formation in the spleen. NKG2I is a novel activating receptor mediating recognition and rejection of allogeneic target cells.

The present study was designed to elucidate the role of Valpha14(+) NKT cells in the host defense against pulmonary infection with Streptococcus pneumoniae using Jalpha281 gene-disrupted mice (Jalpha281KO mice) that lacked this lymphocyte subset. In these mice, pneumococcal infection was severely exacerbated, as shown by the shorter survival time and marked increase of live bacteria in the lung compared to wild-type (WT) mice. The proportion of Valpha14(+) NKT cells, detected by an alpha-galactosylceramide (alpha-GalCer)-loaded CD1d tetramer, increased in thelung after S. pneumoniae infection. This increase was significantly reduced in mice with a genetic disruption of monocyte chemotactic protein (MCP)-1, which was produced in the early phaseof infection in WT mice. In the lungs of Jalpha281KO mice, the number of neutrophils was significantly lower at 12 h than that in WT mice. In support of this finding, macrophage inflammatory protein (MIP)-2 and TNF-alpha synthesis in infected lungs was significantly reduced at 3 h and at both 3 and 6 h, respectively, in Jalpha281KO mice, compared to WT mice. In addition, treatment of mice with alpha-GalCer significantly improved the outcome of this infection. Our results demonstrated MCP-1-dependent recruitment of Valpha14(+) NKT cells and their critical role in early host protection against S. pneumoniae by promoting the trafficking of neutrophils to the site of infection.

Although we have previously demonstrated that IL-12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by IL-12 or by alpha-galactosylceramide (alpha-GalCer) on murine liver regeneration using Valpha 14 NKT knockout (Jalpha 281(-/-)) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jalpha 281(+/+) but not in Jalpha 281(-/-) mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) gamma and tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA) after stimulation with both factors in Jalpha 281(+/+) mice. Either anti-IFN-gamma or TNF-alpha antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF-alpha injection similarly caused injury in hepatectomized livers of both Jalpha 281(+/+) and Jalpha 281(-/-) mice; indeed, adoptively transferred TNF-alpha(+/+) NKT cells enhanced liver injury after hepatectomy in TNF-alpha knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF-alpha synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.

Using natural killer T (NKT) cell-deficient mice, we show here that allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma, does not develop in the absence of V(alpha)14i NKT cells. The failure of NKT cell-deficient mice to develop AHR is not due to an inability of these mice to produce type 2 T-helper (Th2) responses because NKT cell-deficient mice that are immunized subcutaneously at non-mucosal sites produce normal Th2-biased responses. The failure to develop AHR can be reversed by the adoptive transfer of tetramer-purified NKT cells producing interleukin (IL)-4 and IL-13 to Ja281(-/-) mice, which lack the invariant T-cell receptor (TCR) of NKT cells, or by the administration to Cd1d(-/-) mice of recombinant IL-13, which directly affects airway smooth muscle cells. Thus, pulmonary V(alpha)14i NKT cells crucially regulate the development of asthma and Th2-biased respiratory immunity against nominal exogenous antigens. Therapies that target V(alpha)14i NKT cells may be clinically effective in limiting the development of AHR and asthma.

The reductive-oxidative status of tissues regulates the expression of many inflammatory genes that are induced during gram-negative bacterial infections. The cytokine gamma interferon (IFN-gamma) is a potent stimulus for host inflammatory gene expression, and oxidative stress has been shown to inhibit its production in mice challenged with Escherichia coli bacteria. The objective of the present study was to characterize the cells that produced IFN-gamma in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-gamma-expressing lymphocytes following challenge with viable E. coli bacteria. The surface phenotypes of IFN-gamma-expressing hepatic lymphocytes were those of natural killer (NK) cells (NK1.1(+) CD3(-)), conventional T cells (NK1.1(-) CD3(+)), and NK T cells (NK1.1(+) CD3(+)). Treating mice with diethyl maleate to deplete tissue thiols significantly impaired IFN-gamma production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response to E. coli challenge. However, IFN-gamma expression by a subset of NK T cells, which did not bind alpha-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-gamma-expressing cells were also predominantly CD8(+) and bore gamma delta T-cell antigen receptors. The residual IFN-gamma response by NK T cells may explain previous reports of hepatic gene expression following gram-negative bacterial challenge in thiol-depleted mice. The finding also demonstrates that innate immune cells differ significantly in their responses to altered tissue redox status.

Human endometrium is infiltrated by natural killer (NK) cells throughout the menstrual cycle. The number of endometrial NK cells is low in the proliferative phase, but acutely increases after ovulation, and reaches a peak in the late secretory phase, suggesting that endometrium recruits these leukocytes selectively from circulating peripheral blood. We investigated the expression of macrophage inflammatory protein (MIP)-1beta, a potential chemoattractant for NK cells, in the endometrium. RT-PCR and ELISA revealed that MIP-1beta is expressed in the endometrium throughout the menstrual cycle at both the message and protein levels. MIP-1beta expression is stronger in the secretory phase endometrium than in the proliferative phase endometrium. Immunohistochemistry revealed that MIP-1beta is localized in the surface epithelial cells, glandular epithelial cells, and perivascular stromal cells throughout the menstrual cycle. Stromal cells in a wider perivascular area became immunoreactive in the secretory phase. There was a strong correlation between the endometrial MIP-1beta concentration and the number of endometrial NK cells. Progesterone significantly induced MIP-1beta secretion from cultured endometrial stromal cells, whereas 17beta-estradiol had a weak effect. These results suggest that endometrial MIP-1beta may be involved in the recruitment of NK cells from circulating peripheral blood.

Objective: To clarify whether female sex steroids directly affect the bioactivity of the human endometrial CD16neg CD56bright natural killer (NK) cells., Design: In vitro study., Setting: University obstetrics and gynecology department., Patient(s): Thirteen women with histologically normal endometrium who were undergoing hysterectomy and seven women during the first trimester of pregnancy who were undergoing selective termination., Intervention(s): Endometrium or decidua was obtained., Main Outcome Measure(s): The effects of 17beta-estradiol or progesterone (10(-6), 10(-7), and 10(-8) M) on the proliferation, cytolytic activity, and cytokine secretion of the isolated endometrial CD16neg CD56bright NK cells were examined using a 3H-thymidine incorporation assay, 51Cr-releasing assay, and enzyme-linked immunosorbent assay, respectively., Result(s): Neither 17beta-estradiol nor progesterone had significant effects on the proliferation, cytolytic activity, and cytokine secretion of endometrial CD16neg CD56bright NK cells., Conclusion(s): Female sex steroids do not directly affect the bioactivity of the human endometrial CD16neg CD56bright NK cells.

A novel lymphocyte lineage, Valpha14 natural killer T (NKT) cells, is now well established as distinct from conventional alphabeta T cells. Valpha14 NKT cells express a single invariant Valpha14 antigen receptor that is essential for their development. Successful identification of a specific ligand, alpha-galactosylceramide(alpha-GalCer), and the establishment of gene-manipulated mice with selective loss of Valpha14 NKT cells helped elucidate the remarkable functional diversity of Valpha14 NKT cells in various immune responses such as host defense by mediating anti-nonself innate immune reaction, homeostatic regulation of anti-self responses, and antitumor immunity.

Human Valpha24 NKT cells bearing an invariant Valpha24JalphaQ antigen receptor, the counterpart of murine Valpha14 NKT cells, are activated by a specific ligand, alpha-GalCer, in a CD1d-dependent manner. Here, we demonstrate decreased numbers of circulating Valpha24 NKT cells in patients with primary lung cancer compared to healthy volunteers. However, Valpha24 NKT cells and DCs from lung cancer patients were functionally normal, even in the presence of tumor. Furthermore, levels of Valpha24 NKT cells in surgically resected lung tissue appeared to be equivalent to those of Valpha14 NKT cells in the mouse lung. Levels of Valpha24 NKT cells in the tumor tissue itself were increased about 2.5 times. Administration of alpha-GalCer-pulsed DCs expanded Valpha14 NKT cells in the lung more than 10 times, and the increased levels were sustained for 1 week. This may explain the previous finding that alpha-GalCer-pulsed DCs exerted strong antitumor activity in mouse lung tumor metastatic models. The potential use of alpha-GalCer-pulsed DCs for immunotherapy aimed at activating endogenous Valpha24 NKT cells in the lung of cancer patients is discussed., (Copyright 2002 Wiley-Liss, Inc.)

The important role played by CD8(+) T lymphocytes in the control of parasitic and viral infections, as well as tumor development, has raised the need for the development of adjuvants capable of enhancing cell-mediated immunity. It is well established that protective immunity against liver stages of malaria parasites is primarily mediated by CD8(+) T cells in mice. Activation of natural killer T (NKT) cells by the glycolipid ligand, alpha-galactosylceramide (alpha-GalCer), causes bystander activation of NK, B, CD4(+), and CD8(+) T cells. Our study shows that coadministration of alpha-GalCer with suboptimal doses of irradiated sporozoites or recombinant viruses expressing a malaria antigen greatly enhances the level of protective anti-malaria immunity in mice. We also show that coadministration of alpha-GalCer with various different immunogens strongly enhances antigen-specific CD8(+) T cell responses, and to a lesser degree, Th1-type responses. The adjuvant effects of alpha-GalCer require CD1d molecules, Valpha14 NKT cells, and interferon gamma. As alpha-GalCer stimulates both human and murine NKT cells, these findings should contribute to the design of more effective vaccines against malaria and other intracellular pathogens, as well as tumors.

To gain a better understanding of the pathological role of natural killer (NK) T cells in murine tuberculosis, NKT knockout (KO) mice (J(alpha)281(-/-)mice) were utilized. Eight-week-old NKT KO mice of BALB/c origin and wild-type (WT) mice were infected with Mycobacterium tuberculosis Kurono strain by the airborne route using an airborne infection apparatus, and their capacity to control mycobacterial growth, granuloma formation, and cytokine production were examined. The NKT KO mice developed granulomatous lesions in the lungs; there was no statistically significant difference in pulmonary granuloma size between NKT KO and WT mice (p<0.01). The average CFU values increased 3 weeks post-infection, but decreased 9 and 11 weeks post-infection, in the lungs of NKT KO mice. When stimulated with Kurono strain in vitro, splenic cells from NKT KO mice produced less IFN-gamma than did those from WT mice. Expression of mRNA for IL-2, IL-4, IL-6, IL-10 and IL-12 p40 was slightly lower in NKT KO mice compared with WT mice. Our data indicate that NKT cells play a detrimental role in late-phase mycobacterial infection, although Th1 cells are essential in early-phase mycobacterial infection.

We elucidated the contribution of Valpha14 NKT cells to Th1 response and host resistance against mycobacterial infection. In Valpha14 NKT cell-deficient mice, host defense and DTH response to Mycobacterium bovis BCG were not different from wild-type mice after pulmonary infection. There was no significant difference in the lung concentrations of IFN-gamma between the two strains of mice. In addition, host defense to systemic infection with M. tuberculosis was similar to that of M. bovis. Our results indicate that Valpha14 NKT cells play only a marginal role, if any, in the Th1 response and host resistance to mycobacterial infection.

Trypanosoma cruzi is a protozoan parasite that chronically infects many mammalian species and in humans causes Chagas' disease, a chronic inflammatory disease. The parasite expresses glycophosphoinositol (GPI), which potently stimulates interleukin 12 (IL-12) production. During T. cruzi infection IL-12, and possibly GPI, might stimulate NK T cells to affect the protective and chronic inflammatory responses. Here we report that during T. cruzi infection CD1d-restricted NK T cells are stimulated as NK T-cell-deficient mice have greater parasitemia. Furthermore, during T. cruzi infection the percentages of NK T cells in the liver and spleen become decreased for prolonged periods of time, and in vitro stimulation of NK T cells derived from livers of chronically infected mice, compared to uninfected mice, results in increased gamma interferon and IL-4 secretion. Moreover, in NK T-cell-deficient mice the chronic-phase antibody response to a GPI-modified surface protein is decreased. These results indicate that, during the acute infection, NK T cells limit parasitemia and that, during the chronic phase, NK T cells augment the antibody response. Thus, during T. cruzi infection the quality of an individual's NK T-cell response can affect the level of parasitemia and parasite tissue burden, the intensity of the chronic inflammatory responses, and possibly the outcome of Chagas' disease.

To elucidate the role of NKT cells in the host defense to cryptococcal infection, we examined the proportion of these cells, identified by the expression of CD3 and NK1.1, in lungs after intratracheal infection with Cryptococcus neoformans. This population increased on day 3 after infection, reached a peak level on days 6-7, and decreased thereafter. In Valpha14 NKT cell-deficient mice, such increase was significantly attenuated. The proportion of Valpha14 NKT cells, detected by binding to alpha-galactosylceramide-loaded CD1d tetramer, and the expression of Valpha14 mRNA increased after infection with a similar kinetics. The delayed-type hypersensitivity response and differentiation of the fungus-specific Th1 cells was reduced in Valpha14 NKT cell-deficient mice, compared with control mice. Additionally, elimination of this fungal pathogen from lungs was significantly delayed in Valpha14 NKT cell-deficient mice. Production of monocyte chemoattractant protein (MCP)-1 in lungs, detected at both mRNA and protein levels, increased on day 1, reached a peak level on day 3, and decreased thereafter, which preceded the increase in NKT cells. Finally, the increase of total and Valpha14(+) subset of NKT cells after infection was significantly reduced in MCP-1-deficient mice. Our results demonstrated that NKT cells, especially Valpha14(+) subset, accumulated in a MCP-1-dependent manner in the lungs after infection with C. neoformans and played an important role in the development of Th1 response and host resistance to this fungal pathogen.