The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specific cytotoxic T lymphocytes

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.     

Cytokine production and migration of in vitro-expanded NK1.1(-) invariant Valpha14 natural killer T (Valpha14i NKT) cells using alpha-galactosylceramide and IL-2

Mouse natural killer T cells with invariant Valpha14 rearrangement (Valpha14i NKT cells) can rapidly produce both Th1 and Th2 cytokines and regulate various immune responses, such as autoimmunity and tumor immunity. In this study, we describe the phenotypical and functional characterization of in vitro-expanded mouse Valpha14i NKT cells from spleen using a combination of alpha-galactosylceramide (alpha-GalCer) and IL-2. The expanded Valpha14i NKT cells retained the memory/activated (CD44(+)CD69(+)CD62L(-)) and CD4(+) or CD4(-)8(-) double negative phenotypes but modulated or lost the classical NKT cell marker, NK1.1. The expanded Valpha14i NKT cells continuously released IL-4 and IFNgamma and induced NK cell IFNgamma production in vitro. Furthermore, the expanded Valpha14i NKT cells migrated into the liver and spleen after adoptive transfer into lymphopenic SCID mice, and they were able to rapidly produce IL-4 and IFNgamma after alpha-GalCer injection. Our findings suggest that the intrinsic characteristics of the cytokine secretion of Valpha14i NKT cells were equivalent to that of in vitro-expanded Valpha14i NKT cells. In vitro-expanded Valpha14i NKT cells are considered to be useful for NKT cell defect-related diseases, such as autoimmunity and cancer.     

Enhancement of the synthetic ligand-mediated function of liver NK1.1Ag+ T cells in mice by interleukin-12 pretreatment

We previously reported that mouse NK1.1 Ag+ T (NKT) cells activated by interleukin-12 (IL-12) act as anti-tumour/anti-metastatic effectors. However, IL-12 reportedly induces a rapid disappearance of liver NKT cells by activation-induced apoptosis. In the present study, however, we show that injection of IL-12 into mice merely down-regulates the NK1.1 expression of liver NKT cells and Vbeta8+ intermediate T-cell receptor cells and CD1d/alpha-galactosylceramide (alpha-GalCer)-tetramer reactive cells in the liver remained and did not decrease. Furthermore, when IL-12-pretreated (24 hr before) mice were injected with alpha-GalCer, not only serum interferon-gamma but also serum IL-4 concentrations increased several-fold in comparison to the control alpha-GalCer-injected mice. However, IL-12 pretreatment markedly up-regulated serum ALT levels and Fas-ligand expression on NKT cells after alpha-GalCer injection in middle-aged mice only. Consistently, the liver mononuclear cells (MNC) from IL-12-pretreated mice stimulated with alpha-GalCer in vitro produced much greater amounts of interferon-gamma and IL-4, and also showed a more potent cytotoxicity against tumour targets than those from mice pretreated with phosphate-buffered saline. Liver MNC from middle-aged mice, but not from young mice pretreated with IL-12, also showed increased cytotoxicity following in vitro alpha-GalCer stimulation against cultured hepatocytes. Furthermore, IL-12 treatment of middle-aged mice enhanced tumour necrosis factor receptor 1 mRNA expression in liver Vbeta8+ T cells, and in vitro experiments also revealed that IL-12 pretreatment of liver MNC from middle-aged mice enhanced their tumour necrosis factor-alpha production after alpha-GalCer stimulation. Synthetic ligand-mediated functions of NKT cells, including IL-4 production, are thus enhanced by IL-12 pretreatment.     

In vivo activation of invariant V alpha 14 natural killer T cells by alpha-galactosylceramide sequentially induces Fas-dependent and -independent cytotoxicity

The present study was designed to clarify the cytotoxic capacities of invariant V alpha 14 natural killer T (iNKT) cells activated in vivo. We found that as early as 2 h after a single injection of alpha-galactosylceramide (alpha-GalCer), sorted iNKT splenocytes from treated mice kill Fas-transfected target cells. The implication of the Fas pathway in this lysis was strengthened by both the blockage of cytotoxicity in the presence of anti-Fas ligand (FasL) monoclonal antibody (mAb) and the up-regulation of FasL expression on iNKT cells. Sorted NK cells did not participate in the lytic activity at this time point. Yet, they became cytotoxic later on, 24 h post-treatment, when target cell lysis was mainly independent of the Fas pathway. This type of cell killing was predominant at this later time point, even though iNKT cells conserved a slight Fas-dependent cytotoxicity. NK cells failed to acquire the ability to kill target cells when IFN-gamma production in alpha-GalCer-injected mice was blocked by anti-IFN-gamma mAb, underscoring the major role of this cytokine. In conclusion, our findings provide the first direct evidence that iNKT cells can exert Fas-dependent cytotoxicity very shortly after in vivo alpha-GalCer activation and later, through IFN-gamma secretion, enable NK cells to kill target cells in a Fas-independent pathway.     

Phenotypical and functional alterations during the expansion phase of invariant Valpha14 natural killer T (Valpha14i NKT) cells in mice primed with alpha-galactosylceramide

Invariant Valpha14 natural killer T (Valpha14i NKT) cells are a unique immunoregulatory T-cell population that is restricted by CD1d. The glycolipid alpha-galactosylceramide (alpha-GalCer) is presented by CD1d and causes robust Valpha14i NKT-cell activation. Three days after injection of alpha-GalCer, Valpha14i NKT cells vigorously increase in number and then gradually decrease to normal levels. In the present study, we found that the re-administration of alpha-GalCer into mice primed 3 days earlier causes a marked increase in serum interleukin-4 and interferon-gamma. Intracellular staining revealed that the only expanded Valpha14i NKT cells are responsible for the enhanced cytokine production. The enhanced cytokine production was correlated with an increased number of Valpha14i NKT cells after priming. Additionally, primed Valpha14i NKT cells produced larger amounts of cytokine as compared with naive Valpha14i NKT cells when cultured with alpha-GalCer-pulsed dendritic cells. Thus, we considered that a subset of expanded Valpha14i NKT cells acquired a strong ability to produce cytokines. In contrast to mice primed 3 days earlier, cytokine production is markedly diminished in mice primed 7 days earlier. The expanded Valpha14i NKT cells altered the surface phenotype (NK1.1- CD69-) and contained intracellular interferon-gamma. Additionally, we found that primed Valpha14i NKT cells did not disappear or down-regulate surface TCR expression when re-injected with alpha-GalCer as compared with naive Valpha14i NKT cells. These results demonstrate that the function and surface phenotype of Valpha14i NKT cells is dramatically altered after alpha-GalCer priming.     

The natural killer T cell ligand alpha-galactosylceramide prevents or promotes pristane-induced lupus in mice

Systemic lupus erythematosus is a systemic autoimmune disease characterized by inflammation in organs such as kidneys and presence of autoantibodies against nuclear antigens. We have previously shown that CD1d deficiency in BALB/c mice exacerbates lupus nephritis and autoantibody production induced by the hydrocarbon oil pristane. Here, we have tested the impact of activating CD1d-restricted natural killer T (NKT) cells on pristane-induced lupus-like autoimmunity in BALB/c and SJL mice. Repeated in vivo treatment of pristane-injected BALB/c mice with the NKT cell ligand alpha-galactosylceramide (alpha-GalCer) prior to the onset of florid disease suppressed proteinuria, in a manner that was dependent on CD1d and IL-4 expression. In sharp contrast, however, similar treatment of pristane-injected SJL mice with alpha-GalCer resulted in increased proteinuria. Consistent with these dichotomous effects of NKT cell activation on the development of lupus-like autoimmunity, NKT cells in BALB/c and SJL/J mice exhibited a mixed Th1/Th2 and a Th1-biased cytokine production profile, respectively. These findings demonstrate that NKT cell activation with alpha-GalCer suppresses or promotes pristane-induced lupus-like autoimmunity in mice, in a strain-dependent manner.     

Activation-induced NKT cell hyporesponsiveness protects from alpha-galactosylceramide hepatitis and is independent of active transregulatory factors

NK T (NKT) cells, unique lymphocytes expressing features of NK and T lymphocytes, can specifically be activated with the glycolipid antigen alpha-galactosylceramide (alpha-GalCer). In humans and mice, this activation provokes pronounced cytokine responses. In C57BL/6 mice, alpha-GalCer injection additionally induces NKT-mediated liver injury, representing a model for immune-mediated hepatitis in humans. However, a single alpha-GalCer pretreatment of mice prevented NKT-mediated liver injury, cytokine responses (systemically and locally in the liver), and up-regulation of hepatocellular Fas upon alpha-GalCer rechallenge. As alpha-GalCer is used as a NKT cell-activating agent in clinical trials, an investigation of tolerance induction appears crucial. We demonstrate that alpha-GalCer tolerance does not depend on Kupffer cells, IL-10, Caspase-3-mediated apoptosis, or CD4+CD25+ T regulatory cells (Tregs), which are crucial in other models of immunological tolerance. Amending relevant, earlier approaches of others, we cocultivated highly purified, nontolerized and tolerized liver NKT cells ex vivo and could convincingly exclude the relevance of transdominant NKT Tregs. These results strongly suggest alpha-GalCer-induced tolerance to be exclusively caused by NKT cell intrinsic hyporesponsiveness. Tolerized mice showed specific diminishment of the intrahepatic CD4+ NKT cell subpopulation, with the CD4(-) population largely unaffected, and revealed down-modulation of alpha-GalCer-specific TCR and the NKT costimulator glucocorticoid-induced TNFR-related protein on liver NKT cells, whereas inhibitory Ly49I was increased. In conclusion, alpha-GalCer tolerance could serve as a model for the frequently observed NKT cell hyporesponsiveness in tumor patients and might help to develop strategies for their reactivation. Conversely, approaches to render NKT cells hyporesponsive may constitute new therapeutic strategies for diseases, where aberrant NKT cell activation is causally involved.     

Application of natural killer T cells in antitumor immunotherapy

NKT cells are a unique subset of T cells that recognize glycolipid antigens presented by CD1d molecules. NKT cells have the potential to produce key cytokines of both Th1 and Th2 T cells and are involved in the control of several types of immune response. Furthermore, NKT cells perform spontaneous tumor immunosurveillance. Upon specific activation with alpha-GalCer, NKT cells show strong antitumor immune responses through direct cytotoxicity and indirect activation of a cascade of antitumor effector cells such as natural killer (NK) cells and CD8+ cytotoxic T cells. In addition to alpha-GalCer, many other CD1d ligands, including self and bacterial glycolipids and modified synthetic glycolipid antigens, have also been discovered. Structurally different glycolipid antigens have the distinct ability to activate NKT cells. Thus, it seems that we are now close to a position in which we can control the activation status of NKT cells; this makes NKT cells an ideal target of anticancer immunotherapies. Clinical trials with soluble alpha-GalCer or alpha-GalCer-pulsed dendritic cells aimed at in vivo reconstitution and activation of human NKT cells have provided both promising and challenging results. In this review, we discuss NKT-cell-mediated antitumor immune responses, as well as the early outcomes and implications of recent clinical studies.     

Detection and activation of human Valpha24+ natural killer T cells using alpha-galactosyl ceramide-pulsed dendritic cells

Human CD1d-restricted natural killer T (NKT) cells, which are postulated to regulate the immune response in several clinical settings, can be activated by alpha-galactosylceramide (alpha-GalCer) presented by CD1d molecules on antigen presenting cells (APCs). Simple methods to quantify NKT function in fresh blood will greatly benefit studies targeting NKT cells in humans. Here we show that freshly isolated human NKT cells can be readily quantified by an enzyme-linked immunospot (ELISPOT) assay and have a Th1 profile (secreting interferon-gamma, but not IL-4), after stimulation using alpha-GalCer loaded APCs. Using this assay, we also evaluated APC requirements for human NKT cell activation in fresh blood. Monocyte-derived dendritic cells (DCs) are more effective than monocytes/macrophages for detecting and activating NKT cells in fresh blood, with mature alpha-GalCer pulsed DCs being optimal. DCs are also efficient APCs for expanding NKT cells in culture and generating NKT cell lines. NKT cells expanded with DCs were functional, secreting both IFN-gamma and IL-4, and killing NKT-sensitive targets. Optimal activation of these lines was seen using mature DCs loaded with 10-100 ng/ml of alpha-GalCer. DCs matured with several different stimuli were effective. These data help to establish the conditions for loading DCs with alpha-GalCer for immune therapeutic targeting of NKT cells, and provide a new simple assay to monitor NKT function in humans.     

Modulation of acute graft-versus-host disease and chimerism after adoptive transfer of in vitro-expanded invariant Valpha14 natural killer T cells

Mouse natural killer T cells with an invariant Valpha14-Jalpha18 TCR rearrangement (Valpha14i NKT cells) are able to regulate immune responses through rapid and large amounts of Th1 and Th2 cytokine production. It has been reported that in vivo administration of the Valpha14i NKT cell ligand, alpha-galactosylceramide (alpha-GalCer) significantly reduced morbidity and mortality of acute graft-versus-host disease (GVHD) in mice. In this study, we examined whether adoptive transfer of in vitro-expanded Valpha14i NKT cells using alpha-GalCer and IL-2 could modulate acute GVHD in the transplantation of spleen cells of C57BL/6 mice into (B6xDBA/2) F(1) mice. We found that the adoptive transfer of cultured spleen cells with a combination of alpha-GalCer and IL-2, which contained many Valpha14i NKT cells, modulated acute GVHD by exhibiting long-term mixed chimerism and reducing liver damage. Subsequently, the transfer of Valpha14i NKT cells purified from spleen cells cultured with alpha-GalCer and IL-2 also inhibited acute GVHD. This inhibition of acute GVHD by Valpha14i NKT cells was blocked by anti-IL-4 but not by anti-IFN-gamma monoclonal antibody. Therefore, the inhibition was dependent on IL-4 production by Valpha14i NKT cells. Our findings highlight the therapeutic potential of in vitro-expanded Valpha14i NKT cells for the prevention of acute GVHD after allogeneic hematopoietic stem cell transplantation.     

Immunization with alpha-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis.

We have compared the immune responses of mice immunized either with alpha-galactosylceramide (alpha-GalCer), capable of eliciting a CD1-metiated stimulation of V alpha14+ NK T cells, or with lipoarabinomannan (LAM), a glycophospholipid derived from mycobacteria which is known to be presented by CD1b in humans. Within 24 h, alpha-GalCer induces a burst of IFN-gamma secretion in vivo, and recall with antigen in vitro leads to the synthesis of IL-4 and IL-10 in addition to IFN-gamma. Associated with this in vivo cytokine release is a polyclonal activation of splenic B and T cells. CD1-reactive NK T lymphocytes mediate these events, because none of them are observed in alpha-GalCer-immunized CD1-/- mice. LAM immunization fails to promote similar early responses in vivo. Repeated exposure of mice to alpha-GalCer induces splenic T cells to secrete IL-4 and IL-10 but dramatically reduced levels of IFN-gamma. Such a bias in the cytokine balance triggered by NK T cells stimulated with multiple doses of alpha-GalCer suggests that this compound might be useful in the induction of Th2 immune responses and the prevention of chronic inflammatory conditions mediated by Th1 cytokines.     

Critical contribution of IFN-gamma and NK cells, but not perforin-mediated cytotoxicity, to anti-metastatic effect of alpha-galactosylceramide

The glycolipid alpha -galactosylceramide (alpha -GalCer), which is presented by CD1d and specifically activates Valpha 14 NKT cells, exerts a potent anti-metastatic effect when administered in vivo. In this study, we demonstrated that alpha -GalCer administration led to rapid elimination of NKT cells by apoptosis in the liver and spleen, after they produced IFN-gamma and IL-4. In contrast, a more prolonged secretion of IFN-gamma was observed by liver and splenic NK cells after alpha -GalCer administration. Cytotoxic activity of liver mononuclear cells was not augmented 3h after alpha -GalCer administration, but was increased at 24 h when NKT cells were mostly depleted. The alpha -GalCer-induced cytotoxic activity was abolished in IFN-gamma -deficient and NK cell-depleted mice as well as CD1-deficient mice, suggesting that the alpha -Galcer-induced cytotoxicity was mainly mediated by IFN-gamma -activated NK cells. While the alpha -GalCer-induced cytotoxicity in vitro was mostly perforin dependent, anti-metastatic effect of alpha -GalCer was impaired in NK cell-depleted or IFN-gamma -deficient mice but not in perforin-deficient mice. Collectively, these results indicated that the anti-metastatic effect of alpha -GalCer is mainly mediated by NK cells, which are activated secondarily by IFN-gamma produced by alpha -GalCer-activated NKT cells, in a perforin-independent manner.     

Co-inhibitory roles for glucocorticoid-induced TNF receptor in CD1d-dependent natural killer T cells

Invariant natural killer T (iNKT) cells are a special subset of alphabeta T cells with invariant TCR, which recognize alpha-galactosylceramide (alpha-GalCer) presented by CD1d. In addition to signals through the invariant TCR upon stimulation with alpha-GalCer, costimulatory signals, such as signals through CD28 and OX40, are indispensable for full activation of iNKT cells. In this study, we investigated the functions of a well-known costimulatory molecule, glucocorticoid-induced TNF receptor (GITR), on Ag-induced iNKT cell activation. Unexpectedly, engagement of GITR by agonistic mAb DTA-1 suppressed proliferation and cytokine production of iNKT cells upon alpha-GalCer stimulation. In addition, GITR signals in iNKT cells during only the Ag-priming phase was sufficient to inhibit the iNKT cell activation. Consistent with these results, the GITR-deficient iNKT cells showed enhanced proliferation and increased cytokine production upon alpha-GalCer stimulation both in vitro and in vivo. Furthermore, the in vivo administration of alpha-GalCer suppressed tumor metastasis more efficiently in GITR-deficient mice than in wild-type mice. Collectively, GITR plays a co-inhibitory role in Ag-induced iNKT cell activation.     

Expansion of human Valpha24+ NKT cells by repeated stimulation with KRN7000

Changes in Valpha24+Vbeta11+ NKT cell number and function are associated with human autoimmune diseases and cancer. Restoration of this corresponding NKT cell population in mice or in vivo activation with alpha-galactosylceramide (KRN7000) can prevent or reduce tumor growth and autoimmunity. Although the therapeutic value of these natural killer T (NKT) cells in man remains to be determined, large numbers of functional antigen-specific NKT cells can be expanded in vitro. We show that Valpha24+Vbeta11+ human NKT cells are expanded by repeated stimulation with KRN7000, unfractionated donor peripheral blood mononuclear cells (PBMC), and recombinant human interleukin-2 (rhIL-2). NKT cells were expanded continuously for more than 2 months with a potential yield of >10(12) cells. The expanded NKT cells retained their CD4+ or CD4- phenotype after restimulation and were functional as shown by cytokine secretion, killing of antigen-pulsed target cells, and activation of NK cell cytotoxicity. This expansion method may be useful for proof-of-concept studies involving adoptive transfer of ex vivo-expanded NKT cells as a new therapeutic option for cancer and autoimmune diseases.     

Activation of hepatic NKT cells and subsequent liver injury following administration of alpha-galactosylceramide

It has been established that alpha-galactosylceramide (alpha-GalCer), a glycolipid, is recognized by natural killer T (NKT) cells together with the monomorphic MHC-like antigen, CD1d, in mice and humans. In this study, we examined how NKT cells are modulated by in vivo administration of alpha-GalCer in mice. When 2 microg (or more)/mouse of alpha(-GalCer was injected i.p., the majority of NKT cells disappeared in the liver and spleen, possibly undergoing apoptosis, on day 1. At this time, NKT cytotoxicity seen in liver lymphocytes also disappeared. In parallel with this numerical and functional change of NKT cells, there was always concomitant hepatocyte damage, as shown by histology and elevated levels of transaminases. Subsequently, the number and function of NKT cells continued to increase from day 3 to day 7. The response seen in hepatic (and splenic) NKT cells did not occur in thymic NKT cells. All these phenomena induced in the liver did not appear in NKT-deficient mice such as beta2-microglobulin(-/-) and CD1d(-/-) mice. These results shed further light on the in vivo interaction between NKT cells and alpha-GalCer in mice.     

Enhanced gamma interferon production through activation of Valpha14(+) natural killer T cells by alpha-galactosylceramide in interleukin-18-deficient mice with systemic cryptococcosis

We showed recently that activation of Valpha14(+) natural killer T cells (NKT cells) by alpha-galactosylceramide (alpha-GalCer) resulted in increased gamma interferon (IFN-gamma) production and host resistance to intravenous infection with Cryptococcus neoformans. In other studies, interleukin-18 (IL-18) activated NKT cells in collaboration with IL-12, suggesting the possible contribution of this cytokine to alpha-GalCer-induced IFN-gamma synthesis. Here we examined the role of IL-18 in alpha-GalCer-induced Th1 response by using IL-18KO mice with this infection. In these mice, levels of IFN-gamma in serum and its synthesis in vitro by spleen cells stimulated with live organisms were not reduced, but rather enhanced, compared to those in wild-type (WT) mice, while such production was completely absent in IL-12KO mice. The enhanced production of IFN-gamma correlated with increased IL-12 synthesis but not with reduced production of IL-4, which was rather increased. IFN-gamma synthesis in IL-18KO mice was abolished by neutralizing anti-IL-12 antibody and significantly inhibited by neutralization of endogenous IL-4 with a specific monoclonal antibody. In addition, administration of recombinant IL-4 significantly enhanced the production of IFN-gamma in WT mice. Finally, the enhanced production of IFN-gamma in IL-18KO mice correlated with increased host defense against cryptococcal infection, as indicated by enhancement in alpha-GalCer-related clearance of microorganisms. Our results indicated that in IL-18KO mice, IFN-gamma synthesis was enhanced through overproduction of IL-12 and IL-4 after intravenous infection with C. neoformans and a ligand-specific activation of Valpha14(+) NKT cells.     

alpha-Galactosylceramide, a ligand of natural killer T cells, inhibits allergic airway inflammation

alpha-Galactosylceramide (alpha-GalCer) is a specific ligand of natural killer T cells (NKT cells) that regulates the immune responses such as tumor rejection and autoimmunity by producing interferon (IFN)-gamma and interleukin (IL)-4. However, it has not been determined whether alpha-GalCer-activated NKT cells modulate allergic inflammation. Because alpha-GalCer induces a large amount of IFN-gamma production by NKT cells, we hypothesized that an in vivo administration of alpha-GalCer could inhibit allergic airway inflammation in mice. Strikingly, a single intraperitoneal injection of alpha-GalCer almost completely abrogated an infiltrate with eosinophils in the lung tissue as well as in the bronchoalveolar lavage. This inhibition of allergic inflammation was associated with a significant decrease in the levels of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid and in the number of goblet cells. In addition, this ligand significantly inhibited airway hyperresponsiveness to inhaled methacholine and raised the serum levels of ovalbumin-specific IgG2a with a decrease in those of ovalbumin-specific IgE. In IFN-gamma knockout mice, however, alpha-GalCer failed to exert such inhibitory effects in this asthma model. These results indicate that alpha-GalCer prevents allergic airway inflammation possibly through IFN-gamma production by ligand-activated NKT cells, suggesting the potential therapeutic application of alpha-GalCer in asthma.     

Differential modulating effect of natural killer (NK) T cells on interferon-γ production and cytotoxic function of NK cells and its relationship with NK subsets in Chlamydia muridarum infection

Natural killer T (NKT) cells are a newly identified T-cell population with potential immunomodulatory functions. Several studies have shown modulating effects of NKT cells activated by α-galactosylceramide, a model antigen, on NK cell function. We here report a differential modulating effect of NKT cells on the interferon-γ (IFN-γ) production and cytolytic function of NK cells in a chlamydial infection model, using NKT-cell-deficient mice and antibody blocking (anti-CD1d monoclonal antibody) approaches. Our results showed that both NKT and NK cells became activated and produced IFN-γ following Chlamydia muridarum infection in vitro and in vivo. The NK cells in NKT-cell-deficient mice and CD1d-blocked mice showed decreased CD69 expression, cellular expansion and IFN-γ production but surprisingly showed increased cytolytic activity (degranulation) of immature and more mature NK cell subsets, suggesting an inhibitory role of NKT cells on NK cell killing activity. The results suggest that NKT cells preferentially promote IFN-γ production but are inhibitory for the cytotoxic function of NK cells in this infection model. Furthermore, the differential modulating effect of NKT cells on the IFN-γ production and cytotoxicity of NK cells was observed in immature and mature NK cell subsets, although it was more dramatic in the relatively mature CD11b(high) CD27(high) NK cell subset. This finding demonstrates the complexity of innate cell interactions in infection and the possible differential impact of NKT cells on the variable functional aspects of other cell(s) even in one infection setting.     

Relative contribution of NK and NKT cells to the anti-metastatic activities of IL-12

Conventional T cells, NK cells and NKT cells have been implicated in the anti-tumor activities induced by IL-12. Here we show that IL-12-induced immune responses are partially impaired in T and NKT cell-deficient RAG-2(-/-) mice, and in NKT cell-deficient CD1(-/-) mice. In response to a small dose (<1000 U) of IL-12, RAG-2(-/-) and CD1(-/-) mice demonstrated reduced cytotoxicity, serum IFN-gamma elevation and anti-metastatic activities; in contrast, in response to a high dose (>2000 U) of IL-12, the IL-12-induced immune responses of RAG-2(-/-) and CD1(-/-) mice were indistinguishable from wild-type mice. The defective responses to low-dose IL-12 of RAG-2(-/-) mice were corrected by adoptive transfer of NKT cells but not NK cells. These findings indicate that both NK and NKT cells contribute to the anti-metastatic responses induced by IL-12, and that NKT cells are mostly responsible for the low-dose activities of this cytokine.     

CpG ODN enhance antigen-specific NKT cell activation via plasmacytoid dendritic cells

Human Valpha24+ Vbeta11+ natural killer T cells (NKT cells) are "natural memory" T cells that detect glycolipid antigens such as alpha-galactosylceramide (alpha-GalCer) presented on CD1d. In the present study we found that highly purified Valpha24+ NKT cells lack TLR9 mRNA, and thus are not sensitive towards stimulation with CpG oligodeoxynucleotides (ODN). Within PBMC, however, CpG ODN synergistically activated NKT cells stimulated with their cognate antigen alpha-GalCer. Depletion of plasmacytoid dendritic cells (PDC) or myeloid dendritic cells (MDC) revealed that both DC subsets were necessary for the synergistic activation of NKT cells by alpha-GalCer and CpG ODN. While PDC were responsible for the stimulation of NKT cells with CpG ODN, MDC but not PDC presented alpha-GalCer via CD1d. Partial activation of NKT cells was mediated by PDC-derived IFN-alpha, whereas full activation of NKT cells as indicated by IFN-gamma production required cell-to-cell contact of PDC and NKT cells in addition to IFN-alpha; OX40 was involved in this interaction. We conclude that CpG-activated PDC enhance alpha-GalCer-specific NKT cell activation, and bias activated NKT cells towards a Th1 phenotype. Our results lead to a novel concept of PDC function: to regulate effector activity of antigen-stimulated T cells in a cell contact-dependent manner without the need of simultaneous presentation of the cognate T cell antigen.