A distinct IL-18-induced pathway to fully activate NK T lymphocytes independently from TCR engagement

NK T lymphocytes are characterized by their ability to promptly generate IL-4 and IFN-gamma upon TCR engagement. Here, we demonstrate that these cells can also be fully activated in the absence of TCR cross-linking in response to the proinflammatory cytokine IL-18 associated with IL-12. NK T cells stimulated with IL-18 plus IL-12 proliferated, killed Fas+ target cells, and produced high levels of IFN-gamma without IL-4. In these conditions, IFN-gamma production was at least 10-fold higher than that upon TCR cross-linking. Interestingly, a 2-h pretreatment with IL-12 plus IL-18 sufficed to maintain the high IFN-gamma-producing potential during subsequent stimulation with anti-TCR mAbs or with the specific Ag alpha-galactosylceramide. Similar effects were observed in vivo, because splenic CD4+ NK T cells from MHC class II-deficient mice secreted IFN-gamma without further stimulation when removed 2 h after a single injection of IL-12 plus IL-18. In conclusion, our evidence for activation of NK T lymphocytes in response to IL-18 plus IL-12 in the absence of TCR engagement together with the maintenance of preferential IFN-gamma vs IL-4 production upon subsequent exposure to specific Ags is consistent with the active participation of this cell population in innate as well as acquired cellular immune responses.     

Invariant NKT cells amplify the innate immune response to lipopolysaccharide

NKT cells are thought of as a bridge between innate and adaptive immunity. In this study, we demonstrate that mouse NKT cells are activated in response to Escherichia coli LPS, and produce IFN-gamma, but not IL-4, although activation through their TCR typically induces both IL-4 and IFN-gamma production. IFN-gamma production by NKT cells is dependent on LPS-induced IL-12 and IL-18 from APC. LPS induced IFN-gamma production by NKT cells does not require CD1d-mediated presentation of an endogenous Ag and exposure to a combination of IL-12 and IL-18 is sufficient to activate them. In mice that are deficient for NKT cells, innate immune cells are activated less efficiently in response to LPS, resulting in the reduced production of TNF and IFN-gamma. We propose that in addition to acting as a bridge to adaptive immunity, NKT cells act as an early amplification step in the innate immune response and that the rapid and complete initiation of this innate response depends on the early production of IFN-gamma by NKT cells.     

Critical role for CXC chemokine ligand 16 (SR-PSOX) in Th1 response mediated by NKT cells

The transmembrane chemokine CXCL 16 (CXCL16), which is the same molecule as the scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX), has been shown to mediate chemotaxis and adhesion of CXC chemokine receptor 6-expressing cells such as NKT and activated Th1 cells. We generated SR-PSOX/CXCL16-deficient mice and examined the role of this chemokine in vivo. The mutant mice showed a reduced number of liver NKT cells, and decreased production of IFN-gamma and IL-4 by administration of alpha-galactosylceramide (alphaGalCer). Of note, the alphaGalCer-induced production of IFN-gamma was more severely impaired than the production of IL-4 in SR-PSOX-deficient mice. In this context, SR-PSOX-deficient mice showed impaired sensitivity to alphaGalCer-induced anti-tumor effect mediated by IFN-gamma from NKT cells. NKT cells from wild-type mice showed impaired production of IFN-gamma, but not IL-4, after their culture with alphaGalCer and APCs from mutant mice. Moreover, Propionibacterium acnes-induced in vivo Th1 responses were severely impaired in SR-PSOX-deficient as well as NKT KO mice. Taken together, SR-PSOX/CXCL16 plays an important role in not only the production of IFN-gamma by NKT cells, but also promotion of Th1-inclined immune responses mediated by NKT cells.     

IL-4-secreting NKT cells prevent hypersensitivity pneumonitis by suppressing IFN-gamma-producing neutrophils

Hypersensitivity pneumonitis (HP) is mediated by Th1 immune response. NKT cells regulate immune responses by modulating the Th1/Th2 balance. Therefore, we postulated that NKT cells play a critical role in the development of the HP by modulating the Th1/Th2 response. To address this issue, we explored the functional roles of NKT cells in Saccharopolyspora rectivirgula (SR)-induced HP. In CD1d(-/-) mice, the HP was worse in terms of histological changes, hydroxyproline levels, the CD4:CD8 ratio in bronchoalveolar lavage fluid, and SR-specific immune responses than in control mice. CD1d(-/-) mice showed elevated IFN-gamma production in the lung during the HP, and this was produced mainly by Gr-1+ neutrophils. The blockade of IFN-gamma in CD1d(-/-) mice attenuated the HP, whereas the injection of rIFN-gamma aggravated it. Moreover, the depletion of Gr-1+ neutrophils reduced CD8+ T cell numbers in bronchoalveolar lavage fluid during the HP. The adoptive transfer of IL-4(-/-) mouse NKT cells did not attenuate the HP, whereas wild-type or IFN-gamma(-/-) mouse NKT cells suppressed the HP. In conclusion, NKT cells producing IL-4 play a protective role in SR-induced HP by suppressing IFN-gamma-producing neutrophils, which induce the activation and proliferation of CD8+ T cells in the lung.     

Activation of invariant NKT cells by the helminth parasite schistosoma mansoni

Mouse CD1d-restricted NKT cells, including invariant (i)NKT cells, are innate cells activated by glycolipid Ags and play important roles in the initiation and regulation of immune responses. Through their ability to promptly produce large amounts of Th1 and/or Th2 cytokines upon TCR engagement, iNKT cells exert crucial functions in the immune/inflammatory system during bacterial, protozoan, fungal, and viral infections. However, their roles during metazoan parasite infection, which are generally associated with strong Th2 responses, still remain elusive. In this study, we show that during the course of murine schistosomiasis, iNKT cells exhibit an activated phenotype and that following schistosome egg encounter in the liver, hepatic iNKT cells produce both IFN-gamma and IL-4 in vivo. We also report that schistosome egg-sensitized dendritic cells (DCs) activate, in a CD1d-dependent manner, iNKT cells to secrete IFN-gamma and IL-4 in vitro. Interestingly, transfer of egg-sensitized DCs promotes a strong Th2 response in recipient wild-type mice, but not in mice that lack iNKT cells. Engagement of TLRs in DCs is not necessary for iNKT cell stimulation in response to egg-sensitized DCs, suggesting an alternative pathway of activation. Finally, we propose that self, rather than parasite-derived, CD1d-restricted ligands are implicated in iNKT cell stimulation. Taken together, our data show for the first time that helminths can activate iNKT cells to produce immunoregulatory cytokines in vivo, enabling them to influence the adaptive immune response.     

IL-4 induces in vivo production of IFN-gamma by NK and NKT cells

Although IL-4 and IFN-gamma often have opposite effects and suppress each other's production by T cells, IL-4 can stimulate IFN-gamma production. To characterize this, we injected mice with IL-4 and quantified IFN-gamma production with the in vivo cytokine capture assay. IL-4 induced Stat6-dependent IFN-gamma production by NK and, to a lesser extent, NKT cells, but not conventional T cells, in 2-4 h. Increased IFN-gamma production persisted at a constant rate for >24 h, but eventually declined, even with continuing IL-4 stimulation. This eventual decline in IFN-gamma production was accompanied by a decrease in NK and T cell numbers. Consistent with a dominant role for NK cells in IL-4-stimulated IFN-gamma secretion, IL-4 induction of IFN-gamma was B and T cell-independent; suppressed by an anti-IL-2Rbeta mAb that eliminates most NK and NKT cells; reduced in Stat4-deficient mice, which have decreased numbers of NK cells; and absent in Rag2/gamma(c)-double-deficient mice, which lack T, B, and NK cells. IL-4-induced IFN-gamma production was not affected by neutralizing IL-12p40 and was increased by neutralizing IL-2. IL-13, which signals through the type 2 IL-4R and mimics many IL-4 effects, failed to stimulate IFN-gamma production and, in most experiments, suppressed basal IFN-gamma production. Thus, IL-4, acting through the type 1 IL-4R, induces Stat6-dependent IFN-gamma secretion by NK and NKT cells. This explains how IL-4 can contribute to Th1 cytokine-associated immune effector functions and suggests how IL-13 can have stronger proallergic effects than IL-4.     

Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways

Valpha14 NKT cells produce large amounts of IFN-gamma and IL-4 upon recognition of their specific ligand alpha-galactosylceramide (alpha-GalCer) by their invariant TCR. We show here that NKT cells constitutively express CD28, and that blockade of CD28-CD80/CD86 interactions by anti-CD80 and anti-CD86 mAbs inhibits the alpha-GalCer-induced IFN-gamma and IL-4 production by splenic Valpha14 NKT cells. On the other, the blockade of CD40-CD154 interactions by anti-CD154 mAb inhibited alpha-GalCer-induced IFN-gamma production, but not IL-4 production. Consistent with these findings, CD28-deficient mice showed impaired IFN-gamma and IL-4 production in response to alpha-GalCer stimulation in vitro and in vivo, whereas production of IFN-gamma but not IL-4 was impaired in CD40-deficient mice. Moreover, alpha-GalCer-induced Th1-type responses, represented by enhanced cytotoxic activity of splenic or hepatic mononuclear cells and antimetastatic effect, were impaired in both CD28-deficient mice and CD40-deficient mice. In contrast, alpha-GalCer-induced Th2-type responses, represented by serum IgE and IgG1 elevation, were impaired in the absence of the CD28 costimulatory pathway but not in the absence of the CD40 costimulatory pathway. These results indicate that CD28-CD80/CD86 and CD40-CD154 costimulatory pathways differentially contribute to the regulation of Th1 and Th2 functions of Valpha14 NKT cells in vivo.     

Diesel exhaust particles suppress in vivo IFN-gamma production by inhibiting cytokine effects on NK and NKT cells

Diesel exhaust particles (DEP) have strong, selective Th2 adjuvant activity when inhaled with conventional Ags. We used a novel technique for measuring in vivo cytokine production to investigate possible mechanisms by which DEP might promote a Th2 response. Injection of DEP i.p. stimulated IL-6 secretion, but failed to increase IL-4, IL-10, or TNF-alpha secretion, and decreased basal levels of IFN-gamma. When injected with or before LPS, DEP had little effect on the LPS-induced TNF-alpha responses, but partially inhibited the LPS-induced IL-10 response and strongly inhibited the LPS-induced IFN-gamma response. DEP also inhibited the IFN-gamma responses to IL-12, IL-12 plus IL-18, IL-2, and poly(I.C). DEP treatment had little effect on the percentages of NK and NKT cells in the spleen, but inhibited LPS-induced IFN-gamma production by splenic NK and NKT cells. In contrast, DEP failed to inhibit the IFN-gamma response by anti-CD3 mAb-activated NKT cells. Taken together, these observations suggest that DEP inhibit Toll-like receptor ligand-induced IFN-gamma responses by interfering with cytokine signaling pathways that stimulate NK and NKT cells to produce IFN-gamma. Our observations also suggest that DEP may promote a Th2 response by stimulating production of inflammatory cytokines while simultaneously inhibiting production of IFN-gamma, and raise the possibility that the same mechanisms contribute to the association between DEP exposure and asthma.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.     

CD137-deficient mice have reduced NK/NKT cell numbers and function, are resistant to lipopolysaccharide-induced shock syndromes, and have lower IL-4 responses

CD137, a member of the TNF superfamily, is involved in T cell and NK cell activation and cytokine production. To establish its in vivo role in systems dependent on NK and NKT cells, we studied the response of CD137-/- mice to LPS-induced shock, tumor killing, and their IL-4-controlled Th2 responses. In both high and low dose shock models, all the CD137-deficient mice, but none of the wild-type BALB/c mice, survived. After injection of LPS/2-amino-2-deoxy-D-galactose (D-gal), CD137-/- mice had reduced serum cytokine levels and substantially impaired liver IFN-gamma and TNF-alpha mRNA levels. Phenotypic analysis of mononuclear cells revealed fewer NK and NKT cells in the CD137-/- mice. The knockout mice did not generate a rapid IL-4 response after systemic T cell activation, or effective Ag-specific Th2 responses. In addition, both in vitro and in vivo NK-specific cytolytic activities were reduced. These findings suggest that CD137-directed NK/NKT cells play an important role in the inflammatory response leading to the production of proinflammatory cytokines, LPS-induced septic shock, and tumor killing, as well as IL-4-dependent Th2 responses.     

Engagement of glucocorticoid-induced TNF receptor costimulates NKT cell activation in vitro and in vivo

Glucocorticoid-induced TNF receptor (GITR) is known to provide costimulatory signals to CD4+CD25- and CD4+CD25+ T cells during immune responses in vivo. However, the functional roles of GITR expressed on NKT cells have not been well characterized. In this study, we have explored the functions of GITR as a costimulatory factor on NKT cells. GITR was found to be constitutively expressed on NKT cells and its expression was enhanced by TCR signals. GITR engagement using DTA-1, an agonistic mAb against GITR, in the presence of TCR signals, augmented IL-2 production, the expression of activation markers, cell cycle progression, and the nuclear translocations of NF-kappaB p50 and p65. Furthermore, GITR engagement enhanced the production of IL-4, IL-10, IL-13, and IFN-gamma by NKT cells and the expression level of phosphorylated p65 in NKT cells in the presence of TCR engagement, indicating that GITR provides costimulatory signals to NKT cells. The costimulatory effects of GITR on NKT cells were comparable to those of CD28 in terms of cytokine production. Moreover, the coinjection of DTA-1 and alpha-galactosylceramide into B6 mice induced more IL-4 and IFN-gamma production than the coinjection of control mAbs and alpha-galactosylceramide. In addition, the adoptive transfer of DTA-1-pretreated NKT cells into CD1d(-/-) mice attenuated hypersensitivity pneumonitis more than control IgG pretreated NKT cells in these mice. These findings demonstrate that GITR engagement on NKT cells modulates immune responses in hypersensitivity pneumonitis in vivo. Taken together, our findings suggest that GITR engagement costimulates NKT cells and contributes to the regulation of immune-associated disease processes in vivo.     

4-1BB engagement costimulates NKT cell activation and exacerbates NKT cell ligand-induced airway hyperresponsiveness and inflammation

Multiple studies have demonstrated that 4-1BB (CD137), a member of the TNF receptor superfamily, is expressed on several immune cells including activated T cells. However, the expression and the role of 4-1BB on natural killer T (NKT) cells have not been fully characterized. In this study, it was shown that 4-1BB was not expressed on naive NKT cells but was rapidly induced on activated NKT cells by TCR engagement with alpha-galactosylceramide (alpha-GalCer). Also, 4-1BB signaling provided by 3H3, an agonistic anti-4-1BB mAb, promoted NKT cell activation resulting in enhanced cytokine production of NKT cells driven by alpha-GalCer. When NKT cell-driven airway immune responses were evaluated by intranasal administration of alpha-GalCer, airway hyperresponsiveness (AHR) and lung inflammation were significantly more aggravated in mice treated with 3H3 and alpha-GalCer than in mice treated with alpha-GalCer alone. These aggravations were accompanied by up-regulation of IL-4, IL-13, and IFN-gamma production. Interestingly, AHR was not developed in IL-4Ralpha-deficient mice treated with alpha-GalCer with or without 3H3 but was exacerbated in IFN-gamma-deficient mice. Our study suggests that 4-1BB on NKT cells functions as a costimulatory molecule and exacerbates the induction of NKT cell-mediated AHR, which is dependent on the IL-4Ralpha-mediated pathway.     

IL-12 induces monocyte IL-18 binding protein expression via IFN-gamma

IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-gamma production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-gamma, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-gamma derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-gamma production. The IFN-gamma/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-gamma and probably plays a key role in the modulation of both innate and adaptive immunity.     

Synergistic engagement of an ineffective endogenous anti-tumor immune response and induction of IFN-gamma and Fas-ligand-dependent tumor eradication by combined administration of IL-18 and IL-2

IFN-gamma is a critical component of the endogenous and many cytokine-induced antitumor immune responses. In this study we have shown that the combination of IL-18 and IL-2 (IL-18/IL-2) synergistically enhances IFN-gamma production both in vitro and in vivo, and synergizes in vivo to induce complete durable regression of well-established 3LL tumors in >80% of treated mice. We have observed a nascent, but ineffective, host immune response against 3LL that depends on endogenous IFN-gamma and IL-12 production and the Fas/Fas ligand (Fas-L) pathway. The combined administration of IL-18/IL-2 engages this endogenous response to induce tumor regression via a mechanism that is independent of NK and NKT cells or IL-12, but is critically dependent on CD8(+) T cells, IFN-gamma, and the Fas/Fas-L pathway. These studies demonstrate the importance of IFN-gamma as well as the Fas/Fas-L pathway in both endogenous and cytokine-driven antitumor immune responses engaged by IL-18/IL-2 and provide preclinical impetus for clinical investigation of this potent anti-tumor combination.     

Pro-Th1 cytokines promote Fas-dependent apoptosis of immature peripheral basophils

We have previously characterized immature hemopoietic cells of the basophil lineage as a lin(-)c-kit(-) population, which responds to IL-3 by enhancing its histamine synthesis through histidine decarboxylase activation. Herein, we show both in vitro and in vivo that exposure to the pro-Th1 cytokines IL-12 and IL-18 promotes Fas-dependent apoptosis of these cells in the spleen. This conclusion was supported by the following findings: 1) A 24-h treatment with IL-12 plus IL-18 enhanced Fas expression and annexin staining among basophil precursor-enriched lin(-)c-kit(-) splenocytes. 2) Fas or Fas ligand deficiency in mutant mice abolished the inhibitory effect of IL-12 plus IL-18 on IL-3-induced histamine production. 3) The large spectrum inhibitor of the caspase cascade, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone, significantly reduced the effect of IL-12 plus IL-18. The inhibition of histamine production was mediated through NK cells, since it failed to occur upon stimulation of spleen cells from NK cell-deficient mice or after NK cell depletion. IL-12 plus IL-18 rendered NK cells cytotoxic against Fas-transfected target cells and promoted their production of IFN-gamma and TNF-alpha, which are both essential for sensitizing histamine-producing cells to the Fas death pathway. This is the first evidence that pro-Th1 cytokines can promote apoptosis of immature peripheral histamine-producing cells, thus limiting Th2 immune responses. Comparable in vivo data as well as increased histamine production in the spleen of aged Fas-deficient lpr mice support its physiological relevance.     

IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production

The common gamma-chain cytokine, IL-21, is produced by CD4(+) T cells and mediates potent effects on a variety of immune cells including NK, T, and B cells. NKT cells express the receptor for IL-21; however, the effect of this cytokine on NKT cell function has not been studied. We show that IL-21 on its own enhances survival of NKT cells in vitro, and IL-21 increases the proliferation of NKT cells in combination with IL-2 or IL-15, and particularly with the CD1d-restricted glycosphingolipid Ag alpha-galactosylceramide. Similar to its effects on NK cells, IL-21 enhances NKT cell granular morphology, including granzyme B expression, and some inhibitory NK receptors, including Ly49C/I and CD94. IL-21 also enhanced NKT cell cytokine production in response to anti-CD3/CD28 in vitro. Furthermore, NKT cells may be subject to autocrine IL-21-mediated stimulation because they are potent producers of this cytokine following in vitro stimulation via CD3 and CD28, particularly in conjunction with IL-12 or following in vivo stimulation with alpha-galactosylceramide. Indeed, NKT cells produced much higher levels of IL-21 than conventional CD4 T cells in this assay. This study demonstrates that NKT cells are potentially a major source of IL-21, and that IL-21 may be an important factor in NKT cell-mediated immune regulation, both in its effects on NK, T, and B cells, as well as direct effects on NKT cells themselves. The influence of IL-21 in NKT cell-dependent models of tumor rejection, microbial clearance, autoimmunity, and allergy should be the subject of future investigations.     

The Pro-Th1 cytokine IL-12 enhances IL-4 production by invariant NKT cells: relevance for T cell-mediated hepatitis

IL-12 is essential for invariant NKT (iNKT) cells because it can maintain a functionally active population and promote a cytokine profile that is assumed to be mainly of the pro-Th1 type. We used the murine concanavalin A (Con A)-induced hepatitis model, in which iNKT cells, IL-12, IL-4, and IFN-gamma are equally requisite, to reevaluate this issue. We demonstrate that IL-12 interacts directly with iNKT cells, contributes to their recruitment to the liver, and enhances their IL-4 production, which is essential for disease onset. IL-12-deficient mice were less susceptible to experimental hepatitis and their iNKT cells produced less IL-4 than their wild-type counterpart. A normal response could be restored by IL-12 injection, revealing its importance as endogenous mediator. In accordance with this observation, we found that iNKT cells expressed the IL-12R constitutively, in contrast to conventional T cells. Furthermore, the physiological relevance of our data is supported by the lower susceptibility to disease induction of NOD mice, known for their inherent functional and numerical abnormalities of iNKT cells associated with decreased iNKT cell-derived IL-4 production and low IL-12 secretion. Taken together, our findings provide the first evidence that IL-12 can enhance the immune response through increased IL-4 production by iNKT cells, underscoring once more the functional plasticity of this subset.