Identification of IFN-gamma-producing cells in IL-12/IL-18-treated mice

Both IL-12 and IL-18 have been characterized as effective IFN-gamma-inducing cytokines. Concomitant treatment with IL-12 and IL-18 has been shown to synergistically induce IFN-gamma and may be an effective therapy for treating cancer, allergy, and infectious diseases. To understand the mechanisms underlying the strong induction of IFN-gamma by IL-12/IL-18 in mice, we focused our studies on the IFN-gamma-producing cells in various lymphoid organs and tissues and utilized the intracellular cytokine staining method to detect such cells in situ. After combined treatment with IL-12 and IL-18, IFN-gamma-positive cells in C57BL/6 mice were detected in the liver (12.18%), spleen (0.68%), bone marrow (1.80%), and peritoneum (2.12%), but not in the thymus or lymph nodes (<0.05 and <0.08%, respectively). A two-color staining method revealed that the majority of IFN-gamma-producing cells in the liver were NK1.1(+) cells, while those in the spleen were mostly CD3(+) cells, and to a lesser degree NK1.1(+) cells. Both CD4(+) and CD8(+) cells in the liver and in the spleen produced IFN-gamma. The CD19(+) B cell population was not definitely shown to produce IFN-gamma in our induction experiments. NKT cells, which are a subpopulation of NK1. 1(+) CD3(+) cells, were diminished in the liver and did not seem to contribute to IFN-gamma production arising from IL-12/IL-18 treatment. Further in vitro experiments confirmed the responsiveness of hepatic mononuclear cells to IL-12/IL-18 stimulation. This study is the first to show the IFN-gamma-producing mechanisms of IL-12/IL-18 treatment at the phenotypic level.     

NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice

CD4(+) T cells produce IFN-gamma contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-gamma and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (-/-; no T/NKT cells), B6.TCR Jalpha281(-/-) (NKT cell deficient), alpha-galactosylceramide (alphaGalCer) (anergized NKT cells) injected and IL-12p40(-/-) vs B6 controls were tested. IFN-gamma mRNA was undetectable in RAG-1(-/-)- and alphaGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jalpha281(-/-), alphaGalCer-treated, and IL-12p40(-/-) (activated CD4(+) T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mphi; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-kappaB, and CD1d, and addition of rIFN-gamma potentiated Mphi CD1d levels. Together, these data suggest that Langerhans cell/Mphi recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-gamma production by NKT cells, that IFN-gamma is required to optimally activate NK cells to produce IFN-gamma, and that depletion of both NKT/NK cells results in earlier corneal perforation.     

Activating immunity in the liver. II. IFN-beta attenuates NK cell-dependent liver injury triggered by liver NKT cell activation

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10-100 ng/mouse) of alpha-galactosyl ceramide (alphaGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following alphaGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to alphaGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-gamma response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG(-/-) or HBs-tg/RAG(-/-)) hosts via the portal vein elicited IFN-gamma responses of liver NK cells in situ. IFN-beta down-regulates the pathogenic IL-12/IFN-gamma cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-beta suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (alphaGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-gamma response of the specifically activated liver NKT cells. In vivo, IFN-beta attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.     

Mononuclear phagocyte-derived IL-10 suppresses the innate IL-12/IFN-gamma axis in lung-challenged aged mice

Previously, we reported that IL-10-producing mononuclear phagocytes increase in lungs of aged mice, causing impaired innate cytokine expression. Since dendritic cells (DCs) contribute to innate NK cell and adaptive T cell immunity, we tested the hypothesis that age-related IL-10 might influence DC function with effects on NK and T cell activation. The results showed that DC recruitment to sites of lung inflammation was normal in aged mice (>20 mo). However, IFN-gamma-producing NK cells in LPS-challenged lungs were decreased in aged as compared with young mice, which was associated with increased IL-10(+)CD11b(+)Gr-1(low)CD11c(-) cells consistent with mononuclear phagocytes. In vivo or in vitro blockade of IL-10 signaling restored IFN-gamma-producing NK cells. This restoration was reversed by IL-12 neutralization, indicating that IL-10 suppressed sources of IL-12 in aged mice. To probe DC function in adaptive immunity, we transferred young naive OVA-specific TCR transgenic T cells to old mice. Following challenge with OVA plus LPS, Ag presentation in the context of MHC-I and MHC-II occurred with similar kinetics and intensity in draining lymph nodes of young and old recipients as measured by proliferation. Despite this, aged hosts displayed impaired induction of IFN-gamma(+)CD4(+), but not IFN-gamma(+)CD8(+), effector T cells. Blockade of IL-10 signaling reversed age-associated defects. These studies indicate that the innate IL-12/IFN-gamma axis is not intrinsically defective in lungs of aged mice, but is rather suppressed by enhanced production of mononuclear phagocyte-derived IL-10. Our data identify a novel mechanism of age-associated immune deficiency.     

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.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.     

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.     

Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice

The role of mouse liver NK1.1 Ag(+) T (NKT) cells in the antitumor effect of alpha-galactosylceramide (alpha-GalCer) has been unclear. We now show that, whereas alpha-GalCer increased the serum IFN-gamma concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of alpha-GALCER: Injection of Abs to IFN-gamma also inhibited the alpha-GalCer-induced increase in antitumor cytotoxicity of MNCS: alpha-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas alpha-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCS: The alpha-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-gamma. Depletion of CD8(+) T cells did not affect the alpha-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of alpha-GalCer on the survival of B6 mice. Thus, IFN-gamma produced by alpha-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8(+) T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate alpha-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.     

IL-18, but not IL-12, regulates NK cell activity following intranasal herpes simplex virus type 1 infection

Infection of the respiratory tract with HSV type 1 (HSV-1) can have severe clinical complications, yet little is known of the immune mechanisms that control the replication and spread of HSV-1 in this site. The present study investigated the protective role of IL-12 and IL-18 in host defense against intranasal HSV-1 infection. Both IL-12 and IL-18 were detected in lung fluids following intranasal infection of C57BL/6 (B6) mice. IL-18-deficient (B6.IL-18(-/-)) mice were more susceptible to HSV-1 infection than wild-type B6 mice as evidenced by exacerbated weight loss and enhanced virus growth in the lung. IL-12-deficient (B6.IL-12(-/-)) mice behaved similarly to B6 controls. Enhanced susceptibility of B6.IL-18(-/-) mice to HSV-1 infection correlated with a profound impairment in the ability of NK cells recovered from the lungs to produce IFN-gamma or to mediate cytotoxic activity ex vivo. The weak cytotoxic capacity of NK cells from the lungs of B6.IL-18(-/-) mice correlated with reduced expression of the cytolytic effector molecule granzyme B. Moreover, depletion of NK cells from B6 or B6.IL-12(-/-) mice led to enhanced viral growth in lungs by day 3 postinfection; however, this treatment had no effect on viral titers in lungs of B6.IL-18(-/-) mice. Together these studies demonstrate that IL-18, but not IL-12, plays a key role in the rapid activation of NK cells and therefore in control of early HSV-1 replication in the lung.     

Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-gamma synthesis

Innate cellular production of IFN-gamma is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-gamma production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-gamma synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-gamma in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-gamma without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-gamma in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-gamma when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-gamma production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-gamma, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-gamma production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.     

Interleukin-15 and natural killer and NKT cells play a critical role in innate protection against genital herpes simplex virus type 2 infection

Interleukin-15 (IL-15), natural killer (NK) cells, and NK T (NKT) cells, components of the innate immune system, are known to contribute to defense against pathogens, including viruses. Here we report that IL-15(-/-) (NK(-) and NKT(-/+)) mice and RAG-2(-/-)/gamma(c)(-/-) (NK(-) and NKT(-)) mice that lack all lymphoid cells were very susceptible to vaginal infection with a low dose of herpes simplex virus type 2 (HSV-2). IL-15(-/-) and RAG-2(-/-)/gamma(c)(-/-) mice were 100-fold more susceptible and RAG-2(-/-), CD-1(-/-) (NKT(-)), and gamma interferon (IFN-gamma)(-/-) mice were 10-fold more susceptible to vaginal HSV-2 infection than control C57BL/6 mice. NK and/or NKT cells were the early source of IFN-gamma in vaginal secretions following genital HSV-2 infection. This study demonstrates that IL-15 and NK-NKT cells are critical for innate protection against genital HSV-2.     

Genetic background influences natural killer cell activation during bacterial peritonitis in mice, and is interleukin 12 and interleukin 18 independent

Some mouse strains produce strong pro-inflammatory, T-helper (Th)1 responses (e.g. C57BL/6), or strong anti-inflammatory, Th2 responses (e.g. BALB/c). The exact mechanisms for development of distinct immune responses to infection are not completely understood, although cytokines such as interleukin (IL)-12, IL-18 and IL-4 are known to play roles. Natural killer T (NKT)/natural killer (NK) cells are important regulators of immune responses in infection and non-infection models, and NKT/NK activation is also regulated by IL-12 and IL-18 in many models. We investigated the role of IL-12/IL-18 in NKT/NK activation in murine bacterial peritonitis, as well as differential NKT and NK cell activation in C57BL/6 and BALB/c mice. No differences in NKT or NK cell activation or intracellular interferon (IFN)-gamma were determined between mice given control, anti-IL-12 or anti-IL-18 antibodies or in NKT/NK cell activation in STAT4-/- mice (deficient in IL-12 signaling) or wild type controls. However, there were significant differences in the activation of NKT and NK cells between C57BL/6 mice and BALB/c mice, with NKT/NK cytokine production following Th1 or Th2 lines dependent on strain. This suggests a role for NKT and NK cell activation in the development of Th1 and Th2 responses during bacterial infection independently of IL-12 or IL-18.     

IL-18 enhances IL-4 production by ligand-activated NKT lymphocytes: a pro-Th2 effect of IL-18 exerted through NKT cells

NKT cells are a remarkably versatile population whose functional capacities are determined by cytokines present in their microenvironment. In this study, we provide evidence for a new immunoregulatory effect of the proinflammatory cytokine IL-18 on NKT cells. We found that IL-18, mainly known for its involvement in NK cell activation and in Th 1 immune responses, substantially enhanced IL-4 production as well as the percentage of IL-4(+) cells among NKT lymphocytes activated by their specific ligand alpha-galactosylceramide (alpha-GalCer). The effect of IL-18 on IL-4 production by activated NKT cells took place both in vivo and in vitro and was not affected by IL-12 which increased IFN-gamma secretion in the same conditions. We show that NKT cells are the main targets for IL-18-induced IL-4 production since it occurred neither in NKT-deficient mice nor after stimulation of Th2 lymphocytes. Finally, we provide evidence that the IL-4 promptly generated by NKT cells in response to IL-18 plus alpha-galactosylceramide in vivo can effectively contribute to the adaptive Th2 immune response by up-regulating the early activation marker CD69 on B cells. Our data support the notion that, in contrast to the exclusive IFN-gamma inducer IL-12, IL-18 acts in a more subtle manner as a costimulatory factor in both pro-Th1 and pro-Th2 responses depending on the nature of the stimulation and the target cells.     

Dendritic cell-induced activation of adaptive and innate antitumor immunity

While studying Ag-pulsed syngeneic dendritic cell (DC) immunization, we discovered that surprisingly, unpulsed DCs induced protection against tumor lung metastases resulting from i.v. injection of a syngeneic BALB/c colon carcinoma CT26 or a syngeneic C57BL/6 lung carcinoma LL/2. Splenocytes or immature splenic DCs did not protect. The protection was mediated by NK cells, in that it was abrogated by treatment with anti-asialo-GM1 but not anti-CD8, and was induced by CD1(-/-) DCs unable to stimulate NKT cells, but did not occur in beige mice lacking NK cells. Protection correlated with increased NK activity, and increased infiltration of NK but not CD8(+) cells in lungs of tumor-bearing mice. Protection depended on the presence of costimulatory molecules CD80, CD86, and CD40 on the DCs, but surprisingly did not require DCs that could make IL-12 or IL-15. Unexpectedly, protection sensitive to anti-asialo-GM1 and increased NK activity were still present 14 mo after DC injection. As NK cells lack memory, we found by depletion that CD4(+) not CD8(+) T cells were required for induction of the NK antitumor response. The role of DCs and CD4(+) T cells provides a novel mechanism for NK cell induction and innate immunity against cancer that may have potential in preventing clinical metastases.     

Gene Therapy against Murine Melanoma B16F10-Nex2 Using IL-13Ralpha2-Fc Chimera and Interleukin 12 in Association with a Cyclopalladated Drug

Interleukin 13 (IL-13) is immunoregulatory in many diseases, including cancer. The protective or suppressive role of CD1-restricted natural killer T cells (NKT cells) in tumor immunosurveillance and immunity is well documented. Interleukin 12 (IL-12) can activate type I NKT cells to produce interferon-gamma (IFN-gamma), whereas type II NKT cells may produce IL-13. The high-affinity chain of IL-13Ralpha2 may act as negative inhibitor, suppressing the action of IL-13 and helping to maintain tumor immunosurveillance. We constructed an mIL-13Ralpha2-Fc chimera in a eukaryotic expression vector and confirmed the identity of the recombinant protein by immunoblot analysis and binding to IL-13 in chemiluminescent ELISA. Such DNA vaccine was tested against syngeneic B16F10-Nex2 murine melanoma. In vivo experiments showed a protective effect mediated by high production of IFN-gamma and down-regulation of anti-inflammatory interleukins mainly by NKT 1.1(+) T cells. Biochemoterapy in vivo with plasmid encoding mIL-13Ralpha2-Fc in association with plasmid encoding IL-12 and the 7A cyclopalladated drug led to a significant reduction in the tumor evolution with 30% tumor-free mice. We conclude that IL-12 gene therapy, followed by continuous administration of IL-13Ralpha2-Fc gene along with 7A-drug has antitumor activity involving the high production of proinflammatory cytokines and low immune suppression, specifically by NK1.1(+)T cells producing IL-13 and IL-10.     

Memory CD8+ T cells provide an early source of IFN-gamma

During the non-Ag-specific early phase of infection, IFN-gamma is believed to be primarily provided by NK and NKT cells in response to pathogen-derived inflammatory mediators. To test whether other cell types were involved in early IFN-gamma release, IFN-gamma-producing cells were visualized in spleens and lymph nodes of LPS-injected mice. In addition to NK and NKT cells, IFN-gamma was also detected in a significant fraction of CD8(+) T cells. CD8(+) T cells represented the second major population of IFN-gamma-producing cells in the spleen ( approximately 30%) and the majority of IFN-gamma(+) cells in the lymph nodes ( approximately 70%). LPS-induced IFN-gamma production by CD8(+) T cells was MHC class I independent and was restricted to CD44(high) (memory phenotype) cells. Experiments performed with C3H/HeJ (LPS-nonresponder) mice suggested that CD8(+) T cells responded to LPS indirectly through macrophage/dendritic cell-derived IFN-alpha/beta, IL-12, and IL-18. IFN-gamma was also detected in memory CD8(+) T cells from mice injected with type I IFN or with poly(I:C), a synthetic dsRNA that mimics early activation by RNA viruses. Taken together, these results suggest that in response to bacterial and viral products, memory T cells may contribute to innate immunity by providing an early non-Ag-specific source of IFN-gamma.     

Interleukin-12 activates NK cells for IFN-gamma-dependent and NKT cells for IFN-gamma-independent antimetastatic activity

Mechanisms involved in the antimetastatic effect of IL-12 were analyzed in a mouse model of experimental metastasis with either syngeneic fibrosarcoma cells colonizing the lungs or syngeneic B cell lymphoma cells colonizing the liver. IL-12 pretreatment effectively reduced the number of tumor colonies in both systems. This effect was already manifest 24 hours after tumor cell injection, indicating a T and B cell-independent mechanism. Therefore, the involvement of NK and alphabetaNKT cells was investigated using mice with defective NK and alphabetaNKT cell functions. Mice with impaired NK functions due to NK cell depletion, were less responsive to the antimetastatic IL-12 effect. IL-12 treatment failed to inhibit metastasis in beta2-microglobulin-deficient mice which lack alphabetaNKT cells in addition to having impaired NK cell activity, thus, demonstrating the functional importance of IL-12-activated NK and alphabetaNKT cells. While the IL-12-induced antimetastatic effect of NK cells was dependent on IFN-gamma action, IL-12 activation of alphabetaNKT cells did not involve IFN-gamma. The neutralization of IFN-gamma or the use of IFN-gamma receptor-deficient mice did not alter the IL-12-induced effect in the absence of NK cells. Activation of effector cells of the innate immune system, such as NK and alphabetaNKT cells, seems to be the main mechanism for the antimetastatic effect of IL-12.     

IL-18 bridges innate and adaptive immunity through IFN-gamma and the CD134 pathway

IL-18 induces inflammation resulting in either enhanced protection from pathogens or exacerbation of autoimmunity, and T cells are profoundly activated during these responses. How IL-18 influences T cell activation is unknown, but this study in mice shows that IL-18 boosted Ag-specific T cell clonal expansion of effector T cells and induced a subpopulation of IFN-gamma superproducing T cells. Commitment to IFN-gamma production through IL-18 was independent of NK cells and IL-12 but dependent on host-derived IFN-gamma. To determine how expansion of these effectors occurred, IL-18 was shown to induce OX40L on dendritic cells, whereas peptide stimulation induced CD134 (OX40) on specific T cells. CD134 blockade inhibited T cell effector expansion thereby reducing the number of IFN-gamma superproducers by 12-fold. Thus, independent of IL-12, IL-18 impacts T cell immunity throughout lymphoid and nonlymphoid tissue by bridging the innate and adaptive arms of the immune system through IFN-gamma and the CD134 costimulatory pathway.     

C57BL/6 mice genetically deficient in IL-12/IL-23 and IFN-gamma are susceptible to experimental autoimmune myasthenia gravis, suggesting a pathogenic role of non-Th1 cells

Immunization with Torpedo acetylcholine receptor (TAChR) induces experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 (B6) mice. EAMG development needs IL-12, which drives differentiation of Th1 cells. The role of IFN-gamma, an important Th1 effector, is not clear and that of IL-17, a proinflammatory cytokine produced by Th17 cells, is unknown. In this study, we examined the effect of simultaneous absence of IL-12 and IFN-gamma on EAMG susceptibility, using null mutant B6 mice for the genes of both the IL-12/IL-23 p40 subunit and IFN-gamma (dKO mice). Wild-type (WT) B6 mice served as control for EAMG induction. All mice were immunized with TAChR in Freund's adjuvant. dKO mice developed weaker anti-TAChR CD4(+)T cells and Ab responses than WT mice. Yet, they developed EAMG symptoms, anti-mouse acetylcholine receptor (AChR) Ab, and CD4(+) T cell responses against mouse AChR sequences similar to those of WT mice. dKO and WT mice had similarly reduced AChR content in their muscles, and IgG and complement at the neuromuscular junction. Naive dKO mice had significantly fewer NK, NKT, and CD4(+)CD25(+)Foxp3(+) T regulatory (Treg) cells than naive WT mice. Treg cells from TAChR-immunized dKO mice had significantly less suppressive activity in vitro than Treg cells from TAChR-immunized WT mice. In contrast, TAChR-specific CD4(+) T cells from TAChR-immunized dKO and WT mice secreted comparable amounts of IL-17 after stimulation in vitro with TAChR. The susceptibility of dKO mice to EAMG may be due to reduced Treg function, in the presence of a normal function of pathogenic Th17 cells.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.