Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity.

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

Enhancement of natural killer activity and IFN-γ production in an IL-12-dependent manner by a Brassica rapa L.

Certain food components possess immunomodulatory effects. The aim of this study was to elucidate the mechanism of the immunostimulatory activity of Brassica rapa L. We demonstrated an enhancement of natural killer (NK) activity and interferon (IFN)-γ production in mice that were orally administered an insoluble fraction of B. rapa L. The insoluble fraction of B. rapa L. significantly induced IFN-γ production in mouse spleen cells in an interleukin (IL)-12-dependent manner, and NK1.1+ cells were the main cells responsible for producing IFN-γ. Additionally, the results suggested that the active compounds in the insoluble fraction were recognized by Toll-like receptor (TLR) 2, TLR4, and C-type lectin receptors on dendritic cells, and they activated signaling cascades such as MAPK, NF-κB, and Syk. These findings suggest that B. rapa L. is a potentially promising immuno-improving material, and it might be useful for preventing immunological disorders such as infections and cancers by activating innate immunity.

HemoHIM ameliorates the persistent down-regulation of Th1-like immune responses in fractionated γ-irradiated mice by modulating the IL-12p70-STAT4 signaling pathway.

Whole body irradiated mice appear to experience a down-regulation of the helper T (Th)1-like immune response, and maintain a persistent immunological imbalance. In the current study, we evaluated the effect of HemoHIM (an herbal product made from Angelica Radix, Cnidium officinale , and Paeonia japonica cultivated in Korea) to ameliorate the immunological imbalance induce in fractionated γ-irradiated mice. The mice were exposed to γ rays twice a week (0.5 Gy fractions) for a total dose of 5 Gy, and HemoHIM was administrated orally from 1 week before the first irradiation to 1 week before the final analysis. All experiments were performed 4 and 6 months after their first exposure. HemoHIM ameliorated the Th1- and Th2-related immune responses normally occur in irradiated mice with or without dinitrophenylated keyhole limpet hemocyanin immunization. HemoHIM also restored the natural killer cell activities without changing the percentage of natural killer cells in irradiated mice. Furthermore, the administration of HemoHIM prevented the reduction in levels of interleukin-12p70 in irradiated mice. Finally, we found that HemoHIM enhanced the phosphorylation of signal transducer and activator of transcription (STAT) 4 that was reduced in irradiated mice. Our findings suggest that HemoHIM ameliorates the persistent down-regulation of Th1-like immune responses by modulating the IL-12p70/pSTAT4 signaling pathway.

Involvement of tyrosine kinase-2 in both the IL-12/Th1 and IL-23/Th17 axes in vivo.

Tyrosine kinase-2 (Tyk2), a member of the Jak family of kinases, mediates the signals triggered by various cytokines, including type I IFNs, IL-12, and IL-23. In the current study, we investigated the in vivo involvement of Tyk2 in several IL-12/Th1- and IL-23/Th17-mediated models of experimental diseases, including methylated BSA injection-induced footpad thickness, imiquimod-induced psoriasis-like skin inflammation, and dextran sulfate sodium- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. In these disease models, Tyk2 deficiency influenced the phenotypes in immunity and/or inflammation. Our findings demonstrate a somewhat broader contribution of Tyk2 to immune systems than previously expected and suggest that Tyk2 may represent an important candidate for drug development by targeting both the IL-12/Th1 and IL-23/Th17 axes.

Proanthocyanidins inhibit photocarcinogenesis through enhancement of DNA repair and xeroderma pigmentosum group A-dependent mechanism.

Dietary grape seed proanthocyanidins (GSP) inhibit photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated. As ultraviolet B (UVB)-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) has been implicated in skin cancer risk, we studied whether dietary GSPs enhance repair of UVB-induced DNA damage and, if so, what is the potential mechanism? Supplementation of GSPs (0.5%, w/w) with AIN76A control diet significantly reduced the levels of CPD(+) cells in UVB-exposed mouse skin; however, GSPs did not significantly reduce UVB-induced CPD(+) cells in the skin of interleukin-12p40 (IL-12) knockout (KO) mice, suggesting that IL-12 is required for the repair of CPDs by GSPs. Using IL-12 KO mice and their wild-type counterparts and standard photocarcinogenesis protocol, we found that supplementation of control diet with GSPs (0.5%, w/w) significantly reduced UVB-induced skin tumor development in wild-type mice, which was associated with the elevated mRNA levels of nucleotide excision repair genes, such as XPA, XPC, DDB2, and RPA1; however, this effect of GSPs was less pronounced in IL-12 KO mice. Cytostaining analysis revealed that GSPs repaired UV-induced CPD(+) cells in xeroderma pigmentosum complementation group A (XPA)-proficient fibroblasts from a healthy individual but did not repair in XPA-deficient fibroblasts from XPA patients. Furthermore, GSPs enhance nuclear translocation of XPA and enhanced its interactions with other DNA repair protein ERCC1. Together, our findings reveal that prevention of photocarcinogenesis by GSPs is mediated through enhanced DNA repair in epidermal cells by IL-12- and XPA-dependent mechanisms.

Triterpene esters from Uncaria rhynchophylla drive potent IL-12-dependent Th1 polarization.

Dendritic cells (DC) are key antigen-presenting cells that link innate and adaptive immunity and ultimately activate antigen-specific T cells. In the current study, we demonstrated that two triterpene esters, uncarinic acid C (1) and uncarinic acid D (2), which are isolated from the hooks of Uncaria rhynchophylla, activate phenotypic and cytokine production alterations in DC. We also show that 1 and 2 modulate human DC function in a fashion that favors Th1 cell polarization. The effect of 1 (E configuration at the 2' position) was approximately 20 times more potent than that of 2 (Z configuration at 2'). These results indicated that the configuration of the 2' double bond greatly effects activity. Thus, 1 and 2 may prove useful as DC-based vaccines for cancer immunotherapy.

Green tea phenol extracts reduce UVB-induced DNA damage in human cells via interleukin-12.

Green tea chemoprevention has been a focus of recent research, as a polyphenolic fraction from green tea (GTP) has been suggested to prevent UV radiation-induced skin cancer. Recently, it was demonstrated that GTP reduced the risk for skin cancer in a murine photocarcinogenesis model. This was accompanied by a reduction in UV-induced DNA damage. These effects appeared to be mediated via interleukin (IL)-12, which was previously shown to induce DNA repair. Therefore, we studied whether GTP induction of IL-12 and DNA repair could also be observed in human cells. KB cells and normal human keratinocytes were exposed to GTP 5 h before and after UVB. UVB-induced apoptosis was reduced in UVB-exposed cells treated with GTP. GTP induced the secretion of IL-12 in keratinocytes. The reduction in UV-induced cell death by GTP was almost completely reversed upon addition of an anti-IL-12-antibody, indicating that the reduction of UV-induced cell death by GTP is mediated via IL-12. The ability of IL-12 to reduce DNA damage and sunburn cells was confirmed in "human living skin equivalent" models. Hence the previously reported UV-protective effects of GTP appear to be mediated in human cells via IL-12, most likely through induction of DNA repair.

Laser acupuncture and probiotics in school age children with asthma: a randomized, placebo-controlled pilot study of therapy guided by principles of Traditional Chinese Medicine.

Traditional Chinese Medicine (TCM) postulates an interaction between the lung as a Yin-organ and the large intestine as a Yang-organ. The aim of this pilot study was to investigate in asthmatic school age children whether treatment with laser acupuncture and probiotics according to TCM portends a clinical benefit to standard medical treatment performed according to pediatric guidelines. Seventeen children aged 6-12 yr with intermittent or mild persistent asthma were enrolled in this randomized, placebo-controlled, double-blind pilot study. Eight patients received laser acupuncture for 10 wk and probiotic treatment in the form of oral drops (living non-pathogenic Enterococcus faecalis) for 7 wk. Nine patients in the control group were treated with a laser pen which did not emit laser light and were given placebo drops. Peak flow variability (PFV) and forced expiratory volume in 1 s (FEV(1)) were measured and Quality of Life was assessed by a standardized questionnaire. Laser acupuncture and probiotics significantly decreased mean (standard deviation) weekly PFV as a measurement of bronchial hyperreactivity by -17.4% (14.2) in the TCM group vs. 2.2% (22.5) in the control group (p = 0.034). No significant effect was detected for FEV(1), Quality of Life criteria and additional medication. As an exploratory result, patients in the TCM group had fewer days of acute febrile infections when compared with the control group [1.14 (1.4) vs. 2.66 (2.5), p = 0.18]. In conclusion, this pilot study generates the hypothesis that the interactive treatment of lung and large intestine according to TCM by laser acupuncture and probiotics has a beneficial clinical effect on bronchial hyperreactivity in school age children with intermittent or mild persistent asthma and might be helpful in the prevention of acute respiratory exacerbations. These results should be confirmed by further studies.

Interleukin-12 is associated with the in vivo anti-tumor effect of mistletoe extracts in B16 mouse melanoma.

Viscum album (VA) preparations consist of aqueous extracts of different types of lectins of mistletoe. VA exert cytotoxic and immunomodulatory properties that may be relevant for the inhibition of tumor growth. We addressed the effects of VA preparation VA Qu FrF on growth of B16F1 melanoma implanted in mice and on proliferation and cytokine synthesis of splenocytes. In C57BL6 mice, inhibition of tumor growth by VA was associated with an enhancement of splenocyte proliferation and with an up-regulation of IL-12 secretion. In IL-12-deficient strain of mice the inhibition of melanoma growth by VA and the splenocyte proliferation were abrogated. Results from the present study strongly suggest a crucial role of IL-12 in the anti-tumor properties of VA extracts.

Activated STAT4 has an essential role in Th1 differentiation and proliferation that is independent of its role in the maintenance of IL-12R beta 2 chain expression and signaling.

In this study we demonstrated that CD4(+) T cells from STAT4(-/-) mice exhibit reduced IL-12R expression and poor IL-12R signaling function. This raised the question of whether activated STAT4 participates in Th1 cell development mainly through its effects on IL-12 signaling. In a first approach to this question we determined the capacity of CD4(+) T cells from STAT4(-/-) bearing an IL-12Rbeta2 chain transgene (and thus capable of normal IL-12R expression and signaling) to undergo Th1 differentiation when stimulated by Con A and APCs. We found that such cells were still unable to exhibit IL-12-mediated IFN-gamma production. In a second approach to this question, we created Th2 cell lines (D10 cells) transfected with STAT4-expressing plasmids with various tyrosine-->phenylalanine mutations and CD4(+) T cell lines from IL-12beta2(-/-) mice infected with retroviruses expressing similarly STAT4 mutations that nevertheless express surface IL-12Rbeta2 chains. We then showed that constructs that were unable to support STAT4 tyrosine phosphorylation (in D10 cells) as a result of mutation were also incapable of supporting IL-12-induced IFN-gamma production (in IL-12Rbeta2(-/-) cells). Thus, by two complementary approaches we demonstrated that activated STAT4 has an essential downstream role in Th1 cell differentiation that is independent of its role in the support of IL-12Rbeta2 chain signaling. This implies that STAT4 is an essential element in the early events of Th1 differentiation.

A profibrotic function of IL-12p40 in experimental pulmonary fibrosis.

The p40 subunit of IL-12 (IL-12p40), but not the heterodimeric form IL-12p70, is secreted during the development of silica-induced lung fibrosis in C57BL/6 mice. To delineate the contribution of IL-12p40 to the lung inflammatory and fibrotic processes, we compared the pulmonary responses with silica particles of IL-12p35-deficient mice (IL-12p35(-/-), able to produce IL-12p40) and IL-12p40-deficient mice (IL-12p40(-/-)). IL-12p35(-/-) and IL-12p40(-/-) animals developed strikingly contrasting responses to silica in comparison with wild-type C57BL/6 mice. Although the IL-12p40(-/-) mice exhibited limited inflammatory and fibrotic reactions, the IL-12p35(-/-) mice presented a robust and well-developed pulmonary inflammation and fibrosis. Furthermore, the silica-induced increase in lung IL-12p40 content was significantly higher in IL-12p35(-/-) mice than in wild-type controls, and was associated with extensive lung fibrosis and pulmonary macrophage infiltration. The contrasting responses observed between these two IL-12 subunit-deficient murine strains were not accompanied by a strict type 1 or type 2 polarization as estimated by the measurements of lung IFN-gamma/IgG2a and IL-4/IgG1 content. In vitro proliferation, type I collagen expression, as well as myofibroblast differentiation of purified pulmonary fibroblasts were not affected by treatment with exogenous rIL-12p40. In vivo, supplementation with rIL-12p40 restored the impaired pulmonary fibrotic response and macrophage accumulation in silica-treated IL-12p40(-/-) mice, and also promoted fibrosis and macrophage influx in wild-type mice. Together, our data suggest that IL-12p40 plays an important role in silica-induced pulmonary inflammation and fibrosis, possibly by exacerbating macrophage recruitment.

Curcumin inhibits experimental allergic encephalomyelitis by blocking IL-12 signaling through Janus kinase-STAT pathway in T lymphocytes.

Experimental allergic encephalomyelitis (EAE) is a CD4(+) Th1 cell-mediated inflammatory demyelinating autoimmune disease of the CNS that serves as an animal model for multiple sclerosis (MS). IL-12 is a proinflammatory cytokine that plays a crucial role in the induction of neural Ag-specific Th1 differentiation and pathogenesis of CNS demyelination in EAE and MS. Curcumin (1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a naturally occurring polyphenolic phytochemical isolated from the rhizome of the medicinal plant Curcuma longa. It has profound anti-inflammatory activity and been traditionally used to treat inflammatory disorders. In this study we have examined the effect and mechanism of action of curcumin on the pathogenesis of CNS demyelination in EAE. In vivo treatment of SJL/J mice with curcumin significantly reduced the duration and clinical severity of active immunization and adoptive transfer EAE. Curcumin inhibited EAE in association with a decrease in IL-12 production from macrophage/microglial cells and differentiation of neural Ag-specific Th1 cells. In vitro treatment of activated T cells with curcumin inhibited IL-12-induced tyrosine phosphorylation of Janus kinase 2, tyrosine kinase 2, and STAT3 and STAT4 transcription factors. The inhibition of Janus kinase-STAT pathway by curcumin resulted in a decrease in IL-12-induced T cell proliferation and Th1 differentiation. These findings highlight the fact that curcumin inhibits EAE by blocking IL-12 signaling in T cells and suggest its use in the treatment of MS and other Th1 cell-mediated inflammatory diseases.

Enhancement of anti-cancer immunity by a lipoteichoic-acid-related molecule isolated from a penicillin-killed group A Streptococcus.

We isolated the lipoteichoic-acid-related molecule (OK-PSA) from OK-432, a streptococcal preparation, by affinity chromatography on CNBr-activated Sepharose-4B-bound monoclonal antibody TS-2, which neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. We have previously reported that OK-PSA is a potent inducer of Th1-type cytokines in human peripheral blood mononuclear cells in vitro. In this study, we conducted an animal experiment to examine whether OK-PSA exhibits an anti-tumor effect in vivo by acting as a Th1 inducer in syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. It was found that OK-PSA induced Th1-type cytokines [IFN-gamma, tumor necrosis factor-alpha, interleukin (IL)-2, IL-12 and IL-18] in BALB/c mice bearing Meth-A tumor and caused a marked anti-tumor effect. Although it was suggested by an in vitro study. using spleen cells derived from the animals, that IL-18 plays the greatest role in the induction of the Th1-dominant state and tumor cell killing induced by OK-PSA, the in vivo experiments demonstrated that both IL-12 and IL-18 are essential in the anti-tumor effect exhibited by OK-PSA. These findings strongly suggest that OK-PSA is a major effector molecule of OK-432 and may be a useful immunotherapeutic agent, as a potent Th1 inducer, for cancer patients with a Th2-dominant state.

Novel nonantibiotic therapies for pneumonia: cytokines and host defense.

Effective host defense against bacterial infection is dependent on the activation and recruitment of phagocytic cells. The initiation, maintenance, and resolution of this inflammatory response in the setting of bacterial pneumonia is dependent on the expression of cytokines. As the complexities of the host-pathogen interaction are further dissected and unraveled, immunologic manipulation of cytokine expression will likely become an important adjuvant therapy in the treatment of serious lung infections.

IL-12/IL-13 axis in allergic asthma.

Allergic airway diseases are associated with skewed T(H)2 cytokine production, although the underlying cause of this aberrant immune response is not well understood. Recently, 2 double-digit cytokines, IL-12 and IL-13, have been proposed to play pivotal roles in the T(H)2-polarized immune response to inhaled allergens. IL-12 is a critical determinant of T(H)1-mediated immune responses, and it has been shown that deficiency in this cytokine can lead to T(H)2-polarized immune responses. IL-13, on the other hand, has recently been shown to be a critical mediator of the effector arm of the allergic response. Overproduction of this cytokine has been shown to induce many common features of the allergic diathesis, such as airway hyperresponsiveness, eosinophilic inflammation, IgE production, mucus hypersecretion, and subepithelial fibrosis. Substantial evidence suggests that an imbalance in the production of these 2 critical immunoregulatory cytokines occurs in the lungs of atopic and asthmatic individuals, such that IL-13 is overproduced, and IL-12 production is impaired. As a result of this imbalance, IL-13 production may go unchecked and induce the classical allergic phenotype. Although it is not entirely clear what tips the balance between these 2 cytokines, genetic studies suggest that this could indeed be a primary event. Interestingly, the genes encoding both of these cytokines reside within the chromosomal regions on 5q, which have been associated with asthma in many populations. Although no evidence exists to date to support an association between asthma and polymorphisms in the IL12 gene, evidence is accumulating that supports an association between genetic alterations in the IL13 gene and atopy and asthma. From these observations, we can conclude that an imbalance in the IL-12/IL-13 axis may contribute to the development of allergic diseases, such as asthma. Studies to more fully examine the yin-yang relationship between these 2 critical immunoregulatory cytokines are clearly warranted.

Effects of the Japanese herbal medicine 'Inchinko-to' (TJ-135) on concanavalin A-induced hepatitis in mice.

Inchinko-to (TJ-135) is a herbal medicine consisting of three kinds of crude drugs, and in Japan it is administered mainly to patients with cholestasis. The present study evaluated the effects of TJ-135 on concanavalin A (con A)-induced hepatitis in mice in vivo and con A-induced cytokine production in vitro. When mice were pretreated with oral TJ-135 for 1 week before intravenous con A injection, the activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were significantly decreased 8 h after con A administration (-82%, -96% and -66% respectively). In histological investigations, sub-massive hepatic necrosis accompanying inflammatory cell infiltration was not observed in mice pretreated with TJ-135. Serum levels of interleukin-12 (IL-12), interferon-gamma (IFN-gamma) and IL-2 were significantly lower in mice pretreated with TJ-135 compared with controls, while IL-10 levels were higher in these mice. Intrasplenic IL-12 levels were significantly lower in mice pretreated with TJ-135, while intrasplenic IL-10 levels were higher in these mice. In vitro, IL-10 production by splenocytes was increased by the addition of TJ-135 to the culture medium, whereas the production of IL-12 and IFN-gamma was inhibited. These results suggest that con A-induced hepatitis was ameliorated by pretreatment with TJ-135. With regard to the mechanism of these effects of TJ-135, we speculate that TJ-135 inhibits the production of inflammatory cytokine and enhances the production of anti-inflammatory cytokines. Therefore administration of TJ-135 may be useful in patients with severe acute hepatitis accompanying cholestasis or in those with autoimmune hepatitis.

The role of IL-12 in the induction of late-phase cellular infiltration in a murine model of allergic conjunctivitis.

BACKGROUND: The applied murine model of allergic conjunctivitis mimics human disease, and an immediate hypersensitivity reaction (IHR) and a late-phase cellular reaction typically develop in sensitized mice after topical challenge with the allergen. OBJECTIVE: We investigated the role of IL-4, IFN-gamma, and IL-12 in the early and late phases of ocular allergy with use of cytokine knockout (KO) mice and neutralizing antibodies. METHODS: Ragweed-sensitized wild-type or IL-4KO, IL-12KO, IFN-gamma KO, anti-IL-12 mAb-treated, recombinant murine IL-12-treated, and anti-IFN-gamma mAb-treated mice were challenged with the allergen 10 days after the immunization. IHR, cellular infiltration, lymphoproliferative response, and cytokine production from draining lymph nodes were recorded and compared among groups. RESULTS: We show that IL-12KO mice and anti-IL-12 antibody-treated wild-type animals failed to have a cellular infiltration into the conjunctiva. Treatment with recombinant murine IL-12 also reduced the number of infiltrating PMNs but increased the percentage of mononuclear cells in the conjunctiva compared with controls. IFN-gamma KO mice had a significantly stronger IHR and prolonged infiltration into the conjunctiva after challenge with ragweed than controls. CONCLUSION: Our data suggest that the presence of IL-12, although better known as a T(H)1-inducing cytokine, is important for the development and the regulation of the late-phase pathologic features in ocular allergy. Furthermore, IFN-gamma is a limiting factor in the late phase of allergy and thus may be important in preventing chronic allergic disease.

MHC class I-restricted cytotoxic lymphocyte responses induced by enterotoxin-based mucosal adjuvants.

The ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257-264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257-264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257-264. Both 51Cr release assays and IFN-gamma enzyme-linked immunospot assays indicated that IFN-gamma-/- and IL-12 p40-/- gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-gamma and probably by a mechanism unrelated to cross-priming.

The functional synergy between IL-12 and IL-2 involves p38 mitogen-activated protein kinase and is associated with the augmentation of STAT serine phosphorylation.

IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-gamma, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways.