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T-helper 17 (Th17) cells are characterized by producing interleukin-17 (IL-17, also called IL-17A), IL-17F, IL-21, and IL-22 and potentially TNF-αand IL-6 upon certain stimulation. IL-23, which promotes Th17 cell development, as well as IL-17 and IL-22 produced by the Th17 cells plays essential roles in various inflammatory diseases, such as experimental autoimmune encephalomyelitis, rheumatoid arthritis, colitis, and Concanavalin A-induced hepatitis. In this review, we summarize the characteristics of the functional role of Th17 cells, with particular focus on the Th17 cell-related cytokines such as IL-17, IL-22, and IL-23, in mouse models and human inflammatory diseases.

Interleukin (IL)-27, a member of the IL-6/IL-12 heterodimeric cytokine family, induces pro-inflammatory responses including early T helper (Th)1 differentiation and generation of cytotoxic T lymphocytes, and also anti-inflammatory responses including the differentiation to IL-10-producing regulatory T cells, inhibition of Th2 and Th17 differentiation, and suppression of pro-inflammatory cytokine production. Nitric oxide (NO) is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli including cytokines and microbes. Recently, IL-27 was reported to play an anti-inflammatory role in microglia by blocking oncostatin M-induced iNOS expression and neuronal toxicity. In the present study, we investigated the effects of IL-27 on NO production in thioglycollate-elicited peritoneal macrophages. IL-27 together with lipopolysaccharide (LPS) induced morphological change into more spread and elongated cells and synergistically enhanced NO production. The combined stimulation also enhanced iNOS mRNA expression and the NO production was abrogated by an iNOS inhibitor, NG-monomethyl L-arginine. The synergistic NO production could be attributed to the augmented Toll-like receptor (TLR)4 mRNA expression by the combination. Signal transducer and activator of transcription (STAT)1 was indispensable for the morphological change and NO production. The combination induced nuclear factor κB (NF-κB) translocation into nuclear and phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and their inhibitors suppressed NO production. These results suggest that in contrast to the anti-proinflammatory role in microglia, IL-27 exerts a pro-inflammatory role by enhancing NO production in peritoneal macrophages stimulated with LPS through activation of STAT1, NF-κB and MAPKs.

The interleukin (IL)-6/IL-12 family cytokines have pleiotropic functions and play critical roles in multiple immune responses. This cytokine family has very unique characteristics in that they comprise two distinct subunits forming a heterodimer and each cytokine and receptor subunit shares with each other. The members of this cytokine family are increasing; currently, there are more than six cytokines, including the tentatively named cytokines IL-Y (p28/p40), IL-12 (p35/p40), IL-23 (p19/p40), IL-27 [p28/Epstein–Barr virus-induced protein 3 (EBI3)], IL-35 (p35/EBI3), and IL-39 (p19/EBI3). This family of cytokines covers a very broad range of immune responses, including pro-inflammatory responses, such as helper T (Th)1, Th2, and Th17, to anti-inflammatory responses, such as regulatory T (Treg) cells and IL-10-producing Treg cells. IL-12 is the first member of this family, and IL-12, IL-23, and IL-27 are mainly produced by activated antigen-presenting cells, such as dendritic cells and macrophages. IL-12 plays a critical role in the promotion of Th1 immune responses by inducing interferon-γ production to combat pathogens and malignant tumors. IL-23 induces IL-17 production and is necessary to maintain pathogenic Th17 cells that cause inflammatory and autoimmune diseases. IL-27 was initially reported to play a critical role in promotion of Th1 differentiation; however, subsequent studies revealed that IL-27 has broader stimulatory and inhibitory roles by inducing IL-10-producing Treg cells. IL-35 is produced by forkhead box P3+ Treg cells and activated B cells and has immunosuppressive functions to maintain immune tolerance. The most recently identified cytokine, IL-39, is produced by activated B cells and has pro-inflammatory functions. The cytokine tentatively named IL-Y seems to have anti-inflammatory functions by inhibiting Th1 and Th17 differentiation. In addition, individual cytokine subunits were also shown to have self-standing activities. Thus, promiscuity within the IL-6/IL-12 family cytokines complicates structural and functional clarification and assignment of individual cytokines. A better understanding of the recent advances and expanding diversity in molecular structures and functions of the IL-6/IL-12 family cytokines could allow the creation of novel therapeutic strategies by using them as tools and targeted molecules.

IL-17A, IL-17F, and IL-25 are ligands for IL-17RA. In the current study, we demonstrated that IL-25–deficient mice—but not IL-17A–, IL-17F–, IL-17A/F–, IL-23p19–, or retinoic acid-related orphan receptor (ROR)-γt–deficient mice—showed significant suppression of 1) the number of eosinophils and the levels of proinflammatory mediators in bronchoalveolar lavage fluids, 2) airway hyperresponsiveness to methacholine, and 3) OVA-specific IgG1 and IgE levels in the serum during OVA-induced Th2-type/eosinophilic airway inflammation. The IL-25 deficiency did not affect lung dendritic cell migration or Ag-specific memory–Th2 cell expansion during Ag sensitization. Adoptive transfer of T cells, mast cells, or bone marrow cells from IL-25–deficient mice revealed that induction of Th2-type/eosinophilic airway inflammation was dependent on activation of lung epithelial cells and eosinophils by IL-25 produced by airway structural cells such as epithelial cells but not by such hematopoietic stem-cell-origin immune cells as T cells and mast cells. Therefore, airway structural cell-derived IL-25—rather than Th17 cell-derived IL-17A and IL-17F—is responsible for induction of local inflammation by promoting activation of lung epithelial cells and eosinophils in the elicitation phase of Th2-type/eosinophilic airway inflammation. It is not required for Ag-specific Th2 cell differentiation in the sensitization phase.

Paclitaxel (also known as taxol) is a member of the taxane class of anticancer agents, which has a well‐known mechanism that blocks cell mitosis and kills tumor cells, that is often used in clinics to treat cancer. However, some carcinomas such as breast, ovarian and non‐small‐cell lung cancers are often resistant to paclitaxel treatment. In this study, we used a lentiviral siRNA library against the entire human genomes to identify genes associated with sensitivity to paclitaxel. We isolated two paclitaxel‐resistant clones carrying the siRNA specific to septin 10 (SEPT10) and to budding uninhibited by benzimidazoles 3. The relation of budding uninhibited by benzimidazoles 3 to paclitaxel sensitivity has already been established, but that of SEPT10 remains unknown. Interestingly, overexpression of SEPT10 increased cells’ sensitivity to paclitaxel; we also found that SEPT10 is an important regulator for microtubule stability. Furthermore, we found that paclitaxel‐resistant tumors had decreased expression of SEPT10. Thus, SEPT10 may be a novel candidate molecule that acts as a good indicator of paclitaxel‐resistant carcinomas (Cancer Sci 2012; 103: 821–827)

IL-23 plays a critical role in the expansion of highly proinflammatory Th17 cells secreting IL-17 and IL-22. Recently, we demonstrated that Notch signaling drives IL-22 secretion through the aryl hydrocarbon receptor (AHR) and plays a protective role in Con A-induced hepatitis. In this study, we investigated the role of IL-23 in hepatitis using IL-23p19- and IL-17-deficient mice. In WT mice, the injection of Con A induced the upregulation of various cytokines, which included IL-23, IL-22, IL-17, IFN-γ and TNF-α. In IL-23p19-deficient mice, exacerbated hepatitis was observed and serum IL-22 and IL-17 levels were greatly reduced, whereas in IL-17-deficient mice, ameliorated hepatitis was observed. The injection of exogenous IL-22 protected p19-deficient mice from hepatitis, whereas the injection of exogenous IL-23 significantly increased the serum levels of not only IL-22 but also IL-17, and less effectively protected against hepatitis in IL-17-dependent and -independent manners. Finally, it was revealed that STAT3, STAT4 and Notch contributed to the production of both the cytokines, and that the AHR was important only for IL-22 production in response to Con A and IL-23 in liver mononuclear cells. These results suggest that IL-23 plays a protective role in hepatitis through IL-22 production and also a pathological role via IL-17-dependent and -independent mechanisms.

The receptor activator of NF-κB ligand (RANKL), which is expressed by not only osteoblasts but also activated T cells, plays an important role in bone-destructive diseases such as rheumatoid arthritis. IL-27, a member of the IL-6/IL-12 family cytokines, activates STAT1 and STAT3, promotes early helper T (Th)1 differentiation and generation of IL-10-producing type 1 regulatory T (Tr1) cells, and suppresses the production of inflammatory cytokines and inhibits Th2 differentiation. In addition, IL-27 was recently demonstrated to not only inhibit Th17 differentiation but also directly act on osteoclast precursor cells and suppress RANKL-mediated osteoclastogenesis through STAT1-dependent inhibition of c-Fos, leading to amelioration of the inflammatory bone destruction. In the present study, we investigated the effect of IL-27 on the expression of RANKL in CD4(+) T cells. We found that IL-27 greatly inhibits cell surface expression of RANKL on naive CD4(+) T cells activated by T cell receptor ligation and secretion of its soluble RANKL as well. The inhibitory effect was mediated in part by STAT3 but not by STAT1 or IL-10. In contrast, in differentiated Th17 cells, IL-27 much less efficiently inhibited the RANKL expression after restimulation. Taken together, these results indicate that IL-27 greatly inhibits primary RANKL expression in CD4(+) T cells, which could contribute to the suppressive effects of IL-27 on the inflammatory bone destruction.

Rationale: Since primary pure squamous cell carcinoma of the breast is a rare disease, few reports describe the characteristic findings on performing preoperative imaging that can be used to distinguish it from normal breast cancer. The rapid evolution and lack of an established method of treatment has resulted in several reports of advanced cases of primary pure squamous cell carcinoma of the breast. Patient concerns: Case 1 was a 44-year-old woman with an elastic, hard tumor in the left C region. Ultrasonographic analysis revealed a maximal 11-mm hypoechoic area. Histologically, the tumor was a well-differentiated squamous cell carcinoma with prominent keratinization, and there was prominent inflammatory cell infiltration, necrosis, and fibrosis. Case 2 was a 58-year-old woman with an elastic, hard tumor in the left C/D region. Ultrasonographic analysis revealed a maximal 31-mm hypoechoic area with partially calcified areas and a hyperechoic margin. Histologically, the tumor was a squamous cell carcinoma with prominent keratinization exhibiting an infiltrative growth pattern. The tumor had no connection to the epidermis and partially transitioned into the atypical ductal epithelium in the area surrounding the focus. Diagnoses: The patient in Case 1 was preoperatively diagnosed with T1cN0M0 Stage I cancer of the left breast, but both patients were finally diagnosed with T2N0M0 Stage IIA cancer. Interventions: Case 1: left partial mastectomy and axillary lymph node dissection were performed. The patient was administered 4 courses of FEC100 and 4 courses of DTX as postoperative adjuvant therapy. Case 2: left modified radical mastectomy and axillary lymph node dissection were performed without any postoperative adjuvant therapy. Outcomes: Case 1: no sign of relapse was observed, but the patient moved away from the area to another hospital in March 2014 and eventually died due to relapse in January 2016. Case 2: four years after surgery, no relapse has been observed. Lessons: We should always keep the presence of primary pure squamous cell carcinoma among breast cancers in mind although the crisis rate is very low. Due to its high malignancy, needle biopsy and aspiration biopsy cytology should be performed to make a definitive diagnosis.

Malignant melanoma has been considered a prototypical ‘immunogenic’ tumor through clinical observations, such as the spontaneous regression of primary lesions, their higher incidence in immune-suppressed individuals, and the development of vitiligo after immunotherapy. Among many cytokines, IL-12 is one of the best characterized and the most potent anti-tumor cytokines. Although the systemic application of IL-12 resulted in disappointing results owing to its considerable toxicity, IL-12 is not entirely unusable in the clinical setting. IL-12-related cytokines, IL-23 and IL-27, have also been shown to possess anti-tumor activities in preclinical models. Although belonging to the same cytokine family, IL-12, IL-23 and IL-27 were found to have different anti-tumor mechanisms, adjuvant activity for tumor vaccines and adverse effects in a poorly immunogeneic melanoma model. In addition, their novel activities on melanoma have been clarified. We briefly review the key features of these members of the IL-12 cytokine family and discuss their potential relevance to melanoma immunity and antimelanoma immunotherapy.

CD4+helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4+T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4+T cells increased the production of IL-22 even in the absence of STAT3. CD4+T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4+T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch–AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses.

The worldwide incidence of malignant melanoma has been steadily increasing, and it has become a major public health problem in many countries. Melanoma has been considered as a prototypical “immunogenic” tumor on the basis of clinical observations showing that primary lesions can spontaneously regress and that immunosuppressed individuals have an increased incidence of melanoma. Thus, various immunological therapies have been intensively conducted for the treatment of melanoma. Interleukin(IL)-27 is a IL-12-related heterodimeric cytokine composed of p28 and EBV-induced gene 3 subunits that are structurally related to the p35 and p40 subunits of IL-12, respectively. Recent studies reveal that IL-27 exhibits not only potent antitumor immune activities via cytotoxic T lymphocytes or natural killer cells but also an antiangiogenic effect. We recently clarified that IL-27 possesses an antiproliferative activity on melanoma cells. This review summarizes antitumor responses induced by IL-27 and novel anti-melanoma activities of IL-27.

Cytotoxic T lymphocytes (CTLs) play a critical role in the control of various cancers and infections, and therefore the molecular mechanisms of CTL generation are a critical issue in designing antitumor immunotherapy and vaccines which augment the development of functional and long-lasting memory CTLs. Interleukin (IL)-27, a member of the IL-6/IL-12 heterodimeric cytokine family, acts on naiveCD4+T cells and plays pivotal roles as a proinflammatory cytokine to promote the early initiation of type-1 helper differentiation and also as an antiinflammatory cytokine to limit the T cell hyperactivity and production of pro-inflammatory cytokines. Recent studies revealed that IL-27 plays an important role inCD8+T cells as well. Therefore, this article reviews current understanding of the role of IL-27 inCD8+T cell functions and generation of CTLs.

Effector Th17 cells are a major source of IL-17, a critical inflammatory cytokine in autoimmune diseases and in host defenses during bacterial infections. Recently, splenic lymphoid tissue inducer-like cells have been reported to be a source of T cell independent IL-17. In this study, we report that the immune system contains a unique set of natural occurring IL-17 producing cell, “natural” Th17 (nTh17), which are a memory-like T cell subset. The nTh17 cells can develop in the absence of the IL-6/STAT3 signaling axis required by inducible Th17 cells. The nTh17 cell population is distinct from conventional inducible Th17 cells, since nTh17 cells express substantial amounts of IL-17A (IL-17), but not IL-17F, under the control of the master regulator, RORγt. The nTh17 cells simultaneously produce IFN-γ. DO11.10 transgenic mice with a Rag−/− background (DO11.10 Rag−/−) lack nTh17 cells, and, following intranasal administration of OVA, IL-17-dependent neutrophil infiltration occurs in DO11.10 transgenic mice, but not in DO11.10 Rag−/− mice. The impaired neutrophil-dependent airway response is restored by adaptive transfer of nTh17 cells into DO11.10 Rag−/− mice. These results demonstrate that a novel T cell subset, nTh17, facilitates the early phase of Ag-induced airway responses and host defenses against pathogen invasion before the establishment of acquired immunity.

Cancer is a complex disease with interactions between normal and neoplastic cells. Since current therapies for cancer largely rely on drugs or radiation that kill dividing cells or block cell division, these treatments may have severe side effects on normal proliferating cells in patients with cancer. Therefore, the potential for treatment of cancer patients by immunologic approaches, which may be specific for tumors and will not injure most normal cells, has great promise. Cancer immunotherapy aims to augment the weak host immune response to developing tumors. One strategy is to utilize cytokines such as IL-2. More recently, several exciting new interleukins have been characterized that have considerable promise for future immunotherapy. The promise of cancer immunotherapy largely depends upon the identification of these novel interleukins. This review provides an overview of the antitumor effects of relatively new interleukins as potential therapeutic agents applicable for cancer immunotherapy.

IL-23 and IL-27 are two novel IL-12 cytokine family members who are quite similar to, but yet clearly distinct from IL-12 in their structures and T-cell stimulatory mechanisms. Here, we demonstrated that either IL-27 or IL-23 has potent antitumor activity in murine models of MM45T.Li hepatocellular carcinoma (HCC). These potent antitumor effects were induced primarily by CD8(+)T cells, secreting IFN-gamma while CD4(+)T cells were also involved as a help of antitumor immunity. However, the antitumor response induced by IL-27 was observed from an early stage of tumor growth whereas that of IL-23 was only evident in the late stage of tumor cell proliferation. IL-23 could induce mice to develop a long-term systemic immunologic memory response against parental MM45T.Li tumors cells, an effect IL-27 was not able to accomplish. CTLs specific for MM45T.Li cells were significantly induced by IL-23, whereas antitumor efficacy mediated by IL-27 and IL-12 involved NK cells, which IL-23 failed to activate. Furthermore, we demonstrated that CD40 expression also plays an important role in the induction of antitumor activities by IL-27, IL-23 or IL-12. Together our data suggest that IL-27 and IL-23 may be two novel and attractive candidate agents to apply to cancer immunotherapy.

IL-27, a member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in augmentation of Th1 differentiation and suppression of proinflammatory cytokine production. In the present study, we investigated the role of STAT3 in the IL-27-mediated immune functions. IL-27 induced phosphorylation of STAT1, -2, -3 and -5 in wild-type naive CD4+ T cells, but failed to induce that of STAT3 and STAT5 in STAT3-deficient cohorts. IL-27 induced not only proinflammatory responses including up-regulation of ICAM-1, T-box expressed in T cells, and IL-12Rβ2 and Th1 differentiation, but also anti-inflammatory responses including suppression of proinflammatory cytokine production such as IL-2, IL-4, and IL-13 even in STAT3-deficient naive CD4+ T cells. In contrast, IL-27 augmented c-Myc and Pim-1 expression and induced cell proliferation in wild-type naive CD4+ T cells but not in STAT3-deficient cohorts. Moreover, IL-27 failed to activate STAT3, augment c-Myc and Pim-1 expression, and induce cell proliferation in pro-B BaF/3 transfectants expressing mutant gp130, in which the putative STAT3-binding four Tyr residues in the YXXQ motif of the cytoplasmic region was replaced by Phe. These results suggest that STAT3 is activated through gp130 by IL-27 and is indispensable to IL-27-mediated cell proliferation but not to IL-27-induced Th1 differentiation and suppression of proinflammatory cytokine production. Thus, IL-27 may be a cytokine, which activates both STAT1 and STAT3 through distinct receptor subunits, WSX-1 and gp130, respectively, to mediate its individual immune functions.

Interleukin (IL)-27, one of the most recently discovered IL-6 family cytokines, activates both the signal transducer and activator of transcription (STAT)1 and STAT3, and plays multiple roles in pro- and anti-inflammatory immune responses. IL-27 acts on various types of cells including T, B, and macrophage through the common signal-transducing receptor gp130 and its specific receptor WSX-1, but the effect of IL-27 on hematopoietic stem cells (HSCs) remains unknown. Here, we show that IL-27 together with stem cell factor (SCF) directly acts on HSCs and supports their early differentiation in vitro and in vivo. CD34−/lowc-Kit+Sca-1+lineage marker− (CD34−KSL) cells, a population highly enriched in mouse HSCs, were found to express both IL-27 receptor subunits. In vitro cultures of CD34−KSL cells with IL-27 and SCF resulted in an expansion of progenitors including short-term repopulating cells, while some of their long-term repopulating activity also was maintained. To examine its in vivo effect, transgenic mice expressing IL-27 were generated. These mice exhibited enhanced myelopoiesis and impaired B lymphopoiesis in the bone marrow with extramedullary hematopoiesis in the spleen. Moreover, IL-27 similarly acted on human CD34+ cells. These results suggest that IL-27 is one of the limited cytokines that play a role in HSC regulation.

Cancer immunotherapies such as sipuleucel-T and ipilimumab are promising new treatments that harness the power of the immune system to fight cancer and achieve long-lasting remission. Interleukin (IL)-27, a member of the IL-12 heterodimeric cytokine family, has pleiotropic functions in the regulation of immune responses with both pro-inflammatory and anti-inflammatory properties. Evidence obtained using a variety of preclinical mouse models indicates that IL-27 possesses potent antitumor activity against various types of tumors through multiple mechanisms without apparent adverse effects. These mechanisms include those mediated not only by CD8(+) T cells, natural killer cells and macrophages, but also by antibody-dependent cell-mediated cytotoxicity, antiangiogenesis, direct antiproliferative effects, inhibition of expression of cyclooxygenase-2 and prostaglandin E2 , and suppression of epithelial-mesenchymal transition, depending on the characteristics of individual tumors. However, the endogenous role of IL-27 subunits and one of its receptor subunits, WSX-1, in the susceptibility to tumor development after transplantation of tumor cell lines or endogenously arising tumors seems to be more complicated. IL-27 functions as a double-edged sword: IL-27 increases IL-10 production and the expression of programmed death ligand 1 and T-cell immunoglobulin and mucin domain-3, and promotes the generation of regulatory T cells, and IL-27 receptor α singling enhances transformation; IL-27 may augment protumor effects as well. Here, we review both facets of IL-27, antitumor effects and protumor effects, and discuss the potential clinical application of IL-27 as an antitumor agent.

IL-27, a novel member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in positive and negative regulations of immune responses. We recently demonstrated that IL-27 induces Th1 differentiation through ICAM-1/LFA-1 interaction in a STAT1-dependent, but T-bet-independent mechanism. In this study, we further investigated the molecular mechanisms by focusing on p38 MAPK and ERK1/2. IL-27-induced Th1 differentiation was partially inhibited by lack of T-bet expression or by blocking ICAM-1/LFA-1 interaction with anti-ICAM-1 and/or anti-LFA-1, and further inhibited by both. Similarly, the p38 MAPK inhibitor, SB203580, or the inhibitor of ERK1/2 phosphorylation, PD98059, partially suppressed IL-27-induced Th1 differentiation and the combined treatment completely suppressed it. p38 MAPK was then revealed to be located upstream of T-bet, and SB203580, but not PD98059, inhibited T-bet-dependent Th1 differentiation. In contrast, ERK1/2 was shown to be located downstream of ICAM-1/LFA-1, and PD98059, but not SB203580, inhibited ICAM-1/LFA-1-dependent Th1 differentiation. Furthermore, it was demonstrated that STAT1 is important for IL-27-induced activation of ERK1/2, but not p38 MAPK, and that IL-27 directly induces mRNA expression of growth arrest and DNA damage-inducible 45γ, which is known to mediate activation of p38 MAPK. Finally, IL-12Rβ2 expression was shown to be up-regulated by IL-27 in both T-bet- and ICAM-1/LFA-1-dependent mechanisms. Taken together, these results suggest that IL-27 induces Th1 differentiation via two distinct pathways, p38 MAPK/T-bet- and ICAM-1/LFA-1/ERK1/2-dependent pathways. This is in contrast to IL-12, which induces it via only p38 MAPK/T-bet-dependent pathway.

The interleukin (IL)-27 receptor-alpha WSX-1 is one component of the heterodimeric IL-27 receptor that is expressed on various cell types including macrophages. We previously demonstrated that IL-27 induces STAT-3 and is able to inhibit the production of pro-inflammatory cytokines in activated macrophages suggesting a novel feed-back mechanism by which IL-27 can modulate excessive inflammation. Because IL-4 receptor-alpha (IL-4Ralpha)-induced alternatively activated macrophages have also been described to attenuate pathological inflammatory immune responses, we analyzed the contribution of IL-27 in alternative macrophage activation. In the present study, like IL-10 and IL-4, IL-27 was found to suppress IL-12/23p40 production in activated bone marrow-derived macrophages. Whereas IL-10 induced the upregulation of the IL-4Ralpha on macrophages, receptor expression was not triggered by IL-27. In contrast to IL-4, IL-27 did not induce alternative macrophage activation but IL-4 strongly upregulated the expression of WSX-1 on macrophages and alternative macrophage activation enhanced IL-27-mediated signalling. We therefore conclude from our study that IL-10, IL-4 and IL-27 collaborate in modulating macrophage activation by successive upregulation of the IL-4Ralpha and WSX-1 on alternatively activated macrophages.

It is important for more effective gene therapies to clarify the mechanisms by which cDNA integrated into cells can maintain or lose its function in vivo. We evaluated genetic and epigenetic events leading to alternation of the introduced CD95 (Fas/Apo-1) gene as a model of gene therapy. Solid tumors formed by CD95 cDNA-transfected hepatoma cells (F6b) were almost completely cured by a single treatment of anti-CD95 monoclonal antibody (mAb) but recurred in gld/gld lpr/lpr mice after initial complete response. Recurred tumors were resistant to repeated mAb treatment. The ratio of resistant cells in tumors was estimated as 4.2 cells per 106 cells. The CD95-resistant tumor contained CD95-vanished and CD95-decreased cells. CD95-vanished cells were due to the deletion of CD95cDNA. However, CD95-decreased cells retained CD95cDNA, which was highly methylated when determined with methylation-dependent enzymes and a demethylation reagent, indicating that DNA methylation was responsible for the reduced CD95 expression and resistance to mAb. CD95-decreased cells reduced the CD95 expression further but did not delete cDNA after a second in vivo treatment with anti-CD95 mAb, suggesting that the elimination of cDNA is not induced after its methylation and that cells containing methylated genes became more resistant by further methylation. Thus, the elimination and methylation of integrated cDNA appear to occur through different mechanisms. Our study of resistant tumor cells, which arose by both mutational and epigenetic modifications of the introduced CD95 plasmid, provides important and fundamental information about the fate of introduced cDNA, augmenting the efficiency of gene therapy. © 2006 Wiley-Liss, Inc.

IL-27 is a novel IL-6/IL-12 family cytokine that not only plays a role in the early regulation of Th1 differentiation, but also exerts an inhibitory effect on immune responses, including the suppression of proinflammatory cytokine production. However, the molecular mechanism by which IL-27 exerts the inhibitory effect remains unclear. In this study we demonstrate that IL-27 inhibits CD28-mediated IL-2 production and that suppressor of cytokine signaling 3 (SOCS3) plays a critical role in the inhibitory effect. Although IL-27 enhanced IFN-γ production from naive CD4+ T cells stimulated with plate-coated anti-CD3 and anti-CD28 in the presence of IL-12, IL-27 simultaneously inhibited CD28-mediated IL-2 production. Correlated with the inhibition, IL-27 was shown to augment SOCS3 expression. Analyses using various mice lacking a signaling molecule revealed that the inhibition of IL-2 production was dependent on STAT1, but not on STAT3, STAT4, and T-bet, and was highly correlated with the induction of SOCS3 expression. Similar inhibition of CD28-mediated IL-2 production and augmentation of SOCS3 expression by IL-27 were observed in a T cell hybridoma cell line, 2B4. Forced expression of antisense SOCS3 or dominant negative SOCS3 in the T cell line blocked the IL-27-inudced inhibition of CD28-mediated IL-2 production. Furthermore, pretreatment with IL-27 inhibited IL-2-mediated cell proliferation and STAT5 activation, although IL-27 hardly affected the induction level of CD25 expression. These results suggest that IL-27 inhibits CD28-mediated IL-2 production and also IL-2 responses, and that SOCS3, whose expression is induced by IL-27, plays a critical role in the inhibitory effect in a negative feedback mechanism.

The transcription factor T-bet regulates the differentiation of CD4 + T-helper type 1 (Th1) cells and represses Th2 lineage commitment. Since Th1 cells are crucial in the defense against pathogens, several studies addressed the role of T-bet in immunity to infection using T-bet knockout (T-bet −/− ) mice. Nevertheless, it is still unclear whether T-bet is required for defense. Although vaccinia virus (VV) has extensively been used as an expression vector and the smallpox vaccine, there is only limited knowledge about immunity to VV infection. The urgency to understand the immune responses has been increased because of concerns about bioterrorism. Here, we show that T-bet is critical in the defense against VV infection as follows: (i) the survival rate of T-bet −/− mice was lower than that of control littermates postinfection; (ii) T-bet −/− mice lost more weight postinfection; and (iii) control mice cleared VV faster than T-bet −/− mice. As expected, a significant Th2 shift was observed in CD4 + T cells of T-bet −/− mice. Furthermore, absence of T-bet impaired VV-specific CD8 + cytotoxic T-lymphocyte (CTL) function, including cytolytic activity, antiviral cytokine production, and proliferation. Cytolytic capacity of natural killer (NK) cells was also diminished in T-bet −/− mice, whereas anti-VV antibody production was not impaired. These data reveal that the enhanced susceptibility to VV infection in T-bet −/− mice was at least partially due to the Th2 shift of CD4 + T cells and the diminished function of VV-specific CTLs and NK cells but not due to downregulation of antibody production.

The role of CD95 ligand (FasL/Apo-1L)-expressing tumors in immunosuppression or immunopotentiation is controversial. CD95L-transfected tumors induce immunopotentiation after vigorous neutrophil infiltration. Thus, the induction of neutrophil infiltration by CD95L seems to play an important role in tumor rejection. The mechanism by which CD95L-expressing tumors cause neutrophil infiltration and antitumor immunity has not been well understood. CXC chemokine receptor 2 (CXCR2) knockout (KO) mice are a powerful tool for studying CXC chemokine-mediated neutrophil infiltration. We investigated the roles of CD95L and chemokines in CD95L-induced antitumor activity by using CXCR2 KO mice and CD95LcDNA-transfected MethA (MethA + CD95L) fibrosarcoma. MethA + CD95L cells were completely rejected in wild-type (WT) and even in KO mice. MethA + CD95L cells injected intraperitoneally (i.p.) induced the recruitment of both neutrophils and macrophages in WT but only macrophages in KO mice, although CXC and CC chemokines were released in both mice. Macrophages incubated with MethA + CD95L cells released CXC and CC chemokines. Macrophages derived from WT and KO but not neutrophils from WT mice induced the recruitment of neutrophils when adoptively i.p. transferred with MethA + CD95L cells into CD95L/CD95-deficient mice. The different recruitment of inflammatory cells between WT and KO mice was attributed to bone marrow (BM) cells by BM transfer experiment. Our results demonstrated that CXC chemokines are essential for neutrophil recruitment and that macrophages but not neutrophils play a critical role in the CD95L-induced infiltration of inflammatory cells and the eradication of CD95L-expressing tumor cells.

Searching the sequence databases has revealed two novel cytokines: interleukin-23 (IL-23) and IL-27. These cytokines are quite similar to, but clearly distinct from IL-12 in their structures and T-cell stimulatory fashions. In contrast to IL-12, however, little is known about the roles of IL-23 and IL-27 in the immune regulation. Previously, we evaluated the prime-boost immunization consisting of priming and the first boosting with the hepatitis C virus (HCV)-core expression plasmid, followed by a second boosting with recombinant adenovirus expressing HCV core for induction of HCV core-specific cytotoxic T lymphocytes (CTLs) in BALB/c mice. The present study demonstrates that HCV-specific CTL induction was greatly enhanced by coinoculation of an IL-12 expression plasmid in the prime-boost immunization, indicating the potent adjuvant activity of IL-12. We investigated whether similar adjuvant effects could be exerted by either IL-23 or IL-27 in a prime-boost immunization with HLA-A*0201 transgenic mice. Coadministration of either an IL-23 or an IL-27 expression plasmid, as well as an IL-12 expression plasmid, in a prime-boost immunization enhanced induction of HCV-specific CTLs and led to dramatic increases in the numbers of gamma interferon (IFN-γ)-producing, HCV-specific CD8 + cells. Further, preinjections of IL-12, IL-23, or IL-27 expression plasmids before immunization resulted in great increases in the number of IFN-γ-producing, HCV-specific CD8 + cells in response to immunization with recombinant adenovirus. These data revealed that both IL-23 and IL-27, as well as IL-12, are potent adjuvants for epitope-specific CTL induction. The two novel cytokines might offer new prophylactic and therapeutic strategies against infectious pathogens such as HCV.

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. However, its role in B cells remains unexplored. We here show a role for IL-27 in the induction of T-bet expression and regulation of Ig class switching in B cells. Expression of WSX-1, one subunit of IL-27R, was detected at the mRNA level in primary mouse spleen B cells, and stimulation of these B cells by IL-27 rapidly activated STAT1. IL-27 then induced T-bet expression and IgG2a, but not IgG1, class switching in B cells activated with anti-CD40 or LPS. In contrast, IL-27 inhibited IgG1 class switching induced by IL-4 in activated B cells. Similar induction of STAT1 activation, T-bet expression and IgG2a class switching was observed in IFN-γ-deficient B cells, but not in STAT1-deficient ones. The induction of IgG2a class switching was abolished in T-bet-deficient B cells activated with LPS. These results suggest that primary spleen B cells express functional IL-27R and that the stimulation of these B cells by IL-27 induces T-bet expression and IgG2a, but not IgG1, class switching in a STAT1-dependent but IFN-γ-independent manner. The IL-27-induced IgG2a class switching is highly dependent on T-bet in response to T-independent stimuli such as LPS. Thus, IL-27 may be a novel attractive candidate as a therapeutic agent against diseases such as allergic disorders by not only regulating Th1 differentiation but also directly acting on B cells and inducing IgG2a class switching.

To establish a more efficient treatment for immunotherapy against solid tumors, we have evaluated the antitumor effect by coexpression of a chemokine CCL21/secondary lymphoid tissue chemokine and a costimulatory molecule LIGHT in colon carcinoma C26. C26 cells expressing either CCL21 or LIGHT exhibited a significantly reduced tumor growth in vivo, and mice inoculated with these cells showed a prolonged survival, but eventually all these mice died. In contrast, C26 cells expressing both CCL21 and LIGHT exhibited a minimal tumor growth in vivo, and all these mice survived healthily with a tumor remission and consequently acquired a strong protective immunity. A markedly increased infiltration of mature dendritic cells (DCs), and CD8(+) T cells was observed in the tumor mass, and their spleen cells showed a greatly enhanced cytotoxic T lymphocyte (CTL) activity against C26 tumor and interferon (IFN)-gamma production. Neutralization of IFN-gamma or depletion of CD8(+) or CD4(+) T cells significantly reduced the antitumor activity. These results suggest that the combined treatment with CCL21 and LIGHT is able to induce a synergistic antitumor effect to eradicate tumor completely by greatly enhancing tumor-infiltration of lymphocytes including mature DCs and CD8(+) T cells, resulting in markedly augmented CTL activity and IFN-gamma production.

Objective: Subcutaneous administrations of monosodium glutamate (MSG) to neonatal animals result in obesity and induce the toxicity on the central nervous system, and furthermore, have an effect on entero-pancreatic hormone. The effect of MSG on the cell turnover of organs, especially the pancreas, has received little attention until now. This study was designed to examine the effect of MSG on pancreatic cell turnover by immunohistochemistry and [3H]thymidine autoradiography. Research Methods and Procedures: Male JcI-ICR strain mice were SC injected with MSG (2 mg/g body weight daily) for 5 days after birth, received 112 repeated injections of [3H]thymidine at 6-hour intervals for 28 days after birth, and then were killed immediately thereafter, or 30, 60, or 120 days after the last injection. Autoradiography was performed on sections immunostained for glucagon, insulin, and somatostatin. Results: After continuous labeling, most pancreatic cells were labeled, and thereafter, labeling of cells decreased in control and MSG-treated mice. The mean grain counts of acinar cells in MSG-treated mice decreased more slowly than those in control mice. On the other hand, those of islet cells, including glucagon, insulin, and somatostatin cells, decreased more rapidly in MSG-treated mice than those in control mice. Discussion: Cell turnover of acinar cells was decelerated and that of islet cells including glucagon, insulin, and somatostatin cells was accelerated in MSG-treated mice pancreas. MSG-induced hypothalamic lesions exert the contrary influences on the cell turnover of acinar and islet cells.

We recently cloned mouse Thy28 cDNA (mThy28), which is highly conserved among vertebrates and plants. The mThy28 mRNA is highly expressed in testis, liver, kidney, brain, with moderate expression in thymus, spleen, and heart. In the present study, characteristics of mouse Thy28 protein expression were examined using rabbit anti-mThy28 polyclonal antibody (Ab). Levels of mThy28 protein expression were highest in testis, with moderate expression in liver, spleen, and thymus. The Thy28 protein was mainly located in the nucleus, which was revealed by immunofluorescence microscopy and Western blotting using anti-mThy28 Ab, and transient expression of the mThy28/EGFP fusion gene. Engagement of membrane immunoglobulin with anti-IgM induced down-regulation of human Thy28 expression at both mRNA and protein levels, accompanied by induction of apoptosis in Ramos B lymphoma cells. Expression of protein and mRNA and induction of apoptosis were evaluated by flow cytometry/Western blotting, reverse transcription-polymerase chain reaction, and propidium iodide staining, respectively. Anti-IgM also down-regulated the promoter activity of the mThy28 gene, as demonstrated by luciferase assay. Thus, the anti-IgM-induced down-regulation of the nuclear Thy28 expression appears to correlate with the induction of apoptosis in Ramos B lymphoma cells.

Interleukin (IL)-27 is a multifunctional cytokine that belongs to the IL-6/IL-12 family and has potent antitumor activity through various mechanisms. Our novel findings indicate that IL-27 directly acts on hematopoietic stem cells and promotes their expansion and differentiation into myeloid progenitors to control infection and to eradicate tumors.

Tumor necrosis factor (TNF)-α is known to play a pivotal role in the pathogenesis of psoriasis. TNF-α has been shown to act directly on keratinocytes, thereby inducing the production of various kinds of chemokines, which contributes to the infiltration of leucocytes into the psoriatic lesions. Recent studies have shown that both interleukin (IL)-17 and IL-27 are increased in psoriatic lesional tissue. However, the interactions between TNF-α, IL-17 and IL-27 in chemokine production by keratinocytes have not been fully elucidated. Here, we examined in human keratinocytes how TNF-α, IL-17 and IL-27 affect production of chemokines that are involved in the pathogenesis of psoriasis. We found that IL-17 and IL-27 exert opposite effects on TNF-α-mediated chemokine production. This suggests that lesional balance of IL-17 and IL-27 is involved in the recruitment of T cells, natural killer cells, neutrophils, monocytes or dendritic cells, thereby affecting inflammation in skin diseases.

Junko H Ohyashiki,1 Kazushige Ohtsuki,1 Izuru Mizoguchi,2 Takayuki Yoshimoto,2 Seiichiro Katagiri,3 Tomohiro Umezu,1,4Kazuma Ohyashiki3,4 1Department of Molecular Oncology, Institute of Medical Science, 2Department of Immunoregulation, Institute of Medical Science, 3Department of Hematology, 4Department of Molecular Science, Tokyo Medical University, Tokyo, Japan Background: A subset of patients with chronic myeloid leukemia (CML) can sustain a complete molecular response after discontinuing imatinib mesylate (IM). We focused on microRNAs (miRNAs), with the aim of finding a molecular biomarker to discriminate which patients can safely and successfully discontinue IM use. Methods: To identify miRNAs that showed altered expression in patients who had discontinued IM (STOP-IM group), we first screened miRNA expression of peripheral blood mononuclear cells by using a TaqMan miRNA array on samples from five unselected patients from the STOP-IM group, seven CML patients receiving IM (IM group), and five healthy volunteers. We then performed miRNA quantification in 49 CML patients with deep molecular response. Mann–Whitney U and chi-square tests were used to determine statistical significance for comparisons between the control (healthy volunteers) and test groups (STOP-IM and IM groups). Multiple groups were compared by one-way analysis of variance. Results: Downregulation of miR-148b was noted in patients in the STOP-IM group and in a subset of the IM group. We then subdivided the IM patients into two groups: one with downregulated miR-148b expression (IM-1; less than the cut-off value) and the other without downregulated miR-148b expression (IM-2; greater than the cut-off value). The number of patients who had a sustained stable molecular response was significantly lower in IM-2 group. This group also had a significantly lower percentage of natural killer cells. Conclusion: Downregulated miR-148 may contribute to immune surveillance in STOP-IM patients and may therefore have potential as additive information in managing CML patients undergoing treatment with IM. Keywords: chronic myeloid leukemia, imatinib mesylate, discontinuation, miR-148b&nbsp

We previously found that Mtv-2+ lymph nodes (LN) implanted into Mtv-2− mice underwent marked hyperplasia owing to the influx of lymphocytes. LN grafts infected with exogenous mouse mammary tumour viruses (MMTV), MMTV(FM) transmitted by FM mice and MMTV-2 produced by Mtv-2, also swelled in MMTV-free recipients. Mtv-3 and Mtv-7 also displayed this capability. Mtv-2-induced LN hyperplasia was earlier in onset and greater in extent when major histocompatibility complex (MHC) class II I-E was expressed than unexpressed. Mtv-3-induced LN hyperplasia was suppressed completely by Mtv-3 from a different mouse strain and partially by Mtv-6 slightly different from Mtv-3 in superantigen (SAg) Vβ specificity. LN hyperplasia occurred bidirectionally in LN transplantation between mice carrying Mtv-2 and Mtv-3, which are different SAg Vβ specificity. LN hyperplasia induced by MMTV-2 carrying SAg responsive to Vβ14 alone and MMTV(FM) carrying SAg responsive to Vβ14 and Vβ8.2 was completely but partially suppressed by MMTV(FM) and MMTV-2, respectively. CD4+ T cells were essential for MMTV-induced LN hyperplasia. LN in situ also underwent significant hyperplasia when infected with MMTV. Thus, MMTV SAg may entice circulating lymphocytes into lymphoid organs and contribute to more efficient dissemination MMTV in vivo. Secondary lymphoid tissue chemokine (SLC) may not be directly involved in this event.

Mouse mammary tumor virus transmitted by FM mice (FM-MMTV) encodes a superantigen (SAg) characterized by strong reactivity with TCR Vβ8.2 element and broad spectrum of Vβ reactivity. To investigate...

Delayed-type hypersensitivity (DTH) is an important in vivo manifestation of cell-mediated immunity. We examined the DTH response to methylated bovine serum albumin of a novel mutant strain of mice that have soluble CD4 (sCD4) in their circulation without expression of CD4 on the cell surface. The DTH response of the mutant mice was severely impaired, although the response of CD4 knockout (KO) mice, generated by homologous recombination, was comparable to that of wild-type mice. The response of the mutant mice was restored by the neutralization of sCD4 with anti-CD4, and that of CD4KO mice was markedly reduced by the implantation of a diffusion chamber containing sCD4 cDNA transfectant cells. The restored DTH response of the mutant mice treated with anti-CD4 was abolished by treatment with anti-interferon-gamma (IFN-gamma). IFN-gamma production by CD4 mutant and CD4KO mice was consistent with their DTH response and inversely related to the presence of sCD4 in their circulation, indicating that sCD4 impairs the DTH response by blocking the production of IFN-gamma in our mutant mice. These results raise the possibility that sCD4 could impair cell-mediated immunity. Our mutant mice would provide a useful tool with which to analyse the mechanisms of the DTH reaction.

Interleukin (IL)-16 is a chemoattractant cytokine for CD4(+) leukocytes. Because delayed-type hypersensitivity (DTH) reaction is mediated by T helper 1 (Th1) cells and CD4(+) T cells can be chemoattracted by IL-16, we have investigated the involvement of IL-16 in the DTH reaction. Immunohistochemical analysis revealed the IL-16 expression in infiltrating cells and epithelial cells in the DTH footpads. The IL-16 expression was also detected intracellularly in the infiltrating cells. In addition, markedly increased production of IL-16 was detected in the DTH footpad extracts, but not in the control footpad extracts, by an enzyme-linked immunosorbent assay and also by Western blot analysis. The DTH footpad extracts exhibited a strong chemoattractant activity toward splenic T cells, which was significantly inhibited by the inclusion of neutralizing monoclonal antibody (mAb) against IL-16 in the migration assay. Furthermore, treatment of sensitized mice in vivo with the anti-IL-16 neutralizing mAb significantly suppressed the footpad swelling induced by an antigen challenge, together with decreased infiltration of leukocytes including not only CD4(+) T cells but also CD8(+) T cells and macrophages into the DTH footpads. Decreased production of macrophage inflammatory protein 1alpha was also observed in the DTH footpad extracts by the mAb treatment. These results suggest that IL-16 plays an important role in the recruitment of leukocytes-presumably including antigen-specific Th1 cells, which secrete cytokines and chemokines mediating the following hypersensitivity reaction after activation by the interaction with Langerhans cells carrying the antigen-for the elicitation of DTH response. (Blood. 2000;95:2869-2874)

Fas/Apo-1 (CD95)-Fas ligand (FasL) system has been implicated in the suppression and stimulation of immune responses. We examined the induction of antitumor immunity with neuroblastoma Neuro-2a cells transfected with FasL cDNA (Neuro-2a+FasL). Neuro-2a+FasL cells expressed FasL on the cell surface and secreted soluble FasL. Histologic and flow cytometric analyses revealed that Neuro-2a+FasL cells caused neutrophils to infiltrate into the injected site, resulting in strong inflammation. Neutrophil infiltration was inhibited by treatment with anti-FasL mAb and did not occur in Fas-deficient lpr mice. Normal syngeneic mice rejected Neuro-2a+FasL cells after the inflammation and acquired tumor-specific protective immunity. CD8+ T cells were responsible for the antitumor immunity. Neuro-2a+FasL cells formed tumors after far longer latency compared with mock-transfected Neuro-2a+Neo cells in nude mice, and immune competent mice rejected Neuro-2a cells but not sarcoma S713a cells when they were injected with Neuro-2a+FasL cells in a mixture. These results suggest that neutrophils attracted through the Fas-FasL system may impair tumor cells by inflammation at the initial step, followed by development of CD8+ T cell-dependent tumor-specific antitumor immunity, leading to complete eradication of tumor cells. Importantly, the treatment with Neuro-2a+FasL cells exhibited therapeutic efficacy against growing tumors.

We have examined the roles of gamma interferon (IFN-γ), nitric oxide (NO), and natural killer (NK) cells in the host resistance to infection with the blood-stage malarial parasite Plasmodium berghei XAT, an irradiation-induced attenuated variant of the lethal strain P. berghei NK65. Although the infection with P. berghei XAT enhanced NK cell lytic activity of splenocytes, depletion of NK1.1 + cells caused by the treatment of mice with anti-NK1.1 antibody affected neither parasitemia nor IFN-γ production by their splenocytes. The P. berghei XAT infection induced a large amount of NO production by splenocytes during the first peak of parasitemia, while P. berghei NK65 infection induced a small amount. Unexpectedly, however, mice deficient in inducible nitric oxide synthase (iNOS −/− ) cleared P. berghei XAT after two peaks of parasitemia were observed, as occurred for wild-type control mice. Although the infected iNOS −/− mouse splenocytes did not produce a detectable level of NO, they produced an amount of IFN-γ comparable to that produced by wild-type control mouse splenocytes, and treatment of these mice with neutralizing anti-IFN-γ antibody led to the progression of parasitemia and fatal outcome. CD4 −/− mice infected with P. berghei XAT could not clear the parasite, and all these mice died with apparently reduced IFN-γ production. Furthermore, treatment with carrageenan increased the susceptibility of mice to P. berghei XAT infection. These results suggest that neither NO production nor NK cell activation is critical for the resistance to P. berghei XAT infection and that IFN-γ plays an important role in the elimination of malarial parasites, possibly by the enhancement of phagocytic activity of macrophages.

Lymph node (LN) cells of Fas-mutant mice lpr/lpr (lpr) and lpr(cg)/lpr(cg) (lpr(cg)) express an increased level of Fas ligand (FasL) (CD95L). We examined the antitumor potential of cell-bound FasL on these LN cells against Fas+ tumor cells. Fas+ F6b and Fas- N1d cells were produced from murine hepatoma MH134 (Fas-) by gene transfection. lpr and lpr(cg) LN cells inhibited growth of F6b but not N1d cells in vitro. Neither gld/gld lpr/lpr (gld/lpr) LN cells, which lack both FasL and Fas, nor wild-type LN cells showed growth-inhibitory activities against F6b and N1d cells. The effector cells and molecule were CD4-CD8- T cells and FasL, respectively. The tumor neutralization test and adoptive transfer demonstrated that lpr and lpr(cg), but not gld/lpr, LN cells retarded the growth of F6b cells. Although anti-Fas antibody and FasL cause severe liver failure, wild-type mice injected with lpr LN cells appeared clinically normal. Adoptive transfer of lpr LN cells to F6b-bearing mice exerted the same antitumor activity in wild-type and gld/lpr recipient mice, indicating the applicability of cell-bound FasL for Fas-mediated target therapy of cancer. These results suggest that antitumor activity was dependent on the Fas-FasL system and that lymphoid cells overexpressing FasL can be powerful antitumor effector cells against Fas+ tumor cells.

Although interleukin (IL)-12 was originally purified from an Epstein-Barr (EBV)-transformed B cell line and the high correlation of EBV infection and IL-12 expression has been suggested, no study has reported whether EBV infection is directly linked to IL-12 expression. To address this issue, we have investigated IL-12 expression in B cells during in vitro transformation with EBV. Human peripheral B cells became capable of constitutively producing p40 by in vitro transformation with EBV, coincident with the expression of latent membrane protein 1 (LMP1) of EBV. These B cells expressed p40 and p35 mRNA, and phorbol myristate acetate (PMA) stimulation strongly enhanced p40 and p70 production. Furthermore, transfection with LMP1 expression vector into a human B lymphoma cell line, Daudi, led to p40 production with nuclear factor (NF)-κB activation. These results suggest that transformation of primary B cells with EBV induces IL-12 expression potentially through LMP1 expression.

We studied whether the infection with a blood-stage murine malaria lethal Plasmodium berghei NK65 induces IL-12 production, and if so, how the IL-12 production is involved in the protection or pathogenesis. The infection of C57BL/6 mice enhanced mRNA expression of IL-12 p40 and also IFN-γ, IL-4, and IL-10 in both spleen and liver during the early course of the infection. It also enhanced the mRNA expression of TNF-α, Fas ligand, and cytokine-inducible nitric oxide synthase. Increased IL-12 p40 production was also observed in the culture supernatant of spleen cells and in sera of infected mice. In addition, the infection caused massive liver injury with elevated serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and body weight loss. Treatment of these infected mice with neutralizing mAb against IL-12 prolonged the survival and diminished the liver injury with reduced elevation of serum serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and decreased body weight loss. However, the anti-IL-12 treatment did not affect parasitemia, and all these mice eventually died. Similar results were obtained when infected mice were treated with neutralizing mAb against IFN-γ. Moreover, anti-IL-12 treatment greatly reduced the secretion and mRNA expression of IFN-γ in both spleen and liver. These results suggest that the lethal P. berghei NK65 infection induces IL-12 production and that the IL-12 is involved in the pathogenesis of liver injury via IFN-γ production rather than the protection.

The mechanism of development of host resistance to blood-stage malarial infection was studied by use of an irradiation-induced attenuated variant, Plasmodium berghei XAT, obtained from a lethal strain, P. berghei NK65. The infection enhanced mRNA expression of interleukin (IL)-12 p40 and also of interferon (IFN)-gamma, IL-4, IL-10, and cytokine-inducible nitric oxide synthase (iNOS) in spleen. Treatment of these mice with anti-IL-12 or anti-IFN-gamma led to the progression of parasitemia and fatal outcome. Anti-IL-12 treatment significantly reduced the secretion and mRNA expression of IFN-gamma and greatly diminished the augmentation of iNOS mRNA expression. In addition, recombinant IL-12 administration delayed the onset of parasitemia because of the enhanced IFN-gamma production. These results suggest that blood-stage P. berghei XAT infection induces IL-12 production, which is important for the development of host resistance via IFN-gamma production.

Mutant mice derived from C57BR/cdJ mice were found to have a novel genetic defect in CD4 expression. Flow-cytometric analysis demonstrated that there were no CD4+ cells in either the thymus or the peripheral lymphoid organs of the mutant mice. Thymocytes of the mutant mice expressed an amount of CD4 mRNA comparable to normal mouse thymocytes, but the mutant CD4 mRNA was slightly smaller in size than normal CD4 mRNA. The sequence analysis of the mutant CD4 cDNA obtained from thymic RNA revealed that the defect in the CD4 expression was attributable to the deletion of the entire exon VIII, encoding a transmembrane domain of the CD4 molecule. Moreover, soluble CD4 was detected both in the culture supernatant of thymocytes and sera from mutant mice. The analysis of the genomic DNA sequence elucidated that one thymine was substituted for 14 base pairs at the junction between exon VIII and intron VIII in the mutant mice, which could possibly account for the alternative splicing of CD4 mRNA. These mutant mice showed reduced delayed-type hypersensitivity reactions against sheep red blood cells and antibody production against T-dependent antigen but not against T-independent antigen. Thus, these mutant mice have a novel defect in CD4 expression where CD4 mRNA is alternatively spliced to delete a transmembrane domain, giving rise to secretion of soluble CD4 instead of expression of membrane-bound CD4.