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The current success of mRNA vaccines against COVID-19 has highlighted the effectiveness of mRNA and DNA vaccinations. Recently, we demonstrated that a novel needle-free pyro-drive jet injector (PJI) effectively delivers plasmid DNA into the skin, resulting in protein expression higher than that achieved with a needle syringe. Here, we used ovalbumin (OVA) as a model antigen to investigate the potential of the PJI for vaccination against cancers. Intradermal injection of OVA-expression plasmid DNA into mice using the PJI, but not a needle syringe, rapidly and greatly augmented OVA-specific CD8

The interleukin IL27 promotes expansion and differentiation of hematopoietic stem cells into myeloid progenitor cells. Many tumor-infiltrating myeloid cells exert immunosuppressive effects, but we hypothesized that the myeloid cells induced by IL27 would have antitumor activity. In this study, we corroborated this hypothesis as investigated in two distinct mouse transplantable tumor models. Malignant mouse cells engineered to express IL27 exhibited reduced tumor growth in vivo. Correlated with this effect was a significant increase in the number of tumor-infiltrating CD11b+ myeloid cells exhibiting a reduced immunosuppressive activity. Notably, these CD11b+ cells were characterized by an activated M1 macrophage phenotype, on the basis of increased expression of inducible nitric oxide synthase and other M1 biomarkers. In vivo depletion of these cells by administering anti–Gr-1 eradicated the antitumor effects of IL27. When admixed with parental tumors, CD11b+ cells inhibited tumor growth and directly killed the tumor in a nitric oxide-dependent manner. Mechanistically, IL27 expanded Lineage−Sca-1+c-Kit+ cells in bone marrow. Transplant experiments in Ly5.1/5.2 congenic mice revealed that IL27 directly acted on these cells and promoted their differentiation into M1 macrophages, which mobilized into tumors. Overall, our results illustrated how IL27 exerts antitumor activity by enhancing the generation of myeloid progenitor cells that can differentiate into antitumorigenic M1 macrophages.Significance: These findings show how the interleukin IL27 exerts potent antitumor activity by enhancing the generation of myeloid progenitor cells that can differentiate into antitumorigenic M1 macrophages.Cancer Res; 78(1); 182–94. ©2017 AACR.

This file contains Supplemental Table 1, Supplementary Figure Legends, and Supplementary Figures. Supplemental Table 1 shows primers used in this study. Supplementary Figure Legends describe the legend for each supplementary figure. Figure S1 shows the gating strategy in flow cytometry analysis. Figure S2 shows that IL-27 augments the infiltration of CD11b+ myeloid cells into MC38 tumor. Figure S3 shows that IL-27 promotes the differentiation into M1 macrophages in MC38 tumor-bearing mice. Figure S4 shows that IL-27 much less but significantly augments the infiltration of M1 macrophages into B16F10 tumor when the tumor size in B16F10-IL-27 tumor-bearing mice reaches the similar tumor size to that in control tumor-bearing mice. Figure S5 shows that IL-27 inhibits the differentiation into immunosuppressive macrophages in MC38 tumor-bearing mice. Figure S6 shows the therapeutic potential of the CD11b+ myeloid cells purified from tumors of B16F10-IL-27 tumor-bearing mice on pre-existing tumor model. Figure S7 shows that IL-27-induced MC38 tumor-infiltrating myeloid cells exert direct antitumor effects by killing tumor through NO. Figure S8 shows that the enhanced mRNA expression of immune checkpoint molecules and TGF-β1 in CD11b+ myeloid cells in IL-27-expressing tumor-bearing mice.

Interleukin (IL)-27 is an IL-12 family cytokine playing a pivotal role in the induction of Th1 immune responses, although its action on natural killer (NK) cells has not been fully elucidated. Here, we show that IL-27 is capable of inducing phosphorylation of signal transducers and activators of transcription 1 and 3, as well as expression of T-bet and granzyme B in murine DX-5+ NK cells. IL-27 also enhances cytotoxic activity of NK cells both in vitro and in vivo, while the in vitro viability of NK cells is also improved by this cytokine. Therapeutic administration of the IL-27 gene drastically suppressed the growth of NK-unsusceptible SCCVII tumors that had been preestablished in syngenic mice, resulting in significant prolongation of the survival of the animals. This can likely be ascribed to the antibody-dependent cellular cytotoxicity machinery because IL-27 successfully induced tumor-specific IgG in the sera of the tumor-bearing mice, and supplementation of the sera enabled IL-27–activated NK cells to kill SCCVII cells in an Fcγ receptor III–dependent manner. These findings strongly suggest that IL-27 may offer a powerful immunotherapeutic tool to eradicate head and neck squamous cell carcinoma and other poorly immunogenic neoplasms through activating NK cells and inducing tumor-specific immunoglobulin that may cooperatively elicit antibody-dependent cellular cytotoxicity activity. [Cancer Res 2009;69(6):2523–30]

Supplementary Figures 1-2, Table 1 from Interleukin-27 Activates Natural Killer Cells and Suppresses NK-Resistant Head and Neck Squamous Cell Carcinoma through Inducing Antibody-Dependent Cellular Cytotoxicity

Current worldwide mRNA vaccination against SARS-CoV-2 by intramuscular injection using a needled syringe has greatly protected numerous people from COVID-19. An intramuscular injection is generally well tolerated, safer and easier to perform on a large scale, whereas the skin has the benefit of the presence of numerous immune cells, such as professional antigen-presenting dendritic cells. Therefore, intradermal injection is considered superior to intramuscular injection for the induction of protective immunity, but more proficiency is required for the injection. To improve these issues, several different types of more versatile jet injectors have been developed to deliver DNAs, proteins or drugs by high jet velocity through the skin without a needle. Among them, a new needle-free pyro-drive jet injector has a unique characteristic that utilizes gunpower as a mechanical driving force, in particular, bi-phasic pyrotechnics to provoke high jet velocity and consequently the wide dispersion of the injected DNA solution in the skin. A significant amount of evidence has revealed that it is highly effective as a vaccinating tool to induce potent protective cellular and humoral immunity against cancers and infectious diseases. This is presumably explained by the fact that shear stress generated by the high jet velocity facilitates the uptake of DNA in the cells and, consequently, its protein expression. The shear stress also possibly elicits danger signals which, together with the plasmid DNA, subsequently induces the activation of innate immunity including dendritic cell maturation, leading to the establishment of adaptive immunity. This review summarizes the recent advances in needle-free jet injectors to augment the cellular and humoral immunity by intradermal injection and the possible mechanism of action.

Although several in vitro assays that predict the sensitizing potential of chemicals have been developed, none can distinguish between chemical respiratory and skin sensitizers. Recently, we established a new three-dimensional dendritic cell (DC) coculture system consisting of a human airway epithelial cell line, immature DCs derived from human peripheral monocytes, and a human lung fibroblast cell line. In this coculture system, compared to typical skin sensitizers, typical respiratory sensitizers showed enhanced mRNA expression in DCs of the key costimulatory molecule OX40 ligand (OX40L), which is important for T helper 2 (Th2) cell differentiation. Herein, we established a new two-step DC/T cell coculture system by adding peripheral allogeneic naive CD4+ T cells to the DCs stimulated in the DC coculture system. In this DC/T cell coculture system, typical respiratory sensitizers but not skin sensitizers enhanced mRNA expression of the predominant Th2 marker interleukin-4 (IL-4) and its transcription factor GATA-binding protein 3. To improve the versatility, in place of peripheral monocytes, monocyte-derived proliferating cells called CD14-ML were also used in the DC coculture system. Similar to peripheral monocytes, enhanced mRNA expression of OX40L was observed by typical respiratory sensitizers compared to skin sensitizers. When these cell lines were applied to the DC/T cell coculture system with peripheral allogeneic naive CD4+ T cells, typical respiratory sensitizers but not skin sensitizers enhanced the mRNA expression of IL-4. Thus, this DC/T cell coculture system might be useful for discriminating between respiratory and skin sensitizers by differential mRNA upregulation of IL-4 in T cells.

Haptens are small molecules that only elicit an immune response when bound to proteins. Haptens initially bind to self-proteins and activate innate immune responses by complex mechanisms via inflammatory cytokines and damage-associated molecular patterns and the subsequent upregulation of costimulatory signals such as cluster of differentiation 86 (CD86) on dendritic cells. Subsequent interactions between CD86 and CD28 on T cells are critically important for properly activating naive T cells and inducing interleukin 2 production, leading to the establishment of adaptive immunity via effector and memory T cells. Accumulating evidence revealed the involvement of haptens in the development of various autoimmune-like diseases such as allergic, inflammatory, and autoimmune diseases including allergic contact dermatitis, atopy, asthma, food allergy, inflammatory bowel diseases, hemolytic anemia, liver injury, leukoderma, and even antitumor immunity. Therefore, the development of in vitro testing alternatives to evaluate in advance whether a substance might lead to the development of these diseases is highly desirable. This review summarizes and discusses recent advances in chemical- and drug-induced allergic, inflammatory, and autoimmune diseases via haptenation and the possible molecular underlying mechanisms, as well as in vitro testing alternatives to evaluate in advance whether a substance might cause the development of these diseases.

To clarify the effect of collagen addition to transplanted adipose tissue on angiogenesis, cell proliferation and tissue remodelling process and reveal whether collagen addition contributes to improving transplanted adipose tissue engraftment in rats. Adipose tissue was harvested from the inguinal and injected into the back of the rat, in addition to collagen. Engraftment tissue was harvested, semi-quantitatively evaluated and underwent haematoxylin and eosin or Perilipin staining. Moreover, we evaluated viable adipocyte counts and neovascularisation. Macrophages were evaluated using flow cytometry, and the adiponectin or vascular endothelial growth factor (VEGF) mRNA was detected using real-time polymerase chain reaction. By collagen addition to transplanted adipose tissue, higher engraftment rate semi-quantitatively and a greater number of new blood vessels histologically were identified. Perilipin staining revealed a higher adipocyte number. The total cell, M1 macrophage and M2 macrophage count were higher. There was increased adiponectin mRNA significantly at week 4 compared to that at week 1 after transplantation. Note that the expression levels of VEGF mRNA increased. In rats, adding collagen enhanced cell proliferation, induced M2 macrophages, which are involved in wound healing, and promoted adipocytes and neovascularisation. Therefore, collagen addition to transplanted adipose tissue could increase the engraftment rate of adipose tissue.

The interleukin-6 (IL-6)/IL-12 family of cytokines plays critical roles in the induction and regulation of innate and adaptive immune responses. Among the various cytokines, only this family has the unique characteristic of being composed of two distinct subunits, α- and β-subunits, which form a heterodimer with subunits that occur in other cytokines as well. Recently, we found a novel intracellular role for one of the α-subunits, Epstein-Barr virus-induced gene 3 (EBI3), in promoting the proper folding of target proteins and augmenting its expression at the protein level by binding to its target protein and a well-characterized lectin chaperone, calnexin, presumably through enhancing chaperone activity. Because calnexin is ubiquitously and constitutively expressed but EBI3 expression is inducible, these results could open an avenue to establish a new paradigm in which EBI3 plays an important role in further increasing the expression of target molecules at the protein level in collaboration with calnexin under inflammatory conditions. This theory well accounts for the heterodimer formation of EBI3 with p28, and probably with p35 and p19 to produce IL-27, IL-35, and IL-39, respectively. In line with this concept, another β-subunit, p40, plays a critical role in the assembly-induced proper folding of p35 and p19 to produce IL-12 and IL-23, respectively. Thus, chaperone-like activities in proper folding and maturation, which allow the secretion of biologically active heterodimeric cytokines, have recently been highlighted. This review summarizes the current understanding of chaperone-like activities of EBI3 to form heterodimers and other associations together with their possible biological implications.

T-cell-specific Rap1 deletion causes spontaneous colitis in mice. In the present study, we revealed that Rap1 deficiency in T cells impaired the preceding induction of intestinal RORγt+Treg cells. In the large intestinal lamina propria (LILP) of T-cell-specific Rap1-knockout mice (Rap1KO mice), Th17 cells were found to increase in a microbiota-dependent manner, and the inhibition of IL-17A production prevented the development of colitis. In the LILP of Rap1KO mice, RORγt+Treg cells were scarcely induced by 4 weeks of age. The expression of CTLA-4 on Rap1-deficient Treg cells was reduced and the expression of CD80 and CD86 on dendritic cells was consequently elevated in Rap1KO mice. When cultured under each polarizing condition, Rap1-deficient naïve CD4+T cells did not show biased differentiation into Th17 cells; their differentiation into Treg cells as well as Th1 and Th2 cells was lesser than that of wild-type cells. Rap1-deficient naïve CD4+T cells were found to exhibit the defective nuclear translocation of NFAT and formation of actin foci in response to TCR engagement. These data suggest that Rap1 amplifies the TCR signaling required for Treg-mediated control of intestinal colitogenic Th17 responses.

Poly(acrylic acid) (PAA) is a water-soluble synthetic polymer that exhibits bioadhesive properties and has been applied in various novel medical devices, such as drug-delivery carriers and hemostatic agents. PAA forms a water-insoluble complex when mixed with polyvinylpyrrolidone (PVP). If PAA and PVP are mixed in water, they form an aggregated precipitate, which neither swells nor adheres to tissues. The formation of the hydrophobic complex was caused by hydrophobic interactions between the main chains of both polymers aligned the same as a zipper. To hinder the zipper-like alignment of the polymer main chains, hyaluronic acid (HA), a macromolecular viscous polysaccharide, was added to the PVP solution prior to complex formation. When the initial concentration of PAA was lower than 0.05%, HA effectively prevented the aggregation of PAA/PVP complexes and resulted in a slightly clouded suspension. Freeze-drying of the mixture yielded a soft white sponge, which could immediately swell in water to form a highly bioadhesive hydrogel. The PAA/PVP complex prepared with HA exhibited high hemostatic efficiency in clinical studies, even in patients on antithrombotic drugs.

Objectives: To clarify the effect of impregnating transplanted adipose tissue with collagen on angiogenesis, cell proliferation, and tissue remodeling process and to reveal whether collagen impregnation contributes to improving the engraftment of transplanted adipose tissue in rats.Methods: Adipose tissue was harvested from the inguinal and injected into the back of the rat, in addition to collagen. Engraftment tissue was harvested, semi-quantitatively evaluated and underwent HE or Perilipin staining. Moreover, we evaluated viable adipocyte counts and neovascularization. Macrophages were evaluated using flow cytometry, and the adiponectin or VEGF mRNA was detected using real-time PCR. Results: By impregnating transplanted adipose tissue with collagen, higher engraftment rate semi-quantitatively and a greater number of new blood vessels histologically were identified. Perilipin staining revealed a higher adipocyte number. The total cell, M1 macrophage, and M2 macrophage count were higher. There was increased adiponectin mRNA significantly at week 4 compared to that at week 1 after transplantation. Note that the expression levels of VEGF mRNA increased.Discussion: In rats, collagen impregnation enhanced cell proliferation, induced M2 macrophages, which are involved in wound healing, and promoted adipocytes and neovascularization. Therefore, impregnating transplanted adipose tissue with collagen could increase the engraftment rate of adipose tissue.

In this study, we assessed circulating immune cells and plasma cytokine levels in 15 pediatric patients with drug-resistant epilepsy (DRE). DRE patients had a significantly higher percentage of CD14+ monocytes positive for IL-1β, IL-1 receptor antagonist, IL-6, and TNF-α than controls. Significantly higher intracellular levels of IFN-γ in CD4+ T cells and NK cells were also found in DRE patients. The level of IL-1β+ CD14+ monocytes correlated with seizure frequency, and intracellular levels of IFN-γ in NKT-like cells were negatively correlated with the duration of epilepsy. Peripheral immune cells might be involved in the pathogenesis of DRE.

Hematopoiesis is hierarchically orchestrated by a very small population of hematopoietic stem cells (HSCs) that reside in the bone-marrow niche and are tightly regulated to maintain homeostatic blood production. HSCs are predominantly quiescent, but they enter the cell cycle in response to inflammatory signals evoked by severe systemic infection or injury. Thus, hematopoietic stem and progenitor cells (HSPCs) can be activated by pathogen recognition receptors and proinflammatory cytokines to induce emergency myelopoiesis during infection. This emergency myelopoiesis counterbalances the loss of cells and generates lineage-restricted hematopoietic progenitors, eventually replenishing mature myeloid cells to control the infection. Controlled generation of such signals effectively augments host defense, but dysregulated stimulation by these signals is harmful to HSPCs. Such hematopoietic failure often results in blood disorders including chronic inflammatory diseases and hematological malignancies. Recently, we found that interleukin (IL)-27, one of the IL-6/IL-12 family cytokines, has a unique ability to directly act on HSCs and promote their expansion and differentiation into myeloid progenitors. This process resulted in enhanced production of neutrophils by emergency myelopoiesis during the blood-stage mouse malaria infection. In this review, we summarize recent advances in the regulation of myelopoiesis by proinflammatory cytokines including type I and II interferons, IL-6, IL-27, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and IL-1 in infectious diseases.

The interleukin (IL)-23/IL-17 axis is strongly implicated in the pathogenesis of psoriasis. Previous studies showed that IL-18 was elevated in early active and progressive plaque-type psoriatic lesions and that serum or plasma levels of IL-18 correlated with the Psoriasis Area and Severity Index. However, the mechanism whereby IL-18 affects disease severity remains unknown. In this study, we investigated the effects of IL-18 on a psoriasis-like skin inflammation model induced by recombinant mouse IL-23. We found that IL-18, cooperatively with IL-23, induced prominent inflammation and enhanced psoriasis-like epidermal hyperplasia. In the skin of mice treated with IL-23 plus IL-18, the expression of interferon-γ was significantly upregulated and that of chemokine (C-X-C motif) ligand 9 (CXCL9) was synergistically increased. Histologically, strong positive signals of CXCL9 were observed around the infiltrating inflammatory cells. The current results suggest that IL-18 might synergize with IL-23 to induce a T helper 1 immune reaction, without inhibiting the IL-23/IL-17 axis, and thus may aggravate psoriatic inflammation.

Acute liver failure (ALF) is a life-threatening condition, and liver transplantation is the only therapeutic option. Although immune dysregulation is central to its pathogenesis, the precise mechanism remains unclear. Here, we show that the number of peripheral and hepatic plasmacytoid DCs (pDCs) decrease during acute liver injury in both humans and mice. Selective depletion of pDCs in Siglech(dtr/+) mice exacerbated concanavalin A–induced acute liver injury. In contrast, adoptively transferred BM-derived pDCs preferentially accumulated in the inflamed liver and protected against liver injury. This protective effect was independent of TLR7 and TLR9 signaling, since a similar effect occurred following transfer of MyD88-deficient pDCs. Alternatively, we found an unexpected immunosuppressive role of pDCs in an IL-35–dependent manner. Both Il12a and Ebi3, heterodimeric components of IL-35, were highly expressed in transferred pDCs and CD4(+)CD25(+) Tregs. However, the protective effect of pDC transfer was completely lost in mice depleted of Tregs by anti-CD25 antibody. Moreover, pDCs derived from IL-35–deficient mice had less of a protective effect both in vivo and in vitro even in the presence of Tregs. These results highlight a unique aspect of pDCs in association with Tregs, serving as a guide for immunotherapeutic options in ALF.

Epstein-Barr virus–induced gene 3 (EBI3) is a subunit common to IL-27, IL-35, and IL-39. Here, we explore an intracellular role of EBI3 that is independent of its function in cytokines. EBI3-deficient naive CD4(+) T cells had reduced IFN-γ production and failed to induce T cell–dependent colitis in mice. Similarly reduced IFN-γ production was observed in vitro in EBI3-deficient CD4(+) T cells differentiated under pathogenic Th17 polarizing conditions with IL-23. This is because the induction of expression of one of the IL-23 receptor (IL-23R) subunits, IL-23Rα, but not another IL-23R subunit, IL-12Rβ1, was selectively decreased at the protein level, but not the mRNA level. EBI3 augmented IL-23Rα expression via binding to the chaperone molecule calnexin and to IL-23Rα in a peptide-dependent manner, but not a glycan-dependent manner. Indeed, EBI3 failed to augment IL-23Rα expression in the absence of endogenous calnexin. Moreover, EBI3 poorly augmented the expression of G149R, an IL-23Rα variant that protects against the development of human colitis, because binding of EBI3 to the variant was reduced. Taken together with the result that EBI3 expression is inducible in T cells, the present results suggest that EBI3 plays a critical role in augmenting IL-23Rα protein expression via calnexin under inflammatory conditions.

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.

Immunotherapy has gained special attention due to its specific effects on tumor cells and systemic action to block metastasis. We recently demonstrated that ovalbumin (OVA) conjugated to the surface of nanoparticles (NPs) (OVA‑NPs) can manipulate humoral immune responses. In the present study, we aimed to ascertain whether vaccination with OVA-NPs entrapping IL-7 (OVA-NPs-IL-7) are able to induce antitumor immune responses in vivo. Pretreatment with a subcutaneous inoculation of OVA-NPs delayed the growth of thymic lymphoma cells expressing a model tumor antigen OVA (E.G7-OVA), and OVA-NPs-IL-7 substantially blocked the growth of E.G7-OVA tumor cells, although NPs-IL-7 alone had a meager effect, as assessed by the mean tumor size and the percentage of tumor-free mice. However, pretreatment with OVA-NPs-IL-7 failed to reduce the growth of parental thymic tumor cells, suggesting that the antitumor effect was antigen-specific. A tetramer assay revealed that vaccination with OVA-NPs-IL-7 tended to enhance the proportion of cytotoxic T cells (CTLs) specific for OVA. When the tumor-free mice inoculated with OVA-NPs-IL-7 plus EG.7 cells were rechallenged with E.G7-OVA cells, they demonstrated reduced growth compared with that in the control mice. Thus, a single subcutaneous injection of OVA-NPs-IL-7 into mice induced tumor-specific and also memory-like immune responses, resulting in regression of tumor cells. Antigens on NPs entrapping IL-7 would be a promising carrier to develop and enhance immune responses, including humoral and cellular immunity as well as a method of drug delivery to a specific target of interest.

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.

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.