Toshiaki Koda

The critical role of Th1-dominant immunity in tumor immunology.

Abstract To investigate the precise role of antigen-specific Th1 and Th2 cells in tumor immunity, we developed a novel adoptive tumor-immunotherapy model using OVA-specific Th1 and Th2 cells and an OVA gene-transfected tumor. This therapeutic model demonstrated that both antigen-specific Th1 and Th2 cells had strong antitumor activity in vivo with distinct mechanisms. However, immunological memory suitable for the generation of tumor-specific cytotoxic T lymphocytes was induced only when tumor-bearing mice received Th1 cell therapy, but not Th2 cell therapy. Thus it was strongly suggested that Th1-dominant immunity is critically important for the induction of antitumor cellular immunity in vivo. We also proposed that several immunomodulating protocols using interleukin (IL)-12, IL-12 gene, the natural killer T cell ligand α-galactosylceramide, or Th1 cytokine-conditioned dendritic cells might be useful strategies for the induction of Th1-dominant immunity essential for the development of tumor-specific immunotherapy.

Dtrk, a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule.

We report the identification and molecular characterization of Dtrk, a Drosophila gene encoding a receptor tyrosine kinase highly related to the trk family of mammalian neurotrophin receptors. The product of the Dtrk gene, gp160Dtrk, is dynamically expressed during Drosophila embryogenesis in several areas of the developing nervous system, including neurons and fasciculating axons. gp160Dtrk has structural homology with neural cell adhesion molecules of the immunoglobulin superfamily and promotes cell adhesion in a homophilic, Ca2+ independent manner. More importantly, this adhesion process specifically activates its tyrosine protein kinase activity. These findings suggest that gp160Dtrk represents a new class of neural cell adhesion molecules that may regulate neuronal recognition and axonal guidance during the development of the Drosophila nervous system.

Identification of rabaptin-5, rabex-5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER

The role of rab33b, a Golgi‐specific rab protein, was investigated. Microinjection of rab33b mutants stabilised in the GTP‐specific state resulted in a marked inhibition of anterograde transport within the Golgi and in the recycling of glycosyltransferases from the Golgi to the ER, respectively. A GST‐rab33b fusion protein stabilised in its GTP form was found to interact by Western blotting or mass spectroscopy with Golgi protein GM130 and rabaptin‐5 and rabex‐5, two rab effector molecules thought to function exclusively in the endocytic pathway. A similar binding was seen to rab1 but not to rab6, both Golgi rabs. In contrast, rab5 was as expected, shown to bind rabaptin‐5 and rabex‐5 as well as the endosomal effector protein EEA1 but not GM130. No binding of EEA1 was seen to any of the Golgi rabs.

A Critical Role for Mouse CXC Chemokine(s) in Pulmonary Neutrophilia During Th Type 1-Dependent Airway Inflammation

Ag-specific Th1 and Th2 cells have been demonstrated to play a critical role in the induction of allergic diseases. Here we have investigated the precise mechanisms of Th1-induced airway inflammation. Airway inflammation was induced in BALB/c mice by transfer of freshly induced OVA-specific Th1 or Th2 cells followed by OVA inhalation. In this model, both Th1 and Th2 cells induced airway inflammation. The former induced neutrophilia in airways, whereas the latter induced eosinophilia. Moreover, we found that Th1 cells induced more severe airway hyperresponsiveness (AHR) than Th2 cells. The eosinophilia induced by Th2 cell infusion was almost completely blocked by administration of anti-IL-5 mAb, but not anti-IL-4 mAb. In contrast, Th1-induced AHR and pulmonary neutrophilia were inhibited by the administration of anti-human IL-8R Ab, which blocks the function of mouse CXC chemokine(s). These findings reveal a critical role of mouse CXC chemokine(s) in Th1-dependent pulmonary neutrophilia and AHR.

Critical role of the Th1/Tc1 circuit for the generation of tumor-specific CTL during tumor eradication in vivo by Th1-cell therapy

Th1 and Th2 cells obtained from OVA‐specific T cell receptor transgenic mice completely eradicated the tumor mass when transferred into mice bearing A20‐OVA tumor cells expressing OVA as a model tumor antigen. To elucidate the role of Tc1 or Tc2 cells during tumor eradication by Th1‐ or Th2‐cell therapy, spleen cells obtained from mice cured of tumor by the therapy were restimulated with the model tumor antigen (OVA) for 4 days. Spleen cells obtained from mice cured by Th1‐cell therapy produced high levels of IFN‐γ, while spleen cells from mice cured by Th2‐cell therapy produced high levels of IL‐4. Intracellular staining analysis demonstrated that a high frequency of IFN‐γ‐producing Tc1 cells was induced in mice given Th1‐cell therapy. In contrast, IL‐4‐producing Tc2 cells were mainly induced in mice after Th2‐cell therapy. Moreover, Tc1, but not Tc2, exhibited a tumor‐specific cytotoxicity against A20‐OVA but not against CMS‐7 fibrosarcoma. Thus, immunological memory essential for CTL generation was induced by the Th1/Tc1 circuit, but not by the Th2/Tc2 circuit. We also demonstrated that Th1‐cell therapy is greatly augmented by combination therapy with cyclophosphamide treatment. This finding indicated that adoptive chemoimmuno‐therapy using Th1 cells should be applicable as a novel tool to enhance the Th1/Tc1 circuit, which is beneficial for inducing tumor eradication in vivo.

Indispensable Role for TNF-α and IFN-γ at the Effector Phase of Liver Injury Mediated by Th1 Cells Specific to Hepatitis B Virus Surface Antigen

We report the development and characterization of a novel model of severe hepatitis induced against hepatitis B virus surface Ag (HBsAg). HBsAg was successfully targeted into the liver in soluble form. Using this unique property of HBsAg, we established a liver injury model induced by HBsAg-specific Th1 cells. Severe liver injury was induced in C57BL/6 mice by injection of HBsAg together with HBsAg-specific Th1 cells. Histochemical examination demonstrated extensive necroinflammatory hepatic lesions in these animals. Application of this liver injury model to mutant or gene knockout mice enabled us to define the effector mechanisms of Th1 cells in fulminant hepatitis. When Fas-deficient lpr mice were used as recipients, a similar degree of liver injury was induced as in wild-type mice. Moreover, HBsAg-specific Th1 cells obtained from perforin−/− mice could induce severe liver injury in both wild-type and lpr mice. These results indicated that neither Fas ligand nor perforin are essential for Th1-mediated liver injury in this model. Pretreatment with anti-TNF-α mAb prevented liver injury, whereas severe liver injury was induced in TNF-α−/− mice. Moreover, IFN-γ receptor-deficient mice were resistant to Th1-mediated liver injury. Therefore, TNF-α and IFN-γ, which were produced by HBsAg-specific Th1 cells during the effector phase, appeared to be indispensable in the pathogenesis of fulminant hepatitis.

Potentiation of antitumor effect of NKT cell ligand, alpha-galactosylceramide by combination with IL-12 on lung metastasis of malignant melanoma cells.

The combined therapeutic effect of natural killer T (NKT) cell ligand α-galactosylceramide (α-GalCer) and IL-12 against highly metastatic B16-BL6-HM melanoma cells was investigated. In comparison with a single administration of α-GalCer or IL-12, the combined treatment of tumor-bearing mice with α-GalCer plus IL-12 caused a super-induction of serum IFN-γ levels, though α-GalCer-induced IL-4 production was rather inhibited. In parallel with the augmented IFN-γ production, the natural killing activity against YAC-1 cells and syngeneic B16- BL6-HM melanoma was greatly augmented by the combined therapy. The major effector cells responsible for natural killing activity induced by α-GalCer plus IL- 12 were enriched in both NK1.1+TCRαβ+ NKT cells and NK1.1+TCRαβ− NK cells. The preventing effect of α-GalCer or IL-12 alone against lung metastasis of B16-BL6-HM was also enhanced by the combination therapy. The antitumor activity of α-GalCer was totally abolished in NKT-deficient mice. However, IL- 12-induced antitumor activity was not eliminated in NKT-deficient mice though it was inhibited by anti-asialo GM1 Ab treatment. These findings suggested that α-GalCer synergistically act with IL-12 to activate both NKT cells and NK cells, which may play a critical role in the strong prevention of distant tumor metastasis at early stages of tumor-bearing. These data will provide a novel tool for the prevention of tumor metastasis using NKT-specific ligands α-GalCer and IL-12.

17β-Estradiol increases the receptor number and modulates the action of 1,25-dihydroxyvitamin D3 in human osteosarcoma-derived osteoblast-like cells

It is well known that 17β-estradiol (E2) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) have important roles in bone metabolism. This study was undertaken to examine E2 regulation of 1,25(OH)2D3 receptor (VDR) expression and the biological action of 1,25(OH)2D3 in human osteoblast-like cells. When human osteosarcoma-derived osteoblast-like cells were treated with varying concentrations of E2, the VDR levels increased by up to 100% in a dose-dependent manner. VDR levels significantly increased at 10 nM E2 and this increase plateaued at 100 nM E2. E2-dependent increase of VDR was time dependent, plateauing, at 24 hours and was maintained for at least 48 hours in human osteoblast-like cells. Scatchard analysis showed that E2 increased the number of VDR (12.3±0.4 versus 26.5±0.3 fmol/mg protein; mean ±SE of three independent experiments) rather than the Kd (0.15±0.02 versus 0.16±0.01 nM; mean±SE of three independent experiments). Tamoxifen (50 nM), a specific competitor with E2, completely abolished the E2-induced increase of VDR. The levels of VDR mRNA (4.5 kb) from the cells increased in a dose-dependent manner after E2 treatment. With regard to the biological effects, E2 increased by 10–25% the inhibitory effect of 1,25(OH)2D3 on cell growth. However, E2 did not increase the stimulation of alkaline phosphatase activity by 1,25(OH)2D3. The present study suggests that E2 modulates the biological action of 1,25(OH)2D3 through VDR levels in bone cells.

Functional expression of the TrkC gene, encoding a high affinity receptor for NT-3, in antigen-specific T helper type 2 (Th2) cells

Neurotrophins, including nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 (NT-3) are essential factors for the development of the nervous system. In this report, we demonstrate gene expression of neurotrophins and their receptors in T helper 1 (Th1) and T helper 2 (Th2) cells induced from naïve CD4+ CD45RB+ T cells of ovalbumin-specific DO11.10 T cell receptor transgenic mice. Interestingly, the TrkC gene, which encodes a high affinity receptor for NT-3, was expressed in Th2 cells, but not in Th1 and naïve CD4+ T cells. Expression of the TrkC gene was markedly augmented by addition of anti-IFN-gamma monoclonal antibody (mAb) into the culture, whereas it was blocked by anti-IL-4 mAb. Moreover, NT-3 synergistically enhanced anti-CD3 mAb-induced IL-4 production by Th2 cells, but did not affect IFN-gamma production by Th1 cells. These data suggest that NT-3, through its receptor TrkC, plays a critical role in regulating the Th1/Th2 balance.

Absence of BRINP1 in mice causes increase of hippocampal neurogenesis and behavioral alterations relevant to human psychiatric disorders

BackgroundWe have previously identified BRINP (BMP/RA-inducible neural-specific protein-1, 2, 3) family genes that possess the ability to suppress cell cycle progression in neural stem cells. Of the three family members, BRINP1 is the most highly expressed in various brain regions, including the hippocampus, in adult mice and its expression in dentate gyrus (DG) is markedly induced by neural activity. In the present study, we generated BRINP1-deficient (KO) mice to clarify the physiological functions of BRINP1 in the nervous system.ResultsNeurogenesis in the subgranular zone of dentate gyrus was increased in BRINP1-KO mice creating a more immature neuronal population in granule cell layer. The number of parvalbumin expressing interneuron in hippocampal CA1 subregion was also increased in BRINP1-KO mice. Furthermore, BRINP1-KO mice showed abnormal behaviors with increase in locomotor activity, reduced anxiety-like behavior, poor social interaction, and slight impairment of working memory, all of which resemble symptoms of human psychiatric disorders such as schizophrenia and attention–deficit/hyperactivity disorder (ADHD).ConclusionsAbsence of BRINP1 causes deregulation of neurogenesis and impairments of neuronal differentiation in adult hippocampal circuitry. Abnormal behaviors comparable to those of human psychiatric disorders such as hyperactivity and poor social behavior were observed in BRINP1-KO mice. These abnormal behaviors could be caused by alteration of hippocampal circuitry as a consequence of the lack of BRINP1.