Marimo Sato

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.

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.

Functional skewing of bone marrow-derived dendritic cells by Th1- or Th2-inducing cytokines.

To investigate the functional difference of bone marrow (BM)-derived dendritic cells (DC), BM cells were cultured under three different cytokine conditions and the induced DC were temporarily designated as DC0, DC1, or DC2 cells. DC0 were induced by culture of BM cells with granulocyte macrophage colony-stimulating factor (GMCSF) plus interleukin 3 (IL-3). DC1 were induced by culture with Th1-inducing cytokine (IL-12, gamma-interferon-IFN-gamma) in addition to GMCSF plus IL-3. DC2 were induced by culture with Th2-inducing cytokine (IL-4) in addition to GMCSF plus IL-3. Flow cytometric analysis demonstrated that almost all DC0, DC1 and DC2 cells were stained with anti-CD11c, which reacts with a marker for DC cells. However, interestingly, DC0, DC1 and DC2 cells expressed different amounts of functional molecules on their cell surface. Namely, DC1 cells expressed the highest levels of MHC class I, class II, CD40, B7.1 and B7.2 compared with DC0 and DC2 cells. In terms of IL-12 production, DC1 cells showed enhanced production, while DC2 cells showed reduced production compared with DC0 cells. Moreover, it was shown that both DC0 and DC1 supported the differentiation of IFN-gamma-producing Th1 cells, but not IL-4-producing Th2 cells from TCR-transgenic mouse naive Th cells. However, DC2 cells selectively enhanced the differentiation of IL-4-producing Th2 cells. These data strongly suggested that DC1 cells might be preferable antigen-presenting cells for application to immunotherapy.

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.

The essential role of phorbol ester-sensitive protein kinase C isoforms in activation-induced cell death of Th1 cells

Th1 and Th2 cells, which were induced from naive T cells of TCR‐transgenic mice, showed differential sensitivity to activation‐induced cell death (AICD) triggered by stimulation with anti‐CD3 monoclonal antibody. The Th1 cells showed more rapid AICD than Th2 cells. This accelerated AICD of Th1 cells was strongly blocked by protein kinase C (PKC) inhibitors (H‐7 or GF 109203X). Moreover, long‐term treatment of Th1 cells with phorbol 12‐myristate 13‐acetate (PMA) caused the abrogation of anti‐CD3‐induced AICD in parallel with the disappearance of PMA‐sensitive PKC isoforms such as PKC α, γ, ϵ and θ. Therefore, it was clearly demonstrated that PMA‐sensitive PKC isoforms are essential for AICD of Th1 cells. The different susceptibility to AICD between Th1 and Th2 cells was not due to their differential expression levels of PMA‐sensitive PKC isoforms but appeared to be due to their differential requirement for PMA‐sensitive isoforms in the up‐regulation of Fas ligand which is involved in suicide killing of activated Th1 cells.

Natural Killer T Cell Ligand α-Galactosylceramide Inhibited Lymph Node Metastasis of Highly Metastatic melanoma Cells

The role of natural killer T (NKT) cells in the prevention of multiple tumor metastasis was examined. The i.v. inoculation of a highly metastatic subline of B16‐BL6 (B16‐BL6‐HM) melanoma cells resulted in the formation of metastatic nodules in lymph nodes in addition to lung, intrapleural cavity, and ovary. However, treatment of the mice with the NKT cell ligand α‐galactosylceramide (α‐GalCer) three times from 1 day after B16‐BL6‐HM melanoma inoculation caused a significant inhibition of multiple metastasis. Lymph node metastasis of B16‐BL6‐HM was almost completely blocked by α‐GalCer treatment. This antimetastatic effect of α‐GalCer was abolished in NKT celldeficient mice. These results suggest that α‐GalCer‐activated NKT cells played a critical role in the prevention of lymph node metastasis of melanoma cells.

Th1 Cytokine-Conditioned Bone Marrow-Derived Dendritic Cells Can Bypass the Requirement for Th Functions During the Generation of CD8+ CTL

Bone marrow-derived dendritic cell (BMDC) subsets have distinct immunoregulatory functions. Th1 cytokine-induced BMDC (BMDC1), compared with Th2 cytokine-induced BMDC2, have superior activities for the differentiation and expansion of CTL. To evaluate the cellular interactions between dendritic cells and CD8+ T cells for the induction of CTL, BALB/c-derived BMDC subsets were cocultured with purified CD8+ T cells from C57BL/6 mice. Our results demonstrate that BMDC1 support the generation of allogeneic CD8+ CTL in the absence of CD4+ Th cells. In contrast, BMDC0 (GM-CSF- plus IL-3-induced BMDC) and BMDC2 failed to promote the differentiation of CD8+ CTL. Using Ab-blocking experiments and studies with gene knockout mice, IL-2 and LFA-1 are demonstrated to be critical for BMDC1-induced CTL differentiation. Unexpectedly, BMDC1 were able to induce CTL from CD8+ T cells isolated from IFN-γ−/− and IFN-γ receptor−/− mice. However, BMDC1 produced higher levels of IFN-β than other BMDC subsets, and anti-IFN-β mAb blocked BMDC1-dependent CTL generation. These results indicated an indispensable role of IFN-β, but not IFN-γ, during BMDC1-induced CTL differentiation. We conclude that Th1-cytokine-conditioned BMDC1 can bypass Th cell function for the differentiation of naive CD8+ T cells into CTL.

Interleukin-4-dependent induction of preproenkephalin in antigen-specific T helper-type 2 (Th2) cells

Naive Th cells obtained from OVA(323-339)-specific DO11.10 TCR-Tg mice did not express preproenkephalin (PPE) mRNA. However, culture of naive Th cells with OVA(323-339) peptide (OVA-pep) plus IL-2 under Th2-inducing conditions for 7 days resulted in an induction of PPE mRNA. The PPE mRNA was also induced by culturing with OVA-pep plus IL-2 (neutral condition). However, PPE mRNA induction under neutral conditions was totally abrogated by addition of anti-IL-4 mAb. The existence of methionine-enkephalin was also demonstrated in peptidase-digested peptides derived from Th2 cell lysate. These results demonstrate that IL-4 is a critical factor for the induction of PPE mRNA in freshly expanded antigen-specific Th2 cells.

Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4+ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0 x 10(7) cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-gamma, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4+ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c x C57BL/6 F1 mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-gamma than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-gamma-producing ability but rather derived from their poor responsiveness to IFN-gamma.