Yasushi Ohmi

The restraint stress drives a shift in Th1/Th2 balance toward Th2-dominant immunity in mice

When mice were physically restrained in 50-ml tubes for 24 h, a marked decrease of NK activity was demonstrated in parallel with the elevation of serum corticosterone levels. The release of mice from restraint stress resulted in the recovery of NK activity, with a decrease of serum corticosterone levels within 48 h. Using this stress model, we also investigated the influence of restraint stress on mouse Th1/Th2 balance. Consistent with the decrease of NK activity, IFN-gamma production of mouse spleen cells greatly reduced after suffering from restraint stress. In contrast, the IL-4 producing ability of spleen cells was not so much affected by restraint stress. These results initially indicated that stress may induce the skewing of the Th1/Th2 balance toward Th2-dominant immunity, which stimulates the occurrence of infectious diseases and allergic disorders.

Manipulation of Th1/Th2 balance in vivo by adoptive transfer of antigen-specific Th1 or Th2 cells

We have investigated the possibility that the Th1/Th2 balance in vivo may be modulated by adoptive transfer of Th1 or Th2 cells induced in vitro. Thl cells were induced from I-Ad-binding OVA323-339-specific T-cell receptor-transgenic (TCR-Tg) mouse spleen cells by culturing with OVA323-339 peptide and antigen presenting cells (APC) in the presence of IL-2, IL-12 and anti-IL-4 mAb. Th2 cells were induced from TCR-Tg mouse spleen cells by culturing with IL-2, IL-4 and anti-IL-12 mAb in addition to OVA323-339 plus APC. Immunomodulating activities of both Th1 and Th2 cells were determined by their effect on delayed type hypersensitivity (DTH) responses or cytokine production. No significant DTH responses (footpad swelling) were observed in untreated BALB/c mice following a single injection of OVA323-339-pulsed syngeneic spleen cells. However, adoptive transfer of Th1 cells into BALB/c mice induced strong dose dependent DTH responses in response to I-Ad-bound OVA323-339 but not unrelated peptide. In contrast, only slight DTH responses were detected in BALB/c mice transferred with Th2 cells. In parallel with the DTH responses, increased levels of serum IFN-gamma were demonstrated in mice adoptively transferred with Th1, while no significant increase was observed in Th2-transferred mice. In vitro analysis also demonstrated that both spleen cells and popliteal lymph node cells prepared from Th1-transferred mice showed Th1-type cytokine production, while cells obtained from Th2-transferred mice revealed Th2-dominant cytokine production. Such immune deviation induced by antigen-specific Th1 cells was demonstrated up to three months after cell transfer. Therefore, it may be possible to manipulate the Th1/Th2 balance in vivo by adoptive transfer of antigen-specific Th1 or Th2 cells.

FUNCTIONAL CHARACTERIZATION OF NK1.1 + LY-6C+ CELLS

It was found that NK1.1+ cells were subdivided by their different expression pattern of Ly-6C antigen. To characterize their functional significance in immunoregulation, we separated NK1.1 + Ly6C+ cells and NK1.1 + Ly-6C- cells from C57BL/6 mouse nylon-passed spleen cells by FACStar. Both NK1.1 + Ly-6C+ and NK1.1 + Ly-6C- cells responded to the stimulation with IL-2 plus IL-12 and showed strong cytotoxicity against YAC-1 cells. However, these cells revealed different ability in terms of IFN-gamma production. Only NK1.1 + Ly-6C+ cells, but not NK1.1 + Ly-6C- cells, cultured with IL-12 alone or IL-2 plus IL-12, produced high levels of IFN-gamma. Flow cytometric analysis demonstrated that NK1.1 + Ly-6C+ cells consisted of NK1.1 + CD3-Ly-6C+ NK cells and NK1.1 + CD3 + Ly-6C+ NKT cells. Therefore, we further separated these two populations from NK1.1 + Ly-6C+ cells to define their functions. Although, both NK1.1 + CD3-Ly-6C+ NK cells and NK1.1 + CD3+ NKT cells showed the same level of cytotoxicity. It was clearly demonstrated that NK1.1 + CD3+Ly-6C+ NKT cells were major immunoregulatory cells to produce IFN-gamma in respond to IL-12 alone or IL-2 plus 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.

Essential Role of the Adhesion Receptor LFA-1 for T Cell-Dependent Fulminant Hepatitis

Viral hepatitis affects more than 2 billion people worldwide. In particular, no effective treatment exists to abrogate death and liver damage in fulminant hepatitis. Activation of T cells is an initial and critical event in the pathogenesis of liver damage in autoimmune and viral hepatitis. The precise molecular mechanisms that induce T cell-mediated hepatocyte injury remain largely unclear. In mice, T cell-dependent hepatitis and acute liver damage can be modeled using ConA. In this study, we examined the role of the adhesion receptor LFA-1 in ConA-induced acute hepatic damage using LFA-1−/− (CD11a) mice. Massive liver cell apoptosis and metabolic liver damage were observed in LFA-1+/+ mice following ConA injection. By contrast, LFA-1−/− mice were completely resistant to ConA-induced hepatitis and none of the LFA-1−/− mice showed any hepatic damage. Whereas activated hepatic T cells remained in the liver in LFA-1+/+ mice, activated T cells were rapidly cleared from the livers of LFA-1−/− mice. Mechanistically, T cells from LFA-1−/− mice showed markedly reduced cytotoxicity toward liver cells as a result of impaired, activation-dependent adhesion. Importantly, adoptive transfer of hepatic T cells from LFA-1+/+ mice, but not from LFA-1−/− mice, sensitized LFA-1−/− mice to ConA-induced hepatitis. Thus, LFA-1 expression on T cells is necessary and sufficient for T cell-mediated liver damage in vivo. These results provide the first genetic evidence on an adhesion receptor, LFA-1, that has a crucial role in fulminant hepatitis. These genetic data identify LFA-1 as a potential key target for the treatment of T cell-mediated hepatitis and the prevention of liver damage.

Angiostatin gene therapy inhibits the growth of murine squamous cell carcinoma in vivo

Tumor growth is an angiogenesis-dependent process and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. Angiostatin has been shown to potently inhibit endothelial proliferation in vitro and tumor growth in vivo. We now show that a shift in the balance of tumor angiogenesis by gene transfer of a cDNA coding for mouse angiostatin into mouse squamous cell carcinoma NRS-1 and SCC-VII cells suppresses tumor growth in vivo. The inhibition of an angiostatin-transfected tumor was accompanied by a marked reduction in vascularity and the presence of many apoptotic tumor cells. However, transfected-angiostatin cDNA does not affect the expression of the vascular endothelial growth factor (VEGF) and VEGF-R2 in the vascular endothelium. The inhibition mechanisms of neovascularization may be mediated independent of VEGF:VEGF-R2 complex. Our data may provide a useful approach for human oral cancer therapy by gene therapy with angiostatin.

Reconstitution of immune systems in RAG2−/− mice by transfer with interleukin-12-induced splenic hematopoietic progenitor cells

The administration of a high dose of IL-12 into the mice resulted in the induction of splenomegaly. From the flow cytometry analysis of cellularity in an enlarged spleen, it was demonstrated that Thyl.2-CD45RB-c-Kit + Sca-1 + Lin- hematopoietic progenitor cells markedly increased in IL-12-administered mouse spleen compared with untreated mouse spleen. The IL-12-induced hematopoietic progenitor cells showed a greatly enhanced colony-forming activity in CFU-granulocyte/macrophage (CFU-GM), blast-forming units-erythroid (BFU-E) and CFU-spleen (CFU-S) assay. Moreover, it was initially demonstrated that the transfer of IL-12-induced splenic hematopoietic progenitor cells into immunodeficient RAG2-/- mice caused a complete reconstitution of their immune functions including T- and B-cell-mediated immunity. Thus, the evidence that IL-12 has a capability of inducing hematopoietic progenitor cells possessing stem cell-like activity in vivo, indicated another important immunomodulating activity of IL-12 in immunotherapy.

Accumulation of IL-12-activated antitumor effector cells into lymph nodes of tumor-bearing mice.

Simultaneous administration of high dose of IL-12 into tumor-inoculated mice resulted in a marked reduction of tumor growth in parallel with the augmented generation of cytotoxic T-cells, natural killer (NK) cells and IFN-gamma-producing Th cells. We found that these IL-12-activated antitumor effector cells preferentially accumulated in peripheral lymph nodes concomitantly with lymphadenopathy. However, IL-12 rather induced disappearance of antitumor effector cells including CD4+ T, CD8+ T and NK cells from spleen in spite of inducing splenomegaly. Lymph node cells obtained from IL-12-treated B16F0-bearing mice showed a marked IFN-gamma production in response to not only IL-2, IL-12, anti CD3 mAb but also B16F0 melanoma cells. Moreover, they could lyse B16F0 melanoma cells in a long-term cytotoxicity assay. It was also confirmed that IL-12-activated IFN-gamma producing Th1 cells were accumulated in tumor local site. Thus, IL-12 appeared to have a capability of stimulating selective migration of antitumor cells into lymph nodes and tumor local sites.

Growth of human squamous cell carcinoma xenografts in mice is inhibited by local angiostatin gene therapy

The possibility of inhibiting tumor growth by blocking the formation of new tumor vessels has recently received attention. Antiangiogenic tumor therapies have recently attracted intense interest because of their direct endothelial targeting and the absence of drug resistance. Local antiangiogenic gene therapy for cancer offers a potential way to achieve sustained therapeutic release of antiangiogenic substances. As a step toward this goal, we used liposomes complexed to angiostatin cDNA and targeted to human squamous cell carcinoma cell lines in vivo. Tumor cells expressing angiostatin after local gene transfer showed markedly reduced vascularity and contained many apoptotic tumor cells. These results demonstrate the potential utility of liposome-derived angiostatin for adjuvant therapy of oral cancer in humans.

Tumor-specific targeting of T helper type 1 (Th1) cells by anti-CD3 × anti-c-ErbB-2 bispecific antibody

Abstract T helper type1 (Th1) or type2 (Th2) cells were induced from naive Th cells obtained from ovalbumin-specific T cell receptor (TCR) transgenic mice. Th1 cells producing interferon γ (IFNγ) exhibited stronger antigen-specific cytotoxicity against ovalbumin-(323–339)-peptide-pulsed A20 tumor cells than did Th2 cells. To develop a general method for applying antigen-nonspecific Th1 cells to tumor immunotherapy, we examined the targeting of Th1 cells to tumor cells using a bispecific antibody (bsAb) consisting of anti-(mouse CD3) mAb and anti-(human c-ErbB-2) mAb. When ovalbumin-specific Th1 or Th2 cells were cocultured with c-erbB-2-positive transfectants (CMS7HE), neither type of cell showed significant cytotoxicity or cytokine production in response to tumor cells. However, addition of bsAb resulted in the triggering of both Th1 and Th2 cells. Th1 cells showed higher levels of bsAb-dependent cytotoxicity against CMS7HE tumor cells than did Th2 cells. The targeting of Th1 cells to CMS7HE tumor cells by bsAb also triggered the production of cytokines such as IFNγ, interleukin-2 and tumor necrosis factor α (TNFα). The released TNFα was demonstrated to be a critical cytolytic factor in bsAb-mediated cytotoxicity by Th1 cells. Finally, Th1 cells were demonstrated to show antitumor activity in vivo against human c-erbB-2-positive tumor cells implanted in nude mice. These results suggest that Th1 cells are useful effector cells for the application to adoptive tumor immunotherapy in conjunction with bsAb.