Minoru Nakui

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.

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.

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.

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.

Technique of the double-channel ESD method performed with an EEMR tube

Endoscopic submucosal dissection (ESD) recently has been aggressively performed to treat superficial esophageal cancer; however, it is difficult to secure a good field of view for mucosal dissection, and the technique requires considerable skill. We have developed a double-channel ESD method using an endoscopic esophageal mucosal resection (EEMR) tube that makes it possible to perform the submucosal dissection with a good field of view while applying countertraction. Countertraction is achieved by maneuvering a fine grasping forceps inserted through the side channel of the EEMR tube, and the field of view of the submucosal dissection layer can be easily exposed. This technique can be performed while constantly observing the submucosal dissection layer with a stable field of view, and it is also easy to handle the blood vessels. Moreover, there is no disturbance of the field of view by the dissected mucosa.