New anti-tumor immunotherapy with intratumoral injection of IL-2+IL-12 gene-modified dendritic cells.

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1. We investigated the proportion of T helper type 1 cells (Th1) producing IL-2 and IL-12, which usually activates dendritic cells (DCs), in tumor infiltrating lymphocytes of lung cancer patients.1) The ratio of Th1 to Th2 in tumor infiltrating lymphocytes of lung cancer patients were analyzed with flow cytometry.2) The Th1-to-Th2 ratio was significantly elevated in the tumor tissues compared with peripheral blood. The cases of small size tumor (diameter was less than 3cm) and squamous cell carcinoma were suggested to have the advantage of DCs activation because of their higher proportion of Th1.2. We developed the tumor specific cytotoxic T lymphocytes (CTL) with using the antigen-presenting abilities of DCs.1) Induction of DCsMononuclear cells were separated from HLA-A2 positive health donors matching with human lung adenocarcinoma ell lines (PC16) because of the production of specific immuno-reaction. DCs were induced by incubating for five days with GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) and following for two days with tumor-lysates and TNF-α (1000U/ml).2) Development to CTL against PC14Autologous lymphocytes were stimulated with DCs for a couple of days in medium containing IL-2 and solid-phase anti-CD3 stimulation. After three week stimulation lymphocytes increased their number about a hundred times and developed to CTL.3) Anti-tumor cytotoxic activity of CTLwe evaluated the cytotoxic activity of CTL in mixed tumor and CTL culture using IFN-γ secration assay by ELISA.CTL induced with the stimulation with DCs showed the highest γ-IFN concentration and significantly blocked by anti-HLA-A2 antibody.In conclusion, because there are a lot of Th1 activating DCs by favorable cytokine secretion in lung cancer tissues, the possibility of CTL therapy using autologous DCs in lung cancer patients were suggested.