| Co-Investigator(Kenkyū-buntansha) |
TAKAMI Tsuyoshi Gifu University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (70136943)
OHE Naoyuki Gifu University, Graduate School of Medicine, Research Associate, 大学院・医学系研究科, 助手 (60362159)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
We cloned cDNA of murine Interleukin (IL)-2 and 15, and then the cDNAs were re-cinstituted into pIVEX vector for cell free E. coli extract based protein expression system. The cloned vectors were mixed with cell free protein expression mixture for 24 hours with 900 rpm at 30C. Unfortunately, the yield of the protein in supernatant was very low due to precipitation of the protein. Therefore, we utilized the other cell free protein expression system ; yeast extract based system. The products from yeast extract based IL-2 and IL-15 were soluble, and we could purify the protein by Histidine 6 mer with Ni column. The purified proteins were able to stimulate splenocytes. However the yield of each protein was approximately 100ng per synthesis. Therefore, we could not utilized them in vivo analysis. In turn, we prepared IL-2, soluble form tumor necrosis factor receptor II (sTNFRII) transduced MCA 38 murine colon carcinoma sub-lines and GL261 murine glioma sub-lines ; MCA/mock,MCA/sTNFRII,MCA/I
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L-2,MCA/double,GL261/mock,GL261/sTNFRII,GL261/IL-2,GL261/double. We intra-cranially, and subcutaneously inoculated each sub-lines and analyzed tumor infiltrating CD8^+T-cells (CD8^+TIL) and tumor associated macrophages (TAM). In MCA38, MCA/double showed the longest survival. While, GL261/IL-2 showed the longest survival in vivo in GL261 groups. Although there was not significant characteristic change of CD8^+TIL among groups, almost 90 % of TAM recovered from MCA/double got apoptotic in in vitro culture. By in vivo CD4 or CD8 depletion, tumor growth suppression observed in MCA/double was strongly interfered. These data suggested that acquired immunity could be strongly induced by the combination of IL-2 and blocking of TNF by sTNFRII rather than IL-2 alone. Our data also suggested that balance of acquired immunity and innate immunity for exclusion of tumor cells would demand on the character of tumor cells.
In the future, we would like to explore the mechanism how to mature macrophages and dendritic cells in tumor environment by utilizing the present results and methods. Less
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