| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
In the present project, the following studies were performed. (1) High resolution X-ray crystallographic analysis of γδ TCR (2) Design of γδ TCR tetramers based on the X-ray crystallographic analysis. (3) Preparation of γδ TCR tetramers. (4) Identification of tumor antigens by using γδ TCR tetramers. Regarding (1), the truncated γ-chain at 205, and δ-chain at 232 gave diffraction patterns at high resolution on preliminary X-ray analysis. By employing PEG 4000-based buffer, single crystals were obtained reproducibly, whose space group was ideal for synchrocydotron analysis. On analysis at SPring 8, diffraction patterns were obtained at the resolution limit of 1.9A, which was enough for high resolution crystallographic analysis. After molecular modeling and refinement, no contradiction was obtained for crystal structure at the resolution of up to 2.4A. In (2), a biotinylation sequence was introduced immediate downstream of δ205, which gave the most efficient refolding rate. As for (3), inclusion bodies of the original γ-chain and the biotinylated δ-chain were mixed and refolded, to which biotin was attached. Finally the biotinlated TCR was tetramerized using PE-conjugated strep〓, (4) When analyzing a wide spectrum of tumor cells by using the newly prepared tetramers, RPMI8226 was found to express ligands for the tetramers. Based on these findings, γδ T cells apparently recognize certain antigens expressed on tumor cells. In addition, IRP60 was identified as one of the molecules, which may be involved in the γδ T cell recognition of nontpeptide antigens. On analysis of various kinds of tumor cell lines, the tetramer of this molecule was found to recognize U937. In conclusion, we could take a firm stand to elucidate the mechanism for γδ T cell recognition of nonpeptide antigens.
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