| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
As far as peripheral T cells of unknown specificity are concerned, clones per se could hardly be obtained to render it difficult to propagate T cells, to a degree sufficient for testing. In order to overcome the difficulty, an attempt has been made to stimulate T cells with an immobilized anti-CD3 antibody in the presence of IL-2, but the long-term maintenance of T Cells could not readily achieve. This is supposedly due to lack of physiological response of APC via APC-peptide-T cell interaction. Thus, there is a need for developing a process for proliferating peripheral T cells of unknown specificity for cloning, as well as for effective analysis of epitopes recognized by said T cells
In the previous study, it was observed that some peptide partial agonists support T cell survival (Matsushita, S. et al. , J. Immunol. 1997, 158 : 5685-5691). It was also observed that an agonistic anti-CD29 antibody MAR4 increased the efficiency in establishing T cell clones from PBMC, both suppressing antigen-driven / activation-induced cell death and by enhancing the T cell proliferation, only in the presence of TCR/CD3-mediated stimulation (Tanaka, Y. et al. , Hum. Immunol. 1998, 59 : 343-351). Moreover, it was observed that culture supernatant of antigen-stimulated T cells in the presence of monocytes, increased the efficiency of cloning, when added to culture wells for limiting dilution
Under the circumstances, we constructed combinational randomized peptide library and stimulated peripheral blood- or tissue-derived T cells with this library in the presence of interleukins and agonistic anti-CD29, so that the T cell are proliferated and cloned. The isolated T cell clone is then analyzed for its epitope recognition to identify epitopes recognized by said clone by combinatorial assay with peptide library. Based on the identified peptide sequence, natural peptide ligands recognized by the isolated T cell clone can then be identified by pattern match search with database of sequence
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