| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
To clarify the relationship between functions of lymphocytes and FOXP3 gene expression, which is a master gene for a differentiation of regulatory T cells, these studies were performed.
1) Recombinant virus-producing cells were established by transfection of retrovirus vector including wild-type or mutant human FOXP3 gene into packaging cells. These FOXP3 genes were introduced into T cell line established from a FOXP3-deficient patient. No obvious differences between wild-type and mutant human FOXP3 gene-introduced T cells were detected on T cell-related surface antigens including ativation markers. Cytokine productions after activation also showed no defferences between wild-type and mutant human FOXP3 gene-introduced T cells.
2) FOXP3 gene expressions and FOXP3 products on peripheral blood lymphocytes after activation were investigated, using RT-PCR method and Western blot. No obvious changes of FOXP3 on peripheral blood lymphocytes were detected during activation.
3) To develop a detection system of intra-cytoplasmic FOXP3, polyclonal antibodies against human FOXP3 were produced by immunization of rabbit with synthetic peptides predicted from DNA sequences. Cells were stained with anti-FOXP3 antibody after permealization and fixation of cell membranes, then analyzed on flow cytometory. Intra-cytoplasmic FOXP3 in FOXP3-expressing T cell line was detected by flow cytometory. Intra-cytoplasmic FOXP3 in normal peripheral lymphocytes, however, was hard to detect by flow cytometory. Then, conditions to detect intra-cytoplasmic FOXP3 were further examined and determined.
4) Percentages of FOXP3 (+) CD4 (+)-T cells in peripheral blood cells were compared between various autoimmune diseases, using above-mentioned flow cytometory method. Although small differences between the diseases were shown, no significant differences were detected.
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