Functional analysis of FOXP3 gene in the regulation of lymphocyte activation

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16591003
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Pediatrics
• Research Institution: HOKKAIDO UNIVERSITY
• Principal Investigator: KAWAMURA Nobuaki Hokkaido University, Dept of Pediatrics, Associate Professor, 大学院医学研究科, 助教授 (90301879)
• Project Period (FY): 2004 – 2005
• Keywords: FOXP3 / regulatory T cell / autoimmune disease

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.