Analysis of Regulation of FOXP3 expression and induction of regulatory T cells for therapeutic application to autoimmune diseases.

最近的研究强调了具有调节/抑制特性的T细胞在控制自身免疫性疾病中的重要性。CD4(+)CD25(+)调节性T(T(Reg))细胞在维持外周免疫耐受和预防自身免疫中发挥重要作用。1.探讨类风湿关节炎(RA)和系统性红斑狼疮(SLE)患者外周血中CD4(+)CD25(+)T(Reg)细胞或FOXP3阳性细胞的特征。分析RA、SLE和正常人外周血中CD4(+)CD25(+)T细胞和FOXP3阳性细胞的频率和表型。类风湿关节炎和系统性红斑狼疮患者外周血中表达CD25和FOP3的CD4+T细胞的频率明显低于健康献血者。2.用脂质体转染法将FOXP3表达载体导入COST细胞，用核受体技术将FOXP3表达载体导入外周血淋巴细胞。用Western blotting和FACScan分析证实FOXP3蛋白在两种细胞中的表达。3.研究细胞因子和病毒蛋白对FOXP3基因表达的影响。我们将FOXP3表达载体和荧光素酶报告载体共转染到FOXP3表达载体和荧光素酶报告载体上，利用核受体将FOXP3结合序列的3次串联连接到外周血淋巴细胞。即使在IL-1、2、4、6和肿瘤坏死因子存在的情况下，共转染FOXP3也不能诱导荧光素酶活性。共转染TAX p40或CMV E1+2表达载体不能增加荧光素酶的活性。

Recent studies have emphasized the importance of T cells with regulatory/suppressor properties in controlling autoimmune diseases. CD4(+) CD25(+) regulatory T(T(reg)) cells play a critical role in the maintenance of peripheral tolerance and the prevention of autoimmunity. We developed anti-FOXP3 polyclonal and employed western blotting analysis and flowcytometric analysis using mononuclear cells from autoimmune diseases.1.We have explored the characteristics of CD4(+) CD25(+) T(reg) cells or FOXP3 positive cells in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The frequency and phenotype of CD4(+) CD25(+) T cells as well as FOXP3 positive cells in peripheral blood (PB) from patients with RA, SLE and PBMC from normal controls were analyzed. An decreased frequency of CD4+ T cells expressing CD25 or FOP3 were detected in PB from patients with RA and SLE compared with that in healthy donor.2.We transfected FOXP3 expression vectors to COST cells by lipofection and peripheral lymphocytes using nucleofector. Then expression of FOXP3 protein in both cells were confirmed by western blotting as well as by FACScan analysis.3.We also investigate the effect of cytokines or viral protein including human T lymphocytic leukemia virus tax p40 and cytomegalovirus early antigen-on expression of FOXP3 gene. We co-transfecto FOXP3 expression vector and luciferase reporter vector, which was ligated with 3 times tandem repeats of FOXP3 binding sequences to peripheral lymphocytes using nucleofector. Co-transfection of FOXP3 did not induce luciferase activities even in the presence of cytokines including IL-1,2,4,6 and TNF. Co-transfection of Tax p40 or CMV E1+2 expression vector did not increase of luciferase activities.