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)）细胞在维持外周耐受性和预防自身免疫中起关键作用。我们开发了抗foxp3多克隆，并利用自身免疫性疾病的单核细胞进行了western blotting和流式细胞术分析。我们探讨了CD4(+) CD25(+) T（reg）细胞或FOXP3阳性细胞在类风湿关节炎（RA）和系统性红斑狼疮（SLE）患者中的特征。分析了正常对照RA、SLE和PBMC患者外周血CD4（+）、CD25(+) T细胞和FOXP3阳性细胞（PB）的频率和表型。与健康供者相比，RA和SLE患者外周血中表达CD25或FOP3的CD4+ T细胞频率降低。我们将FOXP3表达载体通过脂质体转染至COST细胞，并利用核因子转染外周淋巴细胞。western blotting和FACScan分析证实两种细胞中FOXP3蛋白的表达。我们还研究了细胞因子或病毒蛋白（包括人T淋巴细胞白血病病毒税p40和巨细胞病毒早期抗原）对FOXP3基因表达的影响。我们将FOXP3表达载体与荧光素酶报告载体共转染，用核因子将FOXP3结合序列串联重复3次连接到外周淋巴细胞上。即使在IL-1、2、4、6和TNF等细胞因子存在的情况下，FOXP3的共转染也不会诱导荧光素酶的活性。共转染Tax p40或CMV E1+2表达载体均未增加荧光素酶活性。

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.