CD4^+CD25^+调节性T细胞AICD机制的研究

Activation induces cells death(AICD) of CD4^+CD25^+ regulatory T cells

目的探讨CD4+CD25+调节性T细胞活化诱导的细胞死亡(AICD)发生的机制。方法CD4+CD25+T细胞以磁性细胞分离器(MACS)从BALB／c小鼠或DO11．10小鼠的静息T细胞分离纯化。体外细胞增殖抑制实验证实其免疫调节作用。CD4+CD25+T细胞的AICD以CD3／CD28单克隆抗体活化或以特异性OVA323-339瑚肽、抗原提呈细胞活化等两种方法获得。CD4+CD25+T细胞凋亡相关基因的表达通过实时定量PCR检测。流式细胞仪检测细胞的凋亡率。进一步观察FasL中和抗体、TRAIL中和抗体及caspase抑制剂zVAD-fmk对CD4+CD25+T细胞凋亡的影响。结果MACS成功分离CD4+CD25+T细胞,纯度可达98％,该细胞可特异性表达Foxp3基因,能明显抑制效应性T细胞的体外增殖。CD3／CD28抗体以及OVA特异性抗原活化8 d的CD4+CD25+调节性T细胞AICD达39％-45％。活化前后的CD4+CD25+调节性T细胞死亡受体家族表达发生明显变化;FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4+CD25+调节性T细胞的凋亡。结论FasL/Fas及其他凋亡相关分子可能参与了CD4+CD25+调节性T细胞的凋亡。

Objective To investigate the mechanism of AICD in CD4^＋CD25^＋ regulatory T cells （Treg）. Methods Murine CD4^＋CD25^＋ T cells from naive BALB/c or DO11. 10 mice were isolated by MACS CD4^＋CD25^＋ Treg cells from BALB/c mice were stimulated with anti-CD3/CD28 antibodies, and those from DO11. 10 mice were incubated with OVA323-339 peptide plus APC respectively. Real time PCR was applied to detect the gene expression pattern in CD4^＋CD25^＋ Treg cells. Flow cytometry was performed to determine the apoptosis of CD4^＋CD25^＋ Treg cells. The influence of anti-FasL, anti-TRAIL neutralizing antibody and zVAD-fmk on AICD of CD4^＋CD25^＋ Treg cells was evaluated. Results CD4^＋CD25^＋ T cells were classified by MACS with 98% purity and specifically express the Foxp3 gene which is the ＇master control gene＇ for Treg cells and could suppress the proliferation of CD4^＋CD25^＋ T cells. The apoptosis of freshly isolated CD4^＋CD25^＋ Treg cells was less than 5%, but could reach to 39%-45% after being activated in vitro for 8 days. The results of real time PCR showed different gene expression pattern of TNF family member in freshly isolated CD4^＋CD25^＋ Treg cells and activated CD4^＋CD25^＋ Treg cells. The apoptosis of CD4^＋CD25^＋ Treg cells which induced by anti-CD3/CD28 antibody or OVA323-339 peptide plus APC was significantly inhibited by either anti-FasL neutralizing antibody, or anti-TRAIL neutralizing antibody and caspase inhibitor zVAD-fmk as well. Conclusion FasL/Fas and other apoptosis-related molecules might be involved in the AICD of CD4^＋CD25^＋ Treg cells.