CD4＋CD25＋Treg细胞对肿瘤特异性CTL杀伤效果的影响

Effects of CD4 ＋ CD25 ＋ Treg on the killing effects of tumor-specific cytotoxicity of CTL

目的 探讨CD4＋CD25＋Treg细胞对肿瘤特异性细胞毒T细胞（CTL）杀伤效果的影响及机制.方法 将C57BL/6小鼠80只随机分为4组,每组20只.A组：树突状细胞（DC）与T细胞共同培养前删除CD4＋CD25＋Treg;B组：DC与T细胞共同培养后删除CD4＋CD25＋Treg;C组：DC与T细胞共同培养时不删除CD4＋CD25＋Treg;对照组：无DC诱导的T细胞.应用脾脏来源DC细胞诱导T细胞制备CTL,在CTL形成的不同时期采用MACS法删除CD4＋CD25＋Treg.应用噻唑蓝（MTY）比色法检测不同组别CTL对B16黑色素瘤细胞的杀伤效果.同时应用酶联免疫吸附试验（ELISA）法检测细胞培养液中白细胞介素（IL）-2、干扰素（IFN）-γ含量变化.结果 3组实验组CTL杀伤率明显高于对照组（P〈0.05）.删除CD4＋CD25＋Treg的A组、B组CTL杀伤率明显高于未删除的C组（P〈0.05）.但CTL形成的不同时期删除CD4＋CD25＋Treg对CTL杀伤率的影响无统计学意义（P〉0.05）.IL-2、IFN-γ含量变化与杀伤率呈现相同的变化趋势.结论 删除CD4＋CD25＋Treg细胞可明显提高CTL的杀伤效果,是消除肿瘤免疫耐受机制的新途径.

Objective To explore the killing effects of CD4 ＋ CD25 ＋ Treg on tumor-specific cytotoxicity of cytotoxic T lymphocyte （CTL） to B16 Melanoma and the action mechanism.Methods Eighty C57BL/6 mice were randomly divided into three experimental groups and one control group （n = 20 each group）.Mononuclear cells were isolated from spleen tissues of the mice,and CTLs were prepared by using DCs loaded with B16 tumor antigens.Tumor inhibition rate was evaluated by using methyl thiazol tetrazolium （MTY） assay in vitro after deletion of CD4 ＋ CD25 ＋ Treg by using MACS,and the secretion levels of interleukin-2 （IL-2） and interferon-γ （IFN-γ） in the supernatant of the cell culture were measured by using enzyme linked immunosorbent assay （ELISA）.Results Tumor inhibition rate in three experiment groups was significantly higher than that in control group （ P 〈 0.05 ）.Tumor inhibition rate in groups A and B with a deletion of CD4 ＋ CD25 ＋ Tregs was significantly increased as compared with group C without deletion of Tregs （ P 〈 0.05 ）,but no significant difference was found between groups A and B with deletion of Tregs before and after the culturing （ P 〉 0.05 ）.The difference in the IL-2 and IFN-γwas the same as the tumor tumor inhibition effeciency of antigen-specific CTLs,which might be a new way to breakdown the tumor immune tolerance.