CFSE和AnnexinV双标记技术检测细胞毒性T淋巴细胞的杀伤效应

CFSE and AnnexinV double staining in assessing lethal effect of cytotoxic T lymphocyte

目的利用CFSE和AnnexinV对靶细胞进行染色，建立一种通过流式细胞仪技术检测细胞毒性T淋巴细胞（CTL）杀伤效应的新方法。方法应用磁珠分选OT—IT细胞受体（TCR）转基因小鼠CD8＋CTL细胞和脾脏树突状细胞（sDC），将2种细胞和鸡卯蛋白（OVA）抗原肽共培养65h后分析其细胞分裂增殖、IFN-γ和IL-4细胞内因子及穿孔素、颗粒酶等效应分子的表达特性，收获具备杀伤效应的CD8＋CTL细胞。利用预先激活的小鼠CD8^＋CTL细胞作为效应细胞，CFSE标记的同系小鼠脾细胞作为靶细胞，体内体外2种条件下利用AnnexinV染色靶细胞检测特异性CTL杀伤效应，并与CFSE^high曲和CFSE^low标记靶细胞检测体内CTL杀伤效应的经典方法进行比较。结果OT—I初始CD8^＋CTL细胞体外活化培养后有4个分裂峰，54．1％的CTL细胞表达IFN-γ，0．78％的细胞表达IL-4，穿孔素、颗粒酶2种CTL效应分子均为阳性表达，CD8＋CTL细胞已经具备CTL杀伤活性。体外实验结果显示OVA^＋靶细胞在共培养条件下，AnnexinV阳性细胞比例明显高于分离培养条件下[（62．4±3．5）％比（28．3±2．2）％，P〈0．01]，OVA的AnnexinV阳性靶细胞比例在不同培养条件下差异无统计学意义[（20．3±4．8）％比（17．3±2．9）％，P〉0．05]。共培养条件下OVA^＋靶细胞其AnnexinV阳性细胞比例也明显高于OVA^-靶细胞（P〈0．01），在分离培养条件下差异则无统计学意义（P〉0．05）。活化的CD8^＋CTL细胞所介导的杀伤效应具有抗原依赖性和部分的细胞接触依赖性。体内CTL实验中，利用CFSE和AnnexinV双标记的方法检测出靶细胞的杀伤率高于未孵育OVA抗原肽的对照组靶细胞[（52．63±8．12）％比（13．84±4．37）％，P〈0．01]。而同等条件下利用CFSE^high和CFSE^low双标记的方法检出靶细胞杀伤率为41%。结论CFSE和AnnexinV双标记靶细胞的方法检测CTL杀伤效应与单纯的使用CFSE^high和CFSE^low标记靶细胞方法有很好的可比性，该策略为利用流式细胞仪技术检测细胞杀伤功能的方法学提供了新的选择。

Objective To establish a novel flow cytometry assay for lethal effect of cytotoxic T lymphocyte （CTL） using CFSE and AnnexinV double staining. Methods CD8^ ＋ CTL cells and spleen dendritic ceils （sDCs） of OT- I T cell receptor （TCR） from transgenic mice were selected by magnetic beads. After these two types of cells were cultured with ovalbumin （OVA） peptide for 65 hours, cell division and proliferation, expression features of intracellular IFN-γ and IL-4, perforin, granzyme effector molecules were analyzed, and CD8^＋ CTL cells with lethal effect were obtained. Pre-aetivated mice CD8^＋ CTL cells were used as effector cells, and pre-CFSE-labeled splenocytes derived from allogenic mice as target ceils. Specific CTL effects were analyzed in vitro and in vivo by AnnexinV staining, results of which were compared with those of classic CTL lethal effect detection method, i. e. CFSE^high and CFSE^low for labeling target cells in vivo. Results At the beginning of OT- I , 4 division peaks were observed after CD8 ^＋ CTL cells activation cultured in vitro, with 54.1% CTI, cells of IFN-γ expression, and 0.78% cells of IL-4 expression. Perforin and granzyme these two CTL effector molecules were positive expression. CD8^ ＋ CTL cells had been CTL killing activity. In in vitro assay, more AnnexinV-positive OVA＋ target ceils were identified under co-culture than under separation culture [ （62.4±3.5）% vs （28.3±2.2）%, P〈0.01 ], while no statistical difference was shown in percentage of AnnexinV-positive OVA target cells under different cultures [ （20.3±4.8）% vs （ 17.3± 2.9）%, P〉0.05] ; under co-culture, more AnnexinV-positive OVA＋ ceils were identified than AnnexinV- positive OVA- cells （P〈0.01）, but no statistical difference was revealed under separation culture （P〉0.05）. Antigen dependency and partial cell-to-cell contact dependency were observed in lethal effect mediated by activated CD8^＋ CTL cell. In the CTL experiment in vivo, the killing rate of target cell by CFSE and AnnexinV double staining was higher than that of control group, in which target cells were not incubated in OVA peptide [ （52.63±8.12） % vs （ 13.84±4.37 ） %, P〈0.01 ]. By CFSEhigh and CFSEl^w double staining under same condition, killing rate of 41% was detected. Conclusion CFSE and AnnexinV double labeling method has fair comparability with CFSE^high and CFSE^low labeling in detecting CTL lethal effect, and may provide a new choice for detecting cytotoxicity with flow cytometry.