超抗原对NK细胞CD226分子表达与功能的调节

目的研究超抗原对NK细胞CD226分子表达与功能的调节。方法以超抗原金黄色葡萄球菌肠毒素A/B(SEA/B)活化PBMC为模型,应用双重免疫荧光染色和流式细胞术分析,观察CD226分子在NK细胞上的变化;采用51Cr释放实验,观察NK细胞在超抗原作用下杀伤功能的改变;利用激光共聚焦显微镜,观察CD226分子在NK细胞杀伤相的分布。结果在静止PBMC中,CD56 NK细胞的百分率为12.3%,CD56 CD226 细胞仅为1.4%。当效靶比为5∶1时,NK细胞对K562细胞的杀伤率为(3.2±0.2)%。当0.1mg/LSEA或SEB刺激PBMC1d后,CD56 NK细胞的百分率分别为13.5%和14.1%,CD226在NK细胞上的表达水平明显升高,且主要表达在CD56dim细胞上。在刺激第2天,SEA组CD56 CD226 占CD56 细胞69.1%,SEB组CD56 CD226 占CD56 细胞64.3%。刺激第3天,CD226在NK细胞上的表达水平均较第2天明显下降。在超抗原0.1mg/L作用3d中,SEA组和SEB组NK细胞杀伤率均明显高于同期未刺激组NK细胞的杀伤率及新鲜分离NK细胞的杀伤率(P<0.05),在作用的第2天,SEA组和SEB组杀伤率均达到峰值分别为(82.3±6.9)%和(80.6±7.5)%。激光共聚焦结果显示,CD226分子与LFA-1分子共定位于NK细胞与K562细胞的接触部位。结论超抗原SEA和SEB可提高NK细胞杀伤活性,可能与其促进CD226分子在NK细胞上的表达相关,CD226分子可能参与NK细胞免疫突触的形成。

Expression and function of CD226 on NK cells activated by Superantigens

AIM: To study the regulation of superantigen in expression and function of CD226 on NK cells. METHODS: Double fluorescent staining and flow cytometry analysis were employed to detect the expression of CD226 on NK cells from human peripheral blood mononuclear cells (PBMC) stimulated by staphylococcus enterotoxin A (SEA) or SEB. (51)Cr release assay was performed to compare the cytotoxicities of NK cells in different activation models. Laser scanning confocal microscope was used to observe the distribution of CD226 on NK cells activated by superantigens at killing stage. RESULTS: In resting NK cells, the percentage of CD56(+) NK cells and CD56(+) CD226(+) cells were 12.3% and 1.4% respectively, and the cytotoxicity of NK cells against K562 cells was (3.2+/-0.2)% at the ratio of 5:1. After PBMC were stimulated by 0.1 microg/mL SEA and SEB for 1 day, the percentage of CD56(+) NK cells was 13.5% and 14.1% respectively, and the percentage of CD56(+) CD226(+) cells were increased. Interestingly, CD226 was mainly expressed on CD56(dim) NK cells. On day 2 after SEA/SEB stimulation, the proportions of CD56(+) CD226(+) cells among CD56(+) cells were 69.1% and 64.3% in SEA and SEB group respectively. On day 3 after SEA/SEB stimulation, the expression of CD226 on NK cells decreased. Furthermore, the cytotoxicity of NK cells stimulated by SEA or SEB for 3 days against K562 cells were much higher than that of the resting NK cells as well as NK cells cultured without SAg for the same culture time (P<0.05), and reached to the peak at day 2 (82.3%+/-6.9% and 80.6%+/-7.5%, respectively). Additionally, we observed that CD226 molecules were colocalized with LFA-1 at the interface of NK cells which contacted K562 cells. CONCLUSION: The cytotoxicity of NK cells enhanced by SEA or SEB may be correlation with the increased expression of CD226 molecule, and CD226 may be involved in synapse formation of NK cells at killing stage.