CD8^+CIK细胞表面NKG2D及TCR抗原识别受体的功能分析  

Function of antigen recognition receptors TCR and NKG2D on CD8^+ CIK cells

作　　者：王耀玲[1] 刘建华[1] WANG Yao-ling;LIU Jian-hua(Department of Obstetrics and Gynecology, Shanghai Ninth People＇s Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200011, China)

机构地区：[1]上海交通大学医学院附属第九人民医院妇产科,上海200011

出　　处：《中国妇幼健康研究》2018年第4期428-435,共8页Chinese Journal of Woman and Child Health Research

摘　　要：目的探讨CD8^+细胞因子诱导的杀伤细胞(CIK细胞)亚群表面T细胞受体(TCR)和NKG2D(即CD314)抗原识别受体在发挥抗肿瘤特别是卵巢癌免疫效应中的功能。方法 CIK细胞由卵巢癌志愿者外周血单个核细胞(PBMCs)制备而成,采用磁性细胞分选(MACS)技术从培养1周的CIK细胞中富集CD8^+亚群并继续培养扩增,获得高度均质性的CD8^+CIK细胞后进行表型检测。将CD8^+CIK细胞分别在CD3抗体和NKG2D抗体包被的培养板中进行培养,以CD137为CD8^+CIK细胞激活标志物,应用流式细胞术(FCM)检测CD137表达,评估CD8^+CIK细胞的激活状况。并检测经CD3抗体、NKG2D抗体刺激或与红白血病细胞株人类白细胞抗原(HLA)-主要组织相容性复合体-Ⅰ类相关分子(MIC)A/B+K562细胞共培养对CD8^+CIK细胞分泌γ-干扰素(IFN-γ)的影响。选用K562细胞作为CD8^+CIK细胞作用的靶细胞,应用NKG2D抗体阻断NKG2D与MICA/B相互作用,测试激活的CD8^+CIK细胞在NKG2D阻断状态下对靶细胞K562的杀伤能力。结果 TCR能够提供CD8^+CIK细胞的激活信号,而NKG2D不具备该功能。外周血单个核细胞(PBMC)中CD8^+细胞占24.2%,该细胞群中NKG2D阳性率为16.3%。CIK培养后CD8^+细胞比例增至88.1%,这些CD8^+CIK细胞均表达NKG2D,但其中仍含10%左右的CD8-细胞;CD3抗体组中CD137阳性率为88.8%;CD3抗体刺激后24h,约50%的CD8^+CIK细胞呈IFN-γ染色阳性,而NKG2D抗体刺激后,IFN-γ阳性率(4.8%和5.6%)高于对照组(2.9%),但明显低于CD3抗体;CD8^+CIK细胞经CD3抗体刺激24h后IFN-γ检测阳性率为41.9%,但与K562细胞按1:1比例共培养后阳性率仅为0.5%。CD8^+CIK细胞对K562细胞具有杀伤功能,即能够以HLA非限制的方式杀伤靶细胞。而在CD3-TCR复合物激活的CD8^+CIK细胞上阻断NKG2D与MICA/B相互作用后,CD8^+CIK细胞杀伤活性受到抑制。结论 CD8^+CIK细胞的激活主要通过CD3-TCR受体复合物,而非通过NKG2D受体。CD8^+CIK细胞的NKG2D受体与靶细胞表面相应�Objective To approach the function of T cell receptor （TCR） and antigen recognition receptor NKG2D （CD314） on the surface of CD8^＋ cytokine induced killer cells （CIK cells） in the antitumor immune response, especially on ovarian cancer. Methods Peripheral blood mononuclear cells （PBMCs） of ovarian cancer volunteers were prepared for CIK cells. Enriched CD8^＋ subgroup cells were gained by sorting from CIK cells cultured for 1 week by means of magnetic-activated cell sorting system （MACS） and further cultured and expanded. Phenotyping detection was done after collection of highly homogeneous CD8^＋ CIK cells. CD8^＋ CIK cells were respectively cultured at culture plates coated with TCR antibody or NKG2D antibody, and CD137 was taken as marker of activation status of CD8^＋ CIK cells. Expression of CD137 was detected by flow cytometry （FCM） for evaluation of activation status of CD8^＋ CIK cells. Then influence of stimulation with CD3 antibody, NKG2D antibody and co-culture with major histocompatibility complex-I related molecule cells （MIC） A/B^＋ K562 of human leukocyte antigen （HLA） in erythroleukemia cell line on IFN-γ secretion of CD8^＋ CIK cells were detected. K562 cells were selected as target cells of CD8^＋ CIK cells, and interaction between NKG2D and MICA/B was blocked through NKG2D antibody. Killing ability of activated CD8^＋ CIK cells to target cells K562 under blocking of NKG2D was detected, Results TCR could provide activating signals for CD8^＋ CIK cells, while NKG2D could not. Proportion of CD8^＋ cells in peripheral blood mononuclear cells （PBMC） was 24.2%, and positive rate of NKG2D in this cell group was 16.3%. Proportion of CD8^＋ cells increased to 88.1~ after culturing with CIK. NKG2D were expressed in all these CD8^＋ CIK cells, but they still contained about 10% of CD8^- ceils. Positive rate of CD137 in CD3 antibody group was 88.8%. About 50% of CD8^＋ CIK ceils showed positive IFN-γ staining after stimulation with CD3 antibody for 24 h

关 键 词：细胞因子诱导的杀伤细胞 CD8^+CIK T细胞受体 NKG2D 

分 类 号：R173[医药卫生—妇幼卫生保健]