基于ICS技术的NK细胞ADCC效应评价方法建立及应用  

Intracellular cytokine staining （ICS） for detection of NK cell-mediated antibody-dependent cell-mediated cytotoxicity （ ADCC ）

作　　者：谢喆[1] 李渊韬 吕琳婷 马尔马尔·托乎塔尔别克 沈弢[1] Xie Zhe, Li Yuantao, Lyu Linting, Maermaer. Tuohutaerbieke, Shen Tao(Department of Microbiology, Peking University Health Science Center, Beijing 100191, Chin)

机构地区：[1]北京大学医学部病原生物学系,100191

出　　处：《中华微生物学和免疫学杂志》2018年第1期1-6,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家“十三五”重大科技专项（2017ZX10202101-003,2018ZX10731101-001-008,2018ZX09739002-003）;传染病预防控制国家重点实验室课题（2015SKuD506）

摘　　要：目的探究细胞因子的胞内染色（intracellular cytokine staining，ICS）技术是否可以评价自然杀伤细胞（natural killer cell，NK细胞）的抗体依赖性细胞介导的细胞毒效应（antibody-dependent cellular cytotoxicity，ADCC）以及在HCV、HIV单独感染和HCV/HIV合并感染情况下ADCC效应的变化。方法采用流式细胞术分析NK细胞的频数、表面受体的表达；ImageStreamX MarkⅡ流式成像技术鉴定CD56brightNK细胞和CD56dimNK细胞表面CD3/CD56/CD32/CD16的单细胞表达状态；通过P815细胞抗原抗体复合物模型（P815＋Ab）以及细胞内因子染色技术评价NK细胞的脱颗粒能力以及细胞因子释放能力；通过流式检测细胞内因子表达和靶细胞杀伤比较HIV、HCV、HIV/HCV慢性感染与健康对照者的NK细胞介导ADCC效应的差异。结果本研究证实，NK细胞介导ADCC，CD107a＋和干扰素γ（interferon-γ，IFN-γ）细胞频数与CD16平均荧光强度（mean fluorescence intensity，MFI）降低呈正相关；CD56dimNK细胞高表达CD16且CD107a＋和IFN-γ＋细胞比例较CD56brightNK高；利用ICS法检测NK细胞CD107a和IFN-γ的表达与快速荧光法检测靶细胞裂解率呈正相关；慢性HIV感染时，NK细胞介导ADCC效应受损。结论研究表明ICS技术可以评价NK细胞的ADCC效应。在HCV、HIV和HIV/HCV慢性感染时，NK细胞介导的ADCC效应减弱。ObjectiveTo analyze the possibility of using intracellular cytokine staining （ICS） to evaluate NK cell-mediated antibody-dependent cellular cytotoxicity （ADCC） and to detect the changes in ADCC activity among patients with chronic HIV and/or HCV infection.MethodsFlow cytometry was performed to determine the percentages of NK cells and the expression of NK cell receptors. ImageStreamX MarkⅡ system was used to identify the expression of CD3, CD56, CD16 and CD32 on CD56brightNK and CD56dimNK subsets. Degranulation process and cytokine production in NK cells were detected using an antigen-antibody complex model of P815/Ab in combination with ICS. Differences in NK cell-mediated ADCC were evaluated among patients infected with HIV and/or HIV and healthy subjects by flow cytometry.ResultsThe percentages of CD107a＋ and IFN-γ＋ NK cells were positively correlated with the decrease of mean fluorescence intensity （MFI） of CD16. ICS assay revealed a positive correlation between the secretion of CD107a and IFN-γ by NK cells. CD16 was highly expressed in CD56dimNK cells. The ADCC mediated by CD56dimNK cells was stronger than that mediated by CD56brightNK cells. The rate of target cell lysis detected by rapid fluorescence assay was positively correlated with the percentage of CD107a＋ /IFN-γ＋ NK cells measured by ICS. NK cell-mediated ADCC was suppressed in patients with chronic HIV and/or HCV infection.ConclusionThis study suggests that ICS assay could be used to evaluate NK cell-mediated ADCC. It also reveals that NK cell-mediated ADCC is suppressed in patients with chronic HIV and/or HCV infection.

关 键 词：NK细胞 ADCC效应 HIV HCV CD107a IFN-Γ 

分 类 号：R392[医药卫生—免疫学]