淋巴细胞功能相关抗原-1／细胞间黏附分子-1介导的细胞因子诱导的杀伤细胞体外抑瘤机制

Mechanism of lymphocyte function-associated antigen-l/intercellular adhesion molecule-1 mediatedanti-neoplastic effects of cytokine-induced killer cells

目的 探讨淋巴细胞功能相关抗原-1（LFA-1）／细胞间黏附分子-1（ICAM-1）介导的细胞因子诱导杀伤细胞（CIK）的体外抑瘤机制。方法从白血病患儿外周血分离淋巴细胞，经过干扰素-y（IFN-1）、抗CD，单克隆抗体（CD，McAb）、白细胞介素-2（IL-2）诱导并与树突状细胞（DC）共培养，获得大量的DC—CIK。在经10、20μg／ml等不同质量浓度小鼠抗人I．FA一1单克隆抗体处理后，采用Mrrr法研究DC—CIK细胞对多种白血病细胞株的杀伤活性，RT—PCR与Westernblotting方法检测GATA-3和T-bet基因表达水平的变化。ELISA方法测定DC—CIK细胞释放细胞因子IL-12、IFN一叮、肿瘤坏死因子-α[（TNF—d）的表达水平。结果诱导后的DC—CIK细胞形态规则，经不同浓度的LFA-1单克隆抗体处理后，M啊结果：20雌g／mlLFA-1单克隆抗体封闭组DC—CIK细胞对B95细胞杀伤作用下降最为明显（t=10．138，P〈0．05）；RT．PCR与Westernblotting结果：20μg／mlLFA-1单克隆抗体封闭的B95细胞组，GATA-3基因mRNA水平和蛋白水平表达增加最为明显（仁16．386，P〈0．05：t=22．652，P〈0．05）；同时T—bet基因mRNA水平和蛋白水平表达降低最为明显（t=17．728，p〈0．05；t=7．452，P〈0．05）；ELISA结果：20μg／mlLFA-1单克隆抗体封闭的B95细胞组中细胞因子IL-12、IFN-γ、TNF—d分泌水平下降最为明显（t=21．621，P〈0．05；t=13．739，P〈0．05；t=15．278，P〈0．05）。结论GATA-3和T—bet基因参与了LFA-1／ICAM-1介导的DC—CIK抑瘤途径，并且通过分泌Th1型细胞因子IL-12、IFN-γ、TNF-α等发挥抑瘤作用。

Objective To investigate the molecular mechanism underlying lymphocyte function- associated antigen-1 （LFA-1） / intercellular adhesion molecule-1 （ICAM-1） mediated anti-neoplastic effects of cytokine induced killer （CIK） cells. Methods Lymphocytes isolated from peripheral blood of children leukemia were induced with interferon-gamma （IFN-20, anti-CD3monoclonal antibody （CD3McAb） and interleukin-2 （IL-2） and co-cultured with dendrite cells （DC） to generate DC-CIK cells. When treated with LFA-1 monoclonal antibody, cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the MTT assay, while RT-PCR and Western blotting were used to determine mRNA and protein expressions of GATA-3 and T-bet in DC-CIK cells, respectively. IL-12, IFN-γ and tumor necrosis factor-α （TNF-α levels released by DC-CIK cells were quantified by ELISA. Results Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. When treated with mouse anti-human LFA-1 monoclonal antibody, the cytotoxicity decreased mostly towards B95 cells under administration of 20 μg/ml LFA-1 monoclonal antibody in comparison with the control group（t =10. 138, P 〈0.05）. It led to a highest elevation of GATA-3 mRNA and protein levels （t =16.386, P 〈 0.05; t =22.652, P 〈 0.05） and a most decrease of T-bet mRNA and protein levels （t =17.728, P 〈0.05; t =17.452, P 〈0.05） under 20 p,g/ml LFA-1 monoclonal antibody in B95 cells group in comparison with the control grouP. The expression levels of IL-12, IFN-T, and TNF-α in supernatant were the lowest under 20 μg/ml LFA-i monoclonal antibody in B95 cells group in comparison with the control group （t =21.621, P 〈0.05; t =13.739, P 〈0.05; t =15.278, P 〈0.05）. Conclusion GATA-3 and T-bet were implicated in the LFA-1/ICAM-1 mediated anti-neoplastic effects of DC-CIK cells via activation of the Thl pathway, with high secretion of Thl cytokines, such as IL-12, IFN-γ and TNF-α.