唑来膦酸对骨肉瘤患者PBMCs来源γδT细胞抗骨肉瘤作用的影响

Effect of zoledronate on the cytotoxicity of γδ T cells from PBMCs of osteosarcoma patients against osteosarcoma

目的探讨唑来膦酸对骨肉瘤患者外周血单个核细胞(PBMCs)来源γδT细胞杀伤骨肉瘤作用的影响。方法使用唑来膦酸联合IL-2体外扩增原发性、复发性和转移性骨肉瘤患者PBMCs来源γδT细胞,LDH法检测γδT细胞的杀伤活性,ELISA法检测γδT细胞分泌IFN-γ情况,并应用不同的抗体确定γδT细胞识别、杀伤骨肉瘤细胞的途径。建立裸鼠HOS骨肉瘤移植瘤模型,尾静脉分别注射生理盐水、唑来膦酸、γδT细胞及唑来膦酸联合γδT细胞,观察并比较不同方法对裸鼠肿瘤生长的抑制作用。结果骨肉瘤患者PBMCs体外经过唑来膦酸联合IL-2刺激可高度选择性扩增γδT细胞,并且扩增后的γδT细胞具有杀伤骨肉瘤细胞的能力。唑来膦酸预处理骨肉瘤细胞后可显著增强γδT细胞的杀伤活性,γδT细胞识别、杀伤唑来膦酸预处理骨肉瘤细胞主要通过TCR途径和穿孔素-纤溶酶途径,NKG2D途径和TRAIL途径发挥作用较小。体内实验表明,唑来膦酸联合γδT细胞治疗对肿瘤生长的抑制作用强烈而持久,显著优于单独唑来膦酸治疗组和单独γδT细胞治疗组。结论唑来膦酸能够促进骨肉瘤患者PBMCs来源γδT细胞的增殖,并能够增强γδT细胞的抗骨肉瘤作用。

Objective To investigate the effect of zoledronate （Zol） on the cytotoxicity of γδT cells from peripheral blood mononuclear cells （PBMCs） of osteosarcoma （OS） patients against OS. Methods γδT cells, derived from PBMCs of patients with primary, recurrent and metastatic OS, were ex vivo expanded by Zol and IL-2. γδT cell cytotoxicity against target cells was analyzed by a standard lactate dehydrogenase （LDH） release assay. IFN-γ secreted from γδT cells was determined by ELISA kits. γδT cells were incubated with different blocking Abs in order to investigate the mechanisms of recognition and killing of OS cells. Nude mice were inoculated with human OS cell line （HOS） by subcutaneous injection to create in vivo OS tumor models, and received normal saline, Zol, γδT cells, or γδT cells ＋ Zol by tail vein injection, respec- tively. The inhibition of tumor growth by the different methods was observed and compared. Results γδT cells from PBMCs of patients with OS were selectively expanded by Zol ＋ IL-2 in vitro, and showed cytotoxicity against OS. In addition, γδT cells showed significantly higher cytotoxicity against OS cells after treatment with Zol, and the mechanism of cytotoxicity was largely dependent on TCR pathway and perforin/plasmin pathway, partly on TRAIL pathway and NKG2D pathway. Combina- tion treatment of γδT cells and Zol had stronger and longer-lasting inhibition on tumor growth in vivo, compared with either drug alone. Conclusion Zol can promote γδT cells＇ proliferation from PBMCs of OS patients and enhance γδT cells＇ cytotoxicity against OS.