EBV特异性细胞毒性T淋巴细胞体外诱导培养及杀伤效果鉴定

本研究旨在探讨EB病毒特异性细胞毒性T淋巴细胞（EBV-CTL）体外诱导和扩增培养的方法，并检测其特异性杀伤的效果。采集EBV血清抗体阳性的6例正常供体的外周血单个核细胞（PBMNC），用EBV转化的B淋巴细胞系（BLCL）作为抗原递呈细胞及抗原刺激剂，经辐照灭活后不断刺激培养自体PBMNC，诱导产生EBV-CTL，并将其扩增培养；采用流式细胞仪鉴定其免疫表型，然后检测不同效靶细胞比例条件下EBV-CTL对自体BLCL（autoBLCL）、自体植物血凝素培养的B淋巴母细胞（PHA-blast）、HLA不合供体的BLCL（alloBLCL）、飚62细胞株的杀伤效果。结果表明，6例EBV血清抗体阳性正常供体来源的PBMNC在体外成功筛选并扩增培养了EBV。CTL，扩增效率为PBMNC数的18．6-55．0倍。刺激10次培养后的EBV-CTL对autoBLCL在20：1、10：1、5：1三种效靶细胞比例条件下特异性杀伤效率的平均值分别为59．4％、43．2％、29．O％；对PHA-blast、alloBLCL、K562的非特异性杀伤率在上述三种效靶细胞比例下平均值依次为7．1％、9．4％、10．3％（P〈0．05），6．6％、8．3％、8．1％（P〈0．05），5．4％、7．3％、6．3％（P〈0．05）。结论：EBV-CTL能有效在体外筛选培养并扩增，并能有效杀伤HLA相合的BLCL。可望成为EBV相关性移植后淋巴细胞增殖性疾病的过继免疫治疗的有效方法。

Ex vivo Inducing Cultured Epstein-Barr Virus Specific Cytotoxic T Lymphocytes and Evaluation of Their Killing Effect

This study was aimed to explore the method for induction and expansion of EB virus specific cytotoxic T lymphocytes （EBV-CTL） in vitro, and to detect their killing effect. Peripheral blood mononuclear cells（PBMNC） were collected from 6 EBV seropositive healthy donors, and EBV-transformed B lymphoblastoid cells （BLCL） were used as the antigen-presenting cells and antigen stimulant which was irradiated by 40 Gy 60Co irradiator. The autologous PBMNC and irradiated BLCL were cultured to induce and expand the EBV-CTL, and the immunophenotype was identified by the flow cytometry. The killing effect of the EBV-CTL against the autologous BLCL （ autoBLCL）, the autologous PHA cultured B lymphoblastoid cells（ PHA-BLCL）, the allogeneic BLCL（alloBLCL） and the K562 cells were measured with LDH release assay under different effector-to-target ratio. The results showed that the 6 cell lines of EBV-CTL were induced and expanded from the EBV seropositive healthy donors, the overall increase in cell numbers varied from 18.6 to 55.0 times. After 10 stimulations, the specific killing efficiency of the EBV-CTL for the autoBLCL were 59.4%, 43.2% and 29.0% under the effector-to-target ratio of 20： 1, 10- 1 and 5： 1. The nonspecific killing efficiency for the PHA-blast, alloBLCL and K562 cells were 7. 1%, 9.4% and 10. 3% （P 〈0.05） under the 20：1 ratio; 6. 6%, 8.3% and 8.1% （ P 〈 0.05 ） under 10：1 ; 5.4%, 7.3 % and 6. 3 % （ P 〈 0.05 ） under 5： 1, respectively. It is concluded that the EBV-CTL can be successfully induced and expanded ex vivo for specific killing of HLA matched BLCL and may become a potential treatment for EBV related post-transplant lymphoproliferative disorders.