EB病毒-细胞毒性T细胞免疫治疗改善EB病毒相关非霍奇金淋巴瘤化疗效果的实验研究观察

Effect of EBV specific CTL on the prognosis of chemotherapy in patients with EBV- related non- Hodgkin lymphoma

目的探讨EB病毒(EBV)特异性细胞毒性T细胞(CTL)免疫治疗对EBV相关非霍奇金淋巴瘤(NHL)化疗效果的影响及机制。方法通过细胞学实验成功诱导并扩增出大量的EBV特异性CTL;建立人EBV相关NHL的Balb/c裸雌鼠动物模型,设单纯免疫(EBV-CTL)治疗组、单纯化疗(CHOP)组、免疫治疗联合化疗疫(EBV-CTL+CHOP)治疗组以及空白对照组,5只/组,观察各组动物肿瘤生长情况;治疗结束后剥离肿瘤组织检测瘤内免疫细胞变化情况。结果体外杀伤实验显示伴随效靶比的增加,EBV-CTL杀伤能力逐渐增大:效靶比例分别在2.5∶1、5.0∶1与、10∶1及20∶1时,杀伤效率(%)分别为9.41±1.23、19.45±3.54、50.34±6.77和55.26±7.21(P<0.01);动物实验显示自免疫治疗d3起,与对照组相比,3组实验组抑制淋巴瘤生长率(%)分别为28.57、57.9、76.7(P<0.01)。进一步检测肿瘤相关免疫微环境实验发现,治疗后肿瘤内浸润性CTL及巨噬细胞(TIM)平均比率(%),EBV-CTL+CHOP组、EBV-CTL组、CHOP组及空白对照组分别为13.65±1.48、4.33%±1.04%,10.24±1.2%、3.82±0.54,6.83±0.16、0.95±0.07,6.32±0.18、1.96±0.24(P<0.05)。结论 EBV特异性CTL可能通过激活TIM增殖而间接促进肿瘤免疫应答。EBV-CTL免疫治疗能有效改善EBV相关NHL化疗疗效,或对EBV相关NHL的治疗起到一定指导作用。

Objective To explore the effect of EBV-CTL on conventional EBV-related non-Hodgkin lymphoma chemotherapy and related mechanisms. Methods Large numbers of EBV specific T cells were generated by immune stimulation ex vivo. A murine model was established on human EBV-related non-Hodgkin＇s lymphoma. Tumor growth was observed in a single immunotherapy group,a single chemotherapy group,a combined group and a control group. After the end of treatment,the intact tumor tissue was peeled and digested by collagenase to observe the change in tumor infiltrating immune cells and response. Results The ex vivo experiment showed that EBV-CTL killing ratio boosted as soon as E： T increased. Compared with 2. 5∶ 1 and 5. 0∶ 1,10∶ 1 and 20∶ 1 exhibited more effective killing ability： 4. 41%,9. 45%,17. 34%,25. 26%（ P 〈0. 01）. At the same time,animal model showed that,when compared with any single treatment group,the combined treatment group could more effectively inhibit tumor growth from the 3rd day of treatment（ P 〈0. 01）. In addition,from the tumor associated immune microenvironment,the numbers of tumor infiltrating CTL（ cytotoxicity T lymphocyte） and macrophages were elevated（ CTL ratio = 13. 65%,P〈 0. 03; TIM ratio = 4. 33%,P 〈0. 05） in combined therapy group. The CTL values were 10. 24% ± 1. 2%,6. 83% ± 0. 16% and 6. 32% ± 0. 18% for single immunotherapy group,single chemotherapy group,and control group,respectively. The TIM values were 3. 82% ± 0. 54%,0. 95% ± 0. 07%,and 1. 96% ±0. 24% for single immunotherapy group,single chemotherapy group,and control group,respectively. Conclusion The effects may suggest that EBV-specific CTLs may indirectly promote lymphoma innate immune response through activating tumor infiltrating macrophage proliferation. Our findings may provide a guide in future treatment of EBV-related non-Hodgkin lymphoma.