基因工程Treg细胞对小鼠异基因骨髓移植后GVHD和GVL效应的影响

Influence of the lentiviral vectors-mediated mouse genetic engineering Treg on GVHD and GVL effect after allogeneic bone marrow transplantation in mice

目的 探讨输注慢病毒载体介导的小鼠基因工程调节性T淋巴细胞（Treg细胞）对小鼠异基因骨髓移植后移植物抗宿主病（GVHD）及移植物抗白血病（GVL）效应的影响.方法 利用慢病毒载体介导,将小鼠叉状头螺旋转录因子（Foxp3）基因转导入Balb/c小鼠的CD4＋CD25-T淋巴细胞,即为基因工程Treg细胞.以Balb/c小鼠为供者.C57BL/6小鼠为受者,进行异基因骨髓移植,移植当天受者接受X线直线加速器全身照射.用随机数字表法将受者分为5组,每组10只.（1）单纯照射组：经受者尾静脉输注RPMI 1640培养液0.2 ml;（2）白血病对照组：经受者尾静脉输注供者骨髓细胞5×106个＋C57BL/6小鼠T淋巴细胞白血病/淋巴瘤细胞株（EL4细胞）500个;（3）移植对照组：经受者尾静脉输注供者骨髓细胞5×106个＋脾细胞5×106个＋EL4细胞500个;（4）工程Treg组：经受者尾静脉输注供者骨髓细胞5×106个＋脾细胞5×106个＋EL4细胞500个＋基因工程Treg细胞5×106个;（5）空载体对照组：经受者尾静脉输注供者骨髓细胞5×106个＋脾细胞5×106个＋EL4细胞500个＋空载体转导的CD4＋CD25-T淋巴细胞5×106个.每天观察受者存活情况;记录GVHD及白血病的发生情况;各组均于小鼠濒死前取其肝脏、小肠、皮肤、脾脏等组织,进行病理学观察;取长期存活（超过60 d）受者的骨髓细胞,检测嵌合情况.结果 单纯照射组、白血病对照组、移植对照组、工程Treg组和空载体对照组小鼠存活时间分别为（10.3±1.5）d、（20.7±1.9）d、（26.0±4.3）d、（49.0±17.7）d和（24.4±4.1）d,工程Treg组小鼠存活时间明显长于其他各组,差异有统计学意义（P＜0.05）.白血病对照组小鼠肝、脾组织病理切片均存在白血病细胞浸润表现,移植对照组及空载体对照组小鼠肝脏、皮肤和小肠病理切片存在GVHD病理改变,而工程Treg组长期存活小鼠各组织病理切片结构基本正常,未见GVHD及白血病细胞浸润病理表现,该组GVHD评分明显低于移植对照组及空载体对照组.结论 小鼠异基因骨髓移植时联合输注基因工程Treg细胞可有效减少GVHD的发生并保留GVL效应.

Objective To explore the influence of the lentiviral vectors-mediated mouse genetic engineering regulatory T cells （Treg） infused after allogeneic bone marrow transplantation （alloBMT） on graft-versus-host disease （GVHD） and graft-versus-leukemia （GVL） effect in mice.Methods Lentivirus-mediated expression of Forkhead box P3 （Foxp3） transformed CD4 ＋ CD25- T cells from Balb/c mice into engineered Tregs in vitro. An allo-BMT model of Balb/c→C57BL/6 mice was established. The recipients were given lethal X-ray total body irradiation before transplantation.Mice were randomly assigned into five groups and each group contained 10 recipients： （1） The recipients in radiation group were injected with 0.2 ml RPMI 1640; （2） The recipients in leukemia control group were injected with 5 × 106 donor bone marrow cells and 500 mouse T-cell leukemia/lymphoma cells （EL4 cells）; （3） The recipients in transplantation control group were injected with 5 × 106 donor bone marrow cells and 5 × 106 splenocytes plus 500 EL4 cells; （4） The recipients in engineering Treg group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 genetic engineering Treg; （5） The recipients in empty vector control group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 empty vector-transduced CD4＋ CD25- T cells. Survival time, clinical GVHD score or histopathological analysis （skin, liver and small intestine） were observed after allo-BMT. Chimerism of bone marrow cells from recipients survived for 60 days after transplantation was measured. Results The mean survival time in radiation group, leukemia control group, transplantation control group,engineering Treg group and empty vector control group was （ 10. 3 ± 1.5）, （20. 7 ± 1.9）, （26. 0 ±4.3）, （49. 0 ± 17. 7） and （24. 4 ± 4. 1 ） days respectively. The survival time in engineering Treg group was significantly prolonged as compared with other groups as judged by the log-rank test （P＜0. 05）.Histopathological analysis in several target organs （skin, liver and small intestine） confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD or leukemia were observed in recipients in engineering Treg group and clinical GVHD scores in this group were significantly decreased as compared with transplantation control group and empty vector control group. Conclusion Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD without affecting GVL activity during allo-BMT in mice.