自身反应性CD8^(+)中心记忆性T细胞对实验性自身免疫性脑脊髓炎致病作用

Priliminary study on pathogenic role of autoreactive CD8^(+)central memory T cells in experimental autoimmune encephalomyelitis

目的探讨自身反应性CD8^(+)中心记忆性T细胞(TCM)在实验性自身免疫性脑脊髓炎(EAE)中的致病作用。方法用髓鞘少突胶质细胞糖蛋白(MOG)35-55肽段/完全弗氏佐剂(CFA)乳化液于雌性C57BL/6(B6)小鼠背部4点皮下注射,在0 h(CFA乳化液注射当时)及48 h时间点分别予以百日咳毒素(PTX)腹腔注射,建立EAE模型。于发病高峰期后处死EAE小鼠获取脾脏和淋巴结的单个核细胞(MNC),利用免疫磁珠法经阴性选择纯化CD8^(+)T细胞;进一步采用流式细胞术分选TCM(CD8^(+)CCR7^(+)CD44^(high))。将EAE鼠CD8^(+)T细胞、健康鼠CD8^(+)T细胞分别过继转移(经鼠尾静脉注射)至雌性Rag-1^(-/-)小鼠体内(2×10^(6)/只,n=6),进行Kono评分及病理学检查,以确证CD8^(+)T细胞的致病性;进一步对EAE鼠CD8^(+)T细胞不同亚群进行致病性研究,分为CD8^(+)TCM常规剂量组(5×10^(5)/只,n=6)、CD8^(+)终末效应记忆性T细胞(Ter TEM,5×10^(5)/只,n=6)组、磷酸缓冲盐溶液(PBS,n=6)组及CD8^(+)TCM高剂量组(1.5×10^(6)/只,n=3),分别将不同剂量CD8^(+)TCM、Ter TEM以及PBS相应细胞/溶液注射于Rag-1^(-/-)小鼠;予以MOG35-55/CFA和PTX诱导,进行神经功能评分;于过继转移后第40 d处死小鼠,检测其病理学改变。结果将EAE鼠CD8^(+)T细胞过继转移至Rag-1^(-/-)小鼠体内,在27 d~29 d后出现双后肢瘫痪症状,病理学显示脑/脊髓内炎性细胞浸润和脱髓鞘改变,而健康鼠CD8^(+)T细胞过继转移后小鼠无瘫痪症状,病理学检查亦未见EAE相关病理学改变。EAE鼠CD8^(+)TCM常规剂量组及高剂量组、Ter TEM组、PBS组注射后持续观察40 d则均未出现发病症状,脑/脊髓亦未见及EAE相关的病理学改变。结论EAE自身反应性CD8^(+)T细胞的过继转移可诱导Rag-1^(-/-)小鼠发病,而自身反应性CD8^(+)TCM的过继转移则未能诱导出EAE病症及其相应的病理学改变,提示CD8^(+)TCM缺乏明显的EAE致病效应。

Objective The aim of this study was to investigate the pathogenic role of autoreactive CD8^(+)central memory T cells(TCM)in experimental autoimmune encephalomyelitis(EAE).Methods In order to establish an EAE model,female C57BL/6(B6)mice(6~10 weeks,17~20 g)were immunized with emulsified myelin oligodendrocyte glycoprotein(MOG)35-55 peptide and complete Freund's adjuvant(CFA),followed by a stepwise injection of pertussis toxin(PTX)at the time point of 0 h and 48 h later,respectively.The mice were all sacrificed after their clinical symptoms reached the peak.Thereafter,their spleen and lymph nodes were separated through Cell Strainer to further obtain mononuclear cells(MNC)by using density gradient centrifugation.CD8^(+)T cells were purified with immunomagnetic beads;furthermore,central memory T cells(TCM,CD8^(+)CCR7^(+)CD44^(high))were acquired by using a cell sorter via flow cytometry.The following experiments included two parts:(1)The purified EAE-derived CD8^(+)T cells were injected intravenously into female Rag-1^(-/-)mice(2x10^(6) per mouse,n=6)with adoptive transfer.Same amount of CD8^(+)T cells obtained from healthy mice were transferred to animals in control group(n=6).(2)CD8^(+)TCM(5×10^(5) per mouse,n=6),CD8^(+)terminal effector memory T cells(Ter TEM,n=6),phosphate buffer saline(PBS,n=6),and higher dosage of CD8^(+)TCM(1.5×10^(6) per mouse,n=3)were injected intravenously into Rag-1^(-/-)mice,respectively.At the same time,MOG35-55/CFA and PTX were injected into all of the recipient mice as mentioned before.The Rag-1^(-/-)mice were then observed with continuous assessment of the clinical scores,and sacrificed after 40 days,so as to determine the pathological changes in their brain and spinal cord using hematoxylin-eosin(HE)and Luxol fast blue(LFB)staining.Results The mice in the EAE group showed clinical symptoms within 13~20 days after active immunization,and the symptoms reached the peak 17~25 days later with the neurologic scores of 3.3±0.6.After adoptive transfer with the EAE-derived CD8^(+)T cells,all the recipient Rag-1^(-/-)mice exhibited complete hindquarter paralysis within 27~29 days as well as inflammatory infiltration and demyelination in their brain or spinal cord.After injection with EAE-derived CD8^(+)TCM,CD8^(+)Ter TEM by using same methods,none of the recipient Rag-1^(-/-)mice presented with any clinical signs of EAE or EAE-related pathological changes within 40 days.Conclusions In this study,the adoptive transfer of EAE-derived autoreactive CD8^(+)T cells induced EAE symptomatically and pathologically in Rag-1^(-/-)mice,while the adoptive transfer of autoreactive CD8^(+)TCM failed to induce marked EAE symptoms or EAE-related pathology.Hence our preliminary findings indicate the lack of obvious pathogenic effect of autoreactive CD8^(+)TCM on EAE,and we speculate that the cell population may be predominantly involved in the maintainence of chronic inflammation in CNS,rather than the induction of MS-related immune response.