Abstract: Objective　To investigate the effects of Echinococcus multilocularis infection on levels of memory T (Tm) cells and their subsets in lymph nodes of mice at different stages of infection, so as to provide new insights into immunotherapy for alveolar echinococcosis.  Methods　Twenty⁃four C57BL/6J mice aged 6 to 9 weeks were randomly divided into the infection group and the control group, of 12 mice in each group. Mice in the infection group were administered with 3 000 E. multilocularis protoscoleces via portal venous injection, while animals in the control group were administered with an equal volume of physiological saline. Three mice from each group were sacrificed 4, 12 weeks and 24 weeks post⁃infection, and lymph nodes were sampled and stained with hematoxylin and eosin (HE) to investigate the histopathological changes of mouse lymph nodes in the infection group. The expression and localization of T lymphocyte surface markers CD3, CD4, and CD8 were observed in mouse lymph nodes using immunohistochemical staining. In addition, lymphocyte suspensions were prepared from mouse lymph nodes in both groups at different time points post⁃infection, and the levels of Tm cell subsets and their secreted cytokines were detected using flow cytometry.  Results　HE staining showed diffuse structural alterations in the subcapsular cortical and paracortical regions of mouse lymph nodes in the infection group 4 weeks post⁃infection with E. multilocularis. Immunohistochemical staining detected CD3, CD4 and CD8 expression in mouse lymph nodes in both groups. Flow cytometry revealed higher proportions of CD4+ Tm cells [(55.3 ± 4.8)% vs. (38.8 ± 6.1)%; t = -4.259, P < 0.05] and CD4+ tissue⁃resident Tm (Trm) cells [(57.7 ± 3.7)% vs. (34.1 ± 11.2)%; t = -3.990, P < 0.05] in mouse lymph nodes in the infection group than in the control group 4 weeks post⁃infection, and higher proportions of CD4+ Tm cells [(34.6 ± 3.2)% vs. (23.3 ± 7.5)%; t = -2.764，P < 0.05] and CD4+ Trm cells [(44.0 ± 1.9)% vs. (31.2 ± 1.5)%; t = -4.039，P < 0.05] in mouse lymph nodes in the infection group than in the control group 24 weeks post⁃infection. The proportions of CD8+ Tm cells were higher in the infection group than in the control group 4 weeks [(56.8 ± 2.7)% vs. (43.9 ± 5.2)%; t = -4.416, P < 0.01] and 12 weeks post⁃infection [(25.4 ± 2.7)% vs. (12.0 ± 2.6)%; t = -2.552, P < 0.05], while the proportions of tumor necrosis factor (TNF)⁃α+ CD4+ T cells [(15.7 ± 5.0)% vs. (49.4 ± 6.4)%; t = 7.150, P < 0.01], TNF⁃α+ CD8+ T cells [(20.7 ± 5.5)% vs. (57.5 ± 8.4)%; t = -6.694, P < 0.01], and TNF⁃α+ CD8+ Tm cells [7.0% (1.0%)  vs. 31.0% (11.0%); Z = -2.236, P < 0.05] were lower in the infection group than in the control group 24 weeks post⁃infection. Conclusions　Tm cells levels are consistently increased in lymph nodes of mice at different stages of E. multilocularis infection, with Trm cells as the predominantly elevated subset. The impaired capacity of CD8+ Tm cells to secrete the effector molecule TNF⁃α in mouse lymph nodes at the late⁃stage infection may facilitate chronic parasitism of E. multilocularis.

Key words: Alveolar echinococcosis, Echinococcus multilocularis, Lymph node, Memory T cell, Tissue?resident memory T cell 

摘要： 目的　探讨不同阶段多房棘球蚴感染对小鼠淋巴结记忆性T（memory T，Tm）细胞及其亚群表达水平的影响，为多房棘球蚴病免疫治疗提供新思路。方法　24只6 ~ 9周龄C57BL/6J小鼠随机分成感染组和对照组，每组12只；感染组小鼠经肝门静脉注射3 000 个原头节，对照组小鼠注射相同体积生理盐水。于感染后第4、12、24周，每组小鼠分别各随机取3只处死，取淋巴结组织后采用苏木精⁃伊红（hematoxylin and eosin，HE）染色法观察感染组小鼠淋巴结组织病理变化。通过免疫组织化学染色观察小鼠淋巴结组织中T淋巴细胞表面标志物CD3、CD4和CD8分子表达及定位。提取不同感染时间两组小鼠淋巴结淋巴细胞悬液，采用流式细胞术检测Tm细胞亚型及其分泌的细胞因子水平。结果　多房棘球蚴感染后4 周，HE染色显示感染组小鼠淋巴结被膜下的皮质和副皮质区域结构呈弥漫性改变。免疫组织化学染色结果显示，感染组和对照组小鼠淋巴结组织中均有CD3、CD4和CD8分子表达。流式细胞术检测结果显示，感染后4周，感染组小鼠淋巴结中CD4+ Tm［（55.3 ± 4.8）% vs.（38.8 ± 6.1）%；t = -4.259，P < 0.05］、CD4+ 组织驻留记忆性T（tissue⁃resident memory T，Trm）细胞比例［（57.7 ± 3.7）% vs. （34.1 ± 11.2）%；t = -3.990，P < 0.05］均显著高于对照组；感染后24周，感染组小鼠淋巴结中CD4+ Tm［（34.6 ± 3.2）% vs.（23.3 ± 7.5）%；t = -2.764，P < 0.05］、CD4+ Trm细胞比例［（44.0 ± 1.9）% vs.（31.2 ± 1.5）%；t = -4.039，P < 0.05］亦显著高于对照组。感染后 4 周［（56.8 ± 2.7）% vs.（43.9 ± 5.2）%；t = -4.416，P < 0.01］和 12 周［（25.4 ± 2.7）% vs.（12.0 ± 2.6）%；t = -2.552，P < 0.05］，感染组小鼠淋巴结中CD8+ Tm细胞比例均显著高于对照组；感染后24 周，感染组小鼠淋巴结中肿瘤坏死因子（tumor necrosis factor，TNF）⁃α+ CD4+ T［（15.7 ± 5.0）% vs.（49.4 ± 6.4）%；t = 7.150，P < 0.01］ 、TNF⁃α+ CD8+ T细胞比例［（20.7 ± 5.5）% vs.（57.5 ± 8.4）%；t = -6.694，P < 0.01］ 和 TNF⁃α+ CD8+ Tm细胞比例［7.0%（1.0%） vs. 31.0%（11.0%）；Z = -2.236，P < 0.05］均显著低于对照组。结论　多房棘球蚴感染不同阶段小鼠淋巴结组织中Tm细胞均增加，以Trm细胞增加为主；晚期感染阶段，小鼠淋巴结组织中CD8+ Tm细胞分泌效应分子TNF⁃α能力减弱，可能有助于多房棘球蚴慢性寄生。 

关键词: 多房棘球蚴病, 多房棘球蚴, 淋巴结, 记忆性T细胞, 组织驻留记忆性T细胞