Abstract: Objective　To examine the changes in the immune functions of CD8+ T cells in the spleen of mice following Echinococcus multilocularis infections at various doses and at different time points. Methods　The E. multilocularis protoscoleces were collected, and E. multilocularis infection was modeled in mice via the hepatic portal vein at doses of 50 (low?dose), 500 (medium?dose) and 2 000 protoscoleces (high?dose), while physiological saline served as controls. Mouse spleen was isolated 2 (early?stage), 12 (middle?stage) and 24 weeks post?infection (late?stage), and spleen lymphocytes were harvested. The phenotype of memory CD8+ T cells and 2B4 expression were quantified in the mouse spleen, and the secretion of interferon (IFN)?γ, tumor necrosis factor (TNF)?α, interleukin (IL)?17A and IL?10 was measured. Results　A central?memory phenotype was predominant in the CD8+ T cells in the spleen of mice at the early stage of high?dose protoscolece infections, and the proportion of central?memory CD8+ T cells was significantly greater in the high?dose group than in the control group (35.50% ± 2.00% vs. 25.90% ± 2.46%, P < 0.01), while a effector? memory phenotype was predominant in the CD8+ T cells in the spleen of mice at the late stage of medium? and high?dose protoscolece infections, and the proportions of effector?memory CD8+ T cells were significantly greater in the medium? (25.70% ± 4.12%) and high?dose group (28.40% ± 4.12%) than in the control group (10.50% ± 6.45%) (P < 0.05). The proportions of the central?memory CD8+ T cells were significantly higher in the high?dose group than at middle and late stages than at the early stage (P < 0.01), and the proportion of effector?memory CD8+ T cells was significantly greater in the high?dose group at the late stage than at early and middle stages (P < 0.05). The secretion of IFN?γ and IL?17A by spleen CD8+ T cells was elevated in the low? and medium?dose groups at the early stage of infection, and high?dose protoscolece infection promoted the secretion of IFN?γ and TNF?α by spleen CD8+ T cells; however, the levels of IFN?γ and TNF?α were significantly lower at the late stage than at the early and middle stages (P < 0.05). In addition, high 2B4 expression was detected in spleen CD8+ T cells in the middle? and high?dose groups at the late stage of infection, and the 2B4 expression was significantly higher in the medium? (4.73% ± 1.56%) and high?dose groups (4.94% ± 1.90%) than in the low?dose group (2.49% ± 0.58%) and the control group (2.92% ± 0.60%) (P < 0.05). Conclusions　E. multilocularis may be killed and eliminated through the host immune responses at the middle and late stages of low? and medium?dose protoscolece infections, while high?dose protoscolece infections may trigger the upregulation of 2B4 expression in mouse spleen CD8+ T cells at the late stage, which leads to immune exhaustion and the resultant chronic infections.

Key words: Alveolar echinococcoisis, Echinococcus multilocularis, Spleen, CD8+ T cell, Immune exhaustion, 2B4, Mouse

摘要： 目的　观察不同时间、不同剂量泡球蚴感染对小鼠脾脏CD8+ T细胞免疫功能耗竭的影响。方法　采集泡球蚴原头蚴虫体，经肝门静脉建立低（50个原头蚴）、中（500个原头蚴）和高剂量（2 000个原头蚴）泡球蚴感染小鼠模型，并设生理盐水对照组。于感染后早（2周）、中（12周）、晚期（24周）分别取小鼠脾脏，研磨分离淋巴细胞。流式细胞术检测不同实验组小鼠脾脏中记忆性CD8+ T细胞表型、免疫抑制性分子2B4表达水平，及其分泌γ?干扰素（IFN?γ）、肿瘤坏死因子?α（TNF?α）、白细胞介素?17A（IL?17A）和IL?10的能力。结果　在感染早期，高剂量泡球蚴感染诱导小鼠脾脏CD8+ T细胞以中心记忆性表型为主，其比例为（35.50 ± 2.00）%，显著高于对照组（25.90% ± 2.46%）（P < 0.01）；在感染晚期，中、高剂量泡球蚴感染均诱导小鼠脾脏CD8+ T细胞以效应记忆性表型为主，其比例分别为（25.70 ± 4.12）%和（28.40 ± 4.12）%，均显著高于对照组（10.50% ± 6.45%）（P均 < 0.05）。高剂量泡球蚴感染中期和晚期，中心记忆性CD8+ T细胞比例显著低于感染早期（P均 < 0.01）；高剂量泡球蚴感染晚期，效应记忆性CD8+ T细胞比例显著高于感染早期和中期（P均< 0.05）。低、中剂量泡球蚴感染早期，脾脏CD8+ T细胞分泌IFN?γ和IL?17A能力显著增强，而高剂量泡球蚴感染虽然促进小鼠脾脏CD8+ T细胞分泌IFN?γ和TNF?α能力，但感染晚期其分泌IFN?γ和TNF?α能力显著低于早期和中期（P均< 0.05）。此外，中、高剂量泡球蚴感染晚期小鼠脾脏CD8+ T细胞表面免疫抑制性分子2B4呈高表达，其比例分别为（4.73 ± 1.56）%和（4.94 ± 1.90）%，均显著高于低剂量组（2.49% ± 0.58%）和对照组（2.92% ± 0.60%）（P 均< 0.05）。结论　在低、中剂量泡球蚴感染中期和晚期，机体利用自身免疫应答能力对虫体起到杀伤和清除；而高剂量泡球蚴感染晚期可诱导小鼠脾脏CD8+ T细胞上调2B4分子表达，导致免疫耗竭，造成慢性寄生。

关键词: 多房棘球蚴病, 泡球蚴, 脾脏, CD8+ T细胞, 免疫耗竭, 2B4, 小鼠