Abstract: Objective　To investigate the effect of Echinococcus multilocularis infection on Tim3 expression and its co⁃expression with immune checkpoint molecules 2B4 and LAG3 in spleen natural killer (NK) cells of mice. Methods　C57BL/6 mice, each weighing (20 ± 2) g, were randomly divided into a high⁃dose infection group (15 mice), a low⁃dose infection group (13 mice), and a control group (11 mice). Mice in the high⁃ and low⁃dose infection groups were inoculated with 2 000 and 50 Echinococcus multilocularis protoscolices via the hepatic portal vein, while animals in the control group was injected with an equivalent amount of physiological saline via the hepatic portal vein. Mouse spleen cells were harvested 12 and 24 weeks post⁃infection, and Tim3 expression and its co⁃expression with 2B4 and LAG3 in NK cells were detected using flow cytometry. Results　There were significant differences in the proportions of Tim3 expression (F = 13.559, P < 0.001) and Tim3 and 2B4 co⁃expression (F = 12.465, P < 0.001) in mouse spleen NK cells among groups 12 weeks post⁃infection with E. multilocularis, and the proportion of Tim3 expression was significantly higher in mouse spleen NK cells in the low⁃dose infection group [(23.84 ± 2.28)%] than in the high⁃dose infection group [(15.72 ± 3.67)%] and the control group [(16.14 ± 3.83)%] (both P values < 0.01), while the proportion of Tim3 and 2B4 co⁃expression was significantly higher in mouse spleen NK cells in the low⁃dose infection group [(22.20 ± 2.13)%] than in the high⁃dose infection group [(14.17 ± 3.81)%] and the control group [(15.20 ± 3.77)%] (both P values < 0.01). There were significant differences in the proportions of Tim3 expression (F = 5.243, P < 0.05) and Tim3 and 2B4 co⁃expression (F = 4.659, P < 0.05) in mouse spleen NK cells among groups 24 weeks post⁃infection with E. multilocularis infection, and the proportions of Tim3 expression and Tim3 and 2B4 co⁃expression were significantly lower in mouse spleen NK cells in the high⁃dose infection group [(20.55 ± 7.04)% and (20.98 ± 7.12)%] than in the control group [(31.38 ± 3.19)% and (31.25 ± 3.06)%] (both P values < 0.05), and there were no significantly difference between the proportions of Tim3 expression and Tim3 and 2B4 co⁃expression in splenic NK cells in the low⁃dose infection group [(26.80 ± 6.47)% and (26.48 ± 6.48)%]and the control group(both P > 0.05). There were no significant differences in the proportions of Tim3 and LAG3 co⁃expression in mouse spleen NK cells among groups 12 (F = 2.283, P > 0.05) and 24 weeks post⁃infection (F = 0.375, P > 0.05). In the low⁃dose infection group, there were no significant differences in the proportions of Tim3 expression or Tim3 and 2B4 co⁃expression in mouse spleen NK cells 12 (t = −1.137, P > 0.05) or 24 weeks post⁃infection (t = −1.658, P > 0.05), and the proportion of Tim3 and LAG3 co⁃expression increased in mouse spleen NK cells 24 weeks post⁃infection relative to 12 weeks post⁃infection (t = −5.261, P < 0.01). In the high⁃dose infection group, there was no significant difference in the proportion of Tim3 expression in mouse spleen NK cells 12 and 24 weeks post⁃infection (t = −1.546, P > 0.05); however, the proportions of Tim3 co⁃expression with 2B4 and LAG3 increased in mouse splenic NK cells 24 weeks post⁃infection relative to 12 weeks post⁃infection (t = −2.425 and −4.745, both P values < 0.05). Conclusions　The Tim3 expression and Tim3 co⁃expression with LAG3 and 2B4 on spleen NK cells is affected by doses of E. multilocularis infection and disease stages, and present different phenotypes during the course of alveolar echinococcosis. NK cells tend to form an immunosuppressive phenotype with the progression of E. multilocularis infection, which facilitates immune escape and chronic parasitism of E. multilocularis.

Key words: Echinococcus multilocularis, Spleen cell, Tim3, Natural killer cell, Immune checkpoint

摘要： 目的　探讨多房棘球蚴感染对小鼠脾脏自然杀伤（natural killer，NK）细胞Tim3及其与免疫检查点分子2B4和LAG3共表达的影响。方法　取体质量为（20 ± 2）g 的C57BL/6小鼠，随机分为高剂量感染组（15只）、低剂量感染组（13只）和对照组（11只），高、低剂量感染组分别经肝门静脉接种2 000、50个多房棘球蚴原头节，对照组经肝门静脉注射等量生理盐水。分别于感染后12、24周收集小鼠脾脏细胞，采用流式细胞术检测小鼠脾脏NK细胞Tim3表达及其与2B4和LAG3共表达水平。结果　多房棘球蚴感染后12周，各组小鼠脾脏NK细胞Tim3表达比例及其与2B4共表达比例差异有统计学意义（F = 13.559、12.465，P均< 0.01）；低剂量感染组小鼠脾脏NK细胞Tim3表达比例[（23.84 ± 2.28）%]高于高剂量感染组[（15.72 ± 3.67）%]和对照组[（16.14 ± 3.83）%]（P均< 0.01）；低剂量感染组小鼠脾脏NK细胞Tim3和2B4共表达比例[（22.20 ± 2.13）%]高于高剂量感染组[（14.17 ± 3.81）%]和对照组[（15.20 ± 3.77）%]（P均< 0.01）。多房棘球蚴感染后24周，各组小鼠脾脏NK细胞Tim3表达比例及其与2B4共表达比例差异有统计学意义（F = 5.243、4.659，P均< 0.05）；高剂量感染组小鼠脾脏NK细胞Tim3表达比例[（20.55 ± 7.04）%]、Tim3和2B4共表达比例[（20.98 ± 7.12）%]均低于对照组[（31.38 ± 3.19）%、（31.25 ± 3.06）%]（P均< 0.05），低剂量感染组小鼠脾脏NK细胞Tim3表达比例[（26.80 ± 6.47）%]、Tim3和2B4共表达比例[（26.48 ± 6.48）%]与对照组差异均无统计学意义（P均＞ 0.05）。感染后12、24周，各组小鼠脾脏NK细胞Tim3和LAG3共表达比例差异均无统计学意义（F = 2.283、0.375， P均＞ 0.05）。低剂量感染组中，多房棘球蚴感染后12、24周小鼠脾脏NK细胞Tim3表达比例、Tim3和2B4共表达比例差异均无统计学意义（t = −1.137、−1.658， P ＞ 0.05）；感染后24周，小鼠脾脏NK细胞Tim3和LAG3共表达比例较感染后12周上调（t = −5.261， P ＜ 0.01）。高剂量感染组中，多房棘球蚴感染后12、24周小鼠脾脏NK细胞Tim3表达比例差异无统计学意义（t = −1.546，P ＞ 0.05）；但感染后24周，小鼠脾脏NK细胞Tim3和2B4共表达比例、Tim3和LAG3共表达比例均较感染后12周小鼠上调（t = −2.425、−4.745， P均< 0.05）。结论　NK细胞表面Tim3表达及其与LAG和2B4共表达均受多房棘球蚴不同感染剂量及不同病程阶段影响，并在多房棘球蚴病病程中呈现出不同表型。NK细胞在多房棘球蚴感染后随病程进展形成免疫抑制表型趋势，利于多房棘球蚴免疫逃逸及慢性寄生。

关键词: 多房棘球蚴, 脾脏细胞, Tim3, 自然杀伤细胞, 免疫检查点