Objective To investigate the effects of different doses of Echinococcus multilocularis infection on splenic CD4+ T cell subsets and immune functions in mice. Methods Sixty female C57BL/6 mice were randomly divided into 4 groups, 15 in each group, including sham operation group (saline), low-dose infection group (50 protoscoleces/mouse), medium-dose infection group (500 protoscoleces/mouse), and high-dose infection group (2 000 protoscoleces/mouse). Mice were inoculated via the hepatic portal vein under anaesthetia with different doses of protoscoleces in saline, whereas the control mice were injected with the same volume of saline. Spleens were taken from 5 mice of each group at 2, 12, and 24 weeks after infection, and ground to isolate lymphocytes. Flow cytometry was performed to detect the memory phenotype, the proportions of different subsets, and LAG3 expression in splenic CD4+ T cells in different experimental groups. Statistical analysis was performed using Graphad Prism 6.0 software. Results At 2 weeks after infection, the proportions of splenic CD4+IFN-γ+ T cells in the low- and medium-dose groups were (7.54 ± 1.44)% and (7.58 ± 3.17)%, respectively, which were higher than that in the sham operation group [(3.52 ± 1.03)%, P < 0.05]; and the proportions of splenic CD4+TNF-α+ T cells were (39.34 ± 4.19)% and (39.53 ± 10.7) %, respectively, which were higher than that of the sham operation group [(22.62 ± 1.50)%, P < 0.01]. At 12 weeks after infection, the proportions of splenic CD4+IFN-γ+ T cells in the low- and medium-dose groups were (16.52 ± 0.77)% and (22.98± 4.32)%, respectively, which were higher than that in the sham operation group [(16.88 ± 2.49)%, P < 0.05]; the proportions of splenic CD4+TNF-α+ T cells were (27.26 ± 2.12)% and (28.36 ± 5.24)%, respectively, which were higher than that of the sham operation group [(19.72 ± 3.87)%, P < 0.05]; and the proportions of splenic CD4+IL-17A+ T cells were (10.70 ± 1.81)% and (11.52 ± 2.68)%, respectively, which were higher than that of the sham operation group [(5.40 ± 1.32)%, P < 0.01]. In addition, the proportions of splenic CD4+IL-4+ T cells in the low- and medium-dose groups were (2.87 ± 0.84)% and (3.50 ± 0.77)%, respectively, which were higher than that of the sham operation group (1.75 ± 0.83)% (P < 0.01); and the proportions of splenic CD4+IL-10+ T cells were (4.63 ± 0.78)% and(7.09 ± 2.42)%, respectively, which were higher than that of the sham operation group [(3.03 ± 0.79)%, P < 0.01]. At 24 weeks after infection, the proportions of splenic CD4+IFN-γ+ T cells, CD4+TNF-α+ T cells, CD4+IL-4+ T cells, CD4+IL-10+ T cells and CD4+IL-17A+ T cells in the medium- and high-dose groups were all higher than those in the sham operation group (P < 0.05), and the proportion of splenic Treg cells in the high-dose group was higher than that in the sham operation group (P < 0.01). Moreover, at 24 weeks, the proportions of splenic CD4+ Treg cells in the three infection groups were all higher than that in the sham operation group; and the proportions of splenic CD4+LAG3+ T cells in the three infection groups were (16.45 ± 4.89)%, (14.54 ± 4.96)%, and (14.62 ± 2.43)%, respectively, which were higher than that of the sham operation group (8.43 ± 3.46)% (P < 0.05). At 24 weeks after infection, the proportions of LAG3-positive CD4+ T cells sereting IFN-γ and TNF-α in the high dose group were (1.67 ± 0.66)% and (0.69 ± 0.27)%, which were significantly lower than those LAG3-negative CD4+ T cells[(5.11 ± 1.81)% and(31.7 ± 12.1)%, P < 0.01]. Conclusion After infected with low and medium doses of E. multilocularis, the mice may make advantages of T1 and T17 immune responses to kill and clear the worms, while infection with a high dose of E. multilocularis may lead to imbalance of T1/T2 and T17/Treg type immune responses, and the occurance of CD4+ T cells up-regulating the expression of LAG3, resulting in the exhaustion of CD4+ T cells and causing chronic alveolar echinococcosis.
