The Effect Of Adoptive Transfer Of Regulatory T Cells On Adverse Pregnancy With Toxoplasma Gondii Infection

Objective:To investigate the potential role of inhibitory moleculars on regulatory T cells (Tregs) in the abnormal pregnancy outcome of mice induced by T. gondii infection and the effect of adoptive transfer of Tregs on the adverse pregnancy outcome.Methods:To explore the potential role of inhibitory moleculars on Tregs in T. gondii-induced adverse pregnancy outcome, the mice model with T. gondii infection during early pregnancy in vivo were established. The pregnant mice were firstly randomly assigned into infected group and normal control group. Pregnant mice of infected group were injected intraperitoneally with T. gondii tachyzoites on gestation day (GD) 8. Dams were executed on GD14 and the pregnant outcomes were recorded. The apoptosis of Tregs and the expression of CTLA-4 and PD-1 on Tregs in maternal-fetal interface (placenta and uterine tissues) and spleen were detected by flow cytometry; the levels of cytokine TGF-Pβ、IL-10 to IFN-y were analyzed by ELISA. In order to further investigate the effect of adoptive transfer of Tregs on the adverse pregnancy outcome, Tregs from maternal-fetal interface and spleen of normal pregnant mice were isolated and stained with CFSE, and then they were separately adoptively transferred into T. gondii-infected pregnant mice at GD9. Dams were also executed on gestation 14, pregnant outcomes were recorded; the expression of CTLA-4 and PD-1 and the distribution of CFSE+ Tregs from maternal-fetal interface and spleen in recipients were analyzed by flow cytometry; the levels of TGF-β、IL-10 and IFN-γ were analyzed by ELISA.Results:Pregnant mice in normal control group showed agile action, while the infected pregnant mice were droopy, shambling and erect fur. Most fetuses were bloodless, smaller size and the placenta had serious hemorrhage in the infected group compared to uninfected pregnant group. The abortion rate of infected group was significantly higher, and the weights of placentas and fetuses from infected mice were significantly less than those from uninfected mice. Our result showed that T. gondii induced the apoptosis of Tregs, the expression of CTLA-4 and PD-1 on Tregs were decreased in the infected groups compared with controls. The ratios of cytokine TGF-β、IL-10 to IFN-γ were decreased in infected groups compared to normal groups. Additionally, we found that the expression of CTLA-4 and PD-1 on Tregs at fetal-maternal interface were higher than that on Tregs in spleen from normal pregnant mice. After we adoptively transferred Tregs from fetal-maternal interface or from spleens of normal pregnant mice into infected pregnant mice, adverse pregnancy outcomes were improved only when Tregs were transferred from the fetal-maternal interface but not from the spleen. In infected mice transferred Tregs from the fetal-maternal interface, there was a significant improvement of the mouse behavior, a decrease in placental hemorrhage and the abortion rate, and an increase in the weights of fetus and placenta compared with untreated infected mice. However, there were no significant differences in these parameters between infected mice given Tregs from spleens and the untreated infected mice. What’s more, the result showed that the quantity of CFSE+Tregs at the maternal-fetal interface and in the spleen were equivalent whether the transferred Tregs came from the spleens or maternal-fetal interface of donor mice. The expression of CTLA-4 and PD-1 were increased at maternal-fetal interface and in spleens of infected mice treated with Tregs both from fetal-maternal interface and spleen compared to untreated infected mice, whereas the degree of increase is greater in the group treated with Tregs from maternal-interface than with Tregs from spleen. Besides, we found that the ratios of IL-10 to IFN-γ and the ration of TGF-β to IFN-y increased markedly in infected group treated with Tregs from fetal-maternal interface but that these did not differ from infected controls in the infected group treated with Tregs from spleen.Conclusions:The inhibitory receptors of CTLA-4 and PD-1 on Tregs play an important role in the adverse pregnancy outcomes induced by T. gondii infection. Adoptive transfer of Tregs from maternal-fetal interface of normal pregnancy mice counters adverse effects of T. gondii infection on pregnancy.