Preliminary Study Of The Source Of Regulatory T Cells At The Maternal-fetal Interface

Objective:To investigate the sources and mechanisms of regulatory T cells at the maternal-fetal interface.Methods:Thymus,peripheral blood,spleen,lymph nodes and decidua were harvested from mice of different pregnancy periods(E0,E4.5,E7.5,E10.5,E13.5,E16.5 and E19.5).The proportion of CD4~+Foxp3~+Treg cells among CD4~+T cells in each sample was determined by flow cytometry.Real-time PCR and Western blotting were used to detect the m RNA and protein expression levels of chemokines(CCL2,CCL7,CCL12 and CCL20)in the uterine and decidua of non-pregnant and E13.5 pregnant mice.Expression of chemokines receptors CCR2 and CCR6 on the surface of Treg cells in the peripheral blood and spleen of non-pregnant and E13.5 pregnant mice were tested by flow cytometry.The chemotaxis of CCL2,CCL7,CCL12 and CCL20 on spleen-derived Treg cells during pregnancy was assessed by an in vitro chemotaxis assay.Results:Treg cells of CD4~+T cells in the thymus of syngeneic and allogeneic pairing mice increased in E10.5 and E13.5 compared with non-pregnant group,but there was no difference between the two pairing groups;Treg cells of CD4~+T cells in the decidua of allogeneic pairing mice is significantly higher than syngeneic pairing mice,and that of the whole pregnancy period is significantly higher than non-pregnant group.It gradually increased by gestational age at E4.5 and E7.5,reached highest peak at E10.5,and gradually decreased by gestational age at E13.5,E16.5 and E19.5.The m RNA and protein expression levels of CCL2,CCL12,and CCL20 in the E13.5 uterine and decidua of syngeneic and allogeneic pairing group were significantly higher than the non-pregnant group,and the allogeneic pairing mice were significantly higher than syngeneic pairing mice.While the m RNA expression level of CCL7 in the E13.5 uterine and decidua of the syngeneic and allogeneic pairing mice was significantly higher than non-pregnant group,and the allogeneic pairing mice were significantly higher than the syngeneic pairing mice.But there was no difference in the protein expression level of CCL7 among the three groups.The expression of CCR2 and CCR6 on Treg cells in E13.5 peripheral blood of syngeneic and allogeneic pairings mice were significantly higher than non-pregnant group,and that of allogeneic pairing mice were significantly higher than syngeneic pairing mice;The expression of CCR2 and CCR6 on the Treg cells in the E13.5 spleen of both allogeneic and syngeneic pairing mice were not significantly different from the non-pregnant group.According to the chemotaxis assays,all of the exogenous chemokines CCL2,CCL7,CCL12 and CCL20 had obvious chemotactic effects on the Treg cells of the spleen of allogeneic pairing mice at E13.5,and the order of chemotactic effects from strong to weak followed as CCL20,CCL12,CCL2 and CCL7.Conclusion:The thymus may be the primary source of Treg cells at the maternal–fetal interface,and Treg cells arrived there through peripheral blood by chemotaxis.