The Cross-talking Between Decidual γδ T Cells And Trophoblasts In Human Early Pregnancy

The embryo expresses antigens foreign to the mother, which has been viewed as allograft. Induction of the maternal tolerance to the persistence of the alloantigen is a main purpose for the maintenance of pregnancy. It has been proven that the maternal-fetal interface exhibits a Th2 bias, including IL-4, IL-5 and IL-10 cytokines in normal pregnancy, and failure to establish such an immune milieu or Thl bias, such as interferon (IFN)-γand tumor necrosis factor (TNF)-α, is associated with miscarriage.During pregnancy, the maternal-fetal interface is composed of decidual tissue, lymphocytes and trophoblasts. The decidual tissue is the maternal component at the interface that is composed mainly of decidual stromal cells (DSC). The fetal-derived trophoblasts are in closed contact with the decidua. Implantation of human conceptus involves invasion of trophoblast cells into the uterine epithelium and the underlying stroma that undergoes a complex process of proliferation, migration and differentiation of embryo. A large and specific population of immune cells, including gamma-delta T cells, infiltrate constitutively into the decidua, are involved in a series of immune regulations such as production of cytokines, and antigen phagocytosis and presentation.Our previous studies have demonstrated that human first-trimester trophoblasts can produce CXCL16, and theγδT cells express CXCR6 highly. The first-trimester human trophoblast cells have been able to recruitγδT cells homing into decidua by way of CXCR6/CXCL16 interaction.Here we propose that, at the maternal-fetal interface, the gamma-delta T cells play an important role in preventing embryo from maternal rejection. In the present study, we are to further probe into and elucidate the crosstalk between decidualγδT cells and trophoblasts. 1. The decidualγδT cells are increased in human early pregnancy.The decidualγδT cells are obtained from decidua of human early gestation. Fresh decidua tissue were digested with collagenaseⅣ, and then density gradient centrifugation, decidual stromal cells (DSC) and decidual immno-components cells (DIC) were obtained respectively. DecidualγδT cells were isolated and purified byγδTCR MACS Kit. FCM analyzed the phenotype of these cells. We performed FCM for the expression of CD4 and CD8 in the decidualγδT cell population. The enrichedγδT cells were stained with anti-CD4, anti-CD8 and anti-y/8 TCR mAb. Our results showed that the purity of the isolatedγδT cells was above 95% with CD4 and CD8 negative.The proportion ofγδT cells in the peripheral or decidual lymphocyte population was evaluated by flow cytometry in normal pregnancy with non-pregnant controls. The percentage ofγδT cells in CD3+ T cell population was significantly increased both in the periphery and decidua from the normal early pregnancy compared to that of non-pregnancy. The percentages ofγδT cells in decidual CD3+ T cell population were significantly higher than that of the periphery both in normal pregnancy and in non-pregnancy.The cytokine expression feature in the decidualγδT cells from human normal early pregnancy was determined by flow cytometry for a panel of 6 cytokines (IL-2, TNF-α, IFN-γ, IL-4, IL-10, TGF-β1). TheγδT cells did express highly IL-10 and TGF-β, lowly TNF-αand IFN-γ, and little IL-2 and IL-4 in order as follow:IL-10> TGF-β1> TNF-α≥IFN-γ.Therefore, our current results indicate that theγδT cells in human decidua might be key players in the creation of a Th2 bias, which is beneficial for maternal immunotolerance toward the fetus.2. Regulation of decidual gamma-delta T cells on trophoblasts in human early pregnancyIn an attempt to further elucidate the functional regulation of the decidualγδT cells on proliferation, invasion and apoptosis of trophoblasts, we established co-culture of decidualγδT cells and trophoblast cells. The decidual y8 T cells induced human trophoblast proliferation, and neutralizing antibody to IL-10, other than TGF-β, significantly inhibited the stimulatory effect of theγδT cells. When trophoblasts were treated with recombinant human IL-10 or TGF-β, the proliferation was significantly increased by the rhIL-10 treatment, but not by rhTGF-β. Our results demonstrate that decidualγδT cells promot human trophoblast proliferation mainly through secreting IL-10.To test the effects of decidual y8 T cells on human trophoblast invasion, a matrigel-based transwell assay was carried out. The trophoblasts were added into the upper chamber, and the number of cells migrating to the lower surface was counted in 48 h of incubation. The decidualγδT cells increased human trophoblast invasion, and the effect was partially inhibited by anti-IL-10, but not by anti-TGF-β. The rhIL-10 induced human trophoblast invasion, whereas rhTGF-βdid not. Our results suggest that theγδT cell-stimulated increase in trophoblast invasion might partially depend on IL-10 production.To ascertain whether decidualγδT cells affect trophoblast apoptosis, phosphatidylserine externalization was quantified by flow cytometry, using a commercially available annexinⅤ-FITC apoptosis detection kit following the manufacturer's guidelines. It was clearly showed that decidualγδT cells decreased trophoblast apoptosis, and the effect was partially inhibited by anti-IL-10. Moreover, rhIL-10 also decreased trophoblast apoptosis. Our results suggest that the down-regulation of the y8 T cells on trophoblast apoptosis might also partially depend on IL-10 production.3. Regulative role of trophoblasts in decidual gamma-delta T cells in human early pregnancyCo-culture of trophoblasts and decidualγδT cells were established, with treatment of CXCL16 or neutralizing antibody to CXCL16, respectively, for 48 hours. The proliferation ofγδT cells was analyzed by FCM. Trophoblasts or rhCXCL16 increased the proliferation ofγδT cells, and the stimulatory effect could be significantly inhibited by neutralizing antibody to CXCL16. In addition, we performed flow cytometry to analyze granzyme B inγδT cells. The granzyme B was downregulated by trophoblasts or rhCXCL16, respectively, and such effect could be significantly inhibited by neutralizing antibody to CXCL16. Trophoblasts and decidualγδT cells were co-cultured directly or indirectly, respectively, with anti-CXCL16 neutralizing antibody or not. After they were co-cultured for 24 hours, then the surpernatant was collected. We performed ELISA to detect the production of TGF-βand IL-10 in the supernatant. The trophoblasts strengthenedγδT cells to produce TGF-βand IL-10 by direct contact, while anti-CXCL16 neutralizing antibody could abolish the effect.In conclusion, these data above suggest that theγδT cells play a role in the maintenance of pregnancy. DecidualγδT cells are involved in human first-trimester placentation, which favors successful pregnancy.