The high HLA-G expression levels on EVT from pregnancies with a male fetus may serve to control elevated maternal immune responses to male Y chromosome-related antigens such as H-Y that can be detected in large proportions in pregnant women carrying male fetuses (49). induction of regulatory T cells. These methods and insights are useful for understanding of maternalCfetal tolerance and development of pregnancy complications. and = 19 to 24). (< 0.05; **< 0.01; ***< 0.005. HLA-G Expression Is Increased on EVT from Term Pregnancy Samples with a Male Fetus. Many IL1R2 biological and clinical differences have been observed between placentas from pregnancies with male and female fetuses. To determine possible sex-related differences in HLA expression by EVT, PCR analysis L-ANAP for the sex-determining region Y (SRY) was performed on DNA isolated from first trimester villous tissue or umbilical cord blood of the samples used in Fig. 2= 2), HLA-G+ EVT >37 wk (= 3) and HLA-G+ chorionic EVT >37 wk (= 3). Unsupervised hierarchical cluster analysis resulted in a dendrogram where each cell type created a distinct cluster (< 0.05; **< 0.01; ***< 0.005. Term Pregnancy HLA-G+ EVT Do Not Increase HLA Expression in Response to Fibronectin, IFN, or TNF. When first trimester HLA-G+ EVT were cultured on fibronectin for 2 days, a marked increase in the intensity of HLA-C and HLA-G around the cells was observed (Fig. 5). Engagement of EVT expressed integrins with fibronectin or other extracellular matrix (ECM) proteins of the decidua were shown to increase their capacity to proliferate or differentiate (1, 6). However, both HLA-G+ EVT and chorionic EVT at term pregnancy did not increase HLA-C and HLA-G expression, despite their high expression of ITGA5, which L-ANAP forms the fibronectin receptor together with ITGB1 (and and < 0.01; ***< 0.005. Conversation The maternalCfetal interface consists of unique sites where maternal tissues and immune cells directly connect with fetal placental tissues containing several types of invasive and noninvasive trophoblasts. In early pregnancy, invasive HLA-G+ EVT are found at the suggestions of anchoring villi, from where they migrate deep into maternal decidual and uterine tissues (13, 27). While HLA-G+ EVT remain present in the placenta at the site of implantation throughout pregnancy, their properties as well as the surrounding decidual leukocyte types switch as pregnancy progresses (16C18). These changes may accommodate placental development and establishment of immune tolerance in early pregnancy as well as maintenance of immune tolerance and fetal growth in the third trimester of pregnancy. All three EVT types analyzed here stained positive for HLA-C, HLA-E, and L-ANAP HLA-G expression compared to IgG controls using circulation cytometry. The constitutive HLA-C and HLA-E expression was highest on first trimester EVT, while chorionic EVT experienced the lowest expression levels of HLA-C, HLA-E, and HLA-G. The differences in constitutive HLA-C, HLA-E, and HLA-G expression on EVT relates to their capacity to induce Treg (Fig. 4 and SI Appendix, Fig. S5). This is substantiated by a previous statement demonstrating that blocking HLA-C during EVTCT cell cocultures decreased their ability to increase Treg proportions (21). However, further studies are needed to demonstrate the mechanisms of Treg induction by EVT as well as how trophoblast differentiation affects their ability to increase Treg. Of importance here is the engagement and downstream signaling of trophoblast expressed integrins with fibronectin or other ECM protein of the decidua that was shown to increase trophoblast differentiation (6, 28). When EVT were cultured on fibronectin or stimulated with the proinflammatory cytokine IFN?, only the first trimester EVT, but not the term pregnancy EVT, responded with up-regulation of HLA-C and HLA-G. These data may suggest that the three types of EVT have unique capacities for antigen processing and presentation under normal (with fibronectin) and proinflammatory (with IFN?) conditions. IFN?-induced increase in HLA expression levels serves to increase T cell receptor (TCR) binding and activates T cells to generate cytolytic and other proinflammatory responses. Furthermore, high constitutive HLA-C cell surface expression levels were also shown to enhance CD8+ effector T cell (Teff) responses to viral contamination as well as allogeneic Teff responses (29C31), but it is usually uncertain how IFN? activation of EVT would impact cytotoxic T lymphocytes (CTL) or Treg induction. HLA-C cell surface expression levels also influence NK cell maturation and function through HLA-C specific KIR2D receptors (31, 32). While the expression of a polymorphic paternally inherited HLA-C antigen by EVT provides a target for maternal NK cells and T cells to recognize and respond to, the HLA-C cell surface expression levels influence how this response is usually shaped (33). Similarly, differences in cell surface expression levels of HLA-E and HLA-G may influence decidual leukocyte responses through expression L-ANAP of HLA-E receptors (e.g., NKG2A/C) and HLA-G receptors (e.g., KIR2DL4, LILRB1 and LILRB2) expressed by decidual NK cells (dNK) and decidual macrophages. The importance of Treg for pregnancy was first established in murine models (34C36) and Treg were shown to be reduced in human decidual tissues.