POSTER 12Expression of HLA-C molecule by Sw71 blastocyst surrogates

Maternal immune cells must tolerate fetal allo- antigens to establish a healthy pregnancy and remain competent to respond to infections both systemically and in placental tissues. However, the semi-allogeneic embryo is normally accepted, indicating the presence of active mechanisms of immune tolerance and/or fetus strategy to avoid an attack by the maternal immune system. Such a strategy is the lack of HLA class I molecules expression by villous trophoblasts and their atypical expression on extravillous trophoblasts (EVTs) which invade uterine tissues contacting with maternal immune cells. EVTs are the most invasive cells of extra-embryonic origin and express the invariant non-classical HLA class I molecules HLA-G and HLA-E, and the only classical, polymorphic HLA class I molecule HLA-C. Recognition of paternally inherited HLA-C could initiate allo-responses from maternal T and NK cells and the HLA-C cell surface expression levels influence how this response is shaped. We and others have shown expression of HLA-C by EVTs in situ at maternal-fetal interface during early pregnancy. Here we showed the expression of HLA-C molecule by 3D native trophoblast models and by in vitro 3D Sw71 spheroids, resembling human blastocyst during the peri-implantation period. Generated Sw71 spheroids were functional proved by their ability to attach to the epithelial endometrial cells, to migrate and to invade into ECM. We used human first trimester trophoblast cells (Swan 71) and generated stable and functional trophoblast Sw71 spheroids. Early placenta samples obtained from healthy pregnant women directed to elective abortions (6–12 gw, n = 10) were used for preparation of trophoblasts explants. 3D Sw71 spheroids and 2D Sw71 monolayers were cultured in cell culture chambers for 48 hrs. In order to obtain EVTs the primary trophoblast-derived explants were cultured in the same way for 72 hrs. Some of the EVT cultures were propagate for 7 days for spontaneous aggregation and self-organization of spheroid-like structures. Sw71 spheroids and monolayers, as well as expanded EVTs and EVTs-derived spheroids were stained for HLA-C. Both Sw71 monolayers and functional 3D Sw71 spheroids were positive for HLA-C. In line with our findings about HLA-C-positive EVTs in situ at maternal-fetal interface we detected strong HLA-C expression by freshly expanded EVTs. Moreover, EVTs-derived self-organized spheroid-like structures as natural models of human placenta migrated as well and retain intense HLA-C expression. The strong HLA-C expression by 3D Sw71 spheroids which is detected in comparison with primary trophoblast 3D cultures confirm their applicability as models of human blastocyst surrogates to study trophoblasts-maternal immune cells interactions during human implantation.