Gene Set Enrichment Analysis.To ide

Gene Set Enrichment Analysis.To identify the functional differences that reflect the transcriptional differences between EVT and VT, a Gene Set Enrichment Analysis (GSEA) was performed. GSEA is a computational method that uses an a priori-defined set of genes and determines which “functional” sets of genes are up-regulated. GSEA software was used to generate a list of functional gene sets that are specifically overrepresented in VT or EVT. Interestingly, in EVT, 14 functional gene sets of the 20 most significantly enriched gene sets were associated with direct immune and lymphocyte activation (SI Appendix, Table S2). The core genes of these immune-activation–related pathways are cytokines such as Epstein–Barr virus-induced gene-3 (EBI3), tumor growth factor beta-1 (TGF-β1), TGF-β2, IL-8, and the cell-surface molecule CD276 (B7-H3) and cytotoxic and regulatory T-cell molecule (CRTAM), which can all directly influence lymphocyte binding and are potent inhibitors of lymphocyte activation (SI Appendix, Fig. S7). EBI3 is known to dimerize with two α-chains, IL-12(p28) and IL-12(p35), to form the immune-suppressive cytokines IL-27 and IL-35 (30). However, neither of these IL-12 α-chains are expressed by EVT. How EIB3 functions in EVT and whether EBI3 can form homo-dimers or pairs with an unknown α-chain remain to be determined. The functional gene sets enriched in VT were less significant (increased false discovery rate) and more diverse in function (SI Appendix, Table S3).Coculture of EVT with Sample Matched Maternal Leukocytes.To evaluate the implications of the striking immune-activating and -regulating potential of EVT demonstrated in the GSEA, VT- and EVT-enriched cocultures with all major maternal leukocyte subsets (dNK, dMɸ, CD4+, and CD8+ dT cells) were established. For this sample, matched trophoblast and decidual leukocytes or trophoblasts and peripheral blood leukocytes from unrelated nonpregnant donors were used. The FACS sorting strategy for all cell types can be found in SI Appendix, Fig. S8. Microscopy images revealed that EVT adhere to fibronectin with a distinct, large, spread-out morphology and interact with multiple other EVT. Furthermore, HLA-G+ EVT with various sizes and morphology are found in the cell cultures whereas VT are homogenous, small, rounded cells that do not adhere to fibronectin (SI Appendix, Fig. S9A). Light microscopy also revealed clustering of NK, CD4+, and CD8+ T cells around the large EVT. Multiple NK or T cells can interact with one EVT, either at the cell body or at filopodia-like structures that spread out from the EVT cell body (SI Appendix, Fig. S9B). No signs of cytolysis of EVT were observed in any of the trophoblast–leukocyte cocultures.