Marilen Benner

THREE TYPES OF TREG CONTROL PLACENTAL INFLAMMATION 69 3 core facility. Biomark data were processed using Fluidigm Biomark software. Quality control was based on EvaGreen reagent allowing for detection of unspecific PCR product and formation of primer dimers using the BioMark software. Samples of failed reactions were automatically excluded by the software. The resulting values for 66 primers of 48 samples were normalized by subtracting CT values for GAPDH ( Δ CT) and subsequent conversion into fold-change values relative to the median peripheral blood Teff (2 ΔΔ CT). K-means cluster analysis was performed using the fold-change values of the 66 genes in Express Cluster V1.3 in Genepattern https:// cloud. genepattern.org/gp. Differential expression of significantly correlating genes was based on K-means with a minimum of a 1.5-fold change. Isolation of EVT Isolation of EVT and co-culture with CD4+ T cells was performed as described previously (6) and are also described in detail hereafter. 1st trimester villous tissue was gently scraped from the basal membrane and the tissue was digested for 8 min at 37ºC with a trypsin (0.2%) EDTA (0.02%) solution. Trypsin was quenched with DMEM/F12 medium containing 10% Newborn Calf Serum (NCS) and1%Pen/Strep (all from GIBCO) and filtered over a gauze mesh. Filtrate was washed once and layered on Ficoll (GE Healthcare) for density gradient centrifugation (20 min, 2000 rpm). Cells were collected, washed once and incubated for 20 min at 37ºC in a 30mm tissue culture dish for removal of macrophages. To establish untouched (free of antibody staining) EVT cultures, for each sample the percentage CD45 - HLA-G + EVT was determined by FACS analysis for EGFR1, HLA-G and CD45. Samples with > 8% CD45 - HLA-G + cells were stained for CD45 and sorted for viable CD45- large trophoblast cells. 50.000 CD45 - HLA-G + EVT (calculated based on percentage HLA-G + cells and total cell number) were plated in 48 well cell culture plates (Costar) pre-coated with fibronectin (100 mL 20ng/ml 45min, BD), in DMEM/ F12 (GIBCO) supplemented with 10% NCS, pen/strep and glutamine, insulin, transferrin, selenium (GIBCO), 5ng/ml EGF (Peprotech) and 400 units human gonadotropic hormone (Sigma). Trophoblasts were incubated for 2h at 37ºC and thereafter washed 3 times to remove all non-adherent VT. Cultures resulted in 50%–80% HLA-G+ EVT. Co-culture of EVT, decidual macrophages and CD4+ T cells 50.000 EVT or decidual macrophages were co-incubated with 100.000 CD4 + pT from unrelated blood donors in X-Vivo10 medium (Lonza), supplemented with 5% human AB serum and 50 units/ml IL-2 (Biolegend). For blocking experiments, LEAF-purified anti- HLA-G (MoAb 87G; 20 mg/ml), anti-ILT2 (MoAb GHI/75; 20 mg/ml), anti-PDL1 (MoAb 29E.2A3; 20 mg/ml), anti-TGF β (MoAb 19D8; 25 mg/ml), anti-IL-10R (MoAb 3F9; 20 mg/ml), anti-HLA-DR (MoAb L243; 20 mg/ ml), purified anti-HLA-C (MoAb DT-9; 10 mg/ml) and IgG controls (all from Biolegend), or anti- CD3 (MoAb SPV-T3b; 25 mg/ml) (ThermoFisher Scientific) were added 30 min before addition of CD4 + T cells. For transwell assays, 100.000 EVT or decidual macrophages were plated in 24 well cell culture plates pre-coated with fibronectin, non-adherent EVT were removed and 200.000