Marilen Benner

THREE TYPES OF TREG CONTROL PLACENTAL INFLAMMATION 67 3 scraping the decidua parietalis from the chorion. Decidua basalis was macroscopically dissected from the maternal side of the placenta. Collected decidual tissues were washed with PBS, minced and thereafter digested with 0.1% collagenase type IV and 0.01% DNase I (Sigma- Aldrich) gently shaking in a water bath for 75 min at 37ºC. After digestion, released lymphocytes from 1st trimester and term placenta decidua were washed with RPMI 1640 (Life technologies) containing 10% FBS (Atlanta Biologicals) for 8 min at 1800 rpm and filtered through 100 mm, 70 mm and 40 mm sieves (BD, Labware; NJ). Lymphocytes were dissolved in 20 mL 1.023 g/ ml Percoll (GE Healthcare) and layered on a Percoll gradient (10 mL 1.080 g/ml; 15ml 1.053 g/ ml) for density gradient centrifugation (25min, 2000rpm). Decidual lymphocytes were isolated from the 1.080 – 1.053 g/ml interface, and decidual macrophages were isolated from the 1.053 – 1.023 g/ml interface. Cells were washed twice with RPMI and directly stained for flow cytometric analysis on a BD LSR-II or for FACS sort on a BD FACS ARIA-II. Peripheral blood CD4+ T cells were isolated using a RosetteSep human CD4+ enrichment cocktail (Stem Cell Technologies) followed by Ficoll (GE Healthcare) density gradient centrifugation (20 min, 2000 rpm). Flow Cytometry Antibodies used for flow cytometry are listed in the Key Resources Table. For surface staining, cells were stained for 30 min on ice in PBS 1% NCS. For intracellular staining, cells were fixed and permeabilized using the eBioscience FOXP3 staining kit (eBiosciences). For detection of intracellular cytokines, CD4+ T cells were stimulated for 6 hours with phorbol 12-myristate 13-acetate (PMA; 1 mg/ml; Sigma) and Ionomycin (1 mg/ml; Sigma), and Golgistop was added for the last 4 hours (1 ml/ml; BD Biosciences). Cells were fixed and permeabilized using the BD CytoFix/CytoPerm kit (BV Biosciences). Acquisition and analysis was performed on a LSR-II (BD) using FACS Diva software. t-SNE analysis was performed using FlowJo 10 software. Suppression of T cell proliferation assay Purified decidual or peripheral blood CD4 + T cells were sorted on a BD FACS Aria into four fractions (CD4 + CD25 HI , CD4 + PD1 HI , CD4 + TIGIT + and CD4 + Teff) according to the gating strategy described in Figure S1. Decidual CD8 + T cells were obtained from the same decidual sample. For CFSE labeling CD4 + Teff and CD8 + cells were resuspended in PBS at a concentration of 0.2 -1.0 x106 cells per ml. CFSE (Invitrogen) was added in a 1:2500 dilution and cells were incubated for 5 min in a water bath at 37ºC. Cells were washed with X-VIVO 10 supplemented with 50U IL-2 and 5% human AB serum (Corning) for 8 min at 1800 rpm. Cells were resuspended at 0.4 x106 cells per ml in X-VIVO 10 supplemented with 50U IL-2 and 5% human AB serum. Treg were cultured with the CFSE- labeled Teff in a 1:2 ratio of 20.000 Treg: 40.000 Teff cells, with the addition of Dynabeads Human T-Activator CD3/CD28 (1 ml/ml). After four days, cells were collected and stained for CD4-PerCP, CD8-Alexa700 and CD45-PacificOrange. CFSE dilution of CD4 + Teff and CD8 + T cells was analyzed using the proliferation analysis tool of FlowJo v7.6.5 software. The percentage of undivided cells (generation 0) was calculated based on the total