Marilen Benner

UTERINE LYMPHOCYTES GAIN EXPERIENCE DURING PREGNANCY 39 2 conjugated mAbs for intracellular staining were used: IFN- γ -PC7 (4S.B3) and IL-17-APC- AF780 (eBioscience; eBio64DEC17). A minimum of 200.000 cells per staining was applied. Fluorescence minus one (FMO) and isotype controls were used for gate settings. The data were analyzed using Kaluza V1.1 software (Beckman Coulter). A typical gating strategy used for analysis of T cells and NK cells is depicted in Supplementary Figure S1. Functional analysis Peripheral blood (PBMC), menstrual blood (MMC), and decidua parietalis (DPMC) mononuclear cells were stimulated with phorbol-12-myristate-13-acetate (PMA), ionomycin and brefeldin A (respectively, 12.5 ng/ml, 500 ng/ml and 5 µg/ml; Sigma-Aldrich, St. Louis, USA) for 4 hours at 37°C in a humidified 5% CO 2 incubator. Functionality was determined by measuring the intracellular production of IFN- γ and IL-17 by flow cytometry as described above. Differentiation assay MMC were stimulated in vitro with medium as a control, with anti-CD3/anti-CD28 mAb-coated microbeads in a 1:10 bead-to-cell ratio (Invitrogen, Bleiswijk, The Netherlands) alone, rhIL-15 (100 ng/ml; Gibco Life Technologies) alone, and beads together with rhIL-15 in 96-well U-bottom plates. The rationale for using IL-15 is because IL-2 is hardly detected in decidua and after implantation, endometrial NK cells start to differentiate as a result of local IL-15 production (9). After 5 days of culture at 37°C in a humidified 5% CO 2 incubator, cells were harvested and the presence of T cell subsets and differentiation was measured with flow cytometry. Statistical analysis Statistical analyses were performed using GraphPad Prism 5 (Graphpad software Inc., La Jolla, CA, USA). Non-parametric Kruskal-Wallis with Dunns post-hoc test was performed to compare PBMC, MMC, and DPMC samples. Statistical significance was denoted as values of P < 0.05. All indicated values are mean percentages ± SD. ACKNOWLEDGMENTS The authors would wish to thank all the women who participated in this study, Tadeja Kuret and Konstantina Nikolakopoulou who assisted with the sample processing. The authors are grateful for the support from the Nutricia Research Foundation (grant nr. 2015-50).