Marilen Benner

CHAPTER 3 68 number of cells. The division index was calculated by FlowJo software and reflects the average number of divisions per cell. Suppression of T cell cytokine production assay Purified decidual CD4 + CD25 HI , CD4 + PD1 HI , CD4 + TIGIT + , CD4 + Teff and CD8 + T cells were obtained as described above (Figure S1). CD4 + Teff cells were labeled with CFSE (Invitrogen) and CD8 + cells were labeled with CD45-Alexa700. Treg were cultured with CD4 + and CD8 + Teff in a 1:1:1 ratio (20.000 Treg: 20.000 CFSE + CD4 + Teff: 20.000 Alexa 700+ CD8 + Teff). Control cultures without Treg contained CD4 + and CD8 + Teff in a 1:1 ratio (30.000 CFSE + CD4 + Teff: 30.000 Alexa700 + CD8 + Teff). CD3/CD28 Dynabeads (1 ml/ml) were added to all cultures and after three days, cells were re-stimulated with 2.5 ng/mL PMA in combination with 0.1 mg/mL ionomycin for 6h in the presence of 1 mg/mL GolgiStop (BD Bioscience). Cells were collected and stained for cell surface expression of CD4-PerCP, CD8-Alexa700, CD45-Pacific Orange and intracellular expression of IL-10, IFNg and TNFa upon fixation and permeabilization (CytoFix/Cyto PermTM Plus kit (BD)). Acquisition was performed on an LSR-II (BD) using FACS Diva software for analysis following the gating strategy shown in (Figure S4). RNA isolation and QPCR chip analysis Purified CD4 + T cells from four samples of blood, decidua 6-12wk and d.parietalis >37wk (Figure S1A) were resorted into four types CD4 + CD25 HI , CD4 + PD1 HI , CD4 + TIGIT + and CD4 + Teff (Figure S1B) and collected directly into 600 mL Trizol reagent (Life technologies) supplemented with 0.5 mL glycogen (20mg/ml; Affymetrix) and stored at 80ºC until RNA isolation. Total RNA was isolated using the RNAeasy Micro Kit (QIAGEN) per manufacturer’s instruction. RNA was analyzed on a Nanodrop to determine RNA yield and integrity. RNA quality of all samples was further confirmed by performing a QPCR analysis for GAPDH and FOXP3 expression. In short: RNA was reverse transcribed with Stratagene’s AffinityScript QPCR. cDNA Synthesis Kit and amplification of specific PCR products for FOXP3 and GAPDH were detected using the PerfeCTa SYBR Green Super Mix with Low ROX (QuantaBio) in duplicates. Subsequently, high quality samples were run in duplicate on the BioMark Fluidigm QPCR 96.96 chip. 1.25ml DNA was pre-amplified in a 96-well plate using the Fluidigm PreAmp Master mix combined with 500nM forward and reverse primer of each primer pair (2min at 95ºC, 10 thermal cycles of 15sec at 95ºC and 4min at 60ºC) (primers are listed in Table S1). Exonuclease treatment to remove unincorporated primers was carried out using the Exonuclease I at 40U/ml (New England BioLabs). 2 ml of the Exonuclease I dilution was added to each pre-amplification reaction and incubated in a thermal cycler for 30min at 37ºC and 15min 80ºC. A 5-fold dilution was prepared in TE buffer (10mM Tris-HCl, 1.0mM EDTA, TEKnova, PN T0224). Sample pre-mix for the 48 samples was prepared using 2ml of the prepared cDNA with 20X DNA binding dye sample loading reagent (Fluidigm) and 2X SsoFast EvaGreen supermix with low ROX (Bio-Rad). The assay mix was prepared in a separate 96-well plate consisting of 2X assay loading reagent, 1X DNA suspension buffer and 100mM of mixed forward and reverse primers. Chips were primed, loaded with both assay and sample mix and run on BioMark readout instruments as described by the manufacturer, at Harvard University’s