PBMCs were isolated from leukapheresis products of healthy volunteers by density gradient centrifugation. CD4+CD25highCD45RA+ “naïve” Treg cells and CD4+CD25highCD45RA- “memory” Treg cells were isolated by FACS (BD FACSAria, BD Biosciences, Heidelberg, Germany) and expanded in vitro. T cells were cultured on irradiated, huCD32-expressing L929 cells with anti-CD3 (OKT3; Orthoclone, Ortho Biotech (Neuss, Germany)) and anti-CD28 antibodies (CD28.2, BD Biosciences; 100 ng/mL each) in the presence of high-dose IL-2 (Proleukin, Chiron, Amsterdam, Netherlands; 300 U/mL) and restimulated once per week. Cultures were continued for 11-12 days and rested for 3-4 days in medium containing IL-2.
For FOXP3 staining and subsequent RNA-extraction we composed an own staining buffer comprising PBS, 2 % Tryptone (Roth, Karlsruhe, Germany) and 0.1 % DEPC (Roth). The buffer was autoclaved, cooled to 4 °C prior to use and will in the following be referred to as “staining buffer”. After 11 days of culture and resting for 4 days in IL-2 containing medium, surface staining with CD4-FITC (BD Biosciences) was carried out according to the manufacturer’s instructions. Up to 7 E07 cells were one time washed in staining buffer and subsequently resuspended in 2 ml ice-cold 70 % ethanol (J.T. Baker, Deventer, The Netherlands) on a vortex. For fixation, cells were incubated for 15’ at -20 °C. After two times washing in 20 ml staining buffer, the cells were resuspended in 1000 µl staining buffer containing 20 µl rat-serum (eBioscience) and 400-800 U recombinant RNasin (Promega, Madison, WI). Following 5’ incubation at 4 °C 50 µl anti-human FOXP3-APC (clone PCH101, eBioscience) were added and incubated for another 25’ at 4 °C in the dark. Finally, cells were washed in 20 ml staining buffer and resuspended in 600 µl staining buffer for FACS-sort. RNA was isolated from FACS sorted cells using the RNeasy Micro Kit (Qiagen, Hilden, Germany) or TRizol® Reagent (Invitrogen, Darmstadt, Germany) according to the manufacturer’s instructions. When RNA was extracted from less than 1 E06 cells, 20 µg glycogen (Glycogen for molecular biology, Roche GmbH, Mannheim, Germany) were added during the TRizol® procedure. Quality was checked with the Agilent Bioanalyzer RNA 6000 Nano Kit according to the manufacturer's instructions.
50 to 200 ng of high-quality RNA were amplified and Cyanine 3-CTP labeled with the One Color Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions (Version 6.5 May 2010). Labeled and amplified RNA was purified with Qiagen RNeasy mini spin columns. RNALabeling efficiency was controlled using the NanoDrop spectrophotometer.
600 ng labeled cRNA were fragmented and hybridized on the Whole Human Genome Expression Array G4851A (8x60K, Agilent) according to the manufacturer's instructions (Version 6.5 May 2010). In brief, 600ng labeled cRNA were combined with 5 µl 10x blocking agent, brought to 24µl with ddH2O and 1µl of 25x fragmentation buffer was added. The cRNA was the fragmented for exactly 30 minutes at 60°C, cooled on ice for one minute, and 25µl of 2x GEx Hybridisation Buffer HI-RPM were mixed with each sample by pipetting. The mix was centrifuged briefly and hybrydised immediately to the microarrays. Arrays were rotated over night for 17 hours at 65°C at HI-RPM.
GE Wash Buffers were all supplied with 0.005% Triton X-102; Arrays were disassembled in an ozone-filtered room in GE Wash Buffer I, washed in Wash Buffer I for one minute at room temperature and the for another minute with GE Wash Buffer II at elevated temperature (buffer was prewarmed overnight at 37°C). The arrays were scanned immediately: Dye channel: Green; Scan Region: Scan Area (61 x 21.6mm); Scan Resolution: 3 µm; Tiff: 20 bit;
Images were processed using Feature Extraction Software 10.7.3.1 (Agilent) and further analyzed using GeneSpring GX 11.0.2 software. Fluorescence signals were normalized (75 percentile) and baseline transformed to the median of all samples. Probes were discarded when: Feature is not positive and significant; Feature is not above background