3.5x105 cells/well of the freshly isolated CD4+ cells were seeded into 100 µl of medium within a 96-well plate by using a multichannel pipette. Cells were subsequently activated in separate wells by αCD2/αCD3/αCD28 MACSiBead™ particles (T cell activation/expansion kit, human, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Sampling during a total time period of 24 h was conducted, collecting cells of each donor from three separate wells at intervals of 2 h. Cells were pelleted and lysed immediately by QIAzol® Lysis Reagent from miRNeasy Micro Kit (Qiagen, Hilden, Germany). Samples were frozen (-70 °C) for less than 2 months before RNA extraction.
Venous blood samples were obtained from two age and gender matched volunteers (female; Donor 1: Age 26 years; Donor 2: Age 23 years). Samples were collected using lithium heparin containing vacutainer collection tubes (S-Monovette®, Sarstedt AG& Co. KG, Numbrecht, Germany). PBMCs were isolated by Ficoll density gradient centrifugation. CD4+ T cells were isolated by negative selection (Human CD4+ T cell Isolation Kit, Miltenyi Biotech, Bergisch Gladbach, Germany). Cells were resolved and cultured in RPMI 1640 medium (Life Technologies GmbH, Darmstadt, Germany), supplemented with 10 % heat inactivated fetal bovine serum (Biochrom GmbH, Berlin, Germany), penicillin (100 U/mL) and streptomycin (100 μg/mL)
Total RNA was extracted in a semi-automated procedure, using QIAcube robot systems and following the manufacturers` instructions for miRNeasy Micro Kit (Qiagen, Hilden, Germany). The RNA was quantified by Qubit® Fluorometer using Qubit® RNA HS Assay Kit (Thermo Fisher Scientific Inc., Waltham, Massachusetts, United States). RNA integrity was verified using Agilent 2100 Bioanalyzer instrument and RNA Pico Kit (Agilent Technologies, Santa Clara, California, United States). To determine mRNA expression profiles, 25 ng total RNA of each sample was utilized within mRNA One-Color Microarray-Based Gene Expression analysis.
The synthesis of labeled cRNA was performed using Low Input Quick Amp Labeling Kit (One-Color) (Agilent Technologies, Santa Clara, California, United States). The cRNA samples were purified by RNeasy Mini Kit (Qiagen, Hilden, Germany) and concentrations of the purified samples were determined by NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, United States) following the instructions of the labeling kit.
Hybridization was performed using the Gene Expression Hybridization Kit and Human SurePrint G3 Gene Expression Microarrays (V3, G4851C; Agilent Technologies, Santa Clara, California, United States) following the manufacturer’s protocol.
Arrays were scanned with a resolution of 3 µm. Raw data was extracted using Feature Extraction software (Agilent Technologies, Santa Clara, California, United States).
Gene expression after T cell activation (Timepoint 8)
Raw expression values were background corrected, quantile normalized and log2 transformed using the limma R-package (method = normexp, offset = 16).