Nucleic acids used for stimulation of human immune cells

Human PBMCs were isolated from heparinized blood of healthy donors upon informed consent and approval by the local ethics committee by standard Ficoll-Hypaque density gradient centrifugation (Ficoll 1.078 g/ml). PBMCs were resuspended in complete medium prepared of RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 2% heat-inactivated human serum (1 h, 56°C). For stimulation experiments, RNA was encapsulated with DOTAP (N-[1-(2, 3-dioleoyloxy)propyl]-N, N, N-trimethylammonium-205 methylsulfate) (Carl Roth GmbH Karlsruhe, Germany) at a ratio of 3 μl DOTAP per 1 μg of RNA in Opti-MEM reduced serum medium (Life Technologies) and incubation for 10 min at room temperature. DOTAP encapsulation is necessary to deliver RNA into PBMCs. For transfection experiments, cells were stimulated with RNA at final concentrations of 500, 250 and 125 ng/ml. Where indicated, methylated or unmethylated oligoribonucleotides (ORNs) and native tRNA^Lys3 were mixed with bacterial RNA prior encapsulation with DOTAP. As a positive control, PBMCs were stimulated with bacterial RNA (see below) and TLR7/8-agonist R848 (1 μg/ml) (Invivogen, San Diego, USA). All stimulations were performed in duplicate wells per individual donor at a density of 2 × 10⁵ cells/well PBMCs in a 96-well flat bottom plate. Cells were incubated in a humidified 5% CO₂ atmosphere at 37°C for 20 h. Cell-free supernatants were analyzed by sandwich ELISA for secretion of IFN-α (Affymetrix eBioscience, Frankfurt, Germany) according to the manufacturer’s protocol. Where indicated, values were normalized to cytokine production induced by stimulation with bacterial RNA to account for donor variation.