Isolation and Stimulation of PBMC

PBMC were isolated using the standard protocol for Ficoll density gradient centrifugation followed by erythrocyte lysis as described before [13 (link)]. Briefly, PBMC were cultured in “Very low endotoxin” VLE Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 100 U/mL Penicillin, 100 µg/mL Streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 10% (v/v) serum. Cultures of PBMC were set up at least in triplicates (4 × 10⁵ cells/well) in 96-well flat bottom plates (BD Biosciences, Heidelberg, Germany) as described [13 (link)] and rested for 30 min. Where indicated, recombinant human IL-2 (1 ng/mL; Biotechne), IL-12 (1 ng/mL; Biotechne), or both was added before stimulation of the cells with heat-inactivated S. aureus (Pansorbin Cells Standardized, 0.05% (v/v), Calbiochem, Merck, Darmstadt, Germany) 30 min later. Unstimulated PBMC served as negative control. After 16 h, PBMC were harvested for further analyses.
In some experiments, PBMC from healthy donors were cultured in the presence of 4% (v/v) serum from patients. Therefore, the sera from all patients were pooled for each time point.