Whole blood stimulation
Whole blood was stimulated with 10 μg/mL phytohemagglutinin (PHA, Sigma-Aldrich) for 6 h and 24 h. After 6 h, activation markers on T cells were measured using CD69-APC (clone: REA824), CD71-FITC (clone: REA902), CD154-VioBlue (REA238) and CD25-PE (clone: 3G10), CD3-VioGreen (REA613), CD4-APC-Vio770 (REA623), and CD8-PE-Vio770 (REA734) antibodies and propidium iodide as viability dye (all Miltenyi Biotec, Bergisch-Gladbach, Germany) using a MACSQuant 16 analyzer (Miltenyi Biotec). After 24 h, culture supernatants were collected for cytokine analysis.