For intracellular staining (ICS) of AR, PBMC (10 6 per well) were stimulated with medium alone, anti-CD3 (5 mg/ml) + anti-CD28 (1 mg/ml), Staphylococcal enterotoxin B (SEB, 1 mg/ml), PMA (10 ng/mL) + ionomycin (500 ng/mL), influenza H1N1 peptides (H1N1 [New Caledonia/New York], 20 ng/mL/peptide), Fel d1 (50 mg/mL, INDOOR, Charlottesville, VA), Der p1 (50 mg/mL, INDOOR) or Tetanus peptides (3 mg/mL/peptide) in round-bottom 96-well plate (Costar, Corning Inc., Corning, NY). Th1 or Th2 cultures were treated with medium alone or PMA + ionomycin. After 10 hours stimulation (with 2 mM monensin present for the last 8 hours), the cells were first stained with LIVE?DEAD Fixable Yellow Dead Cell Stain Kit, and then stained for cell surface markers CD4, CD8, CD14, CD123, and CD45RA. After cells were fixed and permeabilized using Fix-Perm (BD Bioscience), the cells were stained with anti-AR, anti-IFNc, anti-IL-2, anti-IL-4, anti-IL-17A, anti-TNFa, anti-CD3 and anti-CD69 (or anti-CD154) intracellularly.