Human peripheral blood mononuclear cells (PBMCs) were either left unstimulated (left images) or stimulated with 1 µg/mL soluble CD3 antibodies and 5 µg/mL soluble CD28 antibodies for two days. After incubation cells were washed and incubated with 50 ng/mL IL-4 and 20 ng/mL IL-2 for 4 days. Cells were then restimulated with PMA and ionomycin for 2 hours and brefeldin A was added for additionally four hours. Cells were then fixed and intracellularly stained with Anti-TNF-β antibodies as well as with CD3 antibodies. Flow cytometry was performed using the MACSQuant^® Analyzer. Cell debris were excluded from the analysis based on scatter signals.