Determination of mRNA level of cytokines by real-time PCR. mRNA expression of IFN-γ, IL-2, TNF-α, IL-12, and IL-1β in the cells was evaluated by using specific primers, SYBR Green PCR Master Mix, and a StepOnePlus (Applied Biosystems). Cells were collected on days 1, 3 and 9 after co-culture with or without PBMCs. The y-axis shows the relative quantitative values calculated from comparative Ct values normalized to the control (actin used as calibrator). The standard deviation of the ΔCt value was determined (Applied Biosystems). Representative data from a single donor of three individual experiments are shown.