Th17 cells were generated in vitro and treated with IL-4 to suppress IL-17 expression as described, then washed and put back into culture for one, two or three days to allow cells to regain IL-17 expression. Primary culture = five days Th17 differentiation and two days rest, as described. Secondary culture = two days re-stimulation in Th0 conditions (anti-CD3, anti-IL-4, anti-IFNγ), Th23 conditions (anti-CD3, anti-IL-4, anti-IFNγ, 20ng/mL IL-23) or Th2 conditions (anti-CD3, 10ng/mL IL-4, anti-IFNγ). Tertiary culture = one, two or three days resting culture. After each phase of culture cells were re-stimulated with PMA, ionomycin and brefeldin A and IL-17 expression was assessed by ICS.