Requirement of prolonged IL-12–mediated signaling for integrin 6/β1 upregulation in Th1 cells. (A) Th1 cells were cultured in medium supplemented with IL-2 and stimulated with IL-12 as before. At 0, 1, 2, 4, 8, and 16 hours after stimulation with IL-12, a combination of anti–IL-12R β1 MoAb and mouse p40 homodimer were added to the culture as described in Materials and Methods. After 48 hours from the initial stimulation, the cells were harvested, washed, and stained for surface expression of integrin 6/β1. The expression of integrin 6/β1 measured after 48 hours of stimulation with IL-12 (black bar) was taken as 100% of expression. The experiment shown is representative of 2 performed. (B) Stat4 DNA-binding activity in Th1 cells before (lane 1), or after 30 minutes (lane 2) and 60 minutes (lanes 3-5) of stimulation with IL-12 (lanes 2-5). A combination of anti–IL-12R β1 MoAb and mouse p40 homodimer was added either at the time of addition of IL-12 (lane 4) or 30 minutes after (lane 5). Migration of specific Stat4:DNA complexes is indicated by the arrow.