c-FLIP is highly up-regulated in short-term activated T cells. (A) Freshly prepared human peripheral T cells (d0) were stimulated with either PHA-L (5 μg/mL) or CD3 antibody (coated, 10 μg/mL OKT-3) for 16 hours. Short-term activated T cells (d1) were washed and incubated with 25 U/mL IL-2 for up to 6 days (d6). c-FLIP, XIAP, Bcl-xL, and Bcl-2 expression were determined by Western blot analysis. Erk2 served as a loading control. (B) Human peripheral T cells were treated for 16 hours with 5 μg/mL PHA-L in the presence or absence of 50 nM PD098059 (MEK1 inhibitor), 50 nM SB203580 (p38 inhibitor), 1 μM JNK-II, or 1 μM cyclosporine A (CsA). Subsequently, cells were analyzed by Western blotting. (C) Isolated peripheral T cells were stimulated with 5 μg/mL PHA-L or PHA-L plus 1 μM CsA for 16 hours. T-cell activation was determined by flow cytometric detection of CD69, CD25, and CD95 surface expression.
