Reduced IFN-γ production correlates with suppressed TCR-induced phosphorylation of p70S6K in activated human T cells in the presence of FTI. (A) Donor PBMCs were seeded at 200 000 cells per well in triplicate in 96-well plates, along with SEA (final concentration 0.1 μg/mL) with or without R11577 (20 μM). For control, PBMCs remained unstimulated. Supernatants were collected at 20 hours and assessed for IFN-γ content by ELISA (R&D Systems). Results represent the mean (± SD) of triplicate cultures. Similar results were seen with 3 additional human donors. (B) Human CD4⁺ T cells were enriched from buffy coats using anti-CD4 mAb-coated beats together with column separation systems. Stimulation of enriched CD4⁺ T cells was achieved for the indicated time points by beads coated with anti-CD2/anti-CD3/anti-CD28 antibodies. In FTI treatment, cells were preincubated for 3 hours with SCH 66336 at indicated concentrations and FTI was present during the entire stimulation. After the indicated time points, stimulation of human T cells was stopped by placing the cells on ice, and cell lysates were analyzed by SDS-PAGE for the presence of phospho-p70S6K. Blotting for actin was used as a loading control. The data are representative of 3 independent experiments.