freshly
isolated PBMC were transiently transfected using the nucleofection technology (human T cell Nucleofector kit, 107 cells, 5 µg plasmid DNA, Nucleofector Program U-14, Amaxa Biosystems, Germany), according to the manufacturer's instructions. Immediately after nucleofection, cells were transferred into prewarmed culture medium [500 µl RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine (all from Gibco, Grand Island, NY)]. Four hours post nucleofection, cells were replated in tissue culture plates, which were uncoated or coated with affinitypurified goat anti-mouse immunoglobulin G (HµL; 10 µg/ml, Jackson ImmunoResearch, West Grove, PA), followed by anti-human CD3 monoclonal antibodies (mAb; 1 µg/ml, clone HIT3a, NA/LE, BD PharMingen, San Diego, CA) and anti-human CD28 mAb (1 µg/ml, clone CD28.2., NA/LE, BD PharMingen). Where indicated, T cell activation was performed by treatment with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) and ionomycin (1 µM; both
from Sigma Chemical Co., St. Louis, MO) or by treatment with phytohemagglutinin- P (PHA-P; 1 µg/ml, Sigma Chemical Co.) and human recombinant IL-2 (20 nM, kind gift from Chiron Corp., Emeryville, CA).