Induction of cell death in normal peripheral human T cells. Normal T cells were cultured with PHA (1 μg/mL) for 3 to 5 days as indicated (D3, D4, and D5), followed by stimulation with the CD3 and the CD28 IgM antibodies. (A) PHA-treated cells were seeded for 24 and 48 hours in a 24-well plate in the presence of the indicated antibodies. After this incubation period, cells were collected and determined for cell death as described in the Materials and Methods. Day-5 PHA-treated cells (B), day-5 PHA-treated cells stimulated with the CD3 (C), or the CD28 IgM antibody (D) for 48 hours, were stained with Hoechst and fixed in 2% paraformaldehyde. Shown are representative confocal images. White arrows indicate condensed and fragmented nuclei. (E) Proteins were extracted from day-2 PHA-treated cells stimulated for 72 hours with CD3 (lane 2) or CD28 IgM (lane 3) antibodies, or from day-5 PHA-treated cells left unstimulated (lane 1) or stimulated for 24 hours with CD3 (lane 4) and CD28 IgM (lane 5) antibodies. Extracted proteins were fractionated by SDS-PAGE and immunoblotted with the FasL MoAb.