Expression of FasL and IL-2 mRNA after CD28 and/or CD3 stimulation (A) and the role of Fas in CD28-induced cell death (B). (A) DWT6.11 cells were left untreated (lane 1) or were incubated in the presence of CD3 plus CD5 (lane 2), CD3 plus CD28 IgG (lane 3), or CD28 IgM alone (lane 4) for 2 and 6 hours as indicated. After these incubation periods, cells were harvested and mRNA was extracted, reverse transcribed, and analyzed by PCR using FasL, IL-2, or β2 microglobulin primer pairs as described in the Materials and Methods. Amplification products were analyzed on a 1% agarose gel, followed by ethidium bromide staining. (B) DWT6.11 cells were incubated in the presence of the indicated amount of the Fas blocking MoAb (Jo2), and either left untreated or treated with the CD3 and CD28 IgM MoAb. After an 8-hour incubation period, cells were PI stained followed by cytofluorometry. Results from a representative out of three independent experiments are shown.