DISC formation in PBMC from LGL leukemia patients and from healthy donors. (A) Fas-mediated DISC formation was determined after 45 minutes and after 6 hours of cross-linking with (500 ng/mL) anti-Fas (APO-1, IgG3) antibody (lanes 1, 3, 4, 6-9, 11-14) in samples from 5 patients with LGL leukemia. Cells were cultured for 24 hours with medium in the absence (−, lanes 1, 3, 6, 7, and 11) and presence of 500 IU/mL IL-2 (+, lanes 2, 4, 5, 8-10, 12, 13) for 24 hours, or PHA (1 μg/mL) plus IL-2 (500 IU/mL) for 5 to 7 days (indicated as PHA in lane 14). IgG3 isotype control antibody (500 ng/mL) was added to demonstrate specificity of protein interactions with the FasR (lanes 2, 5, and 10). After immunoprecipitation and gel electrophoresis, Western blot analysis was performed to detect caspase-8 (top panel) and FADD (bottom panel) in these FasR immunoprecipitates. Immunoprecipitation after anti-Fas antibody cross-linking of H9 cells (T-cell leukemia cell line) was used as a positive control. (B) Whole-cell lysates were prepared from a fraction of cells studied in these immunoprecipitation experiments after 1 and 6 hours of cross-linking with anti-Fas (Apo-1) antibody and isotype control (IgG3). Results shown represent the means (± SEM) of caspase-8, and -3/7 activities that were determined by a fluorometric enzyme activity assay. Protein bands for full-length caspase-8 (casp-8), FADD, and an activated cleaved product of caspase-8 (Ac casp-8) are indicated. NS = nonspecific band observed with the anti-caspase-8 antibody.