TCR-independent sensitivity to Fas-mediated apoptosis in PBMCs from LGL leukemia patients. (A) The percentage of apoptotic cells was determined by annexin-V-FITC/7-AAD staining in healthy control subjects treated in the absence of IL-2 and anti-CD3-antibody (dose 0) or in the presence of IL-2 (500 IU/mL) in combination with increasing doses of plate-bound anti-CD3 antibody (0.01, 0.1, 1.0, and 10 μg/mL). (B) Flow cytometric dot plots of PBMCs from a representative control donor (lower row) and from an LGL leukemia patient (upper row). Cells that stained with both annexin-V-FITC (x axis) and 7-AAD (y axis) plus cells stained with annexin-V-FITC alone were considered apoptotic and the values are shown in the upper right hand corner of each dot plot. (C) The percentage of specific apoptosis was determined in cells from 8 patients with LGL leukemia and from 3 healthy donors. Cells were assigned to one of 3 treatment groups: (1) IL-2 (500 IU/mL) for 48 hours, (2) anti-Fas (1.0 μg/mL) for 24 hours, or (3) IL-2 for 24 hours plus anti-Fas for an additional 24 hours. H9 cells and PHA/IL-2-activated normal PBMCs served as positive control. Results shown represent the mean percentage of specific apoptosis in individual samples using the equation described in “Apoptosis assay.” Standard error of the mean (SEM) is indicated for experiments performed in triplicate.