Death of CLL cells depends on exposure time and CD22 expression. (A) To determine the time-course of cytotoxicity, CLL cells were grown for indicated periods in the presence of 1000 ng/mL BL22 or LMB-9 or in the absence of immunotoxin. Cells were then stained with Annexin V/PI and were analyzed by flow cytometry. To calculate apoptosis induction by BL22, the percentage of spontaneously apoptotic cells in the absence of toxin was subtracted as described in “Patients, materials, and methods.” Results represent the means of 3 individual patient samples. (B) Comparison of apoptosis induction by 1000 ng/mL BL22 for 72 hours and CD22 expression in 22 different patient samples. CD22 was determined as described in the legend to Figure 1. (C) Apoptosis induction of 11 samples expressing CD22 at moderate to high levels (containing more than 50% CD22⁺ cells) compared with 11 low-expressing samples (containing less than 50% CD22⁺ cells). Results represent means ± SEM. The Mann-Whitney U test was used for statistical analysis. (D) Effect of short-term exposure to immunotoxin. CLL cells were incubated for 2, 4, or 6 hours or continuously for 72 hours in the presence of BL22 at 1000 ng/mL. CLL cells exposed to BL22 for short periods were harvested, washed 3 times, and transferred to fresh medium without immunotoxin. After a total culture period of 72 hours, cells were analyzed for apoptosis induction by flow cytometry using Annexin V/PI. Shown are results from a representative experiment; a second experiment gave similar results.