Up-regulation of CD95 expression in BCP-ALL blasts after activation via CD40 does not translate into sensitization to apoptosis. (A) Expression of the death receptor CD95 was analyzed by flow cytometry (median; range) in normal peripheral B cells (n = 8) and BCP-ALL blasts (n = 15) after 72-hour culture on nonmodified feeder cells or feeder cells transgenically expressing CD40L. (B) For assessment of receptor-mediated cell death, cells were also removed from feeder cells and stimulated for apoptosis induction with an agonistic CD95 monoclonal antibody (mAb) for 16 hours. Apoptosis was measured by flow cytometry using annexin V and propidium iodide staining (median; range). The increase in spontaneous apoptosis in the absence of anti-CD95 did not exceed 5% regardless of CD40 activation. Despite equivalent up-regulation of CD95 in CD40-activated normal B cells and BCP-ALLs, induction of specific apoptosis is significantly lower in ALL blasts (P = .001). (C) Caspase-8– and caspase-3/7–like proteolytic activities were examined in CD40-activated normal peripheral B cells (n = 4) and BCP-ALL blasts (n = 4) in a luminescent substrate assay after cross-linking with anti-CD95 mAb for 4 hours (caspase-8) and 16 hours (caspase-3/7). The assay was performed in triplicates of 2.5 × 10⁴ cells/well for caspase-8 and 1 × 10⁴ cells/well for caspase-3/7. The increase in CD95-induced caspase activity in normal B cells and BCP-ALL blasts after activation via CD40 is shown. The increase is presented as the ratio of CD40-activated over resting B cells and blasts (mean ± SEM).