BM blasts from an FAB M1 AML patient are lysed efficiently in a RDL reaction by effectors from an unrelated donor, mediated by SPM-2. (A) Patient isolated BMMC were stained with fluorescent antibodies specific for CD33 and CD123, respectively, in comparison to isotype controls. P2: gate used for analysis of live BMMCs allowing their discrimination from dead cells. Mean fluorescence Intensities measured for each antigen were converted to antigen densities by calibrating fluorescence intensities with the QuiFi kit (Methods). (B) MNCs from an unrelated healthy donor, expanded ex vivo in the presence of IL-2 (Methods), were used as effectors at an MNC : target (E:T) ratio of 10 : 1 in a 4 h RDL assay. This corresponds to an E:T ratio of NK cells : targets of 2.5 : 1, because NK cells were 25% of all cells in this expanded MNC population. Targets were BMMCs from the AML patient obtained at diagnosis, pre-labeled with calcein AM. SPM-2 was added at 10 nM concentration (right bar). The control reaction was allowed to proceed without added agents (left bar; NKs alone). Cellular lysis was measured by calcein release and plotted as fraction (%) of maximum release. Error bars: arithmetic means over 3 separate experiments (n = 3). (C) Flow cytometric analysis after an RDL reaction. Patient’s BMMCs were treated with effector cells and SPM-2 in a 4 h RDL experiment as in panel A. At the end of the reaction, cells were stained with a fluorescent-labeled CD34-specific mAb and analyzed by FACS. Numbers of CD34pos cells are plotted against the intensity of CD34-specific fluorescence. Left panel: after reaction with effectors alone; right panel: after reaction with effectors plus SPM-2. Numbers in upper right corner: percent of CD34pos cells surviving at the end of the reaction.