Secretion of TNF-alpha from whole blood or from supernatants of cytolysis reactions after treatment with SPM-2. (A) Release of TNF-alpha into whole EDTA treated blood from the patient in remission versus the healthy twin after addition of SPM-2. 200 μl samples of EDTA-blood were incubated for 6 h at 37°C with a 10 nM dose of SPM-2. Secretion of TNF-alpha was measured with a commercial ELISA kit (Methods). (B) Supernatants from a 4 h RDL assay using 10 nM dose of SPM-2 and the patient’s autologous BMMCs drawn at diagnosis as targets (red). Effector cells were MACs enriched NKs isolated from PBMCs taken from the patient at diagnosis (left), in remission (center), or from the healthy sibling (right). Secreted TNF-alpha was measured as in panel A. Plotted data are the arithmetic means over 6 independent experiments (n = 6). Error bars represent the standard error of the mean (SEM). Statistical significance was reached with p = .002 (*) and .004 (**) for the differences between cells from the healthy twin and the patient in remission versus at diagnosis, respectively. (C) Supernatants from a 3 h ADCC assay using 10 μg/ml dose of Rituximab and Raji leukemia cells as targets were assayed for TNF-alpha secretion using the same NK effector populations as for panel A (blue). Plotted data are mean values over six independent experiments (n = 6). Statistical significance was reached with p = .0001 (*) and .0003 (**), for the differences between healthy twin and patient in remission versus at diagnosis, respectively.