In the absence of DC stimulation, IL-2 and IL-12 induce NK-cell activation and cytotoxicity but differ in their capacity to promote NK-cell proliferation. Size (forward scatter [FSC]), granularity (side scatter [SSC]), and surface expression of the CD56 and CD16 epitopes were quantified by flow cytometry. FSC and SSC were measured on purified NK cells cultured without (NK only) or with 80 IU/mL rIL-2 (NK + IL-2) or 500 pg/mL rIL-12 (NK + IL-12) for 7 days. CD56 and CD16 expression was quantified on gated CD3^- lymphocytes, which lacked autofluorescence in channel FL-3 and were located in the FSC/SSC lymphocyte gate (dotted line). Percentages indicate NK-cell blasts, CD16^-CD56^bright, CD16⁺CD56^bright, and CD16⁺CD56^dim cells. Note that most of the peripheral blood NK cells are CD56^dim but not CD56^- directly after isolation from PBMCs. The data are representative of 3 independent experiments. (B) Purified peripheral blood NK cells were cultured without cytokines (NK only •) or with 80 IU/mL rIL-2 (NK + IL-2 ○) or 500 pg/mL rIL-12 (NK + IL-12 ▾) for 7 days. NK cytotoxicity of the cultures was determined by ⁵¹Cr release assay against the MHC class I-negative target LCL721.221. Radiolabeled LCL721.221 targets were exposed for 4 to 6 hours to the NK-cell effectors, which were the responder lymphocytes from the original or primary DC/NK cocultures. The effectors were added as total lymphocytes, without adjustment for specific numbers of NK-cell blasts, to ascertain differences between the different DCs in stimulating NK cells in the primary cultures. The effector-target ratio is indicated along the X-axis, and specific lysis is plotted against the Y-axis. The data are representative of 3 independent experiments. (C) Proliferation of purified peripheral blood NK cells was determined by ³H-thymidine incorporation after 6 days of culture alone (0) or with 80 IU/mL rIL-2 (+ IL-2) or 500 pg/mL IL-12 (+ IL-12). Data points represent the average of triplicate microwells and their standard deviation. These data are representative of 3 independent experiments.