Functional assessment of NK cell expansion. Fresh single cell suspensions of unfractionated splenocytes were obtained from mice in each of the indicated treatment groups (n = 5 to 6 per group) and then assayed for NK cytotoxicity against YAC-1 targets (200:1 E/T ratio) in a standard 4 hour ⁵¹Cr release assay, without the addition of exogenous cytokines (A). Unfractionated splenocytes were also cultured (2 × 10⁵ cells per well) in rhIL-2 plus rmIL-12 for 72 hours, after which cell-free culture supernatants were harvested and assayed for murine IFN-γ by ELISA (B). Results represent the mean percent cytotoxicity (A) or IFN-γ production (B) ± SEM. For both assays, the percentage NK1.1⁺CD3⁻ cells were 2.9 ± 0.2% NK1.1⁺CD3⁻ cells in the control group, 1.8 ± 0.3% in the SCF group, 1.8 ± 0.1% in the IL-2 group, and 3.2 ± 0.2% in the SCF plus IL-2 group. The SCF plus IL-2 group had a significantly greater increase in percent NK1.1⁺CD3⁻ cells compared to the IL-2 alone group (P < .005). Statistical significance comparing the SCF plus IL-2 group to the IL-2 alone group are indicated by the asterisk, and represent P ≤ .0005 (A), and P ≤ .01 (B).