Pretreatment with IL-12 and response upon NKR activation. (A) Liver lymphocytes were obtained from untreated liver cells from B6 mice. Cells were pretreated with IL-12 at 37°C for specified times. Cells were washed, chilled, and coated with anti-NKRs for 20 minutes. Cells were then washed and cultured for 4 hours. IL-12 was added to the control at the time of assay initiation, and IL-12 remained in assay for 4 hours. (B) Delayed NKR activation. Liver-enriched NK cells (greater than 90% NK1.1⁺) were pretreated at 37°C with IL-12, then held for specified times, and cells were coated with anti-Ly49D or anti-NKG2D at 4°C for 15 minutes, then washed and cross-linked on plastic dishes coated with antirat or antihamster secondary antibodies. Supernatants were collected at 4 to 5 hours.