Lack of IL-15Rα expression on NK cells. (A) Flow cytometry of IL-15Rα expression on ex vivo NK cells. NK cells were expanded from bone marrow precursors from C57BL/6 mice in the presence of 70 nM IL-15 for 10 days. The cells were stripped of any bound cytokine by treating briefly in acetic acid–phosphate-buffered saline (PBS; pH 3)²²  and then stained using anti–IL-15Rα polyclonal antisera (Santa Cruz), followed by a staining with phycoerythrin (PE) anti–goat IgG (Sigma, St Louis, MO). As a positive control, dendritic cells were expanded from bone marrow precursors in the presence of 1 nM GM-CSF (PeproTech) and were stimulated by lipopolysaccharide (LPS; 1μgmL^-1) and interferon-γ (10 nM) for 24 hours to augment their expression of IL-15Rα. (B) Lack of proliferative responses of NK cells to low doses of IL-15. NK cells expanded by IL-15 as described in panel A (> 90% were NK1.1⁺, data not shown) were incubated with the indicated concentrations of IL-15 for 20 hours and pulsed with 1 μCi (0.037 MBq) [³H]-thymidine for 4 hours to measure their DNA synthesis. More than 10 nM IL-15 was needed to induce meaningful proliferative responses in these NK cells, although less than 100 pM IL-2 induced an almost maximum proliferative response from these cells. (C) Induction of NK proliferation by the IL-15trans-presentation. PT-18 cells^24,25  were transfected with a human IL-15Rα expression construct, and clones expressing this molecule were selected using G418. The IL-15Rα⁺ PT-18 cells were first irradiated by 50 Gy of x-ray on the previous day of the experiment and then incubated with indicated concentrations of soluble IL-15 for 60 minutes at 37°C. After an extensive wash with PBS, 100 000 of these IL-15–bearing PT-18 cells were added to equal numbers of NK cells and incubated for 20 hours, followed by a pulse with 1μCi (0.037 MBq)³[H] thymidine for 4 hours. A similar experiment was undertaken by incubating IL-15Rα⁺ MC38 cells with effector NK cells, but those MC38 cells were lysed almost instantaneously, so that we could not observe NK cell proliferation (data not shown). (D) Strong induction of NK-cell mediated killing of IL-15Rα⁺ MC38 cells. IL-15Rα⁺ or IL-15Rα^- (parental) MC38 cells were incubated with 0.5 nM IL-15 for 2 hours concurrent with the ⁵¹Cr labeling prior to the cytotoxicity assay. The cells were then extensively washed in PBS to remove any free IL-15. Twenty-five thousand labeled (with IL-15 and ⁵¹Cr) cells were mixed with various numbers of effector NK cells for 6 hours, and the cell-bound and released radioactivity were measured.