Values represent mean SE, with 5 mice per group. Inhibitory receptors and IL-12 Our previous studies17 demonstrate that IL-12 can override the ability of Ly49G2 to inhibit Ly49D activation. in the nuclear and the cytoplasmic compartment, but mRNA half-life was not affected. Fifteen minutes of IL-12 pretreatment was sufficient to result in maximal synergistic activation, indicating that the response of the cells to the IL-12 signal was rapid and immediate. Thus, our data demonstrate that multiple convergent signals maximize the innate immune response by triggering complementary biochemical signaling pathways. Introduction Murine natural Sodium dichloroacetate (DCA) killer (NK) cells express multiple Ly49 receptors1-5 that either inhibit or activate NK cell functions, including cytolysis and cytokine secretion. A functionally comparable family of molecules exists on human NK Sodium dichloroacetate (DCA) cellsthe killer cell immunoglobulin-like receptors (KIRs). The inhibitory Ly49 receptors (Ly49A, C, G and I) inhibit NK cell function on binding of class 1 ligands on target cells.6-8 These Sodium dichloroacetate (DCA) Ly49 inhibitory receptorsand inhibitory KIRscontain cytoplasmic immune receptor tyrosine-based inhibitory motifs (ITIMs) that are phosphorylated on stimulation, leading to the recruitment of SHP-1 phosphatase and attenuation of intracellular signals. In contrast, the ITAM-associated activating receptors (eg, Ly49D and Ly49H) mobilize intracellular Ca2+, induce cytokine mRNA and protein production, and mediate reverse antibody-dependent cellular cytotoxicity (ADCC) in the presence of specific mAbs.9-12 Circulating NK cells expressing activating Ly49 also express coreceptor paired inhibitory Ly49. Thus, effector cells that express the activating Ly49D receptor that binds H2-Dd as a ligand also coexpress, at very high levels, inhibitory Ly49G2 or Ly49A13-15 receptors that also bind H2-Dd and inhibit the activating function. Based on this coexpression, engagement of activating Ly49 NK receptors in vivo appears constantly at odds with inhibitory forces. Our previous studies exhibited that cross-linking of activating Ly49D murine NK cell receptors can potently synergize with IL-12 for selective and synergistic production of IFN-, both in vitro and in vivo. Importantly, IL-12 was the key signal needed for overriding the inhibitory Sodium dichloroacetate (DCA) receptor blockade for cytokine production. Given that there are numerous coreceptor systems in the T-cell system that require 2 signals to induce sufficient cellular activation, we postulated that other NK cell receptors may require 2 positive signals to override the ever-vigilant inhibitory receptor blockade. Thus, we sought to examine a model in which the secretory function of activating receptors, in addition to the Ly49 family, might be brought on by coreceptor function. Furthermore, as reported here, we have now characterized the biochemical pathways required for the expression of IFN- in response to multiple, yet distinct, extracellular signals. Materials and methods IL-8 antibody Reagents Alpha () GalCer (KRN7000) was graciously provided by Kirin Brewery (Tokyo, Japan). The ceramide reagents were first dissolved in DMSO, then diluted in phosphate-buffered saline (PBS) made up of 0.5% Tween 20. Control diluent or PBS was used as a control for all those studies. MAP kinase inhibitors SB203580 (source) and U0126 (source) were used at a final concentration of 1 1 M. Cell lines B-cell lines (A20 and A20/CD1d, generously provided by M. Kronenberg, La Jolla Institute, San Diego, CA) were pretreated with various reagents for 30 minutes at 37C, washed, and mixed with sorted populations, and supernatants were collected for analysis after specified culture time. NK cell isolation Liver NK cells were isolated from C57BL/6 (B6) mice, as previously described.14 Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985). Liver mononuclear cells were used either untreated (15%-25% CD3-, NK1.1+) or after in vivo IL-2 treatment (35%-70% CD3–, NK1.1+), followed by lineage depletion (with CD3, CD19, and CD24) (greater than 90% CD3-, NK1.1+) or after in vitro expansion with IL-2 (6000 IU/mL recombinant IL-2) (Chiron, Emeryville, CA), as previously described.16 In vivo IL-2 treatment was conducted as previously described using a plasmid containing the murine gene.17 Antibodies Sodium dichloroacetate (DCA) used The monoclonal antibodies 4E5 (Ly49D), 3D10 (Ly49H), and 3A10 (NKG2D) were previously described11 or were provided by Dr Wayne Yokoyama (Washington University,.