(D) Purified NK cells were stimulated with IL12 and/or m157-Tg MEF for 6 hours and stained for IFN protein and mRNA using primeflow RNA assay

(D) Purified NK cells were stimulated with IL12 and/or m157-Tg MEF for 6 hours and stained for IFN protein and mRNA using primeflow RNA assay. to stimulation with primary m157-Tg target cells, which triggers the activation receptor Ly49H, suggesting that NK cells do require multiple signals for IFN production in the context of target cell recognition. Using qPCR and RNA flow cytometry, we found that cytokines, not activating ligands, act on NK cells to express transcripts. Ly49H engagement is required for IFN translational initiation. Results using inhibitors suggest that the Proteasome-Ubiquitin-IKK-TPL2-MNK1 axis was required during Laminin (925-933) activation receptor engagement. Thus, this study indicates that activation receptor-dependent IFN production is regulated on the transcriptional and translational levels. Introduction Natural killer (NK) cells recognize and attack target cells, including cancer and pathogen-infected cells, through a combination of activation and inhibitory receptorCligand interactions. Upon recognition of a target cell through such interactions NK cells can directly induce lysis of the target, but also produces the signature cytokine IFN. Activation receptor dependent IFN production is frequently studied to assess NK cell functionality (1). NK cells can produce IFN in response to cytokines as well, in particular IL-12 in combination with IL-18 results in strong IFN production (2). Yet, unclear is whether these pathways intersect. Production of IFN by NK cells has been shown to contribute to viral control and tumor rejection. For example, NK cells are the main source of IFN during early stages of MCMV infection (3). This IFN produced early during infection contributes to MCMV clearance, particularly in the liver (4). A susceptibility locus on mouse chromosome 10 is associated with impaired MCMV control and decreased NK IFN production, whereas IFN produced by T cells is unaffected (5), providing genetic evidence suggesting NK cell-produced IFN is critical for viral control. IFN production during MCMV infection requires IL-12 and depends on STAT4 (3, 6). In addition, IL-18 synergizes with IL-12 to induce IFN during infection (7). Thus, in the context of MCMV infection the role for cytokines inducing NK cell IFN is well established. NK cell IFN production has been shown to control metastasis formation of B16 melanoma sub-line (8), implicating a role for NK cell IFN in controlling tumors as well. It is well Rabbit Polyclonal to Keratin 19 established that ligation of activation receptors trigger NK cells to produce IFN, but there is a body of evidence suggesting that stimulation through an activation receptor alone is insufficient for optimal IFN production. Stimulation of mouse NK cells with plate-bound antibodies against activation receptors such NK1.1 or Ly49H triggers IFN production (9C11). In contrast, stimulation with soluble antibodies does not induce IFN, whereas soluble anti-Ly49D has been reported to induce phosphorylation of SLP76 and ERK (12). This indicates that soluble antibody is capable to induce NK cell activation but not IFN production. Plate-bound anti-NKG2D dependent NK cell GM-CSF production requires signaling through CD16 (13), suggesting that plate-bound antibody may also trigger Fc receptors. Moreover, antibodies against different receptors synergize for human NK cell IFN and TNF production when coated on the same beads (14) and a combination of activation receptor ligands and adhesion molecules is required on insect target cells to induce IFN by freshly isolated human NK cells (15). Overexpression of activation ligands on certain cell lines induces IFN by resting mouse NK cells, including over-expression of m157 and NKG2D ligands (5, 16, 17). In addition, NK Laminin (925-933) cells stimulated with murine cytomegalovirus (MCMV)-infected macrophages produce IFN in a Ly49H-dependent manner (17). However, transfer of wildtype NK cells into a na?ve host constitutively expressing the Ly49H ligand m157 Laminin (925-933) as a transgene (m157-Tg) did not result in IFN production but rather caused NK cell hypo-responsiveness within 24 hours (18, 19), indicating that additional signals may be required for activation receptor-dependent IFN production. Here we show that activation receptor-mediated IFN production by NK cells indeed requires additional signals which can be provided by cytokines such as IL-12 and IFN. We found that cytokine signaling induces transcription of mRNA, whereas Ly49H signaling resulted in translation of mRNA. Furthermore, efficient IFN production required a specific order of these stimuli. Taken together, this study provides a molecular basis for the requirements of NK activation receptor-induced IFN production. Materials and Methods Mice C57BL/6 (and congenic CD45.1 C57BL/6) mice were purchased from Charles River Laboratories. IL-12R2?/? and RAG1?/? mice were purchased from Jackson Laboratory. DAP12 KI mice were kindly provided by E. Vivier (CNRS-INSERM-Universite de la Mediterranee, France). DAP10?/? mice were kindly provided by M. Colonna. IFNAR1?/? were backcrossed on Laminin (925-933) C57BL/6 background as described previously (20). m157-Tg and Ly49H-deficient B6. BxD8 mice were generated and maintained in-house in accordance with institutional ethical guidelines. Reagents and cell preparation Fluorescent-labeled antibodies used were anti-NK1.1 (clone PK136), anti-NKp46 (29A1.4), anti-CD3 (145C2C11), anti-CD19 (eBio1D3), anti-CD45.1 (A20), anti-CD45.2(104), anti-Eomes (Dan11mag), anti-TNF (MP6-XT22), and anti-IFN Laminin (925-933) (XMG1.2), all from.