J Clin Invest 105:1035C1038. for NKp46/NCR1 and show that NKp46/NCR1 is usually important for the control of reovirus contamination and for successful reovirus-based therapy of tumors. RESULTS The NKp46 receptor recognizes reovirus. NKp46 is usually a receptor particularly important in the recognition of viruses (24, 32, 33). Elobixibat To test if NKp46 recognizes reovirus, we initially incubated Vero cells with reovirus type 3 (Dearing) and decided that the virus adheres to the cells by staining them with an anti-sigma1 monoclonal antibody (MAb) (Fig. 1A). Next, we prepared fusion proteins made up of the extracellular portion of NKp46 fused to human IgG1 and stained Vero cells in the presence or absence of reovirus. NKp46-Ig recognized uninfected Vero cells Elobixibat (Fig. 1B), suggesting that Vero cells express an unknown ligand for NKp46/NCR1. Importantly, following incubation with reovirus, increased NKp46-Ig binding was seen (Fig. 1B). The binding was specific, since little or no increase in the binding of D1-Ig (prepared in a manner similar to that used for NKp46-Ig) was noticed (Fig. 1B, left histogram; the binding of all fusion proteins is usually summarized in panel C). D1-Ig is the membrane-distal Ig-like domain name of NKp46 that is not involved in the binding of NKp46 to its ligands (24). The integrity of the fusion protein was analyzed by Coomassie-stained gels under nonreducing conditions. As expected, NKp46-Ig appears as a single band slightly larger than 250 kDa (Fig. 1D). Open in a separate window FIG 1 NKp46 is usually activated by reovirus. (A) Vero cells were incubated with reovirus for 14 h and stained with anti-sigma1 antibody (open gray histogram). The filled gray histogram depicts the background staining of Vero cells with the secondary MAb in the absence of reovirus. The background staining of Vero cells in the presence of reovirus was comparable and is not shown. The empty black histogram depicts the staining of uninfected Vero cells with anti-sigma1 antibody. (B) FACS staining of Vero cells incubated for 14 h in the presence or absence of reovirus. Staining was performed with D1-Ig and NKp46-Ig, as indicated around the axis. The filled gray histograms depict the background staining of Vero cells with the secondary MAb in the absence of reovirus. The background staining of Vero cells in the Elobixibat presence of reovirus Mouse monoclonal to ELK1 was comparable and is not shown. The empty black histograms depict the staining of uninfected Vero cells with the fusion proteins indicated. The empty gray histograms depict the staining of Vero cells preincubated with reovirus and stained with the fusion proteins indicated. Shown are the results of one representative experiment out of three performed. (C) The median fluorescence intensity (MFI) of anti-sigma1 antibody, D1-Ig, and NKp46-Ig staining of uninfected and reovirus-infected cells in three different experiments. Each error bar represents the standard deviation (SD). Statistically significant differences are indicated. *, 0.05; ns, not significant. (D) Coomassie staining of the NKp46-Ig fusion protein used in panel B after gel electrophoresis under nonreducing conditions. The image was cropped and the background was adjusted for better clarity. (E) FACS staining of BW cells expressing NKp30-zeta (BW NKp30) and NKp46-zeta (BW NKp46). The empty black histograms depict staining with the MAb indicated, and the filled gray histograms depict background staining with the secondary MAb only. (F) The various BW cells expressing the chimeric proteins shown in panel E were cocultured with Vero cells preincubated in the presence or absence of reovirus for 14 h. IL-2 secretion was determined by ELISA. Relative IL-2 secretion, decided as described in Methods and Components, is shown. Mean SD and ideals of 3 3rd party experiments are shown. Statistically significant variations are indicated. *, 0.05; ns, not really significant. (G) Vero cells had been incubated in the lack (specified uninfected) or existence of reovirus for 14 h and cocultured with human being NK cells. The.