Thr225 of NKp46 is important for NKp46 activation. (A) Parental BW cells, or stably transfected BW cells expressing the chimeric receptors CD99-Zeta, NKp46-Zeta, or NKp46T225A-Zeta, were stained with anti-NKp46 mAb (461-G1), followed by FITC-conjugated goat antimouse Fc. Gray histograms represent the background staining of FITC-conjugated goat antimouse Fc. The MFI values of the anti-NKp46 mAb staining are indicated in the figure. Figure shows one representative experiment of 3 performed. (B) 1106mel cells were incubated overnight with or without human influenza virus (Sydney strain). Next, cells (50 000/well) were irradiated (6000 rad [60 Gy]), washed, and incubated with 50 000 cells/well of BW cells expressing CD99-Zeta (BW/CD99Z), wild-type NKp46-Zeta (BW/NKp46Z wild type), mutated NKp46T225A-Zeta (BW/NKp46T225AZ), or parental BW cells. After 48 hours, IL-2 levels in the supernatants were measured by ELISA. Optical density (OD) absorbance (650 nm) was translated into amount of IL-2 (pg/well) according to a standard curve. Results represent one experiment of 2 performed.