Coated uPA inhibits IFN-γ production by NK-cell lines in an Ly49E-dependent manner. (A) IFN-γ staining of KY and NK03 cells incubated in coated wells, in the presence or absence of anti-Ly49E (4D12) F(ab′)2 fragments as indicated. Wells were coated with no (dotted lines) or anti-NK1.1 mAb (solid lines) at 1 and 5 μg/mL as indicated, together with uPA (red lines) or BSA (black lines) at 200 ng/well. In each histogram, the percentage IFN-γ expression, corrected for its background at 0 μg/mL anti-NK1.1, is indicated above (uPA) or below (BSA) the marker line. The percentage inhibition of IFN-γ by uPA compared with BSA is given in parentheses in the top right corner. Representative data from 1 of 3 experiments are shown. (B) KY cells were transduced with a control or Ly49E shRNA construct, both containing the EGFP marker gene. Cells were seeded into wells coated with no or anti-NK1.1 mAb at 1 and 5 μg/mL, together with uPA or BSA at 200 ng/well, as indicated. Thereafter, cells were costained for IFN-γ and Ly49E. The gated EGFP+Ly49E+ populations are indicated by R1 or R2, the gated EGFP+Ly49E− cells by R3. In each dot plot, the percentage IFN-γ expression in the gated regions is given in the top right corner. Representative data from 1 of 4 experiments are shown. (C) The percentage uPA-mediated inhibition of IFN-γ production by the populations and in the conditions as indicated in panel B. The mean plus or minus SD of 4 experiments is shown.
