hKLRL1 inhibits NK cell cytotoxicity. (A) The purified hexKLRL1-Fc protein is expressed as an approximately 37-kDa fusion protein. The fusion protein was harvested from the supernatants of COS-7 cells transiently transfected with the phexKLRL1-Fc expression vector and purified by using Affi-Gel protein A-agarose columns. Protein expression in supernatants of untransfected COS-7 cells and phexKLRL1-Fc-transfected COS-7 cells was examined by Western Blot analysis by using anti-Fc antibody. (B) Pretreatment of target cells with hexKLRL1-Fc fusion protein enhances NK cell cytotoxicity, except in the case of K562 cells. Target cells (A549, HeLa, MCF-7, and K562 cells) were preincubated for 8 hours with hexKLRL1-Fc fusion protein (hexKL-Fc) or control IgG (20 μg/mL). Cell-mediated cytolysis was assayed by using IL-2-stimulated human polyclonal NK cells and NK-92 cells as effector cells. Target cells untreated with mAb were included as an additional control. All data represent median values of triplicate samples and are representative of 3 independent experiments. (C) Potential hKLRL1 ligands were present on the surface of A549, HeLa, and MCF-7 target cells but not on K562 cells. Flow cytometric analysis of A549, HeLa, MCF-7, and K562 cells stained with hexKLRL1-Fc fusion protein.