Expression level of H60 is directly proportional to the level of NKG2D-mediated activation. (A) Expression levels of H60 on target cells were quantified by flow cytometry following staining with sNKG2D-Ig fusion protein. Soluble immunoglobulin Fc (Ig) or the secondary antibody alone (2°) was used to determine background staining levels. (B) Cytotoxicity of B6- and B10.D2-derived NK cells against EL4, ST61, and ST63. Increasing numbers of NK cells were titrated against a constant number of ⁵¹Cr-labeled target cells (5000 per well), and percent cytotoxicity was determined in a conventional 4-hour cytotoxicity assay. Data presented as the mean percent cytotoxicity ± standard deviation (SD) were determined from triplicate wells and are representative of 6 independent experiments. (C) Expression levels of MHC class I molecules in EL4, ST61, and ST63 were tested using anti-H2-K^b (α-K^b) and anti-H2-D^b (α-D^b) mAbs.