Coexpression of H2-D^d on H60-positive target cells significantly inhibits NK cell cytotoxicity. (A) ST63 (H60^hi) cells were transfected with H2-D^d-encoding plasmids to obtain sublines expressing low (ST72) and high (ST77) levels of H2-D^d. Expression levels of MHC class I were quantified by flow cytometric analysis following staining with anti-D^d (α-D^d), anti-K^b (α-K^b), and anti-D^b (α-D^b) mAbs. Secondary antibody alone (2°) was used to determine background staining levels. (B) Comparable levels of H60 on ST63, ST72, and ST77 were verified by flow cytometry following staining with sNKG2D-Ig. Soluble immunoglobulin Fc (Ig) or the secondary antibody alone (2°) was used to determine background staining levels. (C) Effect of H2-D^d expression on NKG2D-mediated cytotoxicity of NK cells derived from B6 (left), B10.D2 (middle), or BALB/c (right) mice was tested using targets that expressed high levels of H60 and were either negative for H2-D^d (ST63 •) or expressed H2-D^d at low (ST72 ○) or high (ST77 ▪) levels. H60-negative targets (EL4 □) were used as controls. Cytotoxicity assays were performed and data presented as described in the legend to Figure 1B and are representative of 5 independent experiments.