Characterization of sB7-H6 isolated from patient sera. (A) Concentrations of sB7-H6 were measured in the pellet at each round of 3 sequential centrifugations at 1200g, 12 000g, and 110 000g of B7-H6⁺ patient sera. Data correspond to a pool of 3 independent experiments. (B) Flow cytometric analysis of pellets purified from serum of 2 sB7-H6⁺ patients and 1 control individual. After purification, pellets were complexed to latex beads and stained with the indicated mAbs (black histograms); histograms obtained with isotype controls are in gray. (C) Flow cytometric analysis of NKp30 cell-surface expression on freshly isolated NK cells stained with mIgG1 isotype control (gray histograms) or NKp30 mAbs in presence of 5 μg of sB7-H6⁺ pellet (dashed line) or 5 μg of sB7-H6⁻ pellet (black line). Data are representative of 3 independent experiments. (D) NKp30⁺ DOMsp30 reporter cells or NKp30⁻ DO11.10 control cells (parental cells) were cocultured with K562 cells that constitutively express B7-H6, in the presence of anti-B7-H6 mAbs (17B1.3), mIgG1 isotype control, 5 μg of pellet from serum of 2 sB7-H6⁺ patients or 5 μg of pellet from serum of 2 sB7-H6⁻ patients. DOMsp30 cell activation was determined by evaluating IL-2 production in the coculture supernatant in a standard CTLL-2 survival assay. Data are representative of 3 independent experiments.