In vitro induction of B7-H6 cell-surface expression on purified myeloid cells and NKp30-dependent cell activation induced by B7-H6+ monocytes. (A) Flow cytometric analysis of B7-H6 cell-surface expression on freshly isolated monocytes stimulated for 48 hours with TNFα, LPS, or IL-1β. B7-H6 expression was analyzed by flow cytometry with directly conjugated 17B1.3 anti–B7-H6 mAbs (black histograms) or mIgG1 isotype control (gray histograms). Data are representative of at least 3 independent experiments. MFI stim represents the value of the MFI obtained with anti–B7-H6 mAbs minus the MFI obtained with mIgG1 isotype control. (B) NKp30+ DOMsp30 reporter cells (NKp30+) or NKp30− DO11.10 control cells (parental cells) were cocultured with unstimulated or IL-1β–stimulated monocytes in the presence of anti–B7-H6 mAbs (17B1.3) or mIgG1 isotype control. 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. ***P < .001. (C) Autologous NK cells were cocultured with unstimulated or IL-1β–stimulated monocytes (E:T, 1:4) in the presence of anti–B7-H6 (17B1.3) F(ab′)2 fragments. NK-cell activation was determined by evaluating the percentage of CD107-positive NK cells. Data correspond to a pool of 6 independent experiments. *P < .05; **P < .01; and ****P < .0001. (D) Flow cytometric analysis of B7-H6 expression on freshly isolated CD24+CD14−HLA−DR− neutrophils left untreated or stimulated with TNFα, LPS, or IL-1β for 24 hours. B7-H6 expression was analyzed by flow cytometry with F(ab)′2 fragments of the B7-H6–specific 17B1.3 mAbs followed by APC-conjugated anti-mouse IgG (black histograms) or with APC-conjugated anti-mouse IgG alone (control gray histograms). Data are representative of at least 3 independent experiments. MFI stim represent the value of the MFI obtained with anti-B7-H6 F(ab)′2 minus the MFI obtained with the control. E:T, effector to target ratio.
