Figure 3.

Intracellular Opn is required for IFN-α production. (a–c) Lentivirus infection of pDCs stimulated with CpG-B (0.2 μg/ml). (a) Extracellular Opn protein measured by ELISA in 6-hour culture supernatants of sorted splenic Opn-deficient pDCs infected with lentivirus to express full-length Opn (fOpn), mutant Opn (ΔOpn) or GFP control (GFP). HIV capsid protein p24, determined by ELISA, indicates the concentration of lentivirus used for infection (horizontal axis). (b) IFN-α in 24-hour culture supernatants of splenic Opn-deficient pDCs transfected with lentiviral reagents (GFP, ΔOpn and fOpn). (c) IFN-α (left vertical axis) or intracellular Opn (right vertical axis) in culture supernatants and cells. ‘Titration’ of mutant Opn lentivirus (ΔOpn-lenti) into bone marrow–derived Opn-deficient pDCs (1 × 10⁶ cells/ml) was followed by CpG-B stimulation (ODN-1668; 0.2 μg/ml), then analysis 24 h later. (d) IFN-α production in 24-hour culture supernatants of Opn wild-type (WT) and Opn-deficient (KO) bone marrow–derived pDCs after a 20-minute pulse with DOTAP in complex with CpG-B (ODN-1668; left) or CpG-A (ODN-D19) with (+) or without (−) DOTAP (right). Data are representative of at least three independent experiments.