Type I IFNs have a dual effect on cross-presenting conventional DCs. (A) Cross-presenting DCs require CD40L for maximal T-cell activation. DCs loaded with exogenous antigen were prepared by cross-presentation of influenza infected apoptotic cells (DCs x/p flu-apoptotic cells) and compared to uninfected apoptotic cells (DCs x/p apoptotic cells). DC groups were cultured with syngeneic purified CD8⁺ T cells. T-cell activation was monitored by IFN-γ ELISPOT and displayed as spot-forming cells (SFCs)/10⁶ CD8⁺ T cells. Recombinant CD40L was added to cultures as indicated. (B) The effects of IFN-α/β were tested on immature and mature DCs, respectively. As illustrated, IFN-α/β was added to iDC during antigen capture and maturation or to mDCs during CD40 cross-linking and CD8⁺ T-cell engagement. (C) DCs cross-presenting uninfected (unfilled bars) or influenza loaded (filled bars) apoptotic cells were treated with media alone (black bars), exposed to 60 IU/mL IFN-α/β during antigen capture (red bars), or 60 IU/mL IFNα/β following maturation and antigen presentation (green bars). Data are representative of 10 experiments with statistical P indicated. Similar results were obtained when using recombinant IFN-α (data not shown). (D) iDCs were exposed to a dose range of IFN-α/β (concentration 0-600 IU/mL). In the case of direct presentation of antigen, mDCs were directly infected with influenza virus (flu DC). For cross-presentation, the iDCs with cocultured with apoptotic influenza-expressing apoptotic cells (DCs x/p flu-AC) during the maturation process and interferon stimulation. In each condition, the CD8⁺ T-cell and CD4⁺ T-cell activation were monitored by IFN-γ ELISPOT. Percent inhibition of T-cell activation is displayed. Maximal stimulation (no IFN-α/β) in the experiment shown is equivalent to 400 spot forming cells/million T cells.