Type I Interferons Mediate the Innate Cytokine Response to Recombinant Fowlpox Virus but Not the Induction of Plasmacytoid Dendritic Cell-Dependent Adaptive Immunity

Antigen-Specific T-Cell Responses to a Recombinant Fowlpox Virus Are Dependent on MyD88 and Interleukin-18 and Independent of Toll-Like Receptor 7 (TLR7)- and TLR9-Mediated Innate Immune Recognition

“…Cells were cultured for 8 days prior to infection or stimulation, with a 50% medium change daily from days 4 to 8 for GM-CSF cultures and on day 4 for FL cultures. The majority of GM-CSF-expanded DCs took on a conventional DC (cDC) phenotype, while FL-expanded DCs consisted of both cDCs and pDCs as previously described (7,24,43) and confirmed by flow cytometric analysis of B220, CD11b, and CD11c expressions (data not shown).…”

“…Indeed, we have recently shown that pDCs are essential for mediating adaptive immunity to an FWPV-encoded antigen (15). This activity most likely results from the ability of pDCs to either directly present or crosspresent antigens rather than from their ability to secrete copious amounts of type I IFNs (43). In this study, we further our understanding of the way in which the innate arm of the mammalian immune system recognizes rFWPV as well as provide new detail on the PRRs involved and their relevance to the formation of innate and adaptive immune responses.…”

“…injection, and splenocyte suspensions were prepared at the peak of the cellmediated response, 5 days postimmunization. Following RBC lysis, cells were incubated in vitro to remove the adherent cell population, with the nonadherent cells subsequently being assessed for potential OVA-specific cytolytic activity starting at a 200:1 effector-to-target cell ratio according to previously described methods (43). The percent specific lysis was determined by using the following equation: [(experimental 51 Cr release Ϫ spontaneous 51 Cr release)/(maximum 51 Cr release Ϫ spontaneous 51 Cr release)] ϫ100.…”

“…3d. Cells were subsequently harvested, with lysates being subjected to polyacrylamide gel electrophoresis, before proteins were transferred onto polyvinylidene difluoride (PVDF) membranes for the assessment of IB␣ phosphorylation as previously described (43).…”

“…Expanded DC cultures from knockout and control mice were infected for 24 h at a multiplicity of infection (MOI) of 1 (unless otherwise indicated) with FWPV WT (for cytokine analysis) or FWPV OVA (for analysis of OVA production), after which cell-free supernatants were harvested and stored at Ϫ20°C until enzyme-linked immunosorbent assay (ELISA) analysis was performed. For analyses of cytokines, matched antibody pairs and standards were used with sandwich ELISA protocols as previously described (43). Ovalbumin secretion was assessed by use of polyclonal goat anti-OVA capture antibody (ICN) and biotinylated rabbit anti-OVA detection antibody (Rockland), followed by streptavidin-horseradish peroxidase (HRP) (Rockland) and substrate detection (Fast OPD; Sigma-Aldrich) performed with OVA protein standards (grade V; Sigma-Aldrich) with a standard sandwich ELISA protocol.…”