Cells were analyzed by circulation cytometry. of influenza-specific CD8+ cytotoxic T lymphocyte (CTL) reactions. Dendritic cells (DCs) from TLR7?/? mice were unable to cross-present WIV-derived antigen to influenza-specific CTLs mice responded directly to WIV activation by surface marker (MHC class I, CD86, CD80, CD40) upregulation and cytokine (IFN and IL12) secretion. In mice depleted of pDCs during immunization, CTL induction and safety against heterosubtypic challenge were impaired. Therefore, TLR7 triggering is essential for the successful induction of cross-protective cellular immunity by WIV. The primary target cells for the vaccine are pDCs, which appear to play an important part in the induction of virus-specific CTLs. Materials Mouse monoclonal to IHOG and Methods Ethics Statement All mouse experiments were performed in stringent accordance with Dutch legislation on animal experiments (Damp op de dierproeven, 1977; revised in 1996 with implementation of the Western recommendations 86/609/EEG and Dierproevenbesluit 1985) and authorized by the Ethics Committee on Animal Research of the University Medical Center Groningen (Permit quantity: 5101B). Disease Strains and Vaccines Egg-derived A/PR/8/34 (H1N1) disease and egg-derived A/New Caledonia/IVR 116 (H1N1) disease were kind gifts from Solvay Biologicals (Weesp, The Netherlands); these viruses were further amplified on eggs relating to standard methods. A/NIBRG-14, a genetic reassortant of A/PR/8/34 and MPI-0479605 A/Vietnam/1194/2004 (H5N1), was from NIBSC (Potters Pub, UK) and cultured on Madine-Darby Canine Kidney (MDCK) cells. WIV vaccine was prepared by inactivation of NIBRG-14 disease for 24 hr with 0.1% Cpropiolactone (BPL; Acros Organics, Geel, Belgium) at space temperature, followed by dialysis for 24 hr against HNE buffer MPI-0479605 (5 mM HEPES, 150 mM NaCl, 0.1 mM EDTA, pH 7.4). Inactivation of the disease was tested by carrying out serial passages on eggs, according to the protocol published in the MPI-0479605 Western Pharmacopeia [16]. Specifically, one vaccine dose comprising 20 g of total viral protein was injected into the allantoic cavity of each of 20 fertilized eggs and eggs were incubated for 3 days at 33C. Subsequently, 1 ml aliquots of allantoic fluid from each egg were pooled, and 200 l was inoculated into each fresh egg. This passage was repeated once more. After the last passage, allantoic fluid was harvested and the absence of replicative disease was demonstrated by a hemagglutination test, as described elsewhere [17]. Mice Female C57Bl/6 and TLR7?/? mice, 8C10 weeks older, were used in immunization and challenge studies. C57Bl/6 mice were merchant sourced (Harlan, The Netherlands) and TLR7?/? mice (a kind gift from S. Akira and C. Reis e Sousa) were bred at the animal facility of the University Medical Center Groningen (Groningen, The Netherlands). All mice were kept under SPF conditions in standard cages and experienced access to food MPI-0479605 and water Cytotoxicity Assay C57Bl/6 and TLR7?/? mice were vaccinated twice (days 0 and 21) with either 25 g of NIBRG-14 WIV (s.c.) or 400 HAU of A/New Caledonia live disease (we.p.); HNE mock-vaccinated (s.c.) mice served as negative settings. On days 7 and 8 after the booster immunization, an cytotoxicity assay was performed as explained previously [6]. Reactivation of Influenza-specific CTLs and Tetramer Staining Naive C57Bl/6 mice were primed by intraperitoneal injection of 400 HAU of A/New Caledonia live disease. Three weeks.