C57BL/6 (H-2b) mice (6-
to 8-wk old) were purchased from Charles River (St. Constant, QC, Canada). The experiments were conducted in accordance to Adriamycin solubility dmso the guidelines of the Canadian Council on Animal Care. HEK293-NP are stably transfected with LCMV-NP 7, 8 and the cell lines BMA and DC2.4 were cultured in RPMI 5% FBS (Invitrogen, ON, Canada) 32, 33. Ribonuclease A from bovin pancrease (RNase A, R4875, 10 μg/mL), lactacystin, BFA, leupeptin, pepstatin A, and chloroquine were purchased from Sigma (Oakvilla, ON, Canada). Murine rmGM-CSF was purchased from Cedarlane Laboratories (Hornby, ON, Canada). Diphenyleneiodonium chloride was purchased from Calbiochem. LCMV-WE was originally obtained from F. Lehmann-Grube (Germany), propagated and titrated as described previously 8, 34. BM from C57BL/6 mice were collected to generate BM-derived Mø or BM-DC as described previously 35. For BM-derived Mø, after 3 days of culturing, the nonadherent cells were removed and fresh conditioned medium containing 20% of L929 supernatant was added. The medium was changed 2 days later and the cells were tested
after 5 days of culture. For BM-DC, cells were processed as described previously 35 and the medium (2 mL) was removed every 2 days and replaced with fresh medium. At day 6, the nonadherent cells were transferred into a new Crizotinib 6-well plate and left for 4 h before the loosely adherent cells (highly enriched CD11c+ MHC-II+) were harvested and used at this day in the assay. HEK293 cells were infected with LCMV-WE at an moi of 1 for different time points at 37°C. After that, the cells were lysed by employing one cycle of freeze/thaw in liquid N2, followed by UVB radiation using
a CL-1000M Selleckchem Verteporfin UV cross-linker (Ultra-Violet Products, Cambridge, UK) at a radiation intensity of 200 000 μJ/cm2 (maximum intensity) for 1 h to inactivate LCMV. Following UV exposure, cells were collected and used directly after treatment. These cells are termed LyUV-ADC. HEK-NP cells were treated as described previously 8. For LCMV-NP detection, LCMV-infected HEK cells were harvested and stained with anti-LCMV-NP as described previously 8. In a similar fashion, the cells were incubated with mouse anti-LCMV-GP (KL25) followed by Alexa488-goat anti-mouse IgG1 (lot 53419A, Invitrogen, OR, USA) to stain for LCMV-GP. For T-cell activation, IFN-γ production by CTL was measured by intracellular cytokine staining (ICS) in peptide restimulation assays as described previously 8. The APC were loaded with one of the following synthetic peptides: GP33, GP276, NP205, and NP396, or an irrelevant peptide control (SIINFEKL). The peptides (purity>90%) were synthesized at CPC Scientific (San Jose, CA, USA). Peptide-specific CTL were generated as described previously 7. Purified splenocytes were then restimulated with peptide-pulsed (10-7 M)γ-irradiated BMA cells in the presence of IL-2 (20 U/mL).