Immunization and Infection Protocols in Mice

Female mice were challenged via tail vein injections with either 2000 colony-forming units (CFU) per mouse LM expressing whole OVA (LM-OVA) or 5 × 10⁶ plaque-forming units (PFU) per mouse VV Western Reserve (VV-WR) strain or the same strain expressing OVA (VV-OVA). For influenza virus infection, WT (IRF4^fl/fl) and IRF4 conditional knockout (cKO; CD8-cre IRF4^fl/fl) mice were infected with influenza A/PR8/34 strain (~200 PFU per mouse). Influenza was inoculated in anesthetized mice through intranasal route as described before (64 (link)). Data were analyzed in lung CD8⁺ T cells at day 9 after influenza infection. Mice were immunized via tail vein injection or intraperitoneal injection with the indicated innate receptor agonist, with or without αCD40 antibody clone FGK4.5 (BioXcell), and 100 to 150 μg of whole chicken OVA (Sigma). OVA protein was detoxified by phase separation (65 (link)) and was lipopolysaccharide- free as determined by a limulus assay. The following adjuvant doses were used for immunizations: Poly I:C/αCD40 (80 μg/40 μg), Pam3Cys/αCD40 (25 μg/50 μg), Poly I:C (50 μg), Pam3Cys (25 μg), flagellin (15 μg), monophosphoryl lipid A (MPL; 40 μg), and Alum (50 μg). Treatment with 2-DG (1 g/kg) or vehicle control [phosphate- buffered saline (PBS)] was administered by daily intraperitoneal injection at 1 to 6 days post-infection/immunization (dpi).