CD4+ T cell depletion

Mice were vaccinated once a week for 3 weeks with PBS as a control, empty-GPs, or 4X-SA-GP (5x10⁷ GPs/dose). Mice were rested for 4 weeks and then received an i.p. injection with an anti-CD4⁺ antibody (clone Gk1.5, BioXCell) 300 μg in 500 μl PBS on day -1 and 100 μg in 500 μl PBS on day 0 as previously described [76]. Control mice were treated with an i.p. injection of the corresponding isotype control antibody (BioXCell) at the same dosage and volume. On day 0, all mice were then inoculated with 2x10⁷ CFU of S. aureus (LAC USA300) via i.p. injection. Mice were euthanized 24 hours after the infection and spleen and kidneys were harvested, homogenized, and plated on blood agar plates. CFUs were enumerated after overnight incubation at 37°C. PBMCs were isolated from the blood of mice and pooled from all groups of mice on day 0 prior to infection and were stained with the surface markers CD3, CD4, and TCRβ and analyzed by flow cytometry to assess the efficacy of the CD4⁺ T cell depletion. MLNs were harvested on day 1, after the infection, and the isolated cells were stained for the surface markers CD3, CD4, and TCRβ and analyzed via flow cytometry to assess that the CD4⁺ T cells were still depleted post-infection.