Quantifying T Cell Immune Responses to VACV

ICS assay was performed to detect antigen-specific CD4+ T cell and CD8+ T cell immune responses [14 (link)]. The splenocytes from each immunized C57BL/6 mice were cultured with RPMI 1640 containing 10% FBS in 96-well flat-bottom plates (2 × 10⁶ cells/well). Meanwhile, 0.1 MOI of VACV was diluted in RPMI 1640 containing 10% FBS. The cultured splenocytes were stimulated with 0.1 MOI of VACV (100 μl/well) for 6 h and then incubated with Golgiplug (BD Biosciences) for an additional 12 h at 37°C, 5% CO2 incubator. The cells were washed with PBS containing 1% BSA to stain with anti-CD3, anti-CD4, and anti-CD8 surface markers (Biolegend) for 30 min at 4°C in the dark. After that, the cells were fixed and permeabilized using a BD Cytoperm fixation/permeabilization solution (BD Biosciences) and then stained with a cocktail of antibodies: IL-2 (Biolegend), IL-4 (Biolegend), INF-γ (Biolegend), TNF-α(Biolegend). Data were acquired on a BD FACSAria III flow cytometer (BD Biosciences) and analyzed with FlowJo 10.6.2.