Quantifying Poxvirus-specific T Cell Responses

Poxvirus-specific T cell responses in murine splenocytes were measured as previously described [20] (link). Briefly, splenocytes isolated from VACV-infected mice (2×10⁶ PFU/mouse) at 8 days p.i. were stimulated with A20 cells infected with CPXV or VACV (MOI = 5, 16 h) in the presence of Brefeldin A for 6 h. Cells were stained overnight at 4°C with αCD3ε and αCD4 Ab's (clones 145-2C11 and RM 4–5, respectively, BD Biosciences), αCD8 Ab (clone 5H10, Life Technologies), Fc Block (BD Biosciences) and mouse IgG (Sigma). The next day, the cells were washed, fixed, and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) followed by intracellular staining with Ab to IFNγ (clone XMG1.2, BD Biosciences PharMingen) and TNFα (clone MP6-XT22, BioLegend, San Diego, CA). The samples were analyzed by flow cytometry as described above. Non-viable cells were excluded using a live cell gate based on Aqua staining, gated for lymphocytes based on forward and side scatter characteristics followed by gating for CD3ε+. Next, CD3ε+ T cells were gated on either CD4+ or CD8+ and IFNγ+TNFα+ T cells were quantified. Background IFNγ+TNFα+ events from uninfected samples were subtracted. T cell responses to CPXV and CPXV deletion mutants were normalized to VACV.