CD8+ T Cell Activation Assay

Splenocytes (0.2–1 × 10⁶) or CD8⁺ T cells (10⁵, CD8a⁺ T Cell Isolation Kit; Miltenyi Biotec) were incubated with the following: (a) transfected cells (see previous paragraph), (b) synthetic peptides, (c) DC2.4 pulsed with synthetic peptides at various concentrations, or (d) DC2.4 infected with VACV (see Viruses and cell lines) in the wells of a 96-well plate at 37°C and 5% CO₂. Synthetic peptides (if used) were added to a final concentration of 0.5 μM; cells (except those transfected in 96-well format) were used at 1–2 × 10⁵ cells per well. 10 μg/ml brefeldin A was added after 1 h, and the incubation continued for another 3–4 h. Plates were spun, medium was removed, and cells were resuspended in 50 μl of α-CD8-PE (clone 53–6.7; BD Biosciences; some experiments used FITC or PE-Cy5) and incubated on ice for 20 min. Cells were washed, resuspended in 50 μl of 1% paraformaldehyde, and incubated at room temperature for 20 min before another two washes and staining with α-IFN-γ–allophycocyanin (clone XMG1.2; BD Biosciences; some experiments used FITC or PE) overnight in PBS with 0.5% saponin at 4°C. Cells were washed once before acquisition and analysis of fluorescence using a FACSCalibur (BD Biosciences). Analysis was done using Flowjo software (Tree Star Inc.); events were gated for live lymphocytes on FSC × SSC followed by CD8⁺ cells using CD8 × SSC and displayed as CD8 × IFN-γ. Data was recorded as IFN-γ⁺, CD8⁺ cells as a percentage of total CD8⁺ cells. Backgrounds as determined using irrelevant peptides, cells transfected with irrelevant constructs or uninfected cells were usually in the order of 0.1% and were subtracted from the values presented for test samples.