Detection of VACV-specific, IFN-γ-expressing CD8+ T cells.

C57BL/6 mice were inoculated i.d. with vK1L, vΔK1L, or PBS (n = 5 mice per experimental group) as described above. At 10 days postinfection, the mice were euthanized, and spleens and livers were harvested. Single-cell suspensions of spleens and lymph nodes were prepared as follows. Spleens were forced through a 70-μm nylon mesh (Thermo Fisher Scientific, Waltham, MA), and then erythrocytes were removed by treating samples with ammonium-chloride-potassium (ACK) red cell lysis buffer (Sigma-Aldrich). Lymph nodes were homogenized by using a BioMasher II disposable microtube homogenizer (Research Products International, Mt. Prospect, IL).

Single-cell suspensions (10⁶ cells) of spleens or lymph nodes from each animal were kept separate, and cells from each spleen or lymph node were either unstimulated or stimulated with VACV-specific peptide (TSYKFESV; B8R_20–27 epitope) for 4 h in the presence of brefeldin A (5 μg/ml; Sigma, St. Louis, MO). Using this approach, only CD8⁺ T cells recognizing VACV peptides become activated. Then, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD8 antibody (clone 53-6.7; 1:100; BioLegend) in cell-staining buffer (BioLegend, San Diego, CA) for 30 min on ice to stimulate VACV-specific T cells. Next, samples were fixed for 20 min at room temperature and permeabilized on ice in fixation and intracellular staining permeabilization buffer (BioLegend, San Diego, CA), respectively. The cells were stained for intracellular IFN-γ as a marker of T cell activation by incubating fixed cells with Alexa 647-conjugated anti-IFN-γ (clone XMG1.2; 1:100; BioLegend, San Diego, CA) overnight at 4°C. Samples were washed in permeabilization buffer. Flow cytometry was performed with a BD Accuri C6 cytometer (BD Bioscience, San Jose, CA), and data were analyzed with FCS Express software version 5 (DeNovo Software, Glendale, CA). Events were gated for live lymphocytes on forward versus side scatter. Dead cells were excluded on the basis of atypical fluorescence. Data were expressed as the percentage of IFN-γ⁺ CD8⁺ T cells, cells that are considered VACV-specific and activated, out of all CD8⁺ T cells. In addition, data were expressed as the total number of IFN-γ⁺ CD8⁺ T cells per lymph node or spleen.