Quantifying Influenza-Specific T-Cell Responses

CD4⁺ and CD8⁺ T-cell recall responses were determined as described previously (36 (link), 37 (link)). The production of type 1 (IFN-γ, TNF-α, and interleukin 2 [IL-2]) and type 2 (IL-4) cytokines of T cells was detected. Splenocytes and cells from BAL fluid (n ≥ 3 fluid samples per group) were harvested at day 7 postinfection. After RBC lysis, isolated lymphocytes were stimulated by PR8 or HK68 virus and coincubated with anti-CD28, anti-CD49d (BD Biosciences), and IL-2 (Roche) for 6 h, followed by an incubation with GolgiPlug (containing brefeldin A; BD Biosciences) overnight. For splenocytes at 3 weeks after vaccination, PR8, HK68, H1N1/Brisbane/07 (H1N1), and HK/MPF461/07 (H5N2) viruses were used to stimulate the cells. Treated cells were first stained with Zombie Aqua (Biolegend) and then with T-cell markers (Fc block by anti-CD16/CD32 [BD Biosciences]; anti-CD4–allophycocyanin/Cy7 and anti-CD8–PerCP/Cy5.5 [Biolegend]). Stained cells were then fixed with Cytofix/Cytoperm buffer (BD), followed by intracellular cytokine staining (IFN-γ, FITC; TNF-α, allophycocyanin; IL-2, PE; and IL-4, PE/Cy7; Biolegend). Signal acquisition was performed on a BD LSR Fortessa cytometer, and data were analyzed with FlowJo.