Enriched T cell populations from spleen and BAL were stimulated with one of the NP 366 , N3A 366 , PA 224 or PB1 703 peptides (AusPep) for 5 hrs at 37uC, 5% CO 2 in the presence of 1mg/ml Golgi-Plug (BD Biosciences Pharmingen) and 10U/ml recombinant human IL-2 (Roche, Germany) (BD Biosciences). Cells were washed twice with FACS buffer, stained with CD8-PerCP Cy5.5 for 30 mins on ice, fixed, permeabilised and stained with anti-IFN-c-FITC (5mg/ml), TNF-a-APC (2mg/ml), and IL-2-PE (2mg/ml) mAbs (Biolegend). Samples were acquired using flow cytometry, and the total cytokine production calculated by subtracting background fluorescence using no peptide controls. In selected experiments, lymphocytes were stimulated with varying concentrations of peptides, three-fold dilutions ranging from 300nM to 0.0008nM to determine the sensitivity specific peptides, defined as 'functional avidity' [52] .