Tetramer and phenotypic staining of CD8 + T cells Lymphocytes from the BAL and spleen were stained with tetramers conjugated to Strepavidin-APC or PE (Molecular Probes, Eugene, OR, USA) at optimal staining concentrations (10 mg/ml D b NP 366 , 40 mg/ml D b N3A 366 , and 10 mg/ml D b PA 224 tetramers) for 1hr at room temperature. Cells were washed twice in FACS buffer, and stained with 1 mg/ml CD8-PerCP Cy5.5, 5 mg/ml CD62L-FITC and 5 mg/ml CD127-APC mAbs (BD Biosciences) for 30 mins on ice, washed twice and analysed by flow cytometry on the FACS Calibur (BD Immunocytometry) and analysed by CellQuest Pro software (BD Immunocytometry). We titrated all the batches of all the tetramers used in this study. We used tetramers at optimal concentrations (10-40mg/ml) based on both the percentage of epitope-specific CD8 + T cells and the mean fluorescence intensity (MFI) of tetramer staining. A Scatchard analysis [51] based on the tetramer dilution assay (Fig. 7 A-C) was also plotted (Fig. S4 ) and confirmed our observations from routine tetramer titrations that the D b NP 366 tetramer displays slightly superior pMHC binding capacities over the NPN3A tetramer at a concentration ,5mg/ml.