CD8 + T cells from spleen were stained with the D b NP 366 and D b -NPN3A tetramers conjugated to Streptavidin-PE (Molecular Probes, Eugene, OR) for 60mins at room temperature. For a tetramer dilution assay, 2-fold dilutions of PE-conjugated tetramers were used at a range of concentrations (0.15-40mg/ ml). For a tetramer dissociation assay, lymphocytes were stained at the optimal concentration of PE-conjugated tetramers as assessed by tetramer titration as determined by both the percentage of tetramer + CD8 + T cells and mean fluorescence intensity (MFI). Cells were washed twice in FACS buffer (10%BSA/0.02% NaAz in PBS), stained with a FITC-conjugated mAb to CD8a (BD Biosciences Pharmingen) for 30mins on ice, washed and analysed by flow cytometry. As a measure of pMHC avidity, splenic T cells were used in tetramer dissociation assay [29] . After staining with tetramer, T cells were washed and incubated in the presence of anti-H2D b antibody at 5mg/ml at 37 0 C to prevent tetramer rebinding. Cells were removed at intervals, stained with the FITCconjugated mAb to CD8a and analysed by flow cytometry. Loss of tetramer + CD8 + T cells at particular time-points was calculated in comparison to tetramer staining at t = 0mins.