To determine the pMHC-I avidity of the responding D b NP 366 + CD8 + and NPN3A + CD8 + T cells, we additionally performed tetramer dilution (Fig. 7A -C) and tetramer dissociation (Fig. 7D-E) assays. While tetramer dissociation assay measured the ''off-rate'' component of pMHC-I avidity, tetramer dilution technique assessed the overall pMHC-I avidity (both the ''on'' and ''off'' rates). Furthermore, we also assessed CD8b-dependence for functional activation (a measure of low avidity CD8 + T cells) of both wt D b NP 366 + CD8 + and the mutant NPN3A + CD8 + T cells by anti-CD8b mAb blocking, followed by IFN-c ICS (Fig. 7G-I) . Our data obtained from those three additional measures of pMHC-I avidity confirmed the results obtained by the peptide titration combined with ICS (functional avidity, Fig. 6 ) and further suggested significantly lower pMHC-I avidity of the wt D b NP 366 + CD8 + (generated by the wt HK infection) for NPN3A variant.