To determine whether such decreased CD8 + responses elicited after heterologous challenge would affect influenza virus clearance, we performed experiments to examine the protective efficacy of cross-reactive CD8 + T cell repertoires. We performed prime-andchallenge studies in mMT mice lacking B cells to ensure that antibody responses did not mask any possible inhibitory effects of ''suboptimal'' TCRs on viral clearance. As suggested by CD8 + T cell data, assessment of lung viral titres showed delayed viral clearance on d6 after the secondary infection in case of heterologous prime-and-boost (PR8-.X31-NPN3A and PR8-NPN3A-.X31) compared to homologous infections (PR8-.X31 or PR8-NPN3A-.X31-NPN3A) (Fig. 5B) . These results indicate that recall of ''suboptimal'' TCRs for a single T cell specificity can lead to delayed viral clearance, despite the presence of other influenza CD8 + T cell responses (D b PA 224 These patterns of complete, or partial, cross-stimulation following in vivo virus challenge were reflected in the results found for in vitro measurements of ''functional avidity'' (Fig 6) . Pulsing immune spleen cells recovered directly ex vivo with graded doses of wt or mutant peptide in the ICS assay showed comparable levels of IFN-c induction (Fig 6AC) in every situation but one, the exposure of wt-primed T cells to the NPN3A peptide ( Fig 6B) . Thus, while the immune repertoire selected by D b NP3A shows evidence of equivalent avidity following stimulation with the NPN3A or NP 366 peptides, D b NP 366 induces a response that is of higher avidity for the wt than the mutant epitope. The same effect was seen even more clearly when three (all expressing the SGGGNTGQL CDR3b) T cell hybridoma lines [37] specific for D b NP 366 were stimulated with the two peptides ( Fig 6DE) . This result is in accord with findings from both the TCR repertoire analysis of crossreactive clonotypes, assessed by tetramer binding (Table 2) , and the response magnitudes determined following homologous and heterologous virus challenge (Fig 5) . Thus, priming and recall of cross-reactive CD8 + T cells recognising mutant viral epitopes reflects functional (defined as responsiveness to a peptide) pMHC-TCR avidity.