Response characteristics following heterologous challenge and TCR/pMHC-I avidity As there was substantial cross-reactivity in vitro (Fig. 2) for the D b NP 366 and D b NPN3A-specific responses, it was important to determine whether memory T cells that cross-react for the D b NP 366 and D b NPN3A epitopes are preferentially recalled by secondary infection with the heterologous virus. Mice that were primed with the PR8NPN3A and then challenged with either the wt HK or mutant HKNPN3A viruses showed equivalent recall of D b NPN3A + CD8 + T cells during the acute phase of the secondary response. This was detected by IFN-c production ( Fig 5A) and tetramer staining (data not shown) and is consistent with the TCR CDR3b analysis (Table 2) . Conversely, when mice were firstly primed with wt PR8, then later infected i.n. with either the wt HK or mutant HK-NPN3A, the D b NP 366 + CD8 + T cells were differentially recalled indicating that (in the absence of primary selection from the naïve repertoire by the mutant epitope) only some of the D b NP 366 + CD8 + memory T cells that were expanded by heterologous challenge bind D b NPN3A (Fig. 5A) . Interestingly, wt priming and challenge (1 0 PR8-.2 0 X31) resulted in significantly higher CD8 + T cells numbers (Fig. 5B ) than were found for any secondary CD8 + T cell responses after NPN3A priming (1 0 PR8-NPN3A-.2 0 X31-NPN3A and 1 0 PR8-NPN3A-.2 0 X31). These results lead to two main conclusions: (i) priming with the wt virus elicits CD8 + T cells that respond relatively well to a subsequent infection with cross-reactive variant (ie 1 0 PR8-.2 0 X31-NPN3A = 1 0 PR8-NPN3A-.2 0 X31-NPN3A); (ii) priming with the cross-reactive variant can be detrimental as the diminished primary response may limit the full expansion of CD8 + T cells that are able to respond to the subsequent wt infection (ie 1 0 PR8-NPN3A-.2 0 X31 is lower than 1 0 PR8-.2 0 X31) and skew the TCR usage. Thus, using NPN3A for either priming or the challenge gives an equally poor response.