Despite the decreased D b NPN3A + CD8 + T cell numbers generated following primary infection (Fig 2A-C) , the recall response was substantial following HKNPN3A challenge of PRNPN3A-immune mice ( Fig 2DE) and the NP.PA immunodominance hierarchy that has long been recognized for secondary responses to wt influenza A viruses in H2 b mice [25] was maintained ( Fig 2DE) . Similarly, the total cell numbers for memory D b PA 224 -specific T cells on d28 were comparable for those primed and boosted within the wt and mutant virus combinations (Fig 2F) , while the D b NPN3A + CD8 + set was 8-fold smaller than the wt D b NP 366 + CD8 + population (p,0.005). Again, the results following secondary challenge support the view that, at least in the earlier (d28) stages of memory, T cell numbers reflect the extent of clonal expansion during the acute phase [26] . In our experiments, we detected D b NP 366 + CD8 + and NPN3A + CD8 + T cells by two techniques, tetramer staining and IFN-c ICS. Both techniques gave us comparable antigen-specific CD8 + T cell numbers, indicating that both tetramers accurately detected epitope-specific populations (Fig. 2GH) .