Diversity of epitope and cytokine profiles for primary and secondary influenza A virus-specific CD8(+) T cell responses

Screening with the flow cytometric IFN-gamma assay has led to the identific ation of a new immunogenic peptide (SSYRRVPGI) from the influenza PB1 polym erase (PB1(703-711)) and a mimotope (ISPLMVAYM) from the PB2 polymerase (PB 2(198-206)). CD8(+) T cells specific for K(b)B1(703) make both IFN-gamma an d TNF-alpha following stimulation with both peptides. The CD8(+) K(b)PB1(70 3)(+) population kills PB2(198)-pulsed targets, but cell lines stimulated w ith PB2(198) neither bind the K(b)PB1(703) tetramer nor become CTL. This CD 8(+)K(b)PB1(703)(+) population is prominent in the primary response to an H 3N2 virus, although it is much less obvious following secondary challenge o f H1N1-primed mice. Even so, we can now account for >40% of the CD8(+) T ce lls in a primary influenza pneumonia and >85% of those present after H3N2 - -> H1N1 challenge. Profiles of IFN-gamma and TNF-alpha staining following i n vitro stimulation have been traced for the four most prominent influenza peptides through primary and secondary responses into long-term memory. The (DNP366)-N-b epitope that is immunodominant after the H3N2 --> H1N1 challe nge shows the lowest frequencies of CD8(+) IFN-gamma (+)/TNF-alpha (+) cell s for >6 wk, and the intensity of IFN-gamma staining is also low for the fi rst 3 wk. By 11 wk, however, the IFN-gamma /TNF-alpha profiles look to be s imilar for all four epitopes. At least by the criterion of cytokine product ion, there is considerable epitope-related functional diversity in the infl uenza virus-specific CD8(+) T cell response. The results for the K(b)PB1(70 3) epitope and the PB2(198) mimotope also provide a cautionary tale for tho se using the cytokine staining approach to identity antigenic peptides.