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 identification of a new immunogenic peptide (SSYRRVPGI) from the influenza PB1 polymerase (PB1(703-711)) and a mimotope (ISPLMVAYM) from the PB2 polymerase (PB2(198-206)). CD8(+) T cells specific for K(b)B1(703) make both IFN-gamma and TNF-alpha following stimulation with both peptides. The CD8(+) K(b)PB1(703)(+) population kills PB2(198)-pulsed targets, but cell lines stimulated with PB2(198) neither bind the K(b)PB1(703) tetramer nor become CTL. This CD8(+)K(b)PB1(703)(+) population is prominent in the primary response to an H3N2 virus, although it is much less obvious following secondary challenge of H1N1-primed mice. Even so, we can now account for >40% of the CD8(+) T cells in a primary influenza pneumonia and >85% of those present after H3N2 --> H1N1 challenge. Profiles of IFN-gamma and TNF-alpha staining following in 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 challenge shows the lowest frequencies of CD8(+) IFN-gamma (+)/TNF-alpha (+) cells for >6 wk, and the intensity of IFN-gamma staining is also low for the first 3 wk. By 11 wk, however, the IFN-gamma /TNF-alpha profiles look to be similar for all four epitopes. At least by the criterion of cytokine production, there is considerable epitope-related functional diversity in the influenza virus-specific CD8(+) T cell response. The results for the K(b)PB1(703) epitope and the PB2(198) mimotope also provide a cautionary tale for those using the cytokine staining approach to identity antigenic peptides.