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We have used intracellular cytokine staining and MHC class I tetramer binding in conjunction with granzyme B protease expression and in vivo BrdU uptake to characterize the primary murine CD8(+) T cell response to pulmonary influenza virus infection. We have observed that the majority (&gt;90%) of the CD8(+) T cell response to the A/Japan/305/57 virus in the lung at the peak of the response (days 9-11) is directed to four epitopes (three dominant and one subdominant). Using induction of granzyme B as a surrogate to identify specific activated CD8(+) T cells, we found that an unexpectedly large fraction ( approximately 70%) of lung-infiltrating CD8(+) T cells expressed granzyme B on day 6 of infection when estimates by MHC tetramer/intracellular cytokine staining yielded substantially lower frequencies ( approximately 30%). In addition, by using intranasal administration of BrdU during infection, we obtained evidence for proliferative expansion of activated CD8(+) T cells in the infected lung early (days 5-7) in the primary response. These results suggest that the frequency and number of specific CTL present in the lung early in infection may be underestimated by standard detection methods, and primary CD8(+) T cell expansion may occur in both secondary lymphoid organs and the infected lung.

Frequency, Specificity, and Sites of Expansion of CD8+ T Cells during Primary Pulmonary Influenza Virus Infection