Numerous experimental studies have demonstrated that CD8⁺ T cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8⁺ T cells. Here we use a novel computational approach to test for selection in CD8⁺ T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8⁺ T-cell responses and then compare the evolution of these epitopes in parallel lineages of human and swine influenza viruses that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8⁺ T-cell epitopes in NP of human versus swine influenza virus, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions in human influenza virus NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza virus NP, sites in T-cell epitopes evolve more slowly than do nonepitope sites, presumably because these epitopes are under stronger inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8⁺ T cells in human influenza virus NP and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint.

IMPORTANCE There is a strong interest in correlates of anti-influenza immunity that are protective against diverse virus strains. CD8⁺ T cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza virus evolution. Although many studies have shown that T cells limit viral replication in animal models and are associated with decreased symptoms in humans, no studies have proven with statistical significance that influenza virus evolves under positive selection to escape T cells. Here we use comparisons of human and swine influenza viruses to rigorously demonstrate that human influenza virus evolves under pressure to fix mutations in the nucleoprotein that promote escape from T cells. We further show that viruses with these mutations have a selective advantage since they are preferentially located on the “trunk” of the phylogenetic tree. Overall, our results show that CD8⁺ T cells targeting nucleoprotein play an important role in shaping influenza virus evolution.