FIG 6.

Activation kinetics of M1_58–66-specific CD8⁺ T cells after stimulation with cells infected with isogenic influenza A viruses with gene segment 7 of human or avian influenza A viruses. (A) Gating strategy used to assess the upregulation of activation markers on M1_58–66-specific CD8⁺ T cells. Dot plots gate the lymphocytes, single cells, viable cells, and CD3⁺ CD8⁺ cells, followed by gating for the upregulation of the activation markers CD137, CD69, and CD107a. (B) Gating strategy used to determine infection efficiency of the target cells. Dot plots gate the target cells, the viable cells, and finally the influenza A virus-positive cells. Expression of the activation markers CD137 (C), CD69 (D), and CD107a (E) by M1_58–66-specific CD8⁺ T cells after stimulation with A549-HLA-A*0201⁺ cells infected with recombinant virus A/Puerto Rico/8/1934 with gene segment 7 of avian virus A/Vietnam/1194/2004 (H5N1) (WT avian) or human virus A/Netherlands/178/1995 (H3N2) (WT human) or pulsed with M1_58–66 peptide (GILGFVFTL) or untreated (mock) is shown. (F) Percent infected A549-HLA-A*0201⁺ cells at each time point (without T cells). The x axis represent hours postinfection. A549-HLA-A*0201⁺ cells were infected and peptide pulsed for 1 h and then used to stimulate M1_58–66-specific CD8⁺ T cells. Data points represent means and error bars indicate SD for triplicates (n = 3). *, the differences between avian and human derived viruses were statistically significant after correction for multiple hypothesis testing using an FDR of 0.01.