CpG-A and CpG-B enhance the expansion of IFN-γ–producing, cytotoxic influenza peptide–specific CD8⁺ T cells. PBMCs (3 × 10⁶) from HLA-A2–positive donors were enriched for CD8⁺ T cells and stimulated with the HLA-A0201–restricted Flu matrix_58-66 peptide in the presence or absence of CpG ODN (6 μg/mL). After 10 to 14 days, cells were harvested and further analyzed. (A-C) After staining with HLA-A2/Flu matrix_58-66 tetramers-PE, anti-CD8⁺ PerCP, and Topro-3 (for exclusion of dead cells), the percentage of peptide-specific cells within all CD8⁺ cells was determined by flow cytometry (upper right quadrant: numbers indicate the percentage of peptide-specific CD8 T cells). Results of one exemplary experiment (A) and the mean ± SEM of 16 donors (B), respectively, are depicted. *P < .05; **P < .01. (C) ODN 2137 and ODN 2243 are GC control ODN to ODN 2006 and ODN 2216, respectively. Mean ± SEM of 7 (ODN 2137) and 5 (ODN 2243) donors are shown. *P < .05. (D-E) After restimulation with the Flu matrix_58-66 peptide or an HLA-A2–restricted control peptide (HIV Pol), the percentage of IFN-γ–producing cells within all CD8⁺ cells was measured by intracellular cytokine staining and flow cytometry (indicated by the numbers on the dot plots). Results from a representative experiment (D) and the mean ± SEM (E) of 13 donors are shown. *P < .05. (F) Cells were harvested and used as effector cells in a standard ⁵¹Cr lysis assay against T2 cells pulsed either with the Flu matrix_58-66 peptide or a control peptide derived from the HIV pol protein. The results in peptide-specific percentage specific lysis represent the mean of triplicate measurements from which the nonpeptide-specific lytic activity, defined as percentage specific lysis of T2 cells pulsed with the control peptide, was subtracted. Results from 1 of 4 experiments for the comparison of ODN 2006 and ODN 2216 and 1 of 2 experiments for the comparison of ODN 2006 and ODN 2137 are shown. *P < .05.