CpG-B but not CpG-A promotes priming of IFN-γ–producing, cytotoxic Melan-A peptide–specific CD8⁺ T-cells. PBMCs (3 × 10⁶) from HLA-A2–positive donors were enriched for CD8⁺ T cells and stimulated with the HLA-A*0201–restricted Melan A_26-35 A27L peptide in the presence or absence of CpG ODN (6 μg/mL). After 10 to 14 days, the cells were harvested and further analyzed. (A-C) After staining with HLA-A2/Melan A_26-35A27L 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. Results of a representative experiment (A; numbers indicate frequency of peptide-specific CD8 T cells in the upper right quadrant), the mean ± SEM of 16 donors (B) and the mean ± SEM of 3 donors (C), respectively, are depicted. **P < .01. ODN 2137 is a GC control ODN to ODN 2006. (D) After restimulation with the Melan A_26-35A27L peptide or an HLA-A2–restricted control peptide derived from HIV Pol, the percentage of IFN-γ–producing cells within all CD8⁺ cells was measured by intracellular cytokine staining and flow cytometry. Data from 12 donors are presented as mean ± SEM. *P < .05. (E) Cells were harvested and used as effector cells in a standard ⁵¹Cr lysis assay against T2 cells pulsed with Melan A_26-35A27L peptide or a control peptide derived from the HIV pol protein (1 of 3 experiments).