A CCR4 antagonist combined with STxB-Her2/neu or a DNA-E7 (pgDE7) vaccine enhances Ag-specific CD8⁺ T cells in HLA-A2 or E7 TG mice. (A) HLA-A2 TG mice were immunized twice (day 0 and day 14) subcutaneously with STxB-Her_435-443 or the Her2_435-443 peptide in combination or not with the CCR4 antagonist. One week later, splenocytes were harvested and purified with anti-CD8–coated magnetic beads. Detection of anti-Her2_435-443 CD8⁺ T cells was performed by ELISPOT with the use of CD8⁻ cells because APCs were sensitized with the Her_435-443 or an irrelevant peptide. (B) Mice were immunized at day 0 and day 14 with STxB-gp100_25-33 (30 μg) alone or combined with the CCR4 antagonist (1.5 μg). One week later, splenocytes were harvested, and anti-gp100_25-33 CD8⁺ T cells were detected by IFNγ ELISPOT. The number of spots was calculated after subtracting background (APCs incubated with medium alone). Threshold of positivity was defined at 10 spot-forming cells/2.10⁵ cells. (C) E7 TG mice were immunized twice intramuscularly with DNA-E7 (100 μg) in combination or not with the CCR4 antagonist. One week later, splenocytes were harvested and semipurified with anti-CD8–coated magnetic beads. Detection of anti-E7_49-57 CD8⁺ T cells was performed with E7_49-57 D^b tetramer (top and bottom left) gated on CD8⁺ T cells or by ELISPOT (bottom right). An irrelevant tetramer was used in all experiments (top right), and the number of spots was calculated after subtracting background (APCs incubated with medium alone). Results represent the mean of 4 mice per group and has been reproduced twice. P values were calculated by the Mann-Whitney U test.