Role of the CCR4 antagonist in migration of Tregs to the vaccine-draining LN. Ten million in vitro–generated Tregs from OTII Ly5.2 mice, alone or mixed with the CCR4 antagonist (2 μg), were administered to Ly5.1 B6 mice and immunized 2 hours previously with STxB-OVA (30μg) and αGalcer (1 μg). Twenty-four hours later, vaccine draining LNs were harvested, and cells were stained with anti-CD4 and anti-Foxp3 mAbs. Cells were than gated on CD4⁺Foxp3⁺ cells and stained with anti-Ly5.2 mAb. Isotype controls were included in each experiment. Double-positive Foxp3-Ly5.2 cells correspond to the transferred Tregs. Four mice per group were injected with Tregs with or without the CCR4 antagonist. Experiments shown are representative of 3 series of experiments with similar results. The Mann-Whitney test was used for statistical analysis.