Depletion of CD4⁺CD25⁺ cells in C57BL/6 mice after gene transfer with AAV-ApoE/hAAT-hFIX vector. Shown are plasma levels of IgG anti-hFIX as a function of time after vector administration. Animals were injected intraperitoneally with either rat IgG1 (A, isotype control) or rat antimouse CD25 monoclonal antibody PC61 (B) every 2 weeks (up to week 6) starting on the day of liver-directed gene transfer (asterisk). Mice were challenged with 5 μg hFIX formulated in CFA at week 6. (C) Analyses of peripheral blood lymphocytes by flow cytometry showed depletion of CD4⁺CD25⁺ cells after repetitive anti-CD25 injection in C57BL/6 mice. Antibody staining was carried out using FITC-conjugated anti-CD4 and anti–PE-conjugated CD25.