HA-specific CD4⁺CD25⁺ cells allow transgene engraftment in transduced muscle. BALB/c mice were injected intramuscularly at day 0 with AAV-HA, and were either untreated (A,D) or injected intravenously at days 0 and 4 with 10⁶ CD4⁺CD25⁺ cells from BALB/c mice (B,E) or CD4⁺CD25⁺ cells from TCR-HA mice (C,F). At days 14 (A-C) and 35 (D-F), muscles were frozen and HA expression was assayed by immunohistochemistry using horseradish peroxidase (HRP)/diaminobenzidine (DAB) staining in brown on sections counterstained with methyl green, which stains the nuclei of muscle and infiltrating lymphoid cells in blue/green. Images are representative of 3 mice per group. Original magnification × 40. Quantification of HA expression was assessed by computer-assisted image analysis (Histolab; Microvision Instruments, Evry, France). For each mouse in each condition, 5 to 10 entire transverse sections of tibialis anterior were measured. The total section and HA-positive areas were determined by image texture and HRP/DAB color analysis, respectively. The reproducibility of image analysis was controlled using normal muscle sections as a standard on each slide. Results are expressed as an index of HA-positive area over the total tibialis anterior area and represent the mean ± SEM of 3 mice per group, at days 14 (G) and 35 (H).