Generation of FVIII-specific CAR T cells. (A) Schematic view of transgenes used. FVIII-specific ANS8 CAR, containing the FVIII-specific scFv 1G10 in comparison with the empty Mock control. (B) Retrovirally transduced Mock or ANS8 CAR Tregs were incubated with biotinylated FVIII (1.8 μg/mL) or OVA (1.8 μg/mL). Cells were stained with PE-conjugated streptavidin and analyzed by FACS. GFP⁺ ANS8 CAR Tregs showed specific binding to biotinylated FVIII. (C) Transduced, cell proliferation dye-labeled ANS8 CAR T-effector cells were incubated with FVIII or FVIII in combination with irradiated autologous PBMCs for 6 days and analyzed for cell proliferation. ANS8 CAR–transduced cells strongly proliferated in the presence of FVIII and PBMCs. The histogram shows viable CD4⁺ cells. Similar results were obtained in independent experiments from at least 3 different healthy T-cell donors.