2.8. T cell proliferation assay

Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were labeled with 1 µmol/L carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher Scientific) according to the manufacturer's protocol. CFSE‐labeled PBMCs (1 × 10⁵) were plated into 96‐well plates with transduced HSC‐3 cells (2 × 10⁴) in RPMI medium supplemented with 10% FCS, 100 units/mL penicillin, and 100 μg/mL streptomycin. Following the addition of anti‐CD3/anti‐CD28 stimulus (Treg Suppression Inspector human; Miltenyi Biotec), the cells were incubated for 96 h. After incubation, floating cells were harvested and stained with allophycocyanin (APC)‐CD3 antibody (BD Bioscience) at 4°C for 1 h. Then, 7‐AAD (BD Bioscience) was added 10 min before the analysis. The proliferation of CD3‐positive 7‐AAD‐negative cells was measured by dilution of CFSE staining using flow cytometry.