CD8+ T cell proliferation assay

Untransduced and transduced CD8⁺ T cells were washed with PBS to remove serum that affects staining. Then, cells were suspended in PBS at a concentration of 2.4 × 10⁶ cells/mL and labeled with the CTV Proliferation Kit (5 mM; Thermo Fisher Scientific) at 37°C for 20 min. Subsequently, cells were washed with warm fetal calf serum and resuspended in CTL media. After 24 h, cells were analyzed with flow cytometry to confirm CTV staining. Cells were stimulated with aCD3/CD28 Dynabeads in a ratio 1:8 (beads:T cells) or with a tetramer molecule loaded with pWT1₁₂₆. After 4 and 7 days, T cells were stained with Fixable Viability Dye and analyzed with flow cytometry to detect a decrease of CTV signal.