#213 scFv CAR-T cells react to tumor cell lines endogenously expressing WT1 in an HLA-A*2402–restricted manner. (A) IFN-γ production of CD8⁺ cells of #213 scFv CAR-T cells in response to the indicated tumor cell lines was assayed by intracellular cytokine staining after a 6-hour culture (top) along with HLA-A*2402 (middle) and WT1 mRNA expression of those tumor cell lines (bottom). (B) Cytotoxic activity of #213 scFv CAR-T cells against the indicated tumor cell lines was assessed by 6-hour ⁵¹Cr-release assays in triplicate. (C) Confirmation of epitope specificity of #213 scFv CAR-T cells against peptide derived from endogenously expressing WT1. A standard ⁵¹Cr release assay using purified CD8⁺CAR⁺ T cells as effectors and a MESO-4 cell line as a target at an effector-to-target ratio of 2:1 was conducted in the presence of 40-fold excess of unlabeled T2A24 pulsed with either WT1_236Y or MAGE-A4_143-151. Error bars represent SD of the mean. A representative result of 3 independent experiments is shown.