Design and characterization of a WT1_235-243/HLA-A*2402-specific CAR. (A) Schematic representation of a retroviral vector encoding WT1_235-243/HLA-A*2402-specific CAR. (B) Expression of CAR on human T cells. Human PBMCs stimulated with plate coated anti-CD3 were transduced with the retroviral vector as depicted in panel A. Four days after the transduction, cells were stained with phycoerythrin-labeled HLA-A*2402 tetramers presenting WT1_236Y along with APC anti-CD4 and APC/Cy7 anti-CD8. Mock-transduced T cells served as background staining. (C) Production of IFN-γ by #213 scFv CAR-T cells stimulated with immobilized HLA-A*2402 tetramers loaded with WT1_235-243 or WT1_236Y. Culture supernatants were harvested at 24 hours and subjected to IFN-γ enzyme-linked immunosorbent assay in triplicate. (D) Cytotoxic activity of #213 scFv CAR-T cells against T2A24 cells pulsed with the indicated peptides at 10 μM was assayed by 6-hour ⁵¹Cr-release assays in triplicate. (E) IFN-γ production of #213 scFv CAR-T cells stimulated with T2A24 cells pulsed with the indicated peptides. % IFN-γ–positive cells within CD8⁺ cells of #213 scFv CAR-T cells were assayed by intracellular cytokine staining after a 6-hour culture in single experimental samples. Error bars represent standard deviation (SD) of the mean. A representative result of 3 independent experiments is shown.