Potential cross-reactivity of #213 scFv CAR-T cells to homologous peptides on HLA-A*2402. (A) Alanine substitution analysis identified WT1₂₃₅ peptide residues important for recognition by #213 scFv CAR. The WT1_236Y peptide sequence was substituted with alanine from 1 through 9. T2A24 was pulsed with the indicated peptides at 10 μM and cocultured with #213 scFv CAR-T cells for 6 hours. IFN-γ–positive cells gated on CD8⁺ cells in CAR-T cells were assayed by intracellular cytokine staining. (B) The peptides in panel A were subjected to an HLA stabilization assay to determine the critical amino acids required for HLA-A*2402 binding. T2A24 cells were used as peptide-loading cells and their HLA-A*2402 expression was determined by fluorescence-activated cell sorter (FACS). (C) Validation of potential risk peptides derived from the human proteome. T2A24 cells were pulsed with the indicated peptides (sequences provided in supplemental Table 1) at 10 μM and cocultured with #213 scFv CAR-T cells (black column) or mock-transduced T cells (white column) for 24 hours. (D) Validation of potential cross-reactivity to allo-HLAs. LCLs expressing different HLAs were cocultured with #213 scFv CAR-T cells (black column) or mock-transduced T cells (white column) for 24 hours. T2A24 cells pulsed with 10 μM WT1_236Y served as a positive control. Culture supernatants from these cultures were subjected to IFN-γ enzyme-linked immunosorbent assay in triplicate. Error bars represent SD of the mean. A representative result of 3 independent experiments is shown.