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. 2020 Aug 26;111(10):3516–3526. doi: 10.1111/cas.14602

FIGURE 3.

FIGURE 3

#25‐8 recognizes WT1C but cross–reacts to unknown peptides. A, Binding of #25‐8 to a panel of human cell lines. The indicated human cell lines were stained with 1.0 μg/mL #25‐8 or control mouse IgG, and binding was analyzed by FACS. The integrity of the peptide/HLA‐A*02 expression was confirmed with an antibody (BB7.2) against HLA‐A*02. B, PBMC from HLA‐A*24+ healthy donors stained with #25‐8. A representative gating strategy and #25‐8 histogram compared with a control IgG are shown. C, HLA‐A*02 high cells stained with #25‐8 or control IgG. HeLa, HeLa/A02, or HeLa/A2/WT1 cells were treated with or without IFN‐γ for 12 h, and HLA‐A*02 high cells were gated. The binding ability of #25‐8 was evaluated according to mean fluorescent intensity. The bar in each histogram represents the gate defined as HLA‐A*02 high cells (lower panel). Data are representative of two independent experiments. D, The specificity and cross–reactivity of #25‐8 reflects the cytotoxic activity of #25‐8 CAR T‐cells. #25‐8 CAR or CD19 CAR T‐cells were mixed with HeLa, HeLa/A02, or HeLa/A2/WT1 cells, and the cytotoxic activity of the CAR T‐cells was measured. The values for cell viability are represented as the average ± SD of three replicates (n = 3) of two independent experiments (**P < 0.01).