Evaluation of the recognition of KDR⁺ HRPEpic and CD34⁺ cells by K288.E5. (A) KDR expression by ARPE-19 cells was determined in Western blot. Two HUVEC-cell lysates were used as positive controls. The β-actin control is shown for each extract. (B) Failure of K288.E5 to recognize ARPE-19 cells transfected with the HLA-A2 gene. In contrast, efficient T-cell recognition was observed on peptide-pulsed cells (10 μg/mL). Recognition of HUVEC2 cells is also shown for comparison. (C) KDR expression by ex vivo selected human cord blood CD34⁺ cells analyzed by flow cytometry. Percentages represent the number of KDR⁺ cells in anti-KDR scFv-enriched and -depleted CD34⁺ cell fractions. Results from 1 of 2 representative A^*0201⁺ donors are shown. (D) HLA class 1-restricted recognition of KDR⁺CD34⁺ cells by K288.E5. KDR scFv-enriched (▪) or -depleted () CD34⁺ cells (1 × 10⁴/well) were cocultured overnight with CTLs (2 × 10⁴/well) in the presence (□) or absence of W6/32 (20 μg/mL). IFN-γ released in culture supernatants was determined by IFN-γ ELISA. Each value represents the mean ± SD of 6 cultures from 2 different experiments.