Natural processing and presentation of KDR_288-297 peptide to cloned specific CTLs. (A) KDR protein expression by 293-EBNA and HEK-293T cells transfected with full-length KDR cDNA. Cells were transfected with 24 μg KDR plasmid DNA using LipofectAMINE. KDR expression was determined by Western blot. EBV-B cells and nontransfected cells were used as controls. (B) Recognition of transfectants by cloned CTLs. Cells (1 × 10⁴ 293-EBNA or A^*0201-expressing HEK-293T) were transfected with 100 ng KDR, A^*0201 plasmid DNA, or both using LipofectAMINE. Positive controls were cells pulsed with the exogenous KDR_288-297 peptide at 10 μg/mL. T-cell assays were performed using cloned CTLs K288.E5, K288.G2, and K288.B6 specific for KDR_288-297 peptide at an E/T ratio of 2:1. Cytokine contents in 24-hour culture supernatants were determined by standard IFN-γ ELISA and TNF-β bioassay.²⁰  (C) Antigen-specific recognition of K288.E5 in cold-target inhibition assay. Effector cells (5 × 10⁴/well) were incubated with ⁵¹Cr-labeled KDR/HEK-293T cells (5 × 10³/well) in the presence of cold targets, KDR/HEK-293T, T2 alone, or T2 pulsed with KDR_288-297 or an irrelevant peptide, at an inhibitor-target ratio of 20:1. Lytic activity was measured by ⁵¹Cr release assay. Numbers above bars represent percentage inhibition of lysis. (D) No reactivity of K288.E5 with KDR-negative primary cells and cell lines. Cells loaded with KDR_288-297 peptide (10 μg/mL) were used as positive controls. Shown are the mean ± SD of triplicate wells consisting of 2 × 10⁴ CTLs and equal numbers of individual targets. The lack of KDR protein in these cells was affirmed by Western blot analysis using a primary HUVEC culture and the β-actin marker as positive controls. All experiments were performed at least twice, with similar results.