Recognition of human KDR-derived peptides by CD3⁺CD8⁺ CTLs generated from PBMCs of donors S, M, and P. (A) Cytotoxicity was determined in a 6-hour LDH release assay using T2 cells preincubated for 60 minutes with each peptide at 25 μg/mL (•). T2 alone (○), K562 (▵), and Daudi (□) cells were used as negative controls. Percentage of specific lysis is shown at different E/T ratios. (B) Dose-response recognition of KDR_288-297 peptide by CTL clone K288.E5. Serial dilutions of KDR_288-297 peptide were incubated with 5 × 10⁴ T2 cells for 60 minutes, which were then used as target cells to stimulate T cells. Control peptide KDR_82-90 was also used at various concentrations. This experiment was performed twice; results were similar each time. (C) Assessment of peptide-specific recognition of K288.E5 by HLA/peptide tetramer staining. CTLs specific for KDR_288-297 peptide (K288.E5) or for Melan-A_26-35 epitope (Melan-A CTL) were stained with FITC-anti-CD8 mAb and PE-HLA-A^*0201 tetramers folded around either the KDR_288-297 peptide (TA2.KDR) or a EBV/BMLF1-derived epitope (TA2.EBV). Data shown are representative of 2 independent experiments. (D) T-cell recognition of KDR_288-297-pulsed T2 cells was specifically inhibited by mAbs against HLA class 1 and HLA-A2. Results are expressed as IFN-γ release (picogram per milliliter daily) from 2 × 10⁴ K288.E5 CTLs. All antibodies were used at a final concentration of 20 μg/mL each.