Figure 4

Cytotoxic activity of A2/PBF A2.2 tetramer-positive CTL line 5A9. A. The peptide-specific cytotoxicity of CTL 5A9 was determined using T2 and K562 cells in a 6 h standard ⁵¹Cr release assay. T2 cells were pulsed with 50 μg/ml peptide PBF A2.2 or medium for 1 h at room temperature after labeling with ⁵¹Cr. CTL 5A9 lysed PBF A2.2 peptide-pulsed T2 cells in an effector:target ratio-dependent manner, but not K562 or T2 cells without peptide pulsation. B. T2 cells were incubated with various concentrations of the PBF A2.2 peptide and 5 μM HIV control peptide. The cytotoxicity of CTL 5A9 against peptide-pulsed T2 cells was determined at an effector to target ratio of 30:1. Dotted lines indicate half maximum lysis. C. The cytotoxicity of CTL 5A9 against allogeneic osteosarcoma cell lines U2OS, Saos-2, KIKU, OS2000 and HOS. All cell lines express PBF. U2OS and Saos-2 are HLA-A*0201-positive. KIKU is HLA-A*0201-negative, HLA-A*0206-positive. OS2000 and HOS are HLA-A*0201-negative. D. Autologous tumor cells were derived from fresh-frozen biopsy specimens of Patient 4, from whom CTL 5A9 was also developed. U2OS and K562 were used as positive control target cells and natural killer target cells, respectively.