Figure 2.

Tetramer‐based detection of anti‐PBF A24.2 peptide CTLs in peripheral blood of patients with osteosarcoma. (a) peripheral blood mononuclear cell of Patient 26 were seeded into 62 microwells and stimulated with the papillomavirus binding factor (PBF) A24.2 peptide by mixed lymphocyte peptide culture under limiting dilution conditions (LD/MLPC). The resultant cytotoxic T lymphocytes (CTL) pools were stained with the phycoerythrin (PE)‐conjugated A24/PBF A24.2 tetramer, fluorescein isothiocyanate‐conjugated control tetramer, and a PE‐Cy5‐conjugated anti‐CD8 mAb. Cells reacting with the anti‐CD8 mAb were gated. The reactivity of gated cells with the A24/PBF A24.2 tetramer and the control tetramer are shown. The upper and middle columns display six representative pools with positive reactivity to the A24/PBF A24.2 tetramer. The cells labeled with the A24/PBF A24.2 tetramer and non‐labeled cells with the control tetramer were considered to be tetramer‐positive cells and are boxed to show their proportion among CD8⁺ cells. The bottom row shows three negative pools. (b) CD8⁺ cells (2.5 × 10⁵ cells/well) of Patient 36 were seeded into 37 wells of a 48‐well culture plate and stimulated with irradiated peptide‐pulsed CD8⁻ cells (5 × 10⁵ cells/well) by LD/MLPC. Tetramer analysis was performed on day 14. The results of one of the four tetramer‐positive CTL line (CTL #034) in the 37 pools are shown. Cells reacting with the anti‐CD8 mAb were gated. The tetramer‐positive cells are boxed to show their proportion among CD8⁺ cells. (c) The cytotoxicity of CTL #034 against allogeneic osteosarcoma cell lines (OS2000, HOS, U2OS), LCL‐OS2000 and K562 was assessed by a 6 h standard ⁵¹Cr release assay at the indicated effector:target ratios. OS2000 was assayed in the presence and absence of 48 h‐interferon‐gamma pretreatment. (d) The cytotoxicity of CTL #034 against peptide‐pulsed LCL‐OS2000 and K562 was assessed by a 6 h standard ⁵¹Cr release assay at the indicated effector:target ratios. LCL‐OS2000 was pulsed with 25 µg/mL of PBF A24.2 peptide or HIV control peptide for 2 h at room temperature before labeling with ⁵¹Cr. (e) Cold target inhibition assay. The cytotoxicity of CTL #034 against interferon‐gamma‐treated, ⁵¹Cr‐labeled OS2000 was assessed in the presence and absence of a 100‐fold excess of PBF A24.2 peptide‐pulsed, cold LCL‐OS2000 at a 10:1 effector‐target ratio. ⁵¹Cr‐labeled LCL‐OS2000 cells pulsed with the PBF A24.2 peptide or HIV peptide were also used as control target cells.