Expression of Aur-A in leukemia cell lines and the cytotoxicity of AUR-1 against leukemia cell lines. (A) Expression of Aur-A mRNA and protein in leukemia cell lines and normal PBMCs. Expression levels of Aur-A mRNA in the cells were determined by QRT-PCR as detailed in “Methods.” The level of Aur-A mRNA expression in the K562 leukemia cell line, which strongly expresses Aur-A, is shown as 1.0 and the expression levels in the cells were calculated relative to this value. Aur-A protein expression was examined by Western blotting using anti–Aur-A antibody and anti–β-actin antibody as the control. (B) Cytotoxicity of the Aur-A_207-215–specific CTL line AUR-1 against leukemia cell lines. The cytotoxicity of AUR-1 to HLA-A*0201–positive and HLA-A*0201–negative leukemia cell lines was determined by 4-hour ⁵¹Cr-release assays at E/T ratios of 10:1, 5:1, and 2.5:1. (C) HLA class I restriction of cytotoxicity mediated by AUR-1 against leukemia cells. The cytotoxicity of AUR-1 against leukemia cell lines (GANMO-1 and CMK11-5) was determined by 4-hour ⁵¹Cr-release assays at an E/T ratio of 2.5:1 in the presence or absence of anti–HLA class I MoAb or anti–HLA-DR MoAb. (D) Cold target inhibition assays. ⁵¹Cr-labeled GANMO-1 cells (5 × 10³ cells) were mixed with various numbers of ⁵¹Cr-unlabeled Aur-A_207-215 peptide–loaded autologous LCL cells (○) or with ⁵¹Cr-unlabeled Aur-A_207-215 peptide–loaded HLA-A*0201–negative allogeneic LCL cells (●). The cytotoxicity of AUR-1 to the mixture of ⁵¹Cr-labeled and unlabeled target cells was determined by 4-hour ⁵¹Cr-release assays at an effector-to-⁵¹Cr-labeled target cell ratio of 10:1.