Specificity of allo-restricted CTLs generated against the WT235 peptide.

(A) Killing of T2 cells (HLA-A2⁺, TAP⁻) coated with WT235 peptides (open symbols) and an A2-binding control peptide from the E7 protein of human papilloma virus (solid symbols). Three allo-resticted CTL lines (line 2, 4, 12) were tested in this experiment, and line 4, which expanded best, was used in all subsequent experiments. (B) Killing of T2 targets coated with WT125 or WT235 peptides using the WT235 CTL line 4 (squares) or a previously described allo-restricted CTL line 77 (round symbols) raised against the WT126 peptide (see Gao et al11). (C) Killing activity of WT235 CTL line 4 against T2 loaded with decreasing amounts of WT235 peptide. (D) RT-PCR of a panel of leukemia lines. Shown is RT-PCR amplification of WT1 RNA and the housekeeping abl RNA of 8 leukemia lines. The lymphoblastoid cell line C1R-A2 served as negative control for WT1 expression. RT-PCR with intron-spanning primers of WT1 and abl was performed as described previously.11 (E) Killing of a panel of leukemia lines by the WT235-specific CTL line 4 and by the WT126-specific CTL line 77 (F). The leukemia cell lines LAMA81, BV173, 697, and BAF-3 were HLA-A2⁺, and SD-1, U937, and KU-812 were HLA-A2⁻ (determined by FACS staining with 2 A2-specific antibodies). In panel E, T2 cells were coated with WT235 peptide and in panel F with WT126 peptide. (G) Killing activity of the WT235-specific line 4 against the lymphoblastoid cell line C1R-A2 in the presence and absence of WT235 peptides. (H) Killing activity of the WT235-specific line 4 against purified normal CD34⁺ cells or CML patient-derived CD34⁺ cells. CML patients 1, 3, and 4 were HLA-A2⁺ and CML patient 2 was A2⁻.