Human WT1-peptide–specific CTLs can be expanded in vitro and specifically kill peptide-pulsed cells. (A) Independently in vitro expanded WT1.126 (top row) and WT1.235-specific (bottom row) CD8⁺ T cells were identified on day 31 and day 45 of culture, respectively, by double-labeling using specific–HLA-A*0201/peptide-biotin labeled pentamers with streptavidin-PE and anti–CD8-PE-Cy5 labeled antibody. Negative controls were similarly stained with the opposite and irrelevant HLA-A*0201/peptide pentamer. Numbers indicate the percentage of CD8⁺ cells of whole culture capable of binding pentamer. On day 54 of culture, (B) WT1.37, (C) WT1.126, or (D) WT1.235-peptide–specific CD8⁺ T cells were tested for their ability to lyse HLA-A*0201 autologous PHA-Blasts (B) or the VAL cell line (C and D) pulsed with the indicated peptides at the effector/target (E/T) ratios shown. For each CTL line generated (3 WT1.37-specific, 1 WT1.126-specific, and 2 WT1.235-specific), the assay was performed twice. Data were similar in each case and representative results are shown.