Specificity of allo-restricted CTL generated against the WT1-derived peptide P126.

CTL were isolated by limiting dilution cloning of T-lymphocyte bulk cultures from HLA-A0201⁻ donors stimulated with HLA-A0201⁺ stimulator cells coated with P126 peptide. (A) Isolated CTL lines killed the TAP-deficient T2 target cells coated with the immunizing P126 peptide but not T2 cells coated with the HLA-A0201–binding E7 control peptide. (B) Peptide titration experiments showing that 3 anti-P126 CTL lines were of high avidity recognizing low picomolar concentrations of P126, and that 3 CTL lines were of low avidity because nanomolar P126 concentrations were required for target cell recognition. T2 cells coated with the indicated concentrations of P126 were used as CTL targets. High-avidity CTL were used for all subsequent experiments because low-avidity CTL did not recognize target cells expressing WT1 endogenously. (C) High-avidity CTL killed the HLA-A0201⁺ leukemic cell lines BV173, 697 but not the HLA-A0201⁺, EBV-transformed B-lymphoid cells C1R-A2. Coating of C1R-A2 with P126 resulted in efficient CTL killing. The HLA-A0201⁻ leukemia cell line K562 was not killed by the CTL unless transfected with the HLA-A0201 gene.