PRAME CTLs can be reproducibly generated from HLA-A*02⁺ healthy donors. (A) illustrates the frequency of IFNγ + T cells responding to 2 PRAME peptides (ALY and VLD peptides) for PRAME CTL lines generated from 9 HLA-A*02⁺ healthy donors after 3 stimulations, as assessed by ELIspot assay. PRAME CTLs from donors 1 and 2 also targeted SLL and SLY peptides, respectively (see “Cytokine release”). Fewer than 25 IFNγ⁺ SFC were counted when CTL lines were pulsed with the ELA-irrelevant peptide (indicated by ----). (B) Staining with the ALY-specific tetramer of 2 PRAME CTL lines generated from healthy donors. (C) PRAME CTLs expanded from healthy donors were evaluated for their cytotoxic activity, using a standard 4-hour ⁵¹Cr release assay, against autologous PHA blasts loaded with ALY peptide (▴), ELA-irrelevant peptide (■), or no peptide (●). Data represent the means plus or minus SD of PRAME CTLs from 5 healthy donors. (D) Killing (20:1 E:T ratio) of ALY-loaded autologous PHA blasts by CTLs was significantly inhibited by preincubation of the targets with MHC class I antibody, but not by an isotype control, indicating MHC restricted killing. NT indicates not tested.