ALY-specific CTLs are cytotoxic to PRAME⁺ tumor cell lines. (A) Expression of PRAME in several tumor cell lines as assessed by qPCR. (B) Expression of PRAME in the same cell lines by Western blot analysis. In addition to the predicted band (58 kDa), another band of approximately 75kDa was observed, likely originated from posttranscriptional modifications. (C) Cytotoxic activity of ALY-specific CTLs (■) toward tumor cell lines, evaluated using a standard ⁵¹Cr release assay. As negative controls, we used (i) CTLs specific for another HLA-A2*02 tumor-restricted epitope (ELA from MART-1 protein; ); (ii) autologous PHA blasts loaded with ELA-irrelevant peptide; (iii) the L428 tumor cell line (PRAME⁺ but HLA-A*02⁻). As positive control we used autologous PHA blasts loaded with the ALY peptide. Data at a CTLs:tumor cells ratio of 40:1 are shown. Overall, killing of PRAME⁺/HLA-A*02⁺ was significantly higher than control cell killing. (D) Killing of PRAME⁺/HLA-A*02⁺ cell lines was inhibited by preincubation with MHC class I antibody but not with isotype control, confirming MHC class I restriction.