P435/NLT–specific CTLs are cytotoxic to PRAME+ tumor cell lines. (A) Cytotoxic activity of P435/NLT–specific CTLs toward autologous PHA blasts loaded with NLV-irrelevant peptide or loaded with the NLT peptide. Data represent the means ± SD of PRAME-CTLs from 7 healthy donors. (B) Data at a 20:1 E:T ratio of P435/NLT–specific CTLs against autologous PHA blasts loaded with NLV-irrelevant peptide or loaded with the NLT peptide preincubated with isotype control or an anti–HLA class I antibody to confirm MHC-restricted killing. (C) Cytotoxic activity of P435/NLT–specific CTLs toward PRAME⁺ HLA-A*02⁺ tumor cell lines evaluated using a standard ⁵¹Cr-release assay. As negative controls, we used the L428 tumor cell line (PRAME⁺ but HLA-A*02⁻). Killing of PRAME⁺HLA-A*02⁺ cell lines was significantly higher than that of the PRAME⁺HLA-A*02⁻ L428, and was inhibited by preincubation with an anti–HLA class I antibody but not by an isotype control. (D) IFNγ ELISpot release of P435/NLT–specific CTLs or control/irrelevant CTLs against the indicated PRAME⁺ HLA-A*02⁺ cell lines or the control PRAME⁻ HLA-A*02⁺ cell line.