Functional P435/NLT–specific CTLs can be expanded from HLA-A*02⁺ healthy donors. P435/NLT could be used to generate PRAME-CTLs. (A) IFNγ response of P435/NLT–specific or P4-specific CTLs generated from 10 healthy HLA-A*02⁺ donors. P435/NLT–specific CTLs could be generated from 9 of 10 samples tested. (B) Expansion of P435/NLT–specific or P4-specific CTL lines after 3 rounds of stimulation. (C) Staining with the P435/NLT–specific tetramer of CTL lines generated from 2 representative healthy donors. (D) IFNγ⁺ SFCs in response to P435/NLT-peptide or NLV-pp65 (from the pp65 protein of CMV) peptide of P435/NLT–specific CTLs expanded from sorted naive (CD45RA⁺) and memory (CD45RO⁺) T-cell subsets. T cells producing IFNγ in response to the P435/NLT peptide were significantly higher in CTL lines that originated from the naive (CD45RA⁺) T-cell subset. In contrast, CTLs generated against the viral peptide NVL originated predominantly from the memory (CD45RO⁺) T-cell subset. Shown is 1 of 4 representative donors.