PRAME CTLs derive from the naive T-cell subset. To generate PRAME CTLs from healthy donors, CD8⁺ T cells were stimulated weekly with peptide-loaded K562/aAPCs in the presence of IL-7, IL-12, and IL-15, or primed with peptide-loaded autologous pAPCs in the presence of IL-7 and IL-12 with or without IL-15, then expanded with peptide-loaded K562/aAPCs. (A) Frequency of T cells responding (in an IFNγ ELIspot assay) to PRAME peptides; shown is ALY as representative peptide, ■) or an irrelevant peptide (ELA, ). The number of IFNγ⁺ SFC was negligible when T cells were stimulated weekly only with peptide loaded K562/aAPCs. Although IFNγ⁺ SFC were detectable when T cells were primed with peptide-loaded pAPCs, only the addition of IL-15 to the cytokine cocktail significantly increased the generation of ALY-specific CTLs. Shown is 1 representative of 4 donors. (B) Fold expansion of PRAME CTLs primed with autologous pAPCs and stimulated with aAPCs for 3 weeks. Each symbol represents one of the 8 individual PRAME CTL lines and the horizontal lines represent the mean group value. (C) IFNγ⁺ SFC in response to ALY-peptide (■) or ELA-irrelevant peptide () of ALY-specific CTLs expanded from 3 donors from sorted naive (CD45RA⁺) and memory (CD45RO⁺) T-cell subsets. T cells producing IFNγ in response to the ALY 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 (from the pp65 protein of CMV), were originated predominantly from the memory (CD45RO⁺) T-cell subset (D).