Identification of an immunodominant, HLA-A*02–restricted PRAME epitope. When we minimized subpool 11 in ten 15-mer overlapping peptides, IFNγ production by PRAME-CTL was directed against the P435/NLT peptide. (A) Reactivity of PRAME-CTLs was predominantly directed against two 15-mers, P429 and P433. (B) Five predicted HLA-A*02–binding peptide motifs in this region using 2 computational algorithms (SYFPEITHI and BIMAS; Table 3). (C) IFNγ production by 8 PRAME-CTL lines against the 5 minimized epitopes. Although some IFNγ production was observed against the P429-9mer (HLI), the most significant response was observed for the P435-9mer (NLT). (D) Stabilization assay of the new epitope P435/NLT for the HLA-A*02 molecule using a T2-binding assay. An HLA-A11–restricted peptide was used as a negative control. FL, a hepatitis B virus core antigen with high HLA-A*0201–binding affinity, was used as a positive control. The affinity of P435/NLT was superior to that of the previously described P300/ALY.