Implementing CD137 enrichment for epitope discovery. (A) Experimental outline: After 3 rounds of stimulation with a pool of 110 overlapping 15-mers, CD137⁺ cells were enriched and further expanded. The immunogenic peptides were subsequently identified by step-wise testing of smaller peptide pools. (Bi) An enriched T-cell line was tested for IFNγ production in response to autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) pulsed with pools of peptides (10 or 11 peptides/pool). (ii) Peptides derived from pools 3, 4 (clearly positive), and 7 (potentially positive) were tested in smaller pools containing 2 to 5 peptides, in which the peptides were rearranged by selecting partially overlapping peptide sequences. (iii) Identification of peptides 71 and 72 as the stimulating peptides. (C) Fine mapping of the minimal essential amino acid sequence was performed using a T-cell line generated from a second HLA-Cw07⁺ donor after 3 stimulations with the immunogenic peptide. A panel of peptides was synthesized and tested for recognition of the minimal essential amino acid (see also Table 4) using peptide-pulsed, autologous B-LCLs as stimulators.