Reverse immunology strategy for epitope mapping in cyclin-A1 using ICS for IFN-γ. (A) After 4 stimulations with the peptide library, T-cell lines from both donors consisted of more than 60% specific cells (gated on CD8⁺ cells). (B) The cell lines were subsequently tested against 20 peptide pools with each 15-mer being represented in 2 different pools in the peptide matrix. Shaded rows/columns indicate pools with more than 10% IFN-γ-positive cells from each donor. (C) Peptides, which tested positive in both of its pools (> 10% specificity, marked black), were further analyzed as individual peptides. Black columns represent the peptides for which the minimal immunogenic AA sequence from the initial 15-mer was determined.