Identification of the minimal epitopes of the HLA-class II–restricted mHags. Three partially overlapping 19-mer peptides comprising a region of 14 amino acids upstream and downstream of the identified polymorphisms (top) as well as series of peptides containing N- and C-terminal truncations (middle) and a peptide comprising the polymorphism of the donor (bottom) were synthesized, pulsed onto donor EBV-LCLs, and tested for recognition by the mHag-specific CD4⁺ T-cell clones in IFN-γ ELISA. Indicated are the variant residue (shaded), the minimal epitope (boxed), and the core region of the minimal epitope (underlined). (A) T-cell clone 62 was tested against peptides comprising LB-MR1-1R. The minimal epitope is the 12-mer YFRLGVSDPIRG. (B) T-cell clone 78 was tested against peptides comprising LB-PTK2B-1T. The core region is the 9-mer VYMNDTSPL. (C) T-cell clone 80 was tested against peptides comprising LB-LY75-1K. The minimal epitope is the 10-mer GITYRNKSLM. (D) T-cell clone 87 was tested against peptides comprising LB-MTHFD1-1Q. The minimal epitope is the 12-mer SSIIADQIALKL. Mean release of IFN-γ (ng/mL) in 50-μL supernatants of duplicate wells is shown.