CTL clone RDR173 recognizes an epitope translated in an alternative ORF. (A) Deletion constructs derived from patient and donor ECGF-1 cDNA were tested for recognition by CTL clone RDR173 on transfection into COS/HLA-B7 cells. The smaller fragments were hardly recognized by CTL clone RDR173, although they contained the polymorphism. (B) To investigate whether the epitope was encoded by an alternative ORF construct with the possible alternative CTG start codon at positions 1441 to 1443 mutated into an AAG codon, constructs with a stop codon introduced in the alternative ORF, and deletion constructs with Kozak-ATG sequences in the alternative ORF were transfected into COS/HLA-B7. Transfected cells were cocultured with CTL clone RDR173 for 24 hours. Release of IFN-γ was measured by ELISA.