T-cell assays to determine whether a strongly A33-associated mutation in RT lies in a novel epitope. (A) T-cell lines from A33⁺ donors were established by stimulation with an 18-mer peptide reflecting the consensus sequence (pol 54) and tested for recognition against A33-matched targets pulsed with the overlapping peptides (pol 53 and 55, top) and with truncated peptides (bottom) to define the optimal epitope. (B) Confirmation of HLA-A33 restriction was performed with target cell lines matched only at HLA-33 or lacking HLA-A33. (C) Confirmation that the N447S mutation represents an escape from T-cell recognition was performed with pol 54 peptide-specific T-cell clones tested for recognition of the wild-type and mutant peptides in an ELISPOT assay with 2 different A33-expressing target cells.