Identification of CMVpp65 epitopes with the use of a CMVpp65-peptide library. (A) CTLs (105/well) from CB3 (HLA-A2, A29, B35, B44) were stimulated with a CMVpp65-peptide library pooled into 22 pools. Responses were measured in an 18-hour IFN-γ ELISPOT assay. Shown is mean and SD of duplicate wells. (B) All peptides were divided into 22 pools in such a way that each peptide is present in 2 pools. This method allows determining those single peptides that probably induced responses to the peptide pools. Thus, responses to pools 7, 8, and 13 or 9, 10, and 18 or 10, 11, and 14 may be induced by single peptides 7 and 8 or 69 and 70 or 22 and 23, respectively (C) Testing of these individual 20mers identifies the sequence of peptides 7 and 8 or 69 and 70 as most probably the overlapping 15 amino acids, as the CTL epitope. In addition, the T-cell line mapped to a known HLA A2–restricted epitope (MLNIPSINV) contained in peptides 23 and 24. (D) The polyclonal CB-derived virus-specific CTL line in which this epitope had been identified was stained with an HLA-A*0201 MLNIPSINV tetramer. Indicated is the percentage of tetramer-positive cells within the CD8+ population.
