Analyses of CMV-specific T-cell line of an HLA-A*0201⁺healthy seropositive donor after sensitization with the complete pool of pentadecapeptides overspanning the entire pp65 protein. (A) Capacity of T cells to produce intracellular IFN-γ in response to PBMCs pulsed with 1 of each of the 24 pentadecapeptide pools was analyzed. Percentages of CD8⁺ CD69⁺ IFN-γ⁺ cells are shown in the right upper quadrant. Nonpulsed PBMCs and the majority of pentadecapeptide pools (pool 1, 2, 5, 9-17, 19-22, 24) did not induce intracellular IFN-γ production above background. In contrast, pools 3, 4, and 23, containing the known HLA-A*0201 binding epitope NLVPMVATV, induced intracellular IFN-γ production of 22% to 39% of CD8⁺ cells. In addition, pools 6, 7, and 18 induced intracellular IFN-γ production in 5% to 7.5% of CD8⁺ cells. (B) IFN-γ production in response to single pentadecapeptides (121-132) of peptide-pool 23. Pentadecapeptides 123 and 124, containing the full length of the NLVPMVATV epitope, induced intracellular IFN-γ production in 37% and 23% of the CD8⁺ cells, respectively. Pentadecapeptide 122, containing 5 amino acids of the nonamer, induced IFN-γ production by 7% of CD8⁺ cells. IFN-γ response was negative for the remaining pentadecapeptides of this pool. (C) Intracellular IFN-γ production in response to single pentadecapeptides (61-72) of peptide pool 18. Pentadecapeptides 66 and 67 induced intracellular IFN-γ production by 5.6% and 5.1% of CD8⁺ cells, respectively. The remaining pentadecapeptides did not induce IFN-γ production above background.