Mapping of CMVpp65 peptide epitope specificity and HLA restriction of a representative CMVpp65CTL line. (A) IFN-γ responses of T cells against a matrix of CMVpp65 peptide subpools. CD8⁺ T cells of the CMVpp65CTL line respond selectively to subpools #10 and #19, which uniquely share the QAIRETVEL peptide. Furthermore, these CMVpp65CTLs selectively lyse phytohemagglutinin-stimulated blasts loaded with this peptide that share with the CMVpp65CTLs the HLA-B*3503 and not peptide-loaded phytohemagglutinin-stimulated blasts sharing other HLA alleles in the CMVpp65CTL genotype. This line also contains a nondominant population of IFN-γ⁺ CD8⁺ T cells specific for peptide 44 uniquely shared by subpools 8 and 16 that contains the QWKEPDVYYT epitope that can be presented by the donor’s HLA C0401 allele. However, these T cells were not cytotoxic against peptide-loaded targets sharing any single HLA allele, with donor preventing ascertainment of HLA restriction. Note that a low-level (11%) alloresponse to DQB10503 alone and not against the same cells loaded with CMVpp65 peptides was also seen. (B) This CMVpp65CTL line contains dominant CD4⁺ T cells that selectively generate IFN-γ in response to subpools 3 and 23, which uniquely share the LARNLVPMVATV peptide, and lyse targets loaded with the peptide that selectively share only the HLA-DRB*1-0402 allele with the CMVpp65CTLs. This line also contains 2 other CMVpp65CTL populations: a small population of CD8⁺ T cells restricted by HLA A2601 specific for SHIMLDVAF, a CMVpp65 peptide in 15-mer #72 shared by pools 12 and 18, and an IFN-γ⁺ cytolytic CD4⁺ T-cell population restricted by HLA DQB, 0301, specific for subpools 8 and 20 sharing the 15-mer #92: EHPTFTSQYRIQGKL. E:T, effector-to-target ratio.