Determination of sequence and HLA restriction of a pp65-derived epitope in a seropositive individual with uncommon HLA type after sensitization of T cells with the complete pool. T cells of an individual with the HLA-type A*6801, A*6601 and B*4002, B*5301 were generated as described. (A) Intracellular IFN-γ production in response to PBMCs pulsed with pools 6, 7, 18 and complete pool was significant. Pools 1 and 2, representative for all other pentadecapeptide pools, were negative for intracellular IFN-γ production. (B) Only pentadecapeptides 66 and 67 of pool 18 stimulated T cells effectively. IFN-γ response of neighboring pentadecapeptides 65 and 68, as well as all other pentadepeptides contained within the pool 18 (not shown), was negative. (C) Based on the overlapping principle, the amino acid sequence RPHER NGFTVL, represented within pentadecapeptide 66 and 67, likely contains the candidate nonameric peptide being recognized by the T cells. Three candidate nonamers were synthesized and tested. Only the peptide HERNG FTVL induced a significant T-cell response. (D) The HLA restriction of this nonamer was tested by pulsing BLCLs, each expressing one of the individual's HLA allele only. BLCLs encoding the HLA-B*4002 allele were effectively lysed after pulsing with the HERNG FTVL peptide, whereas BLCLs expressing other alleles, pulsed with the peptide, did not induce cytotoxic T-cell responses. T cells of an individual with HLA-type A*0201, A*0301 and B*4001, B*1501 demonstrated a response to HLA-A*0201-restricted NLVPMVATV peptide and a response to the pentadecapeptides 66 and 67 of the pool 18. (D) B*4001-expressing BLCLs of this individual pulsed with HERNG FTVL peptide also were effectively lysed by T cells of the B*4002-expressing individual above.