L428 is unable to present endogenously synthesized antigens to HLA class I–restricted CTLs. (A) A B35-restricted EBNA 3A-specific CTL clone tested against DH BL, L428, and a B35-matched LCL target. All targets had either been preincubated with the cognate peptide epitope (EBNA 3A residues 458-466) or preinfected with a recombinant vaccinia vector expressing EBNA 3A. As controls, targets were preincubated with an equivalent dilution of DMSO solvent (−peptide) or with the vaccinia vector alone (control vacc). (B) Expression of HLA A2 from a recombinant vaccinia vector was tested in L428 and an HLA A2-negative LCL. Viable cells were analyzed by FACScan for surface expression of HLA A2 using the MoAb BB7.2. Solid line, BB7.2 staining; dotted line, staining with second step antibody alone. (C) A B35-restricted EBNA 3A-specific CTL clone tested against a B35-negative LCL and L428. Targets were infected with vaccinia recombinants expressing HLA B35 and/or EBNA 3A and in one case targets were also preincubated with the cognate peptide epitope (EBNA 3A residues 458-466). (D) An A2-restricted LMP 2-specific CTL clone tested against an A2-negative LCL and L428. Targets were infected with vaccinia recombinants expressing HLA A2 and/or LMP 2 and in one case targets were also preincubated with the cognate peptide epitope (LMP 2 residues 426-434). E:T ratios = 5:1 (▪) and 1:1 (▨). Results of cytotoxicity assays are expressed as % specific lysis + 1 SD and are representative of several repeated assays.