Cis- and trans-presentation of CD8⁺ T-cell epitopes in LMP-1–expressing cells. (A) LMP-1⁻ BJAB-gpt (■) and LMP-1⁺ BJAB-MTLM6 () cells were assessed for HLA class I expression by flow cytometery. Data represent the relative mean fluorescence after staining with anti-HLA-Bw6 and anti-HLA-A2–specific monoclonal antibodies. (B) HLA B35–restricted YPLHEQHGM (EBV-encoded EBNA3), HLA B35–restricted IPSINVHHY (HCMV-encoded pp65), and HLA A2–restricted NLVPMVATV (HCMV-encoded pp65)–specific T cells were incubated with BJAB-gpt (■) and BJAB-MTLM6 () cells infected with recombinant vaccinia viruses encoding EBNA3 or pp65, in the presence of brefeldin A. Cells were incubated with MHC-peptide pentamers and anti-CD8 antibody and then assessed for intracellular IFN-γ production. Data represent the relative increase in IFN-γ⁺ cells after incubation with BJAB-MTLM6 infected cells compared with BJAB-gpt infected cells. (C) CD8⁺ T cells specific for a HLA A2–restricted epitope, YLQQNWWTL (EBV-encoded LMP-1), were incubated in the presence of brefeldin A with BJAB-MTLM6 cells pulsed with and without 1 μg/mL YLQQNWWTL peptide (the first 3 amino acids of the sequences are underlined to indicate the abbreviation used for each epitope in the figure). Data represent the percentage of YLQQNWWTL-specific T cells producing IFN-γ after incubation with BJAB-MTLM6 cells at a responder-to-stimulator ratio of 10:1. (D) HLA class I expression in C33A cells after expression of LMP-1. C33A cells were transfected with EGFP-tagged expression vectors encoding GFP alone (■) or different LMP-1 sequences, including B95.8-LMP-1 (), CAO-LMP-1 (), HS6-LMP-1 (), NPC9-LMP-1 (), and QC-LMP-1 (□). Data represent the relative mean fluorescence, compared with cells transfected with GFP-expression vector, after staining with anti-HLA class I and anti-HLA-A2–specific antibodies. (E) YLQQNWWTL-specific T cells were incubated in the presence of brefeldin A with C33A cells transfected with B95.8-LMP-1 (), CAO-LMP-1 (), HS6-LMP-1 (), NPC9-LMP-1 (), and QC-LMP-1 (□) or B95.8-LMP-1 sensitized with synthetic YLQQNWWTL peptide epitope (■). Cells were incubated with MHC peptide pentamers and anti-CD8 and then assessed for intracellular IFN-γ production. Data represent the percentage of IFN-γ⁺ YLQ-specific T cells. (F) CD8⁺ T cells specific for HLA A2–restricted YLQQNWWTL epitope (EBV-encoded LMP-1) and HLA A2–restricted CLGGLLTMV epitope (EBV-encoded LMP-2A) were incubated with C33A cells cotransfected with vectors encoding GFP and LMP-2A-GFP or LMP-1-GFP and LMP-2A-GFP and then assessed for intracellular IFN-γ production. Data represent the percentage of pentamer-positive cells producing IFN-γ at a responder-to-stimulator ratio of 10:1. (G) CD8⁺ T cells specific for HLA A2–restricted YLQQNWWTL epitope (EBV-encoded LMP-1), HLA B58–restricted IALYLQQNW epitope (EBV-encoded LMP-1 protein), HLA A2–restricted YLLEMWRL epitope (EBV-encoded LMP-1 protein), and HLA A2–restricted CLGGLLTMV epitope (EBV-encoded LMP-2A) were incubated with HLA-matched and -mismatched LCLs in the presence of brefeldin A and then assessed for intracellular IFN-γ production by flow cytometry. Data represent the mean IFN-γ–producing cells after stimulation with 3 different LCLs.