Identification of a DR*0401/BHRF1 epitope recognized by the CD4 T-cell clone 4.19. (A) Recognition of the lytic EBV protein BHRF1 by clone 4.19. CTL cells were tested in a 4-hour TNF-α release assay on autologous B-LCLs expressing individual latent or lytic EBV proteins from vaccinial viral vectors. Results are shown as μg/mL. TNF release was observed at an effector-target ratio of 10:1. (B) Identification of the target epitope of BHRF1-specific clone 4.19 using a panel of peptides (23-mers, overlapping by 12 amino acids [aa's]) spanning the BHRF1 protein. These peptides were used as targets in cytotoxic assays by incubating them with ⁵¹Cr-labeled autologous EBV-B cells for 1 hour at 37°C. Clone 4.19 was added at an E/T ratio of 10:1 and Cr release was measured after 4 hours. Note the significant lysis background observed with unloaded autologous B-LCL (No pep). (C) Stimulation of clone 4.19 by the BHRF1 122-133 peptide (PYY). Cytotoxicity of clone 4.19 to Cr-labeled autologous EBV-B cells loaded with various concentrations of peptides. Clone 4.19 was added at an E/T ratio of 10:1 and Cr release was measured after 4 hours. Data obtained with the 23-mer BHRF1_111-133 peptide are shown as a positive control. Data obtained with an irrelevant peptide (irr peptide) are shown as a negative control.