Cytolytic activity of the DR*0401-restricted BHRF1-specific CD4 T-cell clone A22 toward DR*0401⁺ B-LCL is EBV dependent. (A) Cytolytic activity of clone A22 toward the autologous B-LCL (EBV⁺, DR*0401⁺) or toward the CD8 T-cell clone A2.10 directed against BMLF1/HLA-A*0201 (EBV^–, DR*0401⁺). Unlike the autologous B-LCL (left), clone A2.10 (right) was not killed by the BHRF1-specific CD4 T-cell clone A.22, unless it was loaded with PYY peptide. (B) The CTL clone A22 recognized the DR*0401⁺ B-LCL from donor D3 transformed with the wild-type EBV (WT) but not the same B-LCL transformed with a viral mutant with a KO for BHRF1 gene (KO). The 2 B-LCLs were recognized after loading with PYY peptide. The DR*0401⁺ B-LCL from donor RA1 and the DR4^– B-LCL from donor RA17 were included as positive or negative control, respectively. (C) Clone P4.2 (donor RA17), responding to EBNA3C in the HLA-DR16 context, was used as a control to verify that the BHRF1-KO B-LCL from donor D3 was able to present an epitope from the latent protein EBNA3C. Clone P4.2 did not exhibit spontaneous lysis toward DR16⁺ B-LCL, but it killed its autologous B-LCL, as well as the WT and the BHRF1-KO B-LCLs from donor D3 (DR4⁺, DR16⁺), once loaded with the EBNA3C_100-111 peptide (PHD). Results are expressed as percent specific lysis.