Recognition of the Epstein-Barr virus-encoded nuclear antigens EBNA-4 and EBNA-6 by HLA-A11-restricted cytotoxic T lymphocytes: implications for down-regulation of HLA-A11 in Burkitt lymphoma.

Abstract

Evasion from cytotoxic T-lymphocyte (CTL) surveillance may be an important step in the pathogenesis of Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) as suggested by the consistent down-regulation of all transformation-associated viral antigens, except EBV nuclear antigen 1 (EBNA-1), and of certain HLA class I alleles in BL biopsies and cell lines that maintain the tumor cell phenotype in vitro. The most common HLA class I defect recorded in BL lines is a selective down-regulation of HLA-All. To gain some insight into the role of HLA-All down-regulation in pathogenesis of BL, we have investigated the target specificity of 11LA-Allrstricted CTLs derived by stimulation of lymphocytes from three EBV-seropositive individuals with autologous EBVtransformed lymphoblastoid cell lines. Recombinant vaccinia viruses carrying the coding sequences for EBNA-1, -2A, -2B, -5, -3, -4, and -6 (also known as EBNA-1, -2A, -2B, -LP, -3a, -3b, and -3c, respectively) and EBV latent membrane protein 1 were used to induce high levels of expression of the relevant EBV antigen in fibroblasts derived from HLA class I-matched individuals. EBNA-4-expressing fibroblasts were the predominant target of HLA-All-restricted CTLs in all three donors.A less pronounced and less regular EBNA-6-speciflc cytotoxic component was found in two of the donors.Epstein-Barr virus (EBV), a human herpes virus that asymptomatically infects the majority of adult populations worldwide, causes infectious mononucleosis and is strongly linked with endemic Burkitt lymphoma (BL), nasopharyngeal carcinoma, and the large cell lymphomas arising in immunosuppressed patients. Underlying the association of EBV with lymphoid malignancies is the capacity of the virus to transform B lymphocytes into lymphoblastoid cell lines (LCLs), which constitutively express at least six EBV-encoded nuclear proteins, EBNA-1 to EBNA-6 (also known as EBNA-1, -2, -3a, -3b, -LP, and -3c) and two latent membrane proteins, LMP-1 and LMP-2 (reviewed in ref. 1).Cytotoxic T lymphocytes (CTLs) are thought to play an important role in controlling the proliferative potential of EBV-infected B lymphocytes, which persist for life in healthy virus carriers. EBV-specific CTL precursors can be reactivated in vitro by stimulation of lymphocytes from EBVseropositive individuals with the autologous EBV-transformed LCLs (2). The CTL target was operationally called LYDMA (lymphocyte-detected membrane antigen), but it was soon realized that more than one virally encoded protein could serve this function. The assumption has been recently confirmed by the finding that EBNA-2 (3, 4), -3 (5), and -6 (6) are recognized by EBV-specific effectors. The demonstration of selective interaction between peptides derived by processing of protein antigens and polymorphic residues within the antigen binding groove of major histocompatibility complex (MHC) molecules (7,8) suggests that recognition of these proteins may depend on the repertoire of HLA class I alleles that serve as restriction elements in each indiv...

A Positive Correlation between the Precursor Frequency of Cytotoxic Lymphocytes to Autologous Epstein‐Barr Virus‐Transformed B Cells and Antibody Titer Level against Epstein‐Barr Virus‐Associated Nuclear Antigen in Healthy Seropositive Individuals

“…Namely, specific killer T cells recognize the membrane antigen that is expressed on the surface of EBNApositive B cells and prevent proliferation of the virus-infected cells. Recently, transformation and recombinant vaccinia virus experiments have revealed that a significant proportion of the killer T cells recognize some members of the EBNA family such as EBNA 2, 3A, 3B, and 3C (5,8,14,25,26). Although there was no direct evidence for the recognition of these EBNA family antigens by the cytotoxic effector cells in our study, the present result may be interpreted that our PF assay reflects the degree of continuous EBNA-positive cell destruction followed by anti-EBNA antibody production in the latent stage of EBV infection.…”

Apoptosis of Antigen-Specific T Lymphocytes upon the Engagement of CD8 by Soluble HLA Class I Molecules Is Fas Ligand/Fas Mediated: Evidence for the Involvement of p56lck, Calcium Calmodulin Kinase II, and Calcium-Independent Protein Kinase C Signaling Pathways and for NF-κB and NF-AT Nuclear Translocation

“…1a). To this end, we generated HLA-A11-restricted CTL specific for the IVT peptide derived from EBVencoded nuclear Ag EBNA-4 (HLA-A11 IVT) (22,25). Indeed, this effector population lysed autologous target cells expressing HLA-A11 Ags pulsed with the specific IVT peptide but not those pulsed with the unrelated CLG and YVN peptides (Fig.…”

Epstein–Barr virus encoded nuclear protein EBNA-3 binds XAP-2, a protein associated with Hepatitis B virus X antigen

“…CV1, NIH3T3, and EBNA-3 or EBNA-4 expressing BJAB cells were transfected with NGFP-Full436 construct using polyethylenimine (PEI) or electroporation (230 V, 960 mF, 38 ms in volume of 300 ml 10% FCS containing medium in the presence of 1.25% DMSO) respectively. Infection with recombinant vaccinia viruses was done as described (Gavioli et al, 1992).…”