CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines.
ABSTRACT There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.
Full-textDOI: · Available from: Elise Landais, Jan 08, 2014
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ABSTRACT: The transformation of B cells by Epstein-Barr virus (EBV), into lymphoblastoid cell lines (LCLs) results in the upregulation of STAT1, a key transcription factor in the interferon signalling pathway. Although the mechanism of EBV induction of STAT1 protein expression has been intensively studied, there has been little investigation into the function of STAT1 in EBV-transformed LCLs. In this study, we have implemented a novel strategy to investigate the functional role of STAT1 through the introduction of the simian virus 5 (SV5) V-protein into LCLs by retroviral gene transfer. The V-protein is a virally evolved STAT1 inhibitor that specifically targets STAT1 for proteasomal degradation. Using this in vitro model, we have shown that major histocompatibility complex (MHC) class I and class II molecules are downregulated at the cell surface following a reduction in STAT1 protein expression. With regards to MHC class I, the impairment of the antigen processing machinery renders the cells less recognized by the host EBV-specific immunosurveillance. In addition, downregulation of STAT1 increases the expression of LMP2A and lytic cycle antigens and results in a higher proportion of cells entering the lytic cycle. These results suggest that STAT1 is involved in maintaining the latency III viral program observed in transformed B cells and regulating immunorecognition by EBV-specific T cells.Journal of General Virology 08/2009; 90(9-Pt 9):2239-50. DOI:10.1099/vir.0.011627-0 · 3.53 Impact Factor
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ABSTRACT: Epstein-Barr virus (EBV) is associated with several malignant diseases that can be distinguished by their patterns of viral latent gene expression. We developed here an original peptidic approach to favor the induction of a specific CD4+ T-cell response against EBV latency II malignancies (Hodgkin's lymphoma, nasopharyngeal carcinoma, T/NK lymphoma). Previously, we selected 6 peptides derived from EBV nuclear antigen-1, latency membrane proteins (LMP)-1, and LMP-2 highly promiscuous for major histocompatibility complex class II molecules and showed their ability to induce interferon-γ-secreting CD4+ T cells. We confirmed here that all peptides used in cocktail are recognized by human CD4+ memory T cells from healthy donors, inducing a broad T-helper (Th)1 cytokine secretion interferon-γ, interleukin-2. Furthermore, we have generated EBV-specific CD4+ T-cell lines and proved their cytotoxic potential, not only on original models expressing latency II antigens (EBV-transformed T cell or monocyte), but also on lymphoblastoid cell lines expressing latency III antigens (lymphoblastoid cell lines). In addition, granzyme B enzyme-linked immunospot assays suggested that a part of this specific cytotoxic activity could be linked to the granule lytic pathway. Very importantly, we have showed that neither phenotypical changes nor functional activities of CD4+CD25+CD127(low)-regulatory T cells were observed in response to EBV+ peptides, avoiding any risk of aggravation of the preexisting immunosuppressive environment reported in EBV-associated malignancies. In conclusion, our promiscuous peptide cocktail could be used safely in immunotherapeutic approaches against EBV latency II malignancies, mainly to prevent relapse in high-risk patients further to classic treatments.Journal of immunotherapy (Hagerstown, Md.: 1997) 04/2012; 35(3):254-66. DOI:10.1097/CJI.0b013e31824d72c5 · 3.35 Impact Factor
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ABSTRACT: Epstein Barr virus (EBV) persists as a latent herpes virus infection in the majority of the adult human population. The virus can reactivate from this latent infection into lytic replication for virus particle production. Here, we report that autophagic membranes, which engulf cytoplasmic constituents during macroautophagy and transport them to lysosomal degradation, are stabilized by lytic EBV replication in infected epithelial and B cells. Inhibition of autophagic membrane formation compromises infectious particle production and leads to the accumulation of viral DNA in the cytosol. Vice versa, pharmacological stimulation of autophagic membrane formation enhances infectious virus production. Atg8/LC3, an essential macroautophagy protein and substrate anchor on autophagic membranes, was found in virus preparations, suggesting that EBV recruits Atg8/LC3 coupled membranes to its envelope in the cytosol. Our data indicate that EBV subverts macroautophagy and uses autophagic membranes for efficient envelope acquisition during lytic infection.12/2014; 1(2-3):116-125. DOI:10.1016/j.ebiom.2014.11.007