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.

Nuclear location of an endogenously expressed antigen, EBNA1, restricts access to macroautophagy and the range of CD4 epitope display

“…Focusing on three such epitopes, we found that two (SNP and VYG), were detectably displayed on the surface of unmanipulated LCL cells but at very low levels, whereas a third (PQC) was never detectable; indeed, this held true even when EBNA1 expression was increased by >100-fold from a dox-regulated vector. Such findings reinforce a theme seen in earlier work where EBNA1 epitope display on the LCL surface was generally poor, with several epitopes never detected, compared to epitopes from other endogenously expressed EBV proteins, EBNA2, -3A, and -3C (10,12,13).…”

“…CD4 + T cells against all three epitopes showed strong recognition of HLAmatched, peptide-loaded targets. However, as seen in earlier work (13), assays on unmanipulated LCLs revealed clear interepitope differences. Clones against the PQC epitope consistently failed to recognize such targets, whereas clones against the SNP and VYG epitopes showed low level recognition (typically 1-2% of that seen with peptide loading) that titrated with T cell input and was clearly specific, being restricted to LCLs with the appropriate HLA-DR restricting allele (Fig.…”

“…T cell assays were performed in triplicate by coculturing LCL cells (5 × 10 4 /well) overnight with T cells (10 4 /well unless otherwise stated) in a 96-well V-bottom plate; IFN-γ in the supernatant was then measured by ELISA (13), and the mean ± SD are shown. 3-MA (Sigma-Aldrich) was dissolved in cell medium immediately before use; siRNA targeting the ATG7 gene was purchased from Dharmacon and control siRNA was from Sigma-Aldrich.…”

“…T cell clones were generated and their HLA restriction, functional avidity, and ability to recognize LCLs were determined as described (13). T cell assays were performed in triplicate by coculturing LCL cells (5 × 10 4 /well) overnight with T cells (10 4 /well unless otherwise stated) in a 96-well V-bottom plate; IFN-γ in the supernatant was then measured by ELISA (13), and the mean ± SD are shown.…”

Epstein–Barr virus microRNAs reduce immune surveillance by virus-specific CD8 + T cells

“…That study also provided some evidence that CD4 + T-cell recognition is also regulated later, as the structural protein gp350 could be detected by CD4 + T cells in cells 15 d post infection but only when miRNAs were absent (35). The mechanisms of regulation we identified in that context-that is, regulation of MHC II, lysosomal enzymes, and IL-12-are likely relevant in latency as well and may explain why EBNA-specific CD4 + T cells are generally impaired in recognizing LCLs (46). The present study focused on established latency, but because we observed a strong miRNA regulation of EBNA1 recognition by CD8 + T cells already on days 1, 2, and 3 after infection, the hypothesis that EBV miRNAs generally suppress CD8 + T-cell recognition already in the first days of infection during prelatency (13,22,47) deserves closer investigation in the future.…”