Purpose Epstein-Barr virus (EBV) is a human tumor virus of the gamma-Herpes virus family and transforms B cells in vitro and in vivo. EBV establishes persistent infections in more than 90% of the adult population, and although most remain completely healthy, a small minority of infected individuals develops spontaneous EBV-associated malignancies (such as lymphoma, nasal-pharyngeal carcinoma, and post-transplant lymphoproliferative diseases). Its nuclear antigen 1 (EBNA1) is the only EBV protein expressed in all EBV associated malignancies. This study investigates the subtle differences in immune response to EBNA1 among healthy carriers in comparison to patients afflicted with EBV-associated malignancies.
Methods All healthy volunteers and lymphoma patients were consented to provide peripheral blood samples according to our IRB-approved protocols. EBNA1 specific CD4+ T cells were detected by IFN-gamma production and proliferation upon stimulation with overlapping peptides covering the EBNA1400-641 sequence. In the first evaluation, ex vivo blood samples were immediately subjected to pools of overlapping peptides of EBNA1, followed by intracellular cytokine staining and flow cytometry to quantify and phenotype all EBNA1 specific CD4+ T cells. Peripheral blood mono-nuclear cells (PBMCs) stained with carboxyfluorescein succinimidyl ester (CFSE) and were tested for proliferation (CFSE dilution) in response to the same pools of overlapping peptides of EBNA1.
Results We have determined the frequency and surface marker phenotype of EBNA1 specific CD4+ T cells in peripheral blood of healthy EBV carriers. Healthy carriers (17/19) had detectable CD4+ T cell responses to EBNA1. EBNA1 specific CD4+ T cells were primarily CD45RO+CD45RA-CD27+CD28+ and contained both CD62L+ and CD62L- subpopulations. Although lymphoma patients with EBV-negative tumors had detectable “healthy” EBNA1-specific CD4+ T cell responses (n = 4), none of the patients with EBV-positive lymphomas (n = 4) exhibited a “healthy” EBNA1-specific CD4+ T cell response.
Conclusions Among healthy carriers, an average of 0.03% of CD4+ T cells are EBNA1-specific, however, such cells seem to be absent in patients with EBV-positive tumors. EBNA1 specific CD4+ T cells display a similar phenotype as latent EBV antigen specific CD8+ T cells. The continued study of the CD4+ T cell response to EBNA1 may reveal novel targets for therapies of EBV-associated malignancies.