Development of Immunodominance Hierarchies Concurrent with Enhanced Polyfunctionality in T Cell Immunity Towards Epstein-Barr Virus

Abstract

Background: EBV-specific T-cell immunity is characterised by immunodominant responses towards certain lytic and latent viral proteins. Nevertheless, how such immunodominance hierarchy develops with T-cell functionality from early stage of primary infection to long term persistency remains unclear. This study aims to study i) the difference in CD4+ and CD8+ EBV-specific T cell functions between early and late stage of viral infection; ii) correlation of immunodominant responses to the functionality of T cells. Methods: PBMCs of ten patients with infectious mononucleosis (IM) and four individuals with asymptomatic EBV primary infection (NIM) were collected longitudinally from the onset of disease through 12-month post infection. Cells were stimulated with overlapping peptides of four lytic-(BZLF1, BRLF1, BMLF1, and GP350) and five latent- (EBNA1, EBNA3A- 3C, and LMP2) proteins, followed by a 9-color flow cytometric assay examining the coexpression of three cytokines (interferon-γ [IFN-γ], tumour necrosis factor- α [TNF-α] and interleukin-2 [IL-2]), perforin and CD107a (degranulation marker) in CD4+ and CD8+ T cells. Stimulated T cells were selected and tested for the cytotoxicity against autologous EBV-transformed lymphoblastoid cell lines (LCLs) and the proliferation capacity. Results: In both IM and NIM individuals there was a gradual shift of CD8+ T cell responses from targeting the BZLF1 or BRLF1 proteins in acute phase to the EBNA3 proteins in persistent phase of infection, and a shift of CD4+ T cell responses from a broad range of early and late lytic proteins to the EBNA-1 protein over time. Such establishment of immunodominance concurred with increased proportion of lytic- and latent-protein specific CD4+ and CD8+ T cells with multiple functions, which presented enhanced proliferation capacity and cytotoxicity against lysing LCLs. Conclusions: T cells simultaneously producing multiple cytokines with effective cytotoxicity and proliferation were generated along with establishment of immunodominance hierarchy in EBV-specific T cells. Formation of effective long-term immunity towards EBV requires both sufficient quantity of latent-specific T cells and highly functional T cells.