Virus-specific coexpression of T-bet and Eomes defines highly functional CD8 and CD4 T cells in hepatitis B virus infection

Strong Th1-type T cell response is associated with successful clearance of hepatitis B virus (HBV) infection. Th1 master transcription factors are T-bet and Eomes which trigger IFN-γ production and cytotoxic activity. Little is known about how the balance of these two factors influences the CD8 and CD4 T cell response during the course of HBV infection. Therefore, we aimed to investigate T-bet and Eomes coexpression in acute (n = 24) and chronic (n = 45) HBV infection to dissect the expression patterns which are associated with viral control. In this study, we analyzed the virus-specific CD8 and CD4 T cell response by using HLA-A02 and HLA-DRB01-restricted tetramers targeting immunodominant epitopes from the HBV core region. Enrichment-based Tetramer staining was combined with intracellular assessment of transcription factors. Intracellular cytokine staining (ICS) was performed after in vitro culturing of PBMCs with anti-CD3/CD28, antigen with or without rhIL-2 and rhIL-12. In the ex vivo studies we were able to show that antigen-specific CD8 and CD4 T cells from patients with acute HBV infection are associated with high coexpression of T-bet and Eomes, whereas chronically infected patients failed to express both markers. In order to induce T-bet and Eomes in chronic HBV as this phenotype is mentioned to be associated with viral control we first stimulated PBMC with anti-CD3/CD28 as a strong TCR stimulator. CD8 and CD4 T cells were characterized by upregulation of T-bet+Eomes- (CD8: 60%; CD4: 65%) and T-bet+Eomes+ (CD8: 30.8%; CD4: 11%) T-cells. Notably, T-bet+Eomes+ T cells are associated with improved IFN-γ production (CD8: 13%; CD4: 14%) compared to T-bet+Eomes- (CD8: 1.9%; CD4: 2.2%), T-bet-Eomes- (CD8: 0.4%; CD4: 0.8%) and T-bet-Eomes+ (CD8: 4.5%; CD4: 4.9%) cells. Moreover, HBV-specific CD8 T cell proliferation was especially generated from T-bet+Eomes- and T-bet+Eomes+ cells which can be generally induced by PD-L1/2 blockade. Importantly, increasing MFI of T-bet and Eomes determines [T-bet+Eomes+]^high expressing T cells as highly functional T cells characterized by strong IFN-γ and Perforin production. Finally, we were able to selectively induce this [T-bet+Eomes+]^high CD8 T cell subset in an antigen-specific manner which represents a unique chance for prospective immuno-based antiviral treatment strategies.