Objectives:
T-cell exhaustion represents a distinct T-cell differentiation program associated with chronic viral infections. Numerous studies have demonstrated the heterogeneity of exhausted CD8+ T cells. In the context of chronic HBV infection, we and others have observed significant variations in the phenotype, function, and degree of dysfunction among HBV-specific CD8+ T cells targeting different antigens. The objective of this study was to examine the molecular heterogeneity of HBVcore18- versus HBVpol455-specific CD8+ T cells.
 
Method:
To investigate the subset diversification of HBV-specific CD8+ T cells targeting different antigens, we conducted single-cell RNA sequencing from chronically HBV-infected patients who endogenously control the viral infection and patients under NUC treatment. Phenotypic and functional analyses were performed after tetramer-based enrichment.
 
Results:
Cluster analysis of single-cell transcriptomes revealed a different subset diversification of HBVcore18- versus HBVpol455-specific CD8+ T cells. In particular, HBVcore18-specific CD8+ T cells were mostly comprised of precursor/memory-like exhausted T-cell subsets. Within HBVpol455-specific CD8+ T cells, we could identify a cluster of cells that highly expressed cytotoxic genes including GNLY, GZMB and PRF1. Pseudotemporal diffusion analysis further revealed two diverging branches within HBVpol455-specific CD8+ T cells dependent on the clinical phase of chronic HBV infection. In particular, we observed that the cytotoxic subset is exclusive to HBVpol455-specific CD8+ T cells obtained from patients who endogenously control the virus but still show functional adaption in comparison to classical effector cells. This T-cell attenuation is attributed to an increased TGFβ signaling. In contrast, HBVpol455-specific CD8+ T cells obtained from NUC-treated patients exhibited characteristics of exhaustion with a distinct dysfunctional profile.
 
Conclusion:
In sum, our data highlight diverging mechanisms of HBV-specific CD8+ T-cell dysfunction occurring in parallel to classical T-cell exhaustion in chronic HBV infection and associated with distinct clinical manifestations. This observation might have potential implications for the design of immunotherapeutic approaches in HBV cure.