Temporal transcriptional dynamics in HBV-specific CD8 T cells during long-term NUC therapy in HBeAg- chronic hepatitis B

Background and aims: Virus-specific CD8 T cell dysfunction is a hallmark of chronic hepatitis B (CHB), and its restoration represents a key strategy toward HBV cure. This study aims to define transcriptional changes in HBVspecific CD8 T cells associated with viral persistence and control in HBeAg-negative chronic patients undergoing long-term nucleos(t)ide analogue (NUC) therapy, with the ultimate goal to identify potential targets for efficient T cell restoration. Method: We performed a transcriptome study by low-input RNA-seq on core- and polymerase-specific CD8 T cells, isolated from nine patients with HBeAg-negative chronic hepatitis B and high viral loads, prior to the start of NUC therapy and longitudinally at 1 and 4 years after treatment initiation. Five patients who achieved functional cure with HBsAg loss after long-term NUC treatment (rNUC) were included as control group. Transcriptomic data were analyzed using Weighted Gene Co-expression Network Analysis (WGCNA) to investigate the dynamics of gene cluster expression over time. Virological parameters including HBV RNA, HBcrAg and HBsAg levels were assessed at the same time points. Results: Principal component analysis (PCA) of longitudinal transcriptomic profiles revealed a distinct clustering of core-specific CD8 T cells, with chronic patients after 4 years of therapy aligning with rNUC patients. Conversely, polymerase-specific samples displayed greater heterogeneity and lacked a comparable clustering pattern. WGCNA analysis further highlighted distinct transcriptional profile changes over time in virus-specific CD8 T cells, identifying gene clusters that were modulated early (1 year), late (4 years), or remained unchanged. Conclusion: Our longitudinal analysis identifies distinct gene programs in HBV-specific CD8 T cells that progressively normalize or remain dysregulated during therapy, providing candidate targets for interventions aimed at restoring T cell function and achieving functional cure in CHB.