Transcriptional and functional HBV-specific CD8 T cell changes from disease to functional cure in HBeAg-negative chronic hepatitis B

Background & Aims: In chronic HBV infection, HBV-specific CD8 T cells are dysfunctional and comprise distinct subsets defined by phenotype and antigen specificity. We aimed to characterize the transcriptional and functional features of HBV-specific CD8 T-cell subsets in patients with HBeAg-negative chronic HBV infection who were either viremic (CHB) or had achieved spontaneous or nucleos(t)ide analogue (NUC)–induced HBsAg loss (RES), to better elucidate HBV-specific CD8 T-cell dysfunction and identify potential molecular targets for functional cure. Methods: Gene expression profiles of PD1hiCD127low/- and PD1+CD127+ memory-like (ML) core18-27-specific CD8 T-cell subsets were analyzed by Nanostring, adapted for low-input samples in 5 patients with HBeAg-negative CHB and 6 with RES. An expanded cohort of 23 patients with CHB and 22 with RES was evaluated for phenotypic and functional profiling. Selected deregulated genes were functionally validated in an additional cohort of 14 patients with HLA-A2-negative CHB. Results: Analysis of 84 genes concurrently expressed across all CD8 T-cell subsets identified an 11-gene signature describing a progressive transition from exhaustion-oriented PD1hiCD127low/- CD8 T cells in patients with CHB to memory-oriented ML CD8 cells in patients with RES, representing the two extremes of differentiation. Intermediate stages of memory differentiation were identified among ML CD8 T cells from patients with CHB, with high or low TOX expression (p <0.05 by Spearman's rank correlation). Higher frequencies of TOXlow ML CD8 cells in patients with CHB were associated with a greater serum HBsAg decline during NUC treatment compared to TOXhigh MLCHB (Δ slope p value = 0.003). Targeting selected deregulated genes with specific immune modulators significantly enhanced cytokine production by CD8 T cells, with response rates ranging from 30% to 86% of patients. Conclusions: Distinct exhaustion signatures characterize HBV-specific CD8 T-cell subsets and vary across disease phases. These findings support the development of individualized transcriptional and functional correction strategies and identify novel immune modulators with potential for immune-based anti-HBV therapies. Impact and implications: Exhausted HBV-specific CD8 T cells in chronic HBV infection are not a homogeneous population but comprise distinct subsets with differing capacities to control infection. This study identifies: (i) a transcriptional continuum of HBV-specific CD8 T-cell subsets spanning exhaustion to memory differentiation, reflecting disease progression and recovery in HBeAg-negative CHB; (ii) a core CD8 T-cell exhaustion gene signature characterized by progressively increased expression from memory-oriented to exhaustion-oriented subsets during active and resolution phases of disease; and (iii) the ability of targeted modulation of deregulated genes to restore antiviral CD8 T-cell function, with implications for the development of novel immune-based anti-HBV therapies. Overall, these findings advance our understanding of CD8 T-cell heterogeneity in chronic HBV infection and identify molecular targets for immunomodulatory strategies aimed at restoring CD8 T-cell functionality and achieving functional cure.