Ubiquitinated hepatitis D antigen-induced CD8⁺ T-cell responses inhibit HDV replication in HDV-infected liver organoids

Hepatitis D virus (HDV) infection is the most severe form of human viral hepatitis. A poor virus-specific CD8⁺T cell response may result in persistent HDV infection. We investigated anti-viral effect and mechanisms of ubiquitinated small hepatitis D antigen (Ub-S-HDAg) in HBV/HDV superinfected liver organoids.

Induced pluripotent stem cells were differentiated into hepatocyte-like cells and seeded onto inverted colloidal crystal scaffolds. A Ub-S-HDAg recombinant lentiviral plasmid was constructed and transduced into dendritic cells (DCs), which were then co-cultured with T cells. Cytokine levels secreted by CD8⁺T cells and their antiviral efficacy were evaluated, and transcriptomic sequencing was conducted to elucidate immunoregulatory mechanisms of Ub-S-HDAg.

We successfully established a model that stably expressed HDV. Ub-S-HDAg significantly promoted DC maturation and reduced HDV RNA viral load and HDAg levels. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and gene set enrichment analyses suggested that DCs transduced with Ub-S-HDAg promoted CD8⁺ T cell activation via Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. JAK1, JAK2, STAT1, and STAT4 expressions in Ub-S-HDAg group increased and was blocked by JAK2 inhibitor AG490. Additionally, Ub-S-HDAg induced DC maturation-enhanced cytotoxic T lymphocyte responses and increased IFN-γ, IL-2, IL-12, and TNF-α secretion.

We successfully developed HBV/HDV superinfected liver organoids and suggest that Ub-S-HDAg facilitates DC maturation, inducing CD8⁺ T cell proliferation through the JAK/STAT signalling pathway, leading to a stronger antiviral immune response.