Single-Cell RNA Sequencing Shows T-Cell Exhaustion Landscape in the Peripheral Blood of Patients with Hepatitis B Virus-Associated Acute-on-Chronic Liver Failure.

Background/aims: The occurrence and development of hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF) is closely related to the immune pathway. We explored the heterogeneity of peripheral blood T cell subsets and the characteristics of exhausted T lymphocytes, in an attempt to identify potential therapeutic target molecules for immune dysfunction in ACLF patients.

Methods: A total of 83,577 T cells from HBV-ACLF patients and healthy controls were screened for heterogeneity by single-cell RNA sequencing. In addition, exhausted T-lymphocyte subsets were screened to analyze their gene expression profiles, and their developmental trajectories were investigated. Subsequently, the expression of exhausted T cells and their capacity in secreting cytokines (interleukin 2, interferon γ, and tumor necrosis factor α) were validated by flow cytometry.

Results: A total of eight stable clusters were identified, among which CD4⁺ TIGIT⁺ subset and CD8⁺ LAG-3⁺ subset, with high expression of exhaust genes, were significantly higher in the HBV-ACLF patients than in normal controls. As shown by pseudotime analysis, T cells experienced a transition from naïve T cells to effector T cells and then exhausted T cells. Flow cytometry confirmed that the CD4⁺TIGIT⁺ subset and CD8⁺LAG-3⁺ subset in the peripheral blood of the ACLF patients were significantly higher than those in the healthy controls. Moreover, in vitro cultured CD8⁺LAG-3⁺ T cells were significantly fewer capable of secreting cytokines than CD8⁺LAG-3- subset.

Conclusions: Peripheral blood T cells are heterogeneous in HBV-ACLF. The exhausted T cells markedly increase during the pathogenesis of ACLF, suggesting that T-cell exhaustion is involved in the immune dysfunction of HBV-ACLF patients.