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 het-erogeneity 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 se-creting cytokines (interleukin 2, interferon & gamma;, and tumor necrosis factor & alpha;) 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 expe-rienced a transition from naive T cells to effector T cells and then exhausted T cells. Flow cy-tometry 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. (Gut Liver, Published online June 15, 2023)