Single-cell Rna Sequencing Shows The T Cell Exhaustion Landscape In Peripheral Blood Of HBV Associated ACLF

Objective:The occurrence and development of hepatitis B virus-associated acute chronic liver failure(ACLF)(HBV-ACLF)is closely related to the immune pathway,and T cell exhaustion is an important component of immune dysfunction.Here we explored the heterogeneity of peripheral blood T cell subsets and the characteristics of exhausted T lymphocytes in patients with HBV-ACLF,in an attempt to identify potential therapeutic target molecules for immune dysfunction in ACLF patients.Methods:A total of 83,577 T cells from 6 HBV-ACLF patients and 3 healthy controls were detectioned 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 using pseudotime analysis.Subsequently,the expression of exhausted T cells and their capacity in secreting cytokines(IL-2,TNF-α,and IFNγ)were validated by flow cytometry in 30 HBV-ACLF patients and 30 healthy controls.Results:T lymphocytes in the peripheral blood of HBV-ACLF patients had distinct differentiation trajectories and could be differentiated into eight clusters with different gene characteristics,among which CD4+TIGIT+subset and CD8+LAG-3+subset,with high expression of exhaust genes,were significantly higher in HBV-ACLF patients than in normal controls.As shown by pseudotime analysis,the CD4+TIGIT+T cells and CD8+LAG-3+T cells experienced a transition from na(?)ve T cells to effector T cells and then exhausted T cells in the later stage of T cell differentiation.Flow cytometry confirmed that the CD4+TIGIT+subset and CD8+LAG-3+subset in peripheral blood of ACLF patients were significantly higher than those in healthy controls.Moreover,in-vitro cultured CD8+LAG-3+T cells were significantly less capable of secreting cytokines(IL-2,TNF-α,and IFNγ)than CD8+LAG-3-subset.Conclusion:1.Peripheral blood T cells are heterogeneous in HBV-ACLF.2.The exhausted T cells,featured by CD4~+TIGIT~+T lymphocyte subset and CD8~+LAG-3~+T lymphocyte subset,markedly increase during the pathogenesis of ACLF,suggesting that T cell exhaustion is involved in the immune dysfunction of HBV-ACLF patients.3.These results may help to provide potentially effective target molecules for the treatment of immune dysfunction in ACLF patients.