Characterization of NK Cells Using Single‐Cell RNA Sequencing in Patients With Acute‐On‐Chronic Liver Failure

ABSTRACT Background and Aim Acute‐on‐chronic liver failure (ACLF) is characterized by fast progression and high mortality, with systemic inflammation and immune paralysis as its key events. While natural killer (NK) cells are key innate immune cells, their unique function and subpopulation heterogeneity in ACLF have not been fully elucidated. This study aimed to investigate the characteristics of NK cell subsets in the peripheral blood of patients with ACLF and determine their roles in the inflammatory responses. Methods Circulating NK cells (14 751 cells) from patients with ACLF and healthy controls (HCs) were subjected to single‐cell RNA sequencing (scRNA‐seq). Clustering and annotation were used to identify the features of NK cell subsets and the characteristics of disease progression in ACLF. Results Four NK cell subsets were obtained, including adaptive NK cells, mature NK cells, inflamed NK cells, and CD56 bright NK cells. Compared with the HCs, the patients with ACLF had a significantly lower proportion of Mature NK cells and a higher proportion of Inflamed NK cells. Quasi‐temporal analysis showed that Inflamed NK cells were highly enriched in the late quasi‐temporal sequence, and genes related to pro‐inflammatory were significantly up‐regulated in Inflamed NK cells. In addition, scRNA‐seq and flow cytometry confirmed that the expression level of cell migration inducing hyaluronidase 2 ( CEMIP2 ) in NK cells progressively increased from the HC group to the ACLF survival group and then to the ACLF death group. Conclusions scRNA‐seq reveals that Inflamed NK cell subsets are associated with ACLF progression and poor prognosis. CEMIP2 may be a molecular marker for ACLF progression.