Study On The Mechanism Of NK Cells Inducing CD8⁺T Cell Immune Exhaustion In Patients With Chronic Hepatitis B

Objective:The aim of this study was to investigate the immune status of NK cells and CD8⁺T cells in patients with chronic hepatitis B（CHB）and the molecular mechanism by which NK cells induce CD8⁺T cell exhaustion.Methods:（1）Peripheral blood samples from clinical CHB patients and healthy physical examiners are collected;（2）The serum levels of ALT,AST,HBV DNA,and the five hepatitis B items（HBs Ag,HBs Ab,HBe Ag,HBe Ab and HBc Ab）in the samples are measured;（3）The phenotype and function of NK cells and CD8⁺T cells from CHB patients and HCs are detected using flow cytometry（FCM）;（4）Gal-9⁺NK,Gal-9^-NK and CD8⁺T cells from PBMCs of CHB patients are sorted and purified by FACS and cocultured in vitro,the function of CD8⁺T cells is detected;（5）PBMCs from CHB patients are depleted of NK cells and cultured in vitro,the function of CD8⁺T cells is detected;（6）PBMCs from CHB patients are isolated and cultured in vitro with HBV peptide stimulation,the function of CD8⁺T cells is detected after blocking Gal-9/TIM-3pathway;（7）NK and CD8⁺T cells from PBMCs of CHB patients are sorted and purified by FACS and cocultured in vitro,the function of CD8⁺T cells is detected after blocking Gal-9/TIM-3 pathway.Results:（1）The frequency of Gal-9⁺NK was higher in peripheral blood of CHB patients than HCs,and high Gal-9 expression was associated with functionally abnormal of NK cells in CHB patients;（2）CD8⁺T cells in peripheral blood of CHB patients,especially those in immune active phase,were in a state of functional exhaustion;（3）Gal-9/TIM-3axis promotes CD8⁺T cell immune exhaustion in active CHB patients;（4）Gal-9expression in NK cells induces TIM-3⁺CD8⁺T cell dysfunction during the active phase of CHB infection.Conclusions:High expression of Gal-9 on circulating NK cells in CHB patients with abnormal function induces virus-specific CD8⁺T cell immune exhaustion via Gal-9/TIM-3,revealing the mechanism of NK cell-induced CD8⁺T cell immune exhaustion in CHB patients and providing a new idea to reverse CD8⁺T cell immune exhaustion in CHB patients using NK cells as the target.