Regulatory Effect And Mechanism Of Interleukin 21 And Immune Checkpoints Blockade On NK Cells In Chronic HBV Infection

BackgroundHepatitis B virus（HBV）infection is a heavy disease burden worldwide.Chronic HBV infection often leads to virus persistent while acute infection is usually selflimited,implicating the potential of human immune system to eradicate the virus.Up to date,available therapies targeting chronic HBV infection are limited and the efficacy is far from satisfying.Nucleo（t）side analogs（NAs）is the most commonly applied therapy,which is powerful in promoting ALT normalization and lower the risk of developing HCC.But the HBsAg clearance or seroconversion is hardly achieved,therefore most patients require lifelong treatment.Beside NAs,IFN-α is another widely used drug and works better to induce HBsAg clearance,but it is accompanied with frequently occurred side effects.Thus,efforts are urgently needed to explore a novel strategy applying the natural mechanisms that drive sustained virologic control to terminate chronic HB V infection.Immunotherapy treat diseases by artificially modulating the immune function of the body,such as using cytokines and antibodies.IL-21 produced by activated CD4+T cells and NKT cells is a multifaceted cytokine that act on a variety of immune cells including T cells,B cells and NK cells,and IL-21 plays an important role in chronic HBV infection.Our previous study has demonstrated that IL-21 levels in the serum at 12 weeks after NAs treatment correlated with HBeAg seroconversion in HBeAg positive chronic HBV infected patients.It is found that adeno-associated virus-IL-21 induce HBV cccDNA clearance in mice liver by activating liver infiltrated CD8+ T cells.However,the mechanism by which IL-21 promotes HBV clearance is not well clarified.NK cells,as important effector cells of the innate immune system,can effectively and quickly recognize and clear virus-infected cells without MHC restrictions.NK cells are abundant in liver,and play a pivotal role in control HBV infection.However,the role of IL-21 on NK cells in chronic HBV infection has not been well explored.The recent successes of immune checkpoints blockade therapies in cancer patients have driven an interest in targeting these pathways in chronically infected patients.The most focused checkpoints including cytotoxic T lymphocyte-associated antigen 4（CTLA-4）,programmed cell death protein 1（PD-1）,T-cell immunoglobulin and mucin domain 3（TIM-3）,and T cell immunoglobulin and ITIM domain（TIGIT）.While still in early stages,basic and clinical data suggest that blockade of CTLA-4 and PD-1 can be beneficial in the treatment of chronic HBV infection in some patients.but failed in others with mechanisms unknown.Therefore,understanding the mechanism of immune checkpoints blockade therapies is helpful to broaden the treatment options for chronic HBV infection.T cell exhaustion has been demonstrated to be the cause of chronic infection,and checkpoint inhibitors were proposed to reverse exhausted T cells and invigorate antiviral immunity.However,the role of NK cells has been largely overlooked.Early（viral replication）and late（viral clearance）stages of acute HBV infection are accompanied by robust activation of NK cells,suggesting the strong potential of NK cells to clear the infection.NK cells can remove viruses by a variety of means,such as secreting cytokines,expressing TRAIL or FasL to mediate apoptosis,and expressing FcyRIII to mediate cytotoxicity.NK cells can also facilitate or regulate the functions of other immune cells in antiviral immunity,which bridge between innate and adaptive immune response.More importantly,NK cells are enriched in the liver,the target organ of HBV infection,and NK cells in chronic HBV infection expressed many of the checkpoints with impaired function.Collectively,NK cells are promising targets for clearing HBV infection.However,whether immune checkpoints blockade can clear chronic HBV infection by modulating NK cell function is unclear.Considering that IL-21 and immune checkpoints blockade are potential therapeutic options,it is particularly important to explore the regulatory effects and mechanisms of IL-21 and immune checkpoints blockade on NK cells in chronic HBV infection.ObjectiveIn this study,we investigated the characteristics of NK cells in chronic HBV infection and the changes of NK cells after antiviral treatment by recruiting chronic HBV infected persons,longitudinal cohort receiving telbivudine treatment and healthy volunteers.Through in vitro and animal experiments,we elucidated the regulatory effects of IL-21 and immune checkpoints blockade on the function of NK cells,and reveal its biological mechanism,so as to develop feasible strategies for terminating chronic HBV infection.MethodsA total of 83 patients with chronic HBV infection were recruited,including 10 patients with HBeAg-positive chronic HBV infection who participated in a clinical trial of telbivudine treatment and were studied longitudinally for 48 weeks.The diagnosis of chronic HBV infection was based on established criteria.Seventeen healthy people without HBV infection were enrolled as healthy control.Flow cytometry was used to detect the frequencies of NK Cells and their subpopulations and to compare their phenotypes and functions.To compare the dynamic changes of NK cells in peripheral blood of chronic HBV infected patients treated with telbivudine for 48 weeks.Next,we continued to observe the expression of IL-21 R in NK cells of HC volunteers,HBeAg-positive HBV infected patients,HBeAg-positive hepatitis B patients,HBeAgnegative HBV infected patients and HBeAg-negative hepatitis B patients,and the direct effect of IL-21 on NK cells phenotypes and function.Furthermore,IL-21 R-KO mice and WT mice models of HBV infection were established to explore the effect of IL-21 on the control of HBV infection.Meanwhile,we detected the expression of PD-1,CTLA-4,TIM-3 and TIGIT inhibitory molecules on NK cells of chronic HB V infection by flow cytometry.Finally,we investigated effects and mechanism of immune checkpoint blockade alone or in combination with IL-21 on NK cells in chronic HBV infection.Results1.Characteristics of NK cells in chronic HBV infection patients and changes after treatment with Tibivudine1.1.The total frequency of peripheral blood NK cells in chronic HBV patients received Tibivudine treatment decreased significantly,mainly NK^dim subgroup.1.2 NK cell surface receptor expression was unbalanced in patients with chronic HBV infection,and Tibivudine therapy could not restore its expression.1.3 The function of NK cells secreting cytokines is defective in patients with chronic HB V infection,and the function cannot be restored by Tibivudine treatment.2.The regulatory effect and mechanism of IL-21 on NK cells in chronic HBV infection2.1 IL-21 can promote the expression of activating receptor on NK cell in patients with chronic HBV infection.2.2 IL-21 promotes the proliferation and function of NK cells.2.3 In mouse models,IL-21R-KO slowed HBsAg clearance and IL-21 promoted HBsAg clearance.2.4 IL-21 promotes HBV clearance by promoting increased IFN-y secretion by liver NK cells.3.The expression and functional characteristics of immune checkpoint suppressor molecules’ on NK cells in patients with chronic HBV infection3.1 The expression level of TIGIT in NK cells was significantly increased in patients with chronic HBV infection.3.2 The function of secreting cytokines was exhausted of TIGIT positive NK cells in patients with chronic HBV infection.4.The effects and mechanism of immune checkpoint blockade alone or in combination with IL-21 on NK cells in chronic HBV infection4.1IL-21 enhanced anti-TIGIT in promoting IFN-y production in NK^dim cells from chronic HBV infected patients4.2 IL-21 enhanced anti-TIGIT accelerating the clearance of HBsAg and HBeAg in HBV-carrying mice.4.3 IL-21 enhanced anti-TIGIT in clearing HBV through splenic NK cells.Conclusions1.The proportion of NK^dim subgroup is decreased,the expression of activated NK cell receptor is disturbed,and the secretion of IFN-γ and TNF-α is defective in chronic HBV infected patients.And tibivudine fails to restore NK cell function.2.IL-21 facilitated HBV clearance through promoting NK cell function4.TIGIT is an exhausted biomarker of NK cells in chronic HBV infected patients.5.IL-21 enhanced anti-TIGIT in promoting IFN-y production in NK^dim cells from chronic HB V infected patients.6.IL-21 enhanced anti-TIGIT in clearing HBV through splenic NK cells...