Association Of Natural Killer And Hepatitis B Virus-specific T Cells With Virologic Relapse And Liver Damage After Nucleos(t)ide Analogue Cessation

Background and PurposeHepatitis B virus?HBV?infection is a global public health problem.The disease profile of chronic hepatitis B?CHB?is manifold,manifesting from asymptomatic HBsAg carriers,hepatic flare,liver cirrhosis to hepatic decompensation.Nucleos?t?ide analogue?NA?can effectively inhibit the virus replication,recover liver function to normal,delay the progression of liver cirrhosis and reduce the incidence of liver decompensation or hepatocellular carcinoma,thereby improving the prognosis of some patients.However,NA therapy has no direct inhibitory effect on covalently closed circular DNA?cccDNA?,and it is extremely difficult to eradicate HBV.NA cessation is often tough for the majority of CHB patients,even inducing severe hepatitis and leading to death.According to previous clinical observations,HBsAg carriers or clinically cured HBsAg-negative patients still had an increased risk of HBV reactivation after receiving glucocorticoids,anti-tumor drugs or immunosuppressants,indicating that immune factors play an extremely important role in controlling virologic relapse?VR?.The correlations between VR and immune status in patients with NA cessation remain to be elucidated.It has been documented that both innate and adaptive immune responses are indispensably involved in clearing HBV during acute HBV infection.On the contrary,natural killer?NK?and virus-specific T cell responses are compromised,and their antiviral ability are profoundly weakened in chronic HBV infection.In this study,we investigated the changes of NK and HBV-specific T cell responses after NA cessation,and analyzed the association with VR and liver damage.In order to further elaborate their roles in affecting VR,cellular immunity at the end of treatment?EoT?was also cross-sectionally compared with that in patients with spontaneous viral control—inactive carriers and patients with failed viral control—HBeAg-negative hepatitis?ENEG?.Population and Methods1.Study design:We longitudinally followed up 24 patients with NA cessation,all of whom were HBeAg negative,achieved complete virologic suppression for at least two years and serum HBsAg level>200 IU/mL at the end of treatment?EoT?.Based on the presence or absence of serum HBV DNA level>1×10⁴ copies/mL within one year after NA cessation,patients were classified into two groups:virologic relapse?VR?group and no virologic relapse?NVR?group.We also followed up 19 inactive carriers?IC?with baseline HBsAg level>200 IU/mL.In addition,28 treatment-naive HBeAg-negative hepatitis?ENEG?patients with matched age and sex were enrolled as the control group.3.Peripheral blood samples were collected from outpatient or inpatient department.Peripheral blood mononuclear cells?PBMCs?were separated by density-gradient centrifugation,and plasma was collected for HBsAg measurement.4.Flow cytometry was used to detect the frequency,subpopulation,phenotype,the ability to secrete IFN-?and TNF-?,and the cytotoxic activity of NK cells.5.PBMCs was stimulated by HBV core or S antigen peptide library.After 10 days in vitro culture,flow cytometry was used to detect the frequency of CD4 and CD8 T cells-producing IFN-?and IL-2.6.Enzyme-Linked Immunosorbent Spot?ELISpot?assays ex vivo.Results1.Characteristics of the patient cohort.Fourteen patients with NA cessation experienced VR during 1-year follow-up.Among these,nine patients also experienced clinical relapse?CR?with ALT>80 U/L.The cumulative VR and CR rates at 1-year off-treatment was 58.3%an 37.5%,respectively.However,only two?10.5%?IC patients had serum HBV-DNA level>1×10⁴ copies/mL within 1-year follow-up,and ALT>80 U/L occurred in one of the two patients,which was significantly lower than patients with NA cessation?P<0.05?.2.Serum HBsAg levelsSerum HBsAg levels at EoT were significantly lower in VR and NVR group than those in ENEG group,but were similar to those in IC group.Longitudinally,serum HBsAg titers were relatively stable during 1-year off-treatment in NVR group.However,serum HBsAg titers were markedly increased at the points of VR in comparison to those at EoT in VR group.3.Frequency of NK cells and CD56^bright subsetThe proportion of circulating NK cells and CD56^bright subset at the same time points did not differ between VR group and NVR group,which was also similar to that in IC and ENEG group.Furthermore,in comparison to that at EoT,no significant changes of NK cell and CD56^bright subset frequency were observed during 1-year follow-up,regardless of VR.4.NK cell phenotypeThe expressions of inhibitory receptor—CD96 in total NK cells were obviously higher in VR and ENEG group than those in NVR and IC group?P<0.05?.NK cells from VR group expressed the higher levels of inhibitory receptor—NKG2A than those from IC group?P<0.05?.Likewise,the expressions of CD96 at the 1^th and 6^th month,and NKG2A at the 3^thh month were obviously higher in VR group than those in NVR group.Furthermore,the frequency of total NK cells-expressing CD69 was markedly increased in ENEG group in comparison to that in VR,NVR and IC group?P<0.05?.Longitudinally,the expressions of above surface receptors in total NK cells were relatively stable compared with those at EoT in NVR group.In contrast,the expressions of activating markers—NKp44,NKG2C and CD69 in NK cells were markedly increased at the 6^th and12^th month compared with those at EoT in NVR group.5.NK cell FunctionThe ability of NK cells-producing IFN-?at EoT was partially restored in VR group when compared with that in ENEG group,which was still obviously weaker than that in NVR and IC group,independent of stimulators?P<0.05?.NK cell TNF-?production at EoT was relatively attenuated in VR group in comparison to that in IC group?P<0.05?.NK cell cytotoxicity was evaluated by detecting the expressions of perforin,granzyme B,and CD107a,which did not differ across four groups.In addition,NK cell IFN-?production at the 1^th,3^th,6^th and 12^th month was still significantly lower in VR group than that in NVR group?P<0.05?.In a longitudinal analysis,the frequency of NK cells-expressing IFN-?,perforin,granzyme B,and CD107a was similar at the different time points in NVR group?all P<0.05?.In contrast,the proportion of NK cells-expressing perforin and CD107a was markedly increased at the 6^th and 12^th month in VR group in comparison to that at EoT?P<0.05?.The majority?12/14?of patients in VR group experienced serum ALT levels>40 IU/mL at least once during 1-year follow-up,while only one?1/10?patients had abnormal ALT in NVR group.In order to explore the roles of NK cells in mediating liver damage after NA cessation,NK cell functions were further analyzed based on virus load?>1×10⁴ or<1×10⁴copies?in VR group.Compared with that at EoT and the time points with HBV DNA<1×10⁴ copies,the frequency of NK cells-expressing perforin and CD107a at the time points of VR were markedly increased,while IFN-?production in NK cells was not accordingly improved,suggesting that NK cell functions shifted toward the cytotoxic activity.Importantly,the increased frequency of NK cells-expressing perforin and CD107a was positively correlated with serum ALT levels during VR,respectively.Weak core-specific T cell responses were associated with VR after NA cessationAfter 10 days in vitro expansion,core peptide pool-stimulated T cell responses were determined through detecting the frequency of CD4 and CD8 T cells-expressing IFN-?and IL-2.In response to core peptide pool,IFN-?and IL-2 production by CD4 T cells at EoT was partially restored only in NVR group when compared with that in ENEG group?P<0.05?.Notably,IFN-?production by CD4 T cells at EoT was significantly lower in VR group than that in NVR group?P<0.05?.In addition,compared with that in VR and ENEG group,IFN-?and IL-2 production by CD4 and CD8 T cells was markedly increased in IC group?all P<0.05?.We also observed that IFN-?production by CD4 T cells at the 3th,6th and 12^th month,IFN-?production by CD8 T cells at the 6^thh month,and IL-2 production by CD4 T cells at the 1^th and 6^th month was significantly weaker in VR group than that in NVR group?P<0.05?.HBV core-specific T cell responses ex vivo were detected by the IFN-?ELISPOT in the presence of core peptide pool.The number of spot-forming cells?SFC?was significantly increased in NVR group when compared with that in ENEG group?P<0.05?.IC group had the significantly higher number of SFC than ENEG and VR group?P<0.05?.In addition,SFC number at the 3^th and 6^th month differed markedly between VR group and NVR group?P<0.05?,although the significant difference was not reached at EoT.HBV-specific T cell responses were relatively stable after NA cessationCore and S-specific T cell responses were longitudinally analyzed in VR group and NVR group,respectively.Except for S-specific CD8+T cells at the 6^th month in NVR group,we did not observe the substantial changes of core or S-specific T cell responses in comparison to those at EoT.Furthermore,in VR group,core or S-specific T cell responses at the time points with HBV DNA<1×10⁴ copies were similar to those during VR.Conclusion1.The present study demonstrated that NK cell IFN-?production and core-specific T cell responses were significantly lower in VR group than those in NVR group.2.Patients with NA cessation,especially those who would experience VR,had more compromised NK and HBV-specific T cell responses at EoT than IC patients,which coincided with higher VR and CR rates in the former.3.NK cells exhibited an activated phenotype and increased cytotoxicity during VR, which correlated with liver damage after NA cessation.Scientific merit of our study:Low NK and HBV-specific T cell responses are associated with VR after NA cessation,and the augmented NK cell cytotoxicity correlated with liver damage during VR.Our findings are helpful in better understanding the immunological mechanisms under clinical manifestations after NA cessation,and providing the guidance for safe withdrawal.Immune therapy targeting NK and HBV-specific T cells can reverse the imbalance between virus and host immunity,thereby having the potential to achieve sustained viral suppression and establish a more stable disease state.Our study also provides a promising strategy:if radical treatment is difficult to attain,effective immune reconstitution after the antiviral ability,resembling the state of inactive carriers,may be a safe treatment endpoint.