| BackgroundHepatitis B virus(HBV)infects over 350 million people worldwide.Chronic hepatitis B(CHB)is the major risk fator for end-stage liver diseases including liver cirrhosis,liver decompensation and hepatocellular carcinoma(HCC),which raises a public health concern.The transmission of chronic HBV infection is mainly by maternal-neonatal and infants.Its natural history could be divided into four phases:immune tolerance(IT),immune clearance,inactive carrier(IC)and reactivation.As far as we know,anti-HBV therapy is the most important treatment in inhibiting viral replication,reducing liver inflammation injury,preventing development of cirrhosis and HCC,improving the life-quality and survival rates.No matter induced by therapy or spontaneously,hepatitis B virus e antigen(HBeAg)seroconversion is one of the key endpoints and identified as a relatively stable state of immune control.Although most HBV DNA would become undetectable in CHB patients after therapy,the rates of HBeAg seroconversion are still relatively low.Therefore,most of them would relapse once the treatment discontinued.Some studies have indicated that HBeAg seroconversion is associated with serum levels of interleukin-12(IL-12)and IL-21,functions of plasmacytoid dendritic(pDC)cells,frequencies of CD4+CD25+regulator T(Treg)cells,follicular helper T(Tfh)cells and γδ-double-negative T(DNT)in CHB patients,but the mechanism remains unclear.The reason of ineffective inhibiting HBV might be poor immune response.The T cell responses to HBV in patients with acute self-limited hepatitis B are multi-specific,vigorous and polyclonal while they are narrow-spectrum,weak,oligoclonal and even undetectable in CHB patients.It has been reported that DC,Treg and T cells depletion are involved in the hyporesponsiveness of T cells,especially CD8+T cells.CD8+ T cells could be classified into CD8+ TCRαβ T(CD8+ap T)cells and CD8+TCRγδ T(CD8+y8 T)cells according to T cell receptors(TCR).The CD8+ap T cells account for mostly in CD8+T cells with high expressions of CD8,and are limited by major histocompatibility complex-I(MHC-I),But CD8+γδ T cells account for only about 3%in CD8+ T cells with low expressions of CD8 and being non-MHC-I limited.CD8+ T cell response plays a vital role in inhibiting viral infection.One in vivo study on chimpanzees with acute HBV infection demonstrated instead of CD4+ T cells,CD8+ T cells were the primary effector cells to clear HBV.And the inhibition of HBV by CD8+T cells are mainly mediated by the secretions of interferon-y(IFN-y),tumor necrosis factor-a(TNF-a)and other cytokines.The hyporesponsiveness of HBV-specific CD8+ T cells in CHB patients is associated with the overexpressions of programmed death-1(PD-1),CD244,Bim,cytotoxic T-lymphocyte associated antigen 4(CTLA-4).Nevertheless the functions of CD8+T cells could not be restored totally by blocking these inhibitory molecules.So we speculate that there may be other unidentified ways to mediate the dysfunction of CD8+ T cells.NKG2A,one member of the C-type lectin domain family,could combine with CD94 and form the heterologous dimers CD94/NKG2A.They are expressed in natural killer(NK)cells,NK T cells and y8 T cells and act as inhibitive molecules mediated the suppression signal in itself.The ligand of NKG2A mainly are non-classical MHC-I molecules,including mouse Qa-1 and human leukocyte antigen-E(HLA-E).Previous studies have found that HLA-E is mainly expressed in human blood cells,lung,adrenal gland and placenta.NKG2A expression on human melanoma-specific CD8+ T cells could inhibit the oncolytic effects by themselves.And it could inhibit the early T cells activation in lymphocytic choriomeningitis virus(LCMV)infection as well.Moreover,it could mediate the inhibition of HCV-specific CD8+ T cell response in patients with Hepatitis C virus(HCV)infection.Some viral peptides such as HCV aa35-44 stabilize the expression of HLA-E on liver cells’membrane,which would inhibit HCV-specific CD8+ T cell response.But the response could be enhanced by blocking HLA-E/NKG2A pathway.In summary,our study aims to investigate the association between NKG2A and HBV-specific CD8+ T cell response.AimsTo explore whether NKG2A is involved in the hyporesponsiveness of HBV-specific CD8+ T cell,and its association with CHB patients in different phases.Materials and Methods1.SubjectsThis study was conducted according to the ethical guidelines of the 1975 Declaration of Helsinki,and was approved by the Ethical Committee of Nanfang Hospital.Written informed consent was obtained from all subjects.None of these subjects had received IFN-y treatment,or detected any evidence of HCV,HDV or HIV infections,HCC,autoimmune liver disease,bacterial infection,any malignant tumor or any severe metabolic disease.1.1 Cross-sectional study Forty-three patients with chronic HBV infection in three phases(IT=10,CHB=21,IC=12)were recruited from the Hepatology Clinics and Clinical Follow-up Cohort of Nanfang Hospital,while 18 healthy controls(HC)were recruited from Hepatology Clinics and the student of Southern Medical University.All the recruited patients were Hepatitis B virus surface antigen(HBsAg)positive above 6 months between 18-60 years old.1.2 Longitudinal study Ten HBeAg-positive CHB patients from Nanfang Hospital participated in a phase IV,multi-center,open-label clinical trial(EFFORT,the Efficacy Optimization Research of Telbivudine[LdT]Therapy trial,trial NCT00962533).The inclusion and exclusion criteria were same with the cross-sectional study.PBMC specimens were taken at baseline,12,24 and 52 weeks after therapy.The subjects were classified into either a complete response(CR,n=5)group who have undergone HBeAg seroconversion and achieved serum HBV DNA less than 300 copies/mL by week 52,or a non-complete response(NCR,n=5)who have decreased HBV DNA was reduced but remained HBeAg-positive.1.3 Liver tissue Eleven cases with liver tissue were derived from clinical liver biopsy and surgical excision biopsy specimens,which were the residues after conventional histopathological analysis for diagnosis.1.4 Functional study There were 45 subjects for functional study were recruited from the Hepatology Clinics of Nanfang Hospital.The criteria for inclusion is HBsAg-positive,HBeAg-positive,and anti-HBe-negative above 6 months,while the exclusion criteria were same as CHB patients.Explore the relationship between NKG2A and HBV-specific CD8+αβ T cell in this part:HBV core peptide pool specific CD8+T cell response,or core18-17 peptide pool specific CD8+ T cell response in HLA-A2 positive patients.Screening method is as following:1.4.1 Short peptide synthesis by GL-Biochem Company:HBV C ORF 18-mer overlapping peptides pool(C peptide library covers the HBV C-ORF,adjacent overlapping peptides lOaa, core peptides pool, cores), core18-27(FLPSDFFPSV) as a specific stimulus, the purity of synthetic peptide is more than 95%.1.4.2 Experimental groups 5mL heparin anticoagulanting blood were collected from CHB patients.100μL blood were labeled with anti-HLA-A2, and the other were isolatied for PBMC by Ficoll separation medium (Lymphoprep rM, Axis-Shield, Oslo,Norway) density gradient separation. 4x105 PBMC/200μL amplification medium (containing 10% FBS, 1μg/mL anti-CD28 and 1μg/mL anti-CD49d antibody 1640) were added into the 96 well U-plates, each well were stimulated according to the Table 1, cultured for 10 days, and added IL-2 (25IU/mL) at the fourth day.1.4.3 Detected by intracellular cytokine staining (ICS): Cells were harvested at day 10 after centrifuged, resuspended by containing 10% FBS-RPMI-I640 (RIO),and stimulated according to the Table 1 for I hour, then added brefeldin A (BFA, final concentration 1μg/mL) incubated for Sh. Cells were collected and stained with anti-CD3-FITC, anti-CD8-APC-Cy7 as phenotypic markers, washed, fixed,permeablilized, washed again, then staining with anti-IFN-γ-PE, and analyzed by flow cytometry for IFN-γ producting by the CD8+ap T cells.2. The expression of HLA-E, NKG2A on PBMC and liver-infiltrating lymphocytes (LIL) surfaceConjugated antibodies and fluorescent staining technique by flow cytometry were used to detect the expression of HLA-E or NKG2A on the surface of different cell subsets.Fluorescent antibody included:anti-CD3-APC-Cy7(UCTH1),anti-TCRy8-FITC(11F2),anti-CD8-PerCP,anti-NKG2A-PE and anti-HLA-E-APC.3.The associtation between the expression of NKG2A and the function of CD8+αβT cells:3.1 Stimulation through TCR pathway Anti-CD3 and anti-CD28 antibody were used as non-specific stimulus to compare the production of IL-2,TNF-a,IFN-y and proliferation between NKG2A+CD8+αβ T and NKG2A-CD8+αβ T cells.3.1.1 Fluorescent antibodies(anti-CD3-APC-Cy7,anti-CD8-FITC,anti-NKG2A-APC,anti-IL-2-PE,TNF-a-PE-Cy7 and anti-IFN-y-Percp-Cy5.5)were used for detecting cytokines.3.1.2 CFSE(1.5μmol/L)and fluorescent antibodies(anti-CD3-APC-Cy7,anti-CD8-APC)were used for detecting the proliferation of CD8+αβ T cells.3.2 HBV-specific stimulation HLA-A2(＋)Patients with corel 8-27-specific CD8+αβT cell response were detected for the association between the hyporesponsiveness of HBV-specific CD8+αβ T cell and NKG2A.PBMC from the patients were stimulated with both HBV cores and core 18-27 for 10d,then detected by ICS,including anti-CD3-APC-Cy7,anti-CD8-Percp and HBV core18-27-polymer-PE(pentamer or streptamer,corel 8-27-PE)for surface staining and anti-IFN-y-APC for intracellular staining.The production of IFN-γ compared the functional differences between NKG2A+corel 8-27+CD8+ap T and NKG2A-corel 8-27+CD8+αβ T cells.4.The effect of HLA-E/NKG2A blocking on the function of HBV-specific CD8+αβT cells:4.1 Blockage of γδ T(-)NK(-)PBMC In consideration of the high expression of NKG2A on NK and y8 T cell,y8 T(-)NK(-)PBMC were isolated by magnetic beads(TCRγδ MicroBeads,Miltenyi)sorting kit,NK cells beads(CD56 MicroBeads,Miltenyi)and LD column from 1×107 PBMC.Then 5×105 γδ T(-)NK(-)PBMC were pre-incubated with anti-NKG2A(Z199,10μg/mL)and anti-HLA-E(3D12,0.5mg/mL),and cultured with corel 8-27 peptide for 10d.The production of IFN-y were detected by ICS with antibodies including anti-CD3-APC-Cy7,anti-CD8-PerCP,HBV-corel8-27-PE and anti-IFN-γ-APC.4.2 Blockage of CD8+ap T cells γδ T(-)PBMC were isolated by y8 T magnetic beads sorting kit and LD column from 1×107 PBMC.Then used anti-CD8 magnetic beads(CD8 MicroBeads,Miltenyi)and LS columns to collected CD8+αβ T cells,the other cells were named as y8T(-)CD8(-)PBMC.3×105 CD8+αβ T cells were pre-incubated with anti-NKG2A,and then mixed with 3×105γδ8T(-)CD8(-)PBMC.The cultured method and ICS is same as 4.14.3 Blocking experiments of PBMC 5×105 PBMC were incubated with anti-NKG2A and anti-HLA-E to block the expression of NKG2A and HLA-E,the cultural method and ICS is similar to 4.1.5.Statistical analysis:The serum HBV DNA levels were converted into log10(copies/mL)values for statistical analysis.Data were showed with median(range).Chi-Square Tests(Continuity correction)were used for enumeration data.And appropriate statistical methods were chosen according to different data for measurement data,including Kruskal-Wallis H test,Mann-Whitney U test,Friedman Test and Wilcoxon matched pairs test.All data were analyzed by SPSS13.0,GraphPad Prism 5 or SAS.P value<0.05(two tailed)was considered statistically significant.Results1.The expressions of NKG2A on CD8+ap T cells.The frequencies of NKG2A on CD8+αβ T cells were much higher in LIL than those in PBMC from 8 patients received LdT therapy for 104 weeks(P<0.001).The frequency of NKG2A on core 18-27+CD8+ap T cells were higher than corel 8-27-CD8+αβ T cells in 3 LIL and 5 PBMC of HLA-A2(+)patients.2.The association between the expression of NKG2A and the functions of CD8+αβ T cells.2.1 The association between the expression of NKG2A and non-specific CD8+αβT cells function and proliferation.The production of IFN-y,TNF-a and IL-2 and the proliferation in NKG2A+CD8+ap T cells were less than those in NKG2A-CD8+ap T cells in PBMC(n=4).And the production of IFN-y of NKG2A+ subset was also significantly lower than NKG2A-subset in LIL(n=2).2.2 The association between NKG2A and HBV-specific CD8+αβ T cells function2.2.1 Screening patients with HBV-specific CD8+T cell responses 45 patients from outpatient follow-up group were included Only 12 patients were detected being positive for HBV-specific CD8+T cell response.2.2.2 NKG2A was associated with the hyporesponsiveness of HBV-specific CD8+ap T cells The production of IFN-y of NKG2A+ subset was less than NKG2A"subset in core18-27+CD8+αβ T cells from PBMC(n=5,P =0.043).3 Blocking of HLA-E/NKG2A pathway could promote HBV-specific CD8+αβ T cell response78 T(-)NK(-)PBMC from 3 HLA-A2(+)patients were blocked by anti-NKG2A and anti-HLA-E respectively,the productions of IFN-y in corel8-27+CD8+αβ T cells were increased after lOd-culture.CD8+αβ T cells from 3 HLA-A2(+)patients were pre-incubated with anti-NKG2A to block the expression of NKG2A on CD8+αβ T cells,then mixed withαβ T(-)CD8(-)PBMC and cultured for 10d.The productions of IFN-y in CD8+αβ T cells were increased.Total PBMC from 5 CHB patients with HBV-specific response were blocked by anti-NKG2A and anti-HLA-E respectively and stimulated with cores.The productions of IFN-y in CD8+aP T cells were increased in some patients.4 Cross-sectional study Although there are no significant difference(P=0.120)of the frequencies of NKG2A on CD8+αβ T cells in cross-sectional group,the frequencies of NKG2A in the CHB group(P=0.046)and IC group P=0.017)is significantly higher than the HC group.Additionally the frequencies of HLA-E on lymphocytes in different chronic HBV infection phases and HC are different significantly(P=0.006).The HLA-E frequencies in the CHB group(P=0.001),IC group(P=0.028)and IT group(P=0.045)were much higher than the HC group respectively.But the frequencies of NKG2A on CD8+αβ T cells and HLA-E on lymphocytes in different chronic HBV infection phases are similar.5.The association between the dynamic changes of NKG2A and HLA-E during 52 weeks antiviral therapy and clinical outcomesNKG2A expression on CD8+αβ T cells in the CR group(n=10)at baseline was significantly lower than the NCR group(n=10,P = 0.023)And there were no significant difference in HLA-E expression on lymphocytes in each time point.CONCLUSIONIncreased expression of NKG2A on HBV-specific CD8+αβ T cells and HLA-E on lymphocytes may be associated with hyporesponsiveness of HBV-specific CD8+αβ T cells.HBV specific CD8+αβ T cell response may be enhanced by blocking the HLA-E/NKG2A pathway.NKG2A act as an inhibitory receptors involving in mediating the hyporesponsiveness of HBV-specific CD8+αβ T cells.Clinical studies suggest that NKG2A expression on CD8+αβ T cells at baseline of antiviral therapy may be associated with the efficacy of antiviral therapy. |
