| Background Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in China.The chronic infection with hepatitis B virus(HBV)is the main cause of HCC.The host immune system is hard to eliminate HBV in hepatocytes,and liver tissue is in an uncontrolled inflammatory in a long period.A process characterized by "inflammation-necrosis-proliferation" appears continuously in liver lesion.Meanwhile,HBV achieves immune escape by changing its own CD8+T cell recognition epitope.Viral mutations are gradually selected through incomplete immune selection,and mutant HBV can escape immune recognition and attack,which promotes HCC occurrence.One of the main features of mutant HBV is the deletion of CD8+T cell epitopes,among which the A1762T/G1764 A mutation occurs 10 years before HCC,which is the result of inflammatory immune environment.HLA-DR is an important molecule in the antiviral immune response.HLA-DR binding to HBV presents antigenic peptide epitopes to CD4+T cells and activates CD4+T cells.Activated CD4+T cells promote B cell differentiation to produce virus-specific antibodies and secrete cytokines to promote CD8+T cells and NK cells to kill HBV.T cell receptor(TCR)and B cell receptor(BCR)are important molecules in the adaptive immune response,and HBV stimulation can affect the formation of TCR and BCR diversity.To track dynamic changes in TCR or BCR immune repertoires during the different stages of hepatocarcinogenesis in HBV-infected individuals is helpful to better understand the immune microenvironment during HBV infection.To investigate the effects of immune-related molecules such as HLA-DR in the mutation selection and carcinogenesis of HBV,as well as the dynamic changes of TCR and BCR after HBV infection has important significance in clarifying the mechanism of mutation selection,determining which HBV-infected individuals will develop into HCC,and laying the foundation on specific prophylaxis of HCC.Objective This study was aimed to investigate the associations between HLA-DR single nucleotide polymorphism(SNP)and HBV persistence and HCC occurrence,and interactions between HLA-DR SNP and HBV mutations in HCC occurrence,and to study the dynamic changes of immune repertoires at different stages of hepatocarcinogenesis.Methods Taq Man-MGB probe quantitative PCR assay was used for HLA-DRA(rs3135395,rs3135338 and rs2395178)and HLA-DRB1 rs477515 genotyping in 1240 healthy controls,300 HBV natural clearances,311 asymptomatic HBs Ag carriers(ASCs),936 chronic hepatitis B(CHB)patients,611 liver cirrhosis(LC)patients,and 1190 HCC patients.The nested PCR method was employed to amplify the sequence of HBV Enh II/BCP/PC and pre S regions.Logistic regression model was used to analyze the association between HLA-DR SNPs and disease and multiplicative interactions between the SNPs and HBV mutation in HCC occurrence.Cox proportional hazards regression model was used to analyze the influencing factors of postoperative survival in patients with HCC.Kaplan-Meier and Log-rank tests were used to compare the survivals of HCC patients with different genotypes of HLA-DR SNPs.The effects of SNPs on the function of the HLA-DR gene enhancer regions were examined by the dual luciferase reporter gene system.The TCRs and BCRs of patients with ASCs,CHB,LC and HCC with/without A1762T/G1764 A mutation were detected by immune repertoire sequencing,and the V gene and J gene usages and clonal diversity were compared among different stages.Results 1.We regarded HBV natural clearances as controls to analyze the associations between HLA-DR SNP and HBV chronic infection and natural clearance.The rs3135395 GT genotype and dominant model(GT+TT),rs3135338 TC genotype and dominant model(TC+CC),rs477515 TT genotype and dominant model(CT+TT)significantly reduced the risk of developing chronic HBV infection(rs3135395 GT: AOR(95%CI)=0.63(0.48-0.84),P=0.001,GT+TT: AOR(95%CI)=0.67(0.51-0.88),P=0.004;rs3135338 TC: AOR(95%CI)=0.66(0.50-0.87),P=0.004,TC+CC: AOR(95%CI)=0.73(0.56-0.95),P=0.021;rs477515 TT: AOR(95%CI)=0.67(0.49-0.91),P=0.011,CT+TT: AOR(95%CI)=0.70(0.52-0.93),P=0.014).2.We regarded non-HCC HBV infected patients as controls to analyze the associations between HLA-DR SNP and the risk of HCC occurrence.The rs3135395 TT genotype,rs3135338 CC genotype,rs2395178 CG genotype and dominant model(CG+GG),rs477515 TT genotype significantly reduced the risk of HCC in patients infected with HBV(rs3135395 TT: AOR(95%CI)=0.68(0.46-0.96),P=0.031;rs3135338 CC: AOR(95%CI)=0.67(0.47-0.96),P=0.030;rs2395178 CG: AOR(95%CI)=0.80 (0.68-0.95),P=0.011,CG+GG: AOR(95%CI)=0.80(0.69-0.94),P=0.006;rs477515 TT: AOR(95%CI)=0.78(0.64-0.97),P=0.024).3.In the interaction of HLA-DR SNP and HBV mutations in HCC,we found that there were significant synergistic interactions between rs3135395 GT genotype and A1762T/G1764 A,C1653T,G1719 T,T1753C in HCC occurrence in genotype C infected individuals(A1762T/G1764A: AOR(95%CI)=1.945(1.365-2.773),P=2.32×10-4;C1653T: AOR(95%CI)=1.983(1.161-3.388),P=0.012;G1719T: AOR(95%CI)=1.421(1.021-1.972),P=0.035;T1753C: AOR(95%CI)=2.079(1.190-3.632),P=0.010).There were significant synergistic interactions between rs3135338 TC genotype and A1762T/G1764 A,T1753C have in HCC occurrence(A1762T/G1764A: AOR(95%CI)=1.920(1.330-2.773),P=4.93×10-4;T1753C: AOR(95%CI)=1.842(1.141-2.974),P=0.012).4.Cox multivariate regression analysis showed that HBe Ag-positive(HR(95%CI)=2.944(1.725-5.024),P<0.001),tumor size≥5 cm(HR(95%CI)=2.848(1.715-4.731),P<0.001),and microvascular invasion(HR(95%CI)=3.696(2.217-6.160),P<0.001)were independent risk factors for postoperative survival of HCC patients,and postoperative antiviral therapy(HR(95%CI)=0.233(0.137-0.397),P<0.001)and rs477515 variant genotype(HR(95%CI)=0.565(0.425-0.751),P<0.001)were independent protective factors for postoperative survival of HCC patients.AFP≥20 ng/m L(HR(95% CI)=1.552(1.005-2.397),P=0.048),tumor size≥5cm(HR(95% CI)=1.643(1.062-2.543),P=0.026),and microvascular invasion(HR(95%CI)=2.973(1.941-4.553),P<0.001)were independent risk factors for postoperative recurrence in HCC patients,and postoperative antiviral therapy(HR(95%CI)=0.473(0.298-0.752),P=0.002)and low-frequency genotype of rs477515(HR(95% CI)=0.611(0.401-0.932),P=0.022)were independent protective factors for postoperative recurrence in HCC patients.5.There was no significant difference in the transcriptional activity of HLA-DRA containing the rs3135338-C and rs3135338-T enhancer region fragments in Hep G2 and LO2 cells(Hep G2 cells: P=0.543;LO2 cells: P=0.066).In Hep G2 cells,the transcriptional activity of HLA-DRB1 containing the rs477515-T enhancer region was significantly higher than that containing rs477515-C enhancer region fragment(8.2-fold,P=5.0×10-6),and this effect was repeated in LO2 cell(6.4-fold,P=2.9×10-5).The activity of HLA-DRB1 enhancer containing rs477515-T was significantly increased in Hep G2 cells under IFN-γ stimulation(t=11.360,P=3.0×10-4).The activity of HLA-DRB enhancer fragments containing rs477515-C and rs477515-T were significantly decreased in Hep G2 cells under IL-6 stimulation(rs477515-C: t=6.134,P=0.004;rs477515-T: t= 4.429,P=0.011).The activity of HLA-DRB1 enhancer fragment containing rs477515-C was significantly increased(t=5.734,P=0.005)in Hep G2 cells under IL-4 stimulation.The activity of HLA-DRB1 enhancer containing rs477515-T was significantly increased in LO2 cells under IFN-γ stimulation(t=4.785,P=0.009).The activity of HLA-DRB enhancer fragments containing rs477515-T were significantly decreased in LO2 cells under IL-6 stimulation(t=3.835,P=0.019).There were no significant differences in the activity of enhancer fragments containing rs477515-T and rs477515-C in LO2 cells stimulated by IL-4(P>0.05).6.TCR repertoires sequencing showed that TRBV5-1,TRBV20-1,TRBV29-1,TRBV12-3,TRBV28,and TRBJ2-7,TRBJ2-1,TRBJ2-3,TRBJ2-5,TRBJ1-1 were preferential usage of Vβ and Jβ gene segments,respectively.There was no significant difference in the number of CDR3 between the wild group and mutant groups in ASCs,CHB and LC patients(P>0.05),while the number of CDR3 in wild group was significantly higher compared with mutant group in HCC patients(P=0.006).There were no significant differences in Shannon Wiener index,Simpson index and inverse Simpson index between wild group and mutant group in ASCs,CHB,LC and HCC patients(P>0.05).7.BCR repertoires sequencing showed IGHV3-23,IGHV4-39,IGHV3-21,IGHV3-30,IGHV1-18 and IGHJ4 were preferential usage of Vβ and Jβ gene segments,respectively.The number of CDR3 in wild group was significantly higher compared with mutant group in CHB(P=0.014)and HCC patients(P=0.004).There were no statistically significant differences in number of CDR3 between wild group and mutant group in ASCs and LC patients(P>0.05).There were no significant differences in Shannon Wiener index between wild group and mutant group in ASCs,CHB and LC patients(P>0.05),while the Shannon Wiener index in wild group was significantly higher than mutant group in HCC patients(P=0.018).There were no significant differences in Simpson index and inverse Simpson index between wild group and mutant group in ASCs,CHB,LC and HCC patients(P>0.05).Conclusions 1.The rs3135395 GT genotype,rs3135338 TC genotype and rs477515 TT genotype significantly reduced the risk of chronic HBV infection.The rs3135395 TT genotype,rs3135338 CC genotype,rs2395178 CG genotype and rs477515 TT genotype reduced the risk of HCC occurrence.2.The interactions between the low-frequency genotypes of rs3135395,rs3135338,rs2395178,and rs477515 and HBV mutations including C1653 T,T1753V,and A1762T/G1764 A mutaitons were significant in HCC occurrence.3.The rs477515-T significantly improved HLA-DRB1 gene transcription via up-regulating enhancer activity.4.T cells highly expressing TRBV5-1/J2-1,TRBV5-1/J2-7,TRBV20-1/J2-7,TRBV20-1/J2-7 and TRBV5-1/J2-3 gene segments and B cells highly expressing IGHV3-23,IGHV4-39,IGHV3-21,IGHV3-30,and IGHV1-18 gene segments constitute essential components of the host adaptive immune system after HBV infection.5.The clone diversity of TCR and BCR in CHB patients was more obvious compared with ASCs,LC and HCC patients. |
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