T-cell Immunosenescence And Increased Hepatocellular Carcinoma Risk In Chronic Hepatitis B Patients With Persistently Elevated Serum Total Bile Acid

The age-dependent decrease in immunological competence,as a result of progressive deterioration of both innate and adaptive immune systems,is often referred to as ‘immunosenescence'.The immunosenescence is often associated with poor immune response to vaccines,vulnerable to infection by virus or bacteria,and cancer development in the old people.As a major component of adaptive immune systems,the defects in T-cell immunity are most marked.The T-cell immunosenescence is characterized by multiple alterations in T-cell immune system including decline of naive T-cell number,decreased T-cell receptor(TCR)repertoire diversity,shortened T-cell telomere length,defective memory T-cell function,and etc.Chronic infection with persistent pathogens was suggested to have a negative impact on host immune systems.In this study,we investigated whether the T-cell immunity is impaired in chronic hepatitis B(CHB)patients.We also investigated whether impaired T-cell immunity in CHB patients is associated with hepatocellular carcinoma(HCC)development in those patients.We firstly compared the TCR repertoire of naive T-cell pool between 24 CHB patients and 15 healthy controls by high-throughput sequencing.Similar to previous reports,the diversity of naive T-cell TCR repertoire remained stable in healthy controls with age under 40,while the diversity of naive T-cell TCR repertoire declined significantly with the increase of age in CHB patients with age under 40.This indicated that the long-term interaction between hepatitis B virus(HBV)and T-cell immune systems rather than increased age caused the decreased diversity of naive T-cell TCR repertoire in CHB patients,as all the 24 CHB patients acquired HBV infection by vertical transmission.There was no dominant TCR clonotypes in the naive T-cell pool of CHB patients,indicating that the decreased diversity of naive T-cell TCR repertoire in CHB patients was not a result of over-proliferation of some specific TCR clonotypes but reflected an equal loss of unique TCR clonotypes.Our findings in this part indicated that chronic HBV infection could accelerate the T-cell immunosenescence in CHB patients.The decreased diversity of TCR repertoire in naive T-cell pool indicated the disruption of peripheral T-cell pool maintenance in CHB patients.The maintenance of peripheral T-cell pool is mediated mainly by slow T-cell proliferation in adult human being.Firstly,we analyzed the T-cell proliferation in 146 CHB patients and 53 healthy controls.Compared to healthy controls,the T-cell proliferation in CHB patients was significantly increased.The T-cell proliferation rate in some CHB patients was as high as that reported in human immunodeficiency virus type 1(HIV-1)infection.Serum total bile acid(TBA)level was significantly associated with the increased T-cell proliferation in CHB patients: qualitatively,the T-cell proliferation rate was significantly increased in all the T-cell subsets(both naive T cells and memory T cells)of CHB patients with elevated serum TBA,compared to CHB patients with normal serum TBA or healthy controls;quantitatively,the proliration rate of T-cell subsets was significantly associated with serum TBA level in CHB patients with elevated serum TBA.No such significant association was observed between increased T-cell proliferation and other CHB clinical signatures.Secondly,we sampled 11 CHB patients with persistently elevated TBA at two time points to analyze T-cell proliferation.The T-cell proliferation remained at high level at two time points for CHB patients with persistently elevated serum TBA,indicating that T cells in CHB patients with persistently elevated serum TBA were undergoing persistent over-proliferation.Thirdly,we analyzed the T-cell activation and compared the distribution of TCR V? family between proliferating and resting T cells in 12 CHB patients with elevated serum TBA and 3 healthy controls.Proliferating T cells in CHB patients with elevated TBA displayed a phenotype of immune activation with up-regulated expression of activation marker CD38.Comparably high correlations of TCR V? family distribution between proliferating and resting T cells were observed in both CHB patients with elevated serum TBA and healthy controls.This suggests that the increased T-cell proliferation in CHB patients with elevated serum TBA was mediated by antigen non-specific by-stander activation mechanism.Finally,we analyzed the relative telomere length of naive T cells from 5 healthy controls,21 CHB patients with continuous follow-up for more than 4 years,and 4 HBV-associated HCC patients to assess the effect of persistently elevated serum TBA on T-cell immunosenescence.Compared to healthy controls and CHB patients with no or transient serum TBA elevation during follow-up,the telomere length of the naive T cells from CHB patients with persistent serum TBA elevation during follow-up was shortened to the similar level as that from HBV-associated HCC patients.Our findings in this part indicated a close association between persistent TBA elevation and T-cell immunosenescence in CHB patients as a result of T-cell over-proliferation.To assess the effect of persistent serum TBA elevation on HCC development in CHB patients,we did a retrospective observational cohort study of 3021 CHB patients with data from the Hepatitis Biobank at Southwest Hospital(HBS)Program from 2001 to 2014.Of the 3021 patients eligible for the study,3018(99.9%)patients had records of regular antiviral treatment by interferon and/or nucleoside analogue during the follow-up.The association between elevated TBA and HCC was estimated using Cox proportional hazard models,Kaplan-Meier analyses,and propensity score analyses including information about patients' demographic and clinical characteristics recorded in HBS program.Age,sex,liver cirrhosis,serum alanine aminotransferase(ALT),serum HBV DNA,and serum Hepatitis B e antigen(HBeAg)were commonly recognized as HCC risk factors in CHB patients and were chosen as covariates along with serum TBA in this study.Persistence of elevated TBA was determined by the ratio of patient's follow-up time with elevated TBA to his total follow-up time.Patients were classified into 3 groups: none-low,medium,and high persistence of elevated TBA according to the ratio value.There were 81 HCC cases during a total follow-up of 20813.7 person-years in the retrospective study.None-low,medium,and high persistence of elevated TBA were observed in 2139(70.8%),520(17.2%),and 362(12.0%)patients.The cumulative probability of HCC at the end of follow-up was 2.2%,6.3%,and 19.3% for CHB patient with none-low,medium,and high persistence of elevated serum TBA,respectively(Log-rank P< 0.001).Compared to patients with none-low persistence of elevated TBA,the multivariate adjusted hazard ratios(95% confidence interval)were 1.70(0.88-3.26),and 3.08(1.69-5.60)for patients with medium,and high persistence of elevated TBA.The population attributable risks of HCC development based on the multivariate adjusted hazard ratios were 7.8%,and 29.0% for medium,and high persistence of elevated serum TBA.The Propensity score models analyses demonstrated a similar dose-response relationship between persistence of elevated TBA and HCC.Our findings in this part indicated that persistence of serum TBA elevation is a major independent risk predictor for HCC development in CHB patients receiving regular antiviral treatments.To sum up,our findings indicated that CHB patients with persistently elevated TBA have higher risk for HCC,which might be related to the T-cell immunosenescence in those patients.Practically,our finding might improve the clinical surveillance for hepatocellular carcinoma in CHB patients receiving regular antiviral treatments.Theoretically,our finding might provide us an immunological insight into the HCC development in CHB patients.