| Background: Chronic hepatitis B virus(HBV)infection still remains a worldwide public health problem.It constitutes as a primary cause of developing cirrhosis,hepatocellular carcinoma,and end-stage liver disease.HBV-specific T cell response plays a key role in either clearance of the virus or mediating liver inflammation,which is associated with the clinical outcomes.When HBV infection is acute self-limiting,successful HBV clearance is usually dependent on robust,broad,and polyclonal HBV-specific T cell responses,followed by the generation of memory immunity against HBV.In contrast,chronic HBV infection often shows deficiency of virus-specific T cells in numbers and functions,which is described as“exhausted state”,an important mechanism causing infection persists.Exhausted T cells is characterized by a hierarchical impairment of cytokine production,proliferation and increased cell apoptosis.There are several mechanisms involved in HBV-specific T cell exhaustion,including exposure to high level of viral antigens,up-regulated expressions of co-inhibitory receptors(i.e.programmed death 1),suppressive cytokines and regulatory T cells(Treg).Therefore,restoration of exhausted HBV-specific T cell response is crucial to control and clear the infection.Type 1 regulatory T cell(Tr1)is a cell population derived from peripheral CD4~+ T cells in the presence of antigen stimulation.It plays a role in promotion of immune tolerance and maintenance of homeostasis.Tr1 belongs to memory CD4~+ T cells that co-express both integrin ?2?1(CD49b)and lymphocyte activation gene-3(LAG-3).Tr1 is also believed to have unique cytokine profiles,as well as metabolic transcriptomics.The suppressive functions of Tr1 is mainly dependent on producing high amounts of interleukin(IL)10 and the killing by granzyme B.Tr1 is associated withthe development of T cell-mediated disorders,including autoimmune diseases,graft-versus-host disease,infections,and immune escape of tumors.In a mouse model of HBV infection,intrahepatic Tr1 mediated HBV-specific immune tolerance through producing IL-10 on maturation of follicular T cells and B cell within germinal center of lymph nodes.One study has found that HBV-specific IL-10+ Tr1 could not be a reliable biomarker to discriminate distinct phases of chronic HBV infection.However,this conclusion is limited by inappropriate identification of Tr1 due to poor methodology,which cannot fully reflect real effects of Tr1 in chronic HBV infection.Thus,it is of great importance to explore the suppressive role of Tr1 in chronic HBV infection,as well as to design underlying immunotherapeutic strategies targeted restoration of HBV-specific T cell response.Part one Role of Tr1 in natural history of chronic HBV infection and anti-HBV treatmentObjective: To compare the difference of peripheral and hepatic Tr1 frequencies among patients with chronic HBV infection of different phases,as well as to investigate its location within livers.To explore the correlations of tr1 to clinical parameters,including HBV DNA,viral antigens,and histological scores.To investigate the effects of anti-viral treatment on frequencies of tr1.Methods:1 In this cross-sectional study,110 untreated patients with chronic HBV infection and 23 healthy controls were recruited.Patients were classified into immune tolerant(IT)carriers(n=21),HBeAg+ immune active(IA)hepatitis(n=46),inactive carriers(IC)(n=24),and HBeAg– IA hepatitis(n=19).Another 15 patients with HBeAg+ IA hepatitis received pegylated interferon ?2a plus entecavir were studied longitudinally.The diagnosis of chronic HBV infection was according to AASLD guideline.Anticoagulant blood were isolated in all subjects.Partial proportions of patients underwent liver biopsies.2 Laboratory parameters of serum HBV markers,HBV DNA,HBVgenotype,and liver biochemistry were determined.Liver inflammation and fibrosis was investigated according to Knodell and Ishak scores after H&E or Masson staining.3 Both peripheral blood mononuclear cells(PBMC)and intrahepatic lymphocytes(IHL)were isolated from blood and liver samples,respectively.The frequencies of CD49b+LAG-3+ Tr1 in PBMC and IHL were analyzed by multicolor flow cytometry.Correlation analyses of Tr1 and clinical parameters were also investigated.4 Expression and location of LAG-3 and CD4 within livers of patients from different phases were detected by immunohistochemistry.Spatial connections between LAG-3 and CD4 were also explored.Results:1 Demographic and clinical characteristic of subjects in cross-sectional studyAll recruited patients were infected with HBV genotype B and C.IT and HBeAg+ IA patients were younger than IC and HBeAg– IA patients.IA and IC patients had normal serum level of alanine aminotransferase(ALT)and no significant histological inflammation.In contrast,HBeAg+ IA and HBe Ag–IA patients displayed varying degrees of elevated serum ALT levels accompanied by significant inflammation and fibrosis.HBV replicated actively in IT and HBeAg+ IA patients,but it was undetectable or at lower levels in IC patients.A significant decline of HBsAg was observed in IC patients compared with IT and HBeAg+ IA patients,however,HBeAg– IA patients showed intermediary levels among four groups.2 Peripheral frequencies of CD49b+LAG-3+ Tr1 and their correlations to clinical parametersThe patients with chronic HBV infection showed higher frequencies of peripheral CD49b+LAG-3+ Tr1 compared with healthy controls(P<0.0001).Significantly lower frequencies of Tr1 were observed in IC patients than those in IT and HBeAg+ IA patients(all P<0.0001).Both IT and HBeAg+ IA patients had higher Tr1 frequencies than HBeAg– IA patients(P=0.004 andP=0.005,respectively),but no difference was observed between them.In this cohort,HBeAg+ patients had higher frequencies of Tr1 than HBe Ag– patients according to HBeAg status(P<0.0001).In addition,no difference was observed between male and female patients.Correlation analysis demonstrated that peripheral Tr1 was positively correlated with serum HBV DNA(r=0.543,P<0.001)and HBsAg(r=0.582,P<0.001),but not with serum ALT and histological scores.3 Intrahepatic frequencies of Tr1 and locations of LAG-3 and CD4The patients with chronic HBV infection showed higher amounts of Tr1 within IHL than counterpart PBMC(P=0.005).Immunohistochemistry analysis demonstrated that the expression of LAG-3 were lower in IC patients than each of HBeAg+ IA,HBeAg– IA and IT patients.The locations of LAG-3 were predominantly seen in portal areas,necrotic foci and sinusoids within hepatic lobules.On serial sections,partial overlap of hepatic expressions of LAG-3 and CD4 within the same areas.4 Changes of peripheral Tr1 during anti-HBV treatmentOf 15 patients treated with anti-viral agents,peripheral Tr1 at week 48 was significantly lower than those at baseline and at week 12(P=0.01 and P=0.03,respectively).At week 48,peripheral tr1 relative to baseline showed a heavier decline between patients with HBsAg decline ?0.5Log and <0.5Log(P=0.018).Meanwhile,there was a positive correlation of decrease in Tr1 frequencies to that in HBsAg levels between week 48 and week 0(r=0.662,P=0.007).Summary:1 Peripheral frequencies of Tr1 were increased in patients with chronic HBV infection,suggesting the possible role of Tr1 in persistence of HBV infection.2 Peripheral frequencies of Tr1 were significantly distinct among different phases of chronic HBV infection.In particular,IT patients and HBeAg+ IA patients had higher frequencies of Tr1 than IC patients,indicating capacity of HBV replication and degree of immune tolerance.Thus,Tr1 maybe used as a biomarker for discrimination of clinical phases of chronic HBV infection.3 There is an association between decline of Tr1 and HBs Ag levels in patients who received anti-HBV treatment.Tr1 may be an underlying useful immunological biomarker for prediction of immune response during anti-HBV treatment.Part two Cytokine profile analysis of peripheral Tr1 in patients with chronic HBV infectionObjective: To investigate the characteristics of cytokine profile of Tr1 in the context of chronic HBV infection.Methods: Three cell populations of CD49b+LAG-3+ Tr1,CD4~+CD45RA–CD49b–LAG-3– T and CD4~+CD45RA+ T cells were sorted by flow cytometry,followed by stimulation with combination of phorbol myristate acetate and ionomycin.Cytokine profiles of IL-4,L-10,IL-17 A,interferon ?(IFN-?)and tumor necrosis factor ?(TNF-?)in culture supernatants were detected by multiplex cytokines analysis technology on flow cytometry.Results:CD49b+LAG-3+ Tr1 produced significantly higher amounts of IL-10 than its counterpart CD49b–LAG-3– T cells(P=0.02)and CD4~+CD45RA+ T cells(P=0.03).However,there was no difference of IL-10 amounts between the latter two cell populations.IFN-? produced by CD49b+LAG-3+ Tr1 was much lower than CD49b–LAG-3– T cells(P=0.02),but is similar with CD4~+CD45RA+ T cells.In terms of TNF-?,IL-17 A and IL-4,CD49b+LAG-3+Tr1 could produce absolutely minimal amounts compared with CD49b–LAG-3– T cells(P=0.01,P=0.0002,and P=0.001,respectively).In addition,CD4~+CD45RA+ T cells could produce none or very little amount of IL-17 A and IL-4.The hierarchical clustering analysis confirmed that combination of this cytokines profile could discriminate Tr1 from both CD49b–LAG-3– T cells and CD4~+CD45RA+ T cells.Summary:1 Peripheral CD49b+LAG-3+ Tr1 in patients with chronic HBV infectionshowed a specific cytokines profile with predominance of IL-10 secretion,which differs largely from other memory and na?ve populations of CD4~+ T cells.2 IL-10 produced by Tr1 may participate in regulation process of anti-HBV immune response,however,it still needs to be elucidated.Part three Mechanism of Tr1-mediated suppressive effects on HBV-specific T cellsObjective: To investigate the suppressive role of Tr1 on cytokines production and proliferative activity of HBV-specific T cells through IL-10 pathway.Methods: Tr1 was depleted from PBMC that was isolated from HBeAg+IA patients by flow cytometric sorting,and the reminder cells were defined as responder cells(RC).The experiment was divided into RC + Tr1(i.e.reconstitute Tr1 to RC)and RC groups.All cell populations were enrichment cultured ex vivo in the presence of HBV peptide or CEF(i.e.peptides derived from cytomegalovirus,Epstein-Barr virus and influenza virus)peptide pool stimulation.The productions of IFN-? and TNF-? by CD4~+ and CD8+ T cells were analyzed by enzyme-linked immunospot assay(ELISOPT)and intracellular cytokine staining(ICS).Proliferative activity of CD8+ T cells was detected by CFSE dye on flow cytometry.Furthermore,the suppressive role of Tr1 on HBV-specific CD8+ T cells was evaluated by blockade of IL-10 pathway using specific antibodyResults:1 Tr1 suppress production of IFN-? and TNF-? by HBV-specific CD8+ T cellsThe ELISPOT assay showed that IFN-?+ spot forming cells were more abundant in RC group than RC + Tr1 group after HBV peptide stimulation(P=0.014).Furthermore,depletion of Tr1 from PBMC leaded to increased production of IFN-? and TNF-? by CD8+ T cells(P=0.006 and P=0.034,respectively),but not by CD4~+ T cells.In contrast,the cytokines production either by CD4~+ T cells or CD8+ T cells were comparable between RC groupand RC + Tr1 group.2 Tr1 suppress proliferation of CD8+ T cellsDepletion of Tr1 from PBMC contributed to a significant decrease in percentages of undivided CD8+ T cells after TCR stimulation.When Tr1 and CD8+ T cells were co-cultured at different ratios of 0:1,1:4 and 1:2,the proliferative index of CD8+ T cells declined from 3.00 through 1.85 to 1.45,indicating a dose-dependent manner of Tr1 mediated suppressive role on CD8+ T cells.3 Partial restoration of Tr1-mediated dysfunctions of CD8+ T cells by blockade of IL-10 pathwayBlockade with anti-IL10 significantly increased HBV-specific productions of IFN-? and TNF-? by CD8+ T cells as compared with isotype controls(all P<0.05).The degrees of functional restoration were more evident in RC + Tr1 group than RC group,as indicated by fold increase of cytokines productions(fold of increase for IFN-?: 1.73 ± 0.53 vs 1.16 ± 0.10,P=0.01;fold of increase for TNF-?: 1.94 ± 1.10 vs 1.46 ± 0.50,P=0.039).Although anti-IL-10 treatment could partially restore secreting capacity of CD8+ T cells in the presence of CEF peptide,but the restorative degree had no difference between RC + Tr1 group and RC group.These data indicated that Tr1-mediated HBV-specific suppression of CD8+ T cells through IL-10 pathway.Furthermore,anti-IL-10 could reduce the percentages of CD8+ T cells that did not proliferate in both RC + Tr1 group and RC group as compared with isotypes(all P=0.006).However,the fold changes of IL-10 blockade on CD8+ T cells proliferation had no statistical difference between RC + Tr1 group and RC group.Summary:1 Peripheral Tr1 from patients with chronic HBV infection can suppress cytokine production and proliferation of HBV-specific CD8+ T cells.2 Specific blockade of IL-10 pathway can lead to partial restoration of suppressive role of Tr1 on HBV-specific CD8+ T cells.Part Four Role of CD49 b on CD8 + T cells in patients with chronic HB V infectionObjective: To investigate peripheral and intrahepatic frequencies of CD49b+CD8+ T cells in patients with chronic HBV infection,as well as their associations with degrees of disease.To analyze activation and memory phenotypes of CD49b+CD8+ T cells.Methods: 76 patients with chronic HBV infection(composed of 12 IT patients,25 HBeAg+ IA patients,23 IC patients,and 16 HBeAg– IA patients)and 23 healthy controls were recruited from the cross-sectional cohort used in Part One.The frequencies of CD49b+CD8+ T cells among both PBMC and IHL were determined by flow cytometry.The correlations between peripheral CD49b+CD8+ T cells and clinical parameters were analyzed.Expression of CD45 RA was compared among cell populations of CD49b+CD8+ T,CD49b–CD8+ T,and total CD8+ T cells.Distribution of CD49 b and its relationship with CD8 were detected by immunohistochemistry.Results:1 Peripheral frequencies of CD49b+CD8+ T cells in patients with chronic HBV infectionThe frequencies of peripheral CD49b+CD8+ T cells among CD3+CD8+ T cells were much higher than healthy controls(P=0.0003).Significantly higher peripheral frequencies of CD49b+CD8+ T cells were observed in HBeAg+ IA and HBeAg– IA patients compared with IT(P=0.002 and P=0.004,respectively)and IC patients(P=0.016 and P=0.027,respectively).However,differences were observed between neither the former two groups nor the latters.The patients who had active liver disease(i.e.HBe Ag+ IA patients and HBeAg– IA patients)exhibited higher peripheral frequencies of CD49b+CD8+T cells than those who had no liver inflammation(P=0.0003).In addition,no difference of peripheral CD49b+CD8+ T cells between patients by hierarchical analysis according to HBeAg status or gender.2 Associations between peripheral CD49b+CD8+ T cells and clinical parameters In patients with chronic HBV infection,peripheral CD49b+CD8+ T cells had positive correlations with serum levels of ALT and AST(r=0.39,P=0.0005 and r=0.32,P=0.005,respectively),but not serum levels of HBV DNA and HBsAg.Moreover,peripheral CD49b+CD8+ T cells correlated with histological Knodell scores and Ishak scores in patients who received liver biopsy(r=0.497,P=0.001 for Knodell score and r=0.353,P=0.025 for Ishak score,respectively).3 Expressive levels of CD45 RA on CD49b+CD8+ T cellsAmong HBeAg+ IA patients,percentages of CD45RA+ T cells in CD49b+CD8+ T cells were much lower than CD8+CD49b– T cells(P=0.022)and total CD8+ T cells(P=0.025).The ratio of percentages of CD45RA– T cells to CD45RA+ T cells was higher than CD8+CD49b– T cells(P=0.026)and total CD8+ T cells(P=0.015).4 Amounts and distributions of intrahepatic CD49b+CD8+ T cellsHigher amounts of CD49b+CD8+ T cells were observed in IHL than its mathed PBMC from HBeAg+ IA patients(P=0.0007).Immunohistochemistry analysis demonstrated that the expressions of CD49 b were up-regulated in both HBeAg+ IA and HBeAg– IA patients,however,only minimal CD49 b expressions were detected in IA and IC patients.The distributions of CD49 b were predominantly seen in portal areas and intralobular necrotic foci.Notably,the expression of CD49 b exhibited an extension trend from portal areas to loblues that is similar to properties of fobrotic tissuses.On serial sections,partial overlap of hepatic expressions of CD49 b and CD8 within the same areas.Summary:1 Peripheral CD49b+CD8+ T cells were increased in patients with chronic HBV infection,and is correlated with hepatic inflammatory injury.This cell subset can mobilize from periphery to liver,however,its mechanism is still unclear.2 Increased CD49b+CD8+ T cells from patients with chronic HBV infection display active and memory phenotype.Conclusions:1 The quantities and distributions of Tr1 are quite distinct among patients with chronic HBV infection at different phases of natural history,reflecting degrees of both viral replication and immune tolerance of the host.Our study indicates that Tr1 may be an underlying immunological biomarker for discrimination of different phases of natural history.2 Peripheral Tr1 in patients with chronic HBV infection,with predominance of IL-10 production,has a unique cytokines profile that is distinct form other na?ve and memory CD4~+ T cells.3 Tr1-mediated functional inhibition of HBV-specific T cells though IL-10 pathway may be one of the mechanisms that involved in pathogenesis of immune tolerance during chronic HBV infection.4 Tr1-based immunotherapy may be an underlying candidate for restoration of exhausted HBV-specific T cells.5 Although CD49 b is the specific cell surface biomarker for identification of Tr1 with suppressive properties,its expression on CD8+ T cells predominantly mediates immunopathological injury during chronic HBV infection. |
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