Elsevier

Journal of Hepatology

Volume 72, Issue 1, January 2020, Pages 45-56
Journal of Hepatology

Research Article
TNF-α/IFN-γ profile of HBV-specific CD4 T cells is associated with liver damage and viral clearance in chronic HBV infection

https://doi.org/10.1016/j.jhep.2019.08.024Get rights and content

Highlights

  • TNF-α producing cells are the dominant population of HBV-specific CD4 T cells in chronic HBV infection.

  • Increased frequency/dominance of HBV-specific IFN-γ producing CD4 T cells parallels elevated HBV viral clearance.

  • HBV core-specific TNF-α producing CD4 T cells are associated with liver damage in patients with a hepatitis B flare.

  • HBV-specific IFN-γ producing CD4 T cells are associated with HBV viral clearance in patients with a hepatitis B flare.

  • Some HBV-specific IFN-γ producing CD4 T cells might originate from HBV-specific TNF-α producing CD4 T cells during flare.

Background & Aims

The role of hepatitis B virus (HBV)-specific CD4 T cells in patients with chronic HBV infection is not clear. Thus, we aimed to elucidate this in patients with chronic infection, and those with hepatitis B flares.

Methods

Through intracellular IFN-γ and TNF-α staining, HBV-specific CD4 T cells were analyzed in 68 patients with chronic HBV infection and alanine aminotransferase (ALT) <2x the upper limit of normal (ULN), and 28 patients with a hepatitis B flare. HBV-specific HLA-DRB1*0803/HLA-DRB1*1202-restricted CD4 T cell epitopes were identified.

Results

TNF-α producing cells were the dominant population in patients’ HBV-specific CD4 T cells. In patients with ALT <2xULN, both the frequency and the dominance of HBV-specific IFN-γ producing CD4 T cells increased sequentially in patients with elevated levels of viral clearance: HBV e antigen (HBeAg) positive, HBeAg negative, and HBV surface antigen (HBsAg) negative. In patients with a hepatitis B flare, the frequency of HBV core-specific TNF-α producing CD4 T cells was positively correlated with patients’ ALT and total bilirubin levels, and the frequency of those cells changed in parallel with the severity of liver damage. Patients with HBeAg/HBsAg loss after flare showed higher frequency and dominance of HBV-specific IFN-γ producing CD4 T cells, compared to patients without HBeAg/HBsAg loss. Both the frequency and the dominance of HBV S-specific IFN-γ producing CD4 T cells were positively correlated with the decrease of HBsAg during flare. A differentiation process from TNF-α producing cells to IFN-γ producing cells in HBV-specific CD4 T cells was observed during flare. Eight and 9 HBV-derived peptides/pairs were identified as HLA-DRB1*0803 restricted epitopes and HLA-DRB1*1202 restricted epitopes, respectively.

Conclusions

HBV-specific TNF-α producing CD4 T cells are associated with liver damage, while HBV-specific IFN-γ producing CD4 T cells are associated with viral clearance in patients with chronic HBV infection.

Lay summary

TNF-α producing cells are the dominant population of hepatitis B virus (HBV)-specific CD4 T cells in patients with chronic HBV infection. This population of cells might contribute to the aggravation of liver damage in patients with a hepatitis B flare. HBV-specific IFN-γ producing CD4 T cells are associated with HBV viral clearance. Differentiation from HBV-specific TNF-α producing CD4 T cells into HBV-specific IFN-γ producing CD4 T cells might favor HBV viral clearance.

Introduction

In chronic hepatitis B virus (HBV) infection, HBV-specific T cells plays a pivotal role in viral clearance and liver damage. Studies on a chimpanzee model of HBV acute infection have demonstrated an irreplaceable role of CD8 T cells in HBV clearance and disease pathogenesis.1 In patients with chronic HBV infection, HBV-specific CD8 T cells usually show functional exhaustion or even clonal deletion,[2], [3], [4], [5], [6], [7] which is deemed a major reason for the HBV persistence in those patients.

CD4 T cells, as the main regulating cell population, also play an important role in HBV clearance. A mice model of HBV genome plasmid hydrodynamic transfection indicated a critical role of CD4 T cells in induction of CD8 T cell-mediated HBV clearance.8 Strong HBV-specific CD4 T cell responses have been observed in patients with self-limited acute HBV infection.[9], [10] A lack of HBV-specific CD4 T cell responses has been associated with HBV persistence in HBV/hepatitis C virus coinfection patients despite detectable HBV-specific CD8 T cells in those patients.11 Most of the studies on HBV-specific CD4 T cells have focused on the magnitude of response, and usually found a low frequency of HBV-specific CD4 T cells in patients with chronic HBV infection, resembling the T cell exhaustion in HBV-specific CD8 T cells.[3], [4], [5], [6], [7] As a versatile cell population, detailed functional dissection of HBV-specific CD4 T cells has received less attention in previous studies. The observation of an increased frequency of circulating HBV-specific follicular T helper (Tfh)-like cells in patients with HBV e antigen (HBeAg) seroconversion has directly associated a functional subset of HBV-specific CD4 T cells with HBV viral control.12 Our previous study has identified HLA-DR as the major locus for susceptibility to HBV-related acute-on-chronic liver failure, indicating a role of CD4 T cells in the immune-pathogenesis of HBV-related acute-on-chronic liver failure.13 Compared to HBV-specific CD8 T cell epitopes, fewer HBV-specific CD4 T cell epitopes have been identified,[14], [15] which hinders detailed functional analysis of HBV-specific CD4 T cells.

Persistent exposure to pathogen antigen has been indicated as the main cause of T cell exhaustion in mice with chronic lymphocytic choriomeningitis virus (LCMV) infection.16 A lot of studies have correlated the HBV-specific T cell response with serum HBV antigen in patients with chronic HBV infection,[3], [4], [5], [6], [7] while the discovery of preserved T cell function in young adults in the immune-tolerant phase argues against current knowledge about HBV-specific T cell exhaustion.17 Another scenario not matching current knowledge of HBV-specific T cell exhaustion is hepatitis B flare. Patients with a hepatitis B flare show both high HBV viral load and rising HBV-specific T cell responses.18 Some of these patients would undergo HBeAg or even HBV surface antigen (HBsAg) seroclearance after the flare, which indicates that the rising HBV-specific T cells in those patients is effective at HBV clearance.19 Those discrepancies indicate that some mechanisms, besides T cell exhaustion, might be involved in the regulation of HBV-specific T cell responses.

In this study, we firstly analyzed the IFN-γ/TNF-α functional profile of HBV-specific CD4 T cells in 68 patients with chronic HBV infection with alanine aminotransferase (ALT) <2x the upper limit of normal (ULN). Then, the IFN-γ/TNF-α functional profile of HBV-specific CD4 T cells was analyzed in 28 patients with a hepatitis B flare. Additionally, for samples from HLA-DRB1*0803/HLA-DRB1*1202 patients, HLA-DRB1*0803/HLA-DRB1*1202 restricted CD4 T cell epitopes were identified.

Section snippets

Study design

The selection of patients with chronic HBV infection in this study is illustrated in Fig. S1. A total of 4–8 ml of venous blood was sampled from 210 patients with ALT <2xULN visiting our outpatient clinic. Twenty-seven HLA-DRB1*0803/HLA-DRB1*1202 samples and 41 non-HLA-DRB1*0803/HLA-DRB1*1202 samples were included for analysis of HBV-specific CD4 T cells. Potential HLA-DRB1*0803/HLA-DRB1*1202-restriced CD4 T cell epitopes were identified according to CD4 T cell response data. Another 13

HBV-specific CD4 T cells are dominated by TNF-α producing cells in patients with chronic HBV infection with ALT <2xULN

HBV core and S-specific CD4 T cells were analyzed in 68 patients with chronic HBV infection with ALT <2xULN. IFN-γ and TNF-α are recognized as 2 major cytokines of type 1 helper (Th1) CD4 T cells, we analyzed patients’ HBV-specific CD4 T cells by intracellular cytokine staining of those 2 cytokines. The gating strategy of flow cytometry is illustrated in Fig. S3. Similar to previous reports,3 HBV-specific CD4 T cells in patients with chronic HBV infection could hardly be detected by direct ex

Discussion

In this study, we firstly analyzed the TNF-α/IFN-γ profile of HBV-specific CD4 T cells in patients with chronic HBV infection with ALT <2xULN. We found that both the frequency and the dominance of HBV-specific IFN-γ producing CD4 T cells increased in parallel with the elevated HBV viral clearance level in patients. Scrutinizing previous studies which analyzed HBV-specific CD4 T cell response in HBeAg negative patients with different outcomes (HBsAg loss vs. HBsAg persistence) after

Financial support

This study was supported in part by the National Natural Science Foundation of China (81330038, 81571978), the Chinese State Key Project Specialized for Infectious Diseases (2018ZX10302206), the Chongqing Natural Science Foundation (CSTC-2018jcyjAX0495), and the Southwest Hospital Medical Science Innovation Plan (SWH2017YBXM-05, SWH2018BJKJ-01).

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Please refer to the accompanying ICMJE disclosure forms for further details.

Authors’ contributions

HW, WT, and GD conceived the study questions and designed the study. HW, XW, and HL performed most of the experiments. HW conducted data analysis, and interpreted results. HW and GD drafted the manuscript. XF assisted with flow cytometry analysis. QM advised on the concept and design of the study. XX and JZ were involved in the blood sample collection of patients with alanine aminotransferase <2x upper limit of normal. YZ was involved in the clinical follow-up and blood sample collection of

Acknowledgements

We thank all study nurses of the Department of Infectious Diseases, Southwest Hospital for support in collecting patient samples and all patients that participated in this study. This study was supported in part by the National Natural Science Foundation of China (81330038, 81571978), the Chinese State Key Project Specialized for Infectious Diseases (2018ZX10302206), the Chongqing Natural Science Foundation (CSTC-2018jcyjAX0495), and the Southwest Hospital Medical Science Innovation Plan

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