The Expression, Function, And The Clinical Relevance Of CD28Family Receptors In Chronic Hepatitis B Virus Infection

BackgroundT cell exhaustion is a key characteristic of persistent HBV infection and is caused by long-term, large amounts of antigen stimulation. The previous studies have shown that CD28family receptors, e.g. PD-1, was upregulated on CD8+T cells in chronic hepatitis B patients. CD28family contains two costimulatory receptors (CD28and ICOS) and three three inhibitory receptors (PD-1, CTLA-4, BTLA). All these five receptors have high homology, belong to the immunoglobulin (Ig) superfamily with similar characteristics in structure. The role of these receptors in chronic HIV and HCV persistence infection has been well studied, however, the few study was done in CHB. The present work will focus on investigating the expression of all five CD28family receptors on PBMC of CHB and their correlation with serum viral markers. The effect of blocking PD-1/PD-L1pathway on the expression of the CD28family receptors as well as the T cell functionare also investigated. This study may provide some very useful information for novel HBV immune therapy strategy.Objective1.To investigate the expression of all five CD28family receptors (CD28, ICOS PD-1, CTLA-4, and BTLA) on PBMC of CHB and their correlation with serum viral markers.2. To investigate the effect of blocking PD-1/PD-L1pathway on the expression of all CD28family receptors in CHB patients.3. To understand the effect of blocking PD-1/PD-L1pathway on the function of T cells in CHB patients by analyzing the expression of transcription factors T-bet and GATA-3.Methods1. Forty-eight patients with chronic hepatitis B infection and26healthy subjects were enrolled in this study. All the patients were selected from the Department of Infectious Disease, Union Hospital, Wuhan, China. All patients signed the consent forms.2. Blood was collected with heparin tube and PBMC was separated by Ficoll density gradient centrifugation. The expression of CD28family receptors (CD28, ICOS, PD-1, CTLA-4, BTLA) on total T cell, CD4+T cells and CD8+T cells was analyzed by flow cytometry.3. For assessment the effect of blocking PD-1:PD-L1pathway, PBMC of HLA-A2+patients were cultured in a flat-bottomed96-well plate in the presence of HBV peptide (AA,.18-27, FLPSDFFPSI), anti-PD-L1or control IgG for PD-1:PD-L1blocking test for10days. Flow cytometry were used to investigate the expression of the CD28family receptors on CD4+and CD8+T cells’ surface and the intracellular cytokines IFN-y. RNA was extracted from the collected PBMC treated with anti-PD-L1or control antibody for10days using the RNAiso Plus reagent according to the manufacture’s instruction. The Real-time PCR was used for quantifing the expression of T cell transcription factors:T-bet and GATA-3.4. ELISA kit was used to detect the viral serologic markers, including HBV, HIV, HCV and HDV. PCR was used to determine the allotype of HLA-A2. Real-time PCR was employed to quantify HBV-DNA for blood samples.. 5.. Nonparametric Mann-Whitney, Wilcoxon matched pairs test or Spearman correlation coefficient test were employed for statistical analysis.Results1. Compared to healthy controls, CHB patients had higher expression of PD-1, CTLA-4on the CD4+T cells, and ICOS, PD1and BTLA on the CD8+T cells.2. The relevance of the same receptor expression on both CD4+and CD8+T cells was different:CD28, ICOS, PD-1showed positive correlation in CHB patients, but not in the healthy controls. BTLA presented a good positive correlation.in both healthy controls and CHB patients.3. The relevance of different receptors on the CD4+or CD8+T cells was different: CD28and CTLA-4showed a negative correlation on CD4T cells in CHB patients, but not in the healthy controls. ICOS and PD-1showed a positive correlation on CD4+T cells in both CHB patients and healthy controls. CD28and BTLA on CD8+T cells had a positive correlation both in patients with CHB and in healthy controls.4. The expression of PD-1and BTLA on CD8+T cells of CHB patients had a positive correlation with HBV DNA load, while ICOS had negative correlation with HBV DNA load.5. After blocking PD-1:PD-L1path in vitro:(1) The expression of CD28, ICOS, PD-1and CTLA-4on the CD4+cells in CHB patients was upregulated, and the change of CD28and ICOS was positively correlated, meanwhile, the change of CTLA-4had a positive correlation with CD28、 ICOS、PD-1. Furthermore, the expression of PD-1on CD4+T cells showed a positive correlation with HBV DNA load after the PD-1:PD-L1path was blocked. However, none of the receptors showed a significant expression change on CD8+T cells.(2) The expression of intracellular IFN-y in CD4+T cell was increased. The fold change of IFN-y was positively correlated to the expression of ICOS on CD4+T cells, while negatively correlated with PD-1expression in CD4+T cells. Blocking PD-1:PD-L1path did not affect the expression of IFN-y of CD8+T cells.(3) The expression of T-bet and GATA-3mRNA in PBMC was increased and positively correlated with each other. After blocking th PD-1:PD-L1path, the expression of IFN-y rised when T-bet:GATA-3>1.3.Conclusions1. In CHB, CD28family inhibitory receptors, PD-1, and CTLA-4were upregulated on CD4T cells, PD-1and BTLA were upregulated on CD8T cells. CD28family costimulatory receptors ICOS had a higher expression on CD8T cells. The upregulation of PD-1and BTLA on CD8T cells were positively correlated with HBV DNA load, while ICOS high expression on CD8T cells was negatively correlated with HBV DNA load.2. Blocking PD-1:PD-L1pathway could increase the expression of CD28family costimulation receptors (CD28, ICOS) and inhibitory receptor PD-1on CD4T cells of CHB patients as well as the secretion of intracellular IFN-y from CD4T cells, while it did not present the similar effect on CD8T cells.3. Blocking the PD-1:PD-L1pathway in vitro could significantly increase the expression of T cell transcription factors T bet and GATA3at mRNA level, and a greater ratio of T-bet:GATA-3was beneficial for the expression of IFN-γ in CD4+T cells.