Analysis Of The Phenotype And Function Of HBV Specific CD8 T Cells In Peripheral Blood And Liver

350 million people worldwide have a persistent infection with hepatitis B virus(HBV) and are at risk of developing chronic liver inflammation leading to cirrhosis and hepatocellular carcinoma.Although the pathogenesis of chronic liver disease is not well understood,there is a consensus that liver damage is immune mediated.Since the Virus is preferentially hepatotropic and not cytopathic,it has been assumed that the recognition of HBV-infected hepatocytes by Ag-specific HLA class I-restricted CTLs is causing the liver damage.In HBV infection,the cellular immune response is thought to contribute to both viral clearance and liver cell injury.These two opposing functions have even been attributed to the same cell:upon cognate recognition of viral peptides on MHC class I molecules of HBV-infected cells,CD8 T cells acquire the capacity to either cure HBV infected cells via noncytopathic,cytokine-mediated inhibition of HBV replication or to destroy them via perforin-,Fas ligand(FasL)-,and TNFa -mediated death pathways.Both effector functions have been observed during resolution of acute hepatitis B.HBV-specific T cells are also detectable in chronic hepatitis,although at a lower frequency compared with acute resolving infection.HBV-specific CD8⁺ T-cell lines have also been generated from the human liver.The long-term survival of HBV-specific CD8⁺ T cells in the liver has also been demonstrated by MHC I-pentamer analysis.Other studies have detected pentamer-positive CD8⁺ T cells constituted up to 9%of all intrahepatic CD8⁺ T cells indicating that these cells accumulate at the primary site of infection.The mechanisms by which HBV is able to evade the virus-specific T-cell response that is present in the infected liver are still only poorly understood.Previous studies showed that CD8⁺ T cells change the expression of costimulatory molecules(CD27,CD28,and CD45RA) on their surface according to their differentiation and maturation.Therefore,these molecules have been used for phenotypic classification of human CD8⁺ T cells.Naive,memory,and effector CD8⁺ T cells express CD27⁺CD28⁺CD45RA⁺,CD27⁺CD28⁺CD45RA^-,and CD27^-CD28^-CD45RA⁺ phenotypes, respectively.A previous study further demonstrated that CD8⁺T cells with the CD27^-CD28^-CD45RA^+/- and CD27^lowCD28^-CD45RA^+/- phenotype have the ability to kill target cells.Chemokine receptors play an important role in lymphocyte trafficking.They are also used to define the functional subsets of human CD8⁺ T cells.Previous studies revealed that T cells could be classified based on their expressions of CD45RA and the chemokine receptor CCR7 that is associated with their ability to home in on secondary lymph nodes(11).They were classified into CD45RA⁺CCR7⁺(naive),CD45RA^-CCR7⁺ (central memory),CD45RA^-CCR7^-(effector memory),and CD45RA⁺CCRT(effector).It has been hypothesized that antigen-primed CD8 T cells segregate into a memory population and an effector population with markers like these.However,this simple phenotypic picture may not be true in patients with persistent infection.The differentiation of T cells must be different during chronic infection.Firstly that an epitope hierarchy exists within the HBV-specific CD8 T-cell responses can be altered by viral persistence.The distribution of T cells was changed that these cells can not come back to lymph nodes to acquire memory phenotype but accumulated at local organs.The impact of viral load on antiviral T-cell responses has been precisely characterized in animal models of viral infections(like LCMV),all of which show that sustained presence of viral antigens leads to a progressive functional decline of virus-specific CD8 responses and ultimately leads to virus-specific T-cell deletion.Virus-specific CD8⁺ T-cell populations did not differ from each other on the basis of CD28 and CD27 expression during primary infection,but during chronic infections they were represented in each of the three different phenotypic subsets, EBV as well as HCV-specific CD8+ T cells demonstrated a clear enrichment of the early phenotype(CD27⁺CD28⁺),whereas CMV-specific CD8+ T cells were substantially enriched in the late phenotype cells(CD27^-CD28^-),and HIV-1-specific CD8⁺ T cells showed a substantial enrichment of cells belonging to the intermediate subset(CD27⁺CD28^-).Several different mechanisms could explain the failure of the HCV-specific CD8⁺ T-cell response.For example,during the chronic infection the presence of a multi-specific CD8 T-cell response in the absence of a CD4 T-cell response and that the absence of CD4 T cell help prevented the maturation of a functionally efficient CD8 T-cell response. HBV-specific CD4 T-cell responses with a Th2 profile of cytokine production are detectable in the blood of patients with chronic infections.It is possible that CD4⁺ CD25⁺T cells are also responsible for the weak HBV-specific T-cell response in chronic hepatitis B patients and may inhibit the expansion and function of HBV-specific CD8 T cells,precluding HBV clearance but also limiting immune mediated liver damage.Functional alterations of dendritic cell populations could explain the state of T cell hypo-responsiveness present in chronic hepatitis B patients.Circulating HBV-specific CD8 cells from patients without liver inflammation and with low viral load express the phenotype of Ag-experienced resting cells and exhibit efficient proliferation after reencounter with the viral Ag.These cells can exert antiviral effector functions after such expansion.The frequencies of HBV-specific T cells found to be increased in patients who accepted anti-viral therapy.All of these suggest that HBV-specific T-cell tolerance is not absolute but appears to be regulated mainly by the quantity of HBV replication present in chronic hepatitis B patients.Many recent studies indicate that the PD-1/PD-L1 pathway can play a role in the T cell exhaustion occurring when T cells are chronically exposed to high antigen loads and that blockade of PD-1/PD-L1 interaction can allow restoration of exhausted CD8 cells.Thus,high expression of the inhibitory PD-1 receptor seems to be a signature of functional CD8 exhaustion.PD-1 belongs to the CD28 family and shares 23%amino acid sequence homology with cytotoxic T-lymphocyte-associated antigen 4(CTLA-4).Unlike the expression of CD28 and CTLA-4,which is restricted to T cells,PD-1 is expressed on activated T cells,B cells and myeloid cells.The cytoplasmic region contains an immunoreceptor tyrosine-based inhibitory motif(ITIM) and an immunoreceptor tyrosine-based switch motif(ITSM).The expression and structure of PD-1 suggest that PD-1 has broader roles in immune regulation.The PD-1/PD-L1/PD-L2 pathway is one such negative regulatory pathway that has been implicated in autoimmunity and responses to pathogens.The negative co-stimulatory function of PD-1 was initially suggested because PD-1 knockout mice developed spontaneous autoimmune diseases.Recent studies in mice indicate that the PD-1 pathway is an important regulator of T cell responses to chronic viral infections.PD-1 is upregulated on virus-specific CD8+ T cells during chronic LCMV infection.During chronic LCMV infection the PD-1 pathway plays a prominent role controlling CD8+ T cell exhaustion and preventing optimal antiviral function.The blockade of this pathway in persistently infected mice results in improved T cell function and more efficient viral control. An important role has also been suggested for the PD-1/PD-L1 pathway in inflammation of the liver.â‘ The absence of PD-L1 also allowed susceptibility to experimental autoimmune hepatitis when mice lacking PD-L1 were injected with Con A.â‘¡Additional studies have shown that infection of PD-1 deficient mice with adenovirus resulted in marked expansion of antigen-specific T cells in the liver compared to wild-type mice and led to a rapid abrogation of viral infection.â‘¢PD-L1 expression in hepatocytes was strongly enhanced by viral infection and markedly augmented by further stimulation with typeâ… or typeâ…¡interferons.â‘£HCV-specific CD8 exhaustion may represent a mechanism of HCV persistence.Most HCV-specific CD8 cells expressed PD-1 at the time of acute illness,but the expression declined with the acquisition of a memory phenotype. whereas high levels were maintained when HCV persisted and HCV-specific CD8 cells remained dysfunctional.Blocking PD-1/PDL-1 interaction with an anti PDL-1 antibody improved the capacity of expansion of virus specific-CD8 cells.Although a great deal has been learned about the role of the virus-specific T-cell response in peripheral blood,very little information is currently available about intrahepatic HBV-specific CD8 T cells.In this study we used the MHC I-pentamer technique in combination with functional cytokine assays to analyze and compare the frequency, phenotypes and cytokine production capacity of HLA-A2-restricted intrahepatic and peripheral HBV specific CD8⁺ T cells at the single-cell level.The results of our study indicate:(1) intrahepatic HBV-specific CD8⁺T cells are present in the majority of patients with chronic HBV infection.(2) The phenotype of these cells almost is CD45RA^-CCR7^-CD27⁺CD28^+/-.(3) The frequency of PD-1⁺ cell in HBV-specific CTL is high.(4) A large fraction of intrahepatic HBV-specific CD8⁺ T cells are impaired in their ability to secrete interferon-γ(IFN-γ).In conclusion,intrahepatic HBV-specific CTLs with preterminally differentiated phenotype are exhausted and PD-1 may be the causing.