| Background and aims:Chronic hepatitis B(CHB)infection is a chronic infectious disease caused by hepatitis B virus(HBV)with a high global disease burden.Infants clear HBV at a much lower rate than adults,and adult patients who were infected before age 5 represent the major global reservoir.However,there is no treatment method that can completely eliminate HBV up to now.Immunotherapy can realize the functional cure,which is one of the promising treatment strategies for CHB infection.The immune system is tolerant or exhausted during chronic HBV infection.Immune checkpoints are molecules expressed on the surface of immune cells that can activate or inhibit the immune response.The tolerance can be broken by regulating them.The costimulatory molecules OX40/OX40L and 4-1BB are members of the tumor necrosis factor(receptor)superfamily(TNF(R)SF)and play an important role in the activation of immune responses,especially in the activation of T cells.Programmed cell death 1(PD-1)is a member of the CD28 subfamily of immunoglobulin receptors.As an inhibitory signal molecule,PD-1 is involved in tolerance or exhaustion of the immune system.However,it is not clear whether these immune checkpoints can be used as immunomodulatory targets for the treatment of HBV.Compared with infants and children,adults can produce a strong and effective immune response to eliminate HBV and the immune molecules that play an age-dependent role are not clear.Based on the background of chronic HBV infection,the expression characteristics of OX40/OX40L PD-1 4-1BB in CHB patients were studied to clarify its relationship with age and clinical significance.We studied the influence of activating OX40 on HBV replication in rAAV8/1.3 HBV mouse model and its related mechanism to provide more theoretical basis for the immune target therapy of CHB.Materials and methods:We firstly collected the human specimens to investigate the expression characteristics and clinical significance of immune checkpoints OX40/OX40L,PD-1and 4-1BB in our study.Peripheral blood samples were collected from 64 patients with CHB and 37 healthy volunteers recruited from September 2018 to June 2019 in the First Hospital of Jilin University.CHB patients included 52 patients who were treatment na（？）ve and 12 patients who were receiving entecavir(ETV)while healthy volunteers included 24 adults and 13 children.Liver tissues were collected from 18 patients with CHB and 6 adult patients who had a surgery for a hemangioma.Eighteen patients with CHB included 9 patients who had a disease flared and 9 patients who had a disease not flared.Peripheral blood mononuclear cells(PBMCs)and plasma were isolated from the peripheral blood.Membrane-bound OX40/OX40L(m OX40/OX40L)expressed in PBMCs were detected by flow cytometry;Soluble OX40 and OX40L(s OX40 and s OX40L)in plasma were detected by Elisa;The expression of OX40 and OX40L in liver tissues was detected by IHC.In the following study,the expression characteristics of PD-1 and 4-1BB in CHB patients were studied by using the samples which was used to detect OX40/OX40L expression plus the liver tissue samples of 6 children applied from the Department of Pathology.We isolated the CD4~+and CD8~+T cells from PBMCs by magnetic bead sorting and detected the mRNA levels of PD-1 and 4-1BB in each T cell subpopulation by q RT-PCR.We also measured soluble PD-1 and 4-1BB(s PD-1 and s4-1BB)levels by Elisa in plasma.The expression of PD-1 and 4-1BB in liver tissues was detected by IHC.We related the results with the clinical parameters in patients to explore their clinical significance during CHB infection.Based on the preliminary results from the human specimens,we applied an OX40-activated m Ab to a mouse model of rAAV8/1.3HBV established with adeno-associated virus(AAV)through tail vein injection for the first time.To confirm the effect of activating immune checkpoint OX40 on HBV replication,the levels of hepatitis B surface antigen(HBs Ag)and hepatitis B e antigen(HBe Ag)in serum were detected by automatic electrochemiluminescence analyzer,the levels of HBV DNA were detected by q RT-PCR,HBs Ag and hepatitis B core antigen(HBc Ag)expression in mouse liver tissue was detected by IHC.To clarify liver inflammation caused by the activation of OX40 target,the levels of ALT and AST in the serum of mice were detected by microplate method,and the pathological changes of the liver from the mice were observed by HE staining.In order to observe immunological changes,we isolated infiltrated lymphocytes in the liver of mice and tested the proportion of T cell subsets by flow cytometry.At the same time,we used CBA to measure the Th1,Th2,Th17related cytokines in serum.In order to investigate the mechanism,CD4~+and CD8~+T cells were depleted respectively in rAAV8/1.3HBV mouse model and the virologic levels and serum ALT in the model were detected by previous methods.In order to further study the changes of related mRNA expression from the transcriptome level in the process of activating OX40 target to inhibit HBV,we isolated the lymphocytes in the liver of mice and conducted mRNA-seq.The differentially expressed mRNAs(DEmRNAs)were clustered in the volcanic map and the heatmap,respectively.The enrichment methods including Go、KEGG and Reactome were used to explore the functions and pathways that these DEmRNAs were enriched on.Results:The results showed that the percentage of OX40~+T cells in PBMCs decreased in CHB patients,especially in patients with a high viral load and the disease flare and the percentage of OX40~+T cells was negatively correlated with the serum virus levels.The percentage of OX40L~+B cells and monocytes from PBMCs increased in patients with CHB and the same trend was also evident in the high viral load group and the disease flare group.Besides,the percentage of OX40L~+B cells and monocytes was mainly positively correlated with liver inflammation indicators.We also found that the expression of s OX40/OX40L in plasma of patients with CHB was significantly increased and the trend was more obvious in the high viral load group and the disease flare group.The level of s OX40/OX40L in plasma was related to the clinical parameters including viral replication and liver inflammation.In addition,there was no significant difference in s OX40 before and after ETV treatment.We also found that the expression trend of OX40/OX40L in liver tissue was consistent with that of s OX40/OX40L in plasma.Besides,we found that the percentage of OX40~+cells in total T cells,CD4~+T cells and CD14~+monocytes as well as the levels of s OX40 in plasma were all positively correlated with age in healthy donors.The results of the expression of PD-1 and 4-1BB showed that the PD-1 mRNA levels in both CD4~+T cells and CD8~+T cells in PBMCs from CHB patients were increased compared with the healthy adult group,which was consistent with the expression of PD-1 in the liver tissues.The level of s PD-1 was not significantly different before and after ETV treatment.Besides the levels of s PD-1 in plasma were positively correlated not only with the virological parameters but also with the indicators of liver inflammation.The 4-1BB expression characteristics in CHB patients are roughly similar to PD1.Briefly,the s4-1BB level in plasma of patients with CHB is higher,especially in a high viral load group and disease flare group.The level of s4-1BB was not significantly different before and after ETV treatment.The levels of s4-1BB in plasma were positively correlated only with the virological parameters.The expression trend of 4-1BB in liver tissue was consistent with the s4-1BB in plasma.The level of 4-1BB mRNA in total PBMCs,CD4~+T cells and CD8~+T cells all increased in patients with CHB,but there was no statistical difference.Finally,we observed that there was no significant correlation between the expression of either PD-1 or 4-1BB and age in healthy donors.By analyzing and comparing the clinical significance of the above immune checkpoints,we applied an agonist targeting OX40 in successfully constructed rAAV8/1.3HBV mouse model.The levels of HBs Ag in serum and intrahepatic HBV DNA decreased after activating the OX40 target in the HBV mouse model,and the HBs Ag levels in serum reached its lowest point on day 8 after OX40 agonist administration.Compared with the placebo group,the expression of HBs Ag and HBc Ag in liver tissue also decreased.However,the activation of the OX40 target in HBV mouse model seem to not affect HBV DNA and HBe Ag levels in serum.The inhibition of activating OX40 target on HBV replication was accompanied by the liver inflammation,the levels of ALT and AST in serum increased and the infiltration of inflammatory cells in liver tissue was observed.In addition,the percentage of CD8~+T cells and the level of the Th1,Th2 and Th17 related cytokines were increased.The levels of cytokines all reached the highest values near the time point when serum HBs Ag reached the lowest value.In addition,we found that the administration of OX40agonists induced splenomegaly in mice.We also found that the OX40 agonist applied in this model was dose-dependent,which was mainly reflected in HBs Ag in serum,HBV DNA in liver,the percentage of CD8~+T cells and the extend of splenomegaly.The mechanism of OX40 agonist suppressing HBV was explored.We found that the HBs Ag in serum was higher in CD4~+/CD8~+T cell depleted HBV mouse model compared to the HBV mouse with the intact immune system in day 8 and 12 after administration of OX40 agonist and the levels of HBs Ag in serum increase more obviously in CD8~+T cell depleted HBV mouse.The HBV DNA levels in liver of CD4~+T cell depleted HBV mouse showed no significant difference compared with those in normal immunological group while the HBV DNA level in liver of CD8~+T cell depleted HBV mouse showed a slightly increased trend.The ALT levels in serum were increased in both HBV mouse with intact immune system and CD4~+T cell depleted HBV mouse while the ALT levels in CD8~+T cell depleted HBV mouse have no increasing.Transcriptome sequencing based on mRNA level of the infiltrated lymphocytes in liver of HBV mouse treated with OX40 agonist and placebo showed that 3008 mRNAs were up-regulated and 2269 were down-regulated.After GO function enrichment analysis,we found that the DEmRNAs were mainly enriched on leukocyte differentiation,chromatin and histone modification and regulation in the way of biological processes.In terms of cell composition,the DEmRNAs were mainly enriched on the chromatin,heterochromatin and centrosomes.In terms of molecular functions,the DEmRNAs are mainly enriched on transcription,translation,small molecule GTP-binding,RAS GPT binding and other functions.KEGG pathway enrichment analysis showed that DEmRNAs were mainly enriched on HBV,human T-cell leukemia virus(HTLV),EB virus infection-related pathway,mitogen-activated protein kinase(MAPK),chemokines and NF-Kapa B signaling pathways.The differentially expressed genes analyzed by Reactome pathway enrichment analysis were mainly enriched on neutrophil degranulation,immune system cytokine signal,interleukin signal,transcriptional regulation and other signaling pathways.Conclusions:The expression characteristics of immune checkpoints OX40/OX40L,PD-1 and4-1BB in patients with CHB suggested that these immune checkpoints might be associated with chronic HBV infection.However,only the expression of OX40 was negatively correlated with HBV virology indicators,considering that it was closely related to virus clearance,and only the expression of OX40/OX40L was age-dependent.Therefore,we applied OX40 agonist in rAAV8/1.3 HBV mouse model for the first time,and found that the activation of OX40 target had an inhibitory influence on HBV replication and was a promising immune checkpoint for CHB.However,the activation of OX40 target could not completely clear HBV,so it may need to combine with other therapies in the future.The mechanism of activating OX40 targets to inhibit HBV replication shows that the process is more dependent on CD8~+T cells than on CD4~+T cells.The importance of CD4~+T cells is undeniable,but we should perhaps focus more on CD8~+T cells for immunotherapy of HBV in the future. |
