Immunomodulatory Effect Of IL-21 In Chronic HBV Infection

BackgroundThe eradication or persistent of hepatitis B virus (HBV) infection was mainly dependent on the innate and adaptive immune function of individuals. As an organ with advantage of innate immune response, the liver contains a large amount of innate immune cells, such as natural killer (NK) cells. As an important member of innate immune system, NK cells exert antiviral effect through the production of interferon-gamma (IFN-y) and TNF alpha (TNF-a), meanwhile, NK cells play essential role in the initiation and modulation of adaptive immune response. Previous studies have shown that activation of adaptive immune response, especially antigen specific cytotoxic T lymphocyte (CTL) response, plays an important role in controlling HBV infection through the secretion of IFN-y and TNF-a, which act in a non-cytolytic mechanism with the degradation of viral replication intermediates, transcripts. The host immune function against the virus affects not only the outcome of HBV infection in natural history, but also influences the treatment response to antiviral therapy, especially the achievement of HBeAg seroconversion. HBeAg seroconversion, defined as loss of HBeAg with the appearance of HBeAb, is an important event during the natural course of chronic HBV infection and antiviral treatment. In chronic hepatitis B (CHB) patients with HBeAg positive, HBeAg seroconversion is usually followed by sustained HBV DNA suppression and clinical remission of necroinflammatory activity. Meanwhile, it is also associated with a lower incidence of cirrhosis and hepatocellular carcinoma (HCC) and a higher probability of HBsAg loss and seroconversion. Based on evidence supporting HBeAg seroconversion as a marker of durable remission, current guidelines issued by major liver associations have also adopted HBeAg seroconversion as an appropriate treatment end point for HBeAg-positive CHB patients.The effector cells exert effective function with the aid of helper T (Th) cells. CD4+T cells are best known for their ability to maintain B cell and CD8+T cell responses. As aforementioned, HBeAg seroconversion represents the specific immune response against HBV antigen. Since HBeAg is a T cell dependent antigen, helper CD4+T cells was definitely participate the procedure of the production of HBeAb from B cells. Although the primary CD8+T cell response against pathogen infection can work properly independent of CD4+T cell help, recent studies have indicated that CD4+T cell help is required for the generation of long-lived, functional memory CD8+T cell that respond rapidly upon secondary exposure to pathogen. The regulation of humoral and cellular immune response by CD4+T cells was conducted mainly through the secretion of cytokines. During the course of HBeAg seroconversion in CHB patients with antiviral therapy, many important changes of cytokines have been observed. A substantial increase of bioactive interleukin (IL)-12 and Thl cytokines has been associated with HBeAg seroconversion in HBeAg-positive CHB patients treated with IFN-α. Importantly, the peak level of IL-12 occurred either before or simultaneously with HBeAg seroconversion. Results from the study of the natural history of chronic HBV infection have shown that higher levels of serum IL-12 were associated with early, spontaneous HBeAg seroconversion in HBeAg-positive CHB patients. These studies have provided some clues that, apart from the decisive role in initiating and shaping immune-related pathologic responses to chronic viral infection, cytokines have also been linked to HBeAg seroconversion.CD4+T cell can be classified into Th1, Th2, Th9, Th17, Th22, and Treg subsets according to secreted cytokines and relevant function. Th1 subsets mainly secret IL-2, IFN-γ, and TNF-α, which mediated cellular immune response. Previous studies have suggested that Th2 subtype provide help for B cell proliferation and differentiation into plasma cells predominantly through the role of IL-4, IL-6, and IL-10. However, recent studies have found special helper T cells which resided at lymphoid follicle, named follicular helper T cells (Tfh), can directly provide help for B cells to participate in the humoral immune in germinal center. Tfh cells predominantly secrete IL-21, which has more potent ability to promote B cells differentiate into plasma cells when compared with other Th2 cytokines. In addition, recent studies have shown that CD4+T cell "help" for CD8+T cell responses involves IL-21 as an intermediary. Therefore, during chronic viral infection, IL-21 secreted from CD4+T cells plays an important role in promoting B cell function and maintaining CTL response.IL-21 is a member of a large family of cytokines (IL-2, IL-4, IL-7, IL-9, and IL-15) whose receptors share a common receptor y chain (CD132). This cytokine is produced by activated CD4+T cells, in particular follicular T helper (Tfh) cells, Th17 cells, and activated NKT cells. The effects of IL-21 are pleiotropic because of the broad cellular distribution of IL-21 receptor (IL-21R), especially on B cells. Previous cross sectional data from our group have shown that the inactive carrier (IC) patients, characterized by seropositivity of HBeAb and low or undetectable levels of HBV DNA, showed high levels of serum IL-21 as well as higher frequency of IL-21 secreting CD4+T cell. In combination with substantial data regarding the role of IL-21 in other chronic viral infection, such as LCMV and HIV, we hypothesize that IL-21 could modulate the innate and adaptive immune response through the action with different cell subtypes, which plays an important role in promoting the production of HBV specific antibodies and virus elimination during chronic HBV infection.ObjectiveCross sectional cohort of chronic HBV infection in natural history and longitudinal cohort of CHB patients with antiviral treatment were enrolled in this study. We aimed to analyze the characteristics of IL-21 in chronic HBV infection, explore the effect of IL-21 on B cells, CD8+T lymphocytes, and NK cell, and clarify its role in promoting HBeAg seroconversion and virus elimination. In addition, mouse models carrying HBV were established by injection of engineered HBV expression plasmid into tail veins of C57BL/6 mice to further research the role of IL-21 in HBV infection in vivo. This study could provide the theoretical foundation for individualized treatment based on immunology index, and also provide new clues and strategies for immune therapy in HBV infection.MethodsOne hundred and thirty patients with chronic HBV infection were recruited for the cross-sectional study. They were classified into immune tolerant carrier (IT, n=20), HBeAg positive CHB (n=75), and inactive carrier (IC, n=35) groups. Fifty-five healthy controls (HC) were also enrolled. One hundred and eight patients with HBeAg-positive CHB who participated in independent clinical trial of telbivudine were studied longitudinally. Serum and heparinized blood were collected at 0,12,24, and 52 weeks after starting telbivudine treatment. The subjects were classified into either a complete response (CR) group, if they had undergone HBeAg seroconversion and achieved serum HBV-DNA level less than 2.5 log10 copies/mL by week 52, or a non-complete response (NCR) group, if serum HBV-DNA was reduced but HBeAg remained positivity. All patients in both groups achieved normal alanine aminotransferase (ALT) levels by week 52. In addition, according to HBV DNA levels at week 52, the subjects were classified into virological response group (VR, HBV DNA<2.5 log10 copies/mL) and non-virological response (NVR, HBV DNA>2.5 log10 copies/mL).1. The characteristics of IL-21 in HBV infection1.1 Serum levels of IL-2, IL-4, IL-6, IL-10, TNF-a, and IFN-y were detected using cytometric bead array (CBA), and serum levels of IL-21 was measured using enzyme-linked immunosorbent assay (ELISA) in longitudinal cohort. Serum levels of cytokines at different time point after antiviral therapy were compared between CR and NCR group;1.2 For patients with chronic HBV infection and HCs, the serum levels of IL-21 and transcriptional level of IL-21 in PBMC were quantified using ELISA and quantitative PCR; The expression of IL-21 in liver tissue was detected by immunohistochemistry;1.3 The secretion of IL-21 from Thl, Th2, Th17, and Tfh subsets in HBeAg positive CHB patients were detected using intracellular cytokine staining (ICS), which was conducted with a BD FACSCanto II flow cytometer platform.2. The effect of CXCR5+CD4+T cell, IL-21, and B cell on HBeAg seroconversion2.1 Spleen tissues were mechanically dispersed and lymphocytes were isolated by Ficoll-Hypaque density gradient centrifugation. PBMCs and spleen-derived lymphocytes were stained with fluorescence antibodies at room temperature for 20 minutes and analyzed on a BD FACSCanto II flow cytometer;2.2 Intracellular cytokine staining after stimulation with PMA/ionomycin was performed. To determine the frequency of HBV-specific IL-21 producing CXCR5+CD4+T cells, thawed PBMCs were cultured with or without HBV peptides for 72 hours, and brefeldin A (BFA) was added for the last 12 hours of culture. A response was considered positive if the percentage of IL-21-producing CXCR5+CD4+T cells exceeded that of medium-only control (background) by 0.35% and was at least 2-fold above the background;2.3 Circulating T cells (CXCR5+CD4+ or CXCR5-CD4+) and autologous CD19+B cells were sorted from either CR or NCR patients by BD InfluxTM cell sorter. HBV-specific antibody production was assessed using ELISPOT assay;2.4 The concentration of IL-21 was quantitated in duplicate wells using a commercial human IL-21 ELISA kit in accordance with the manufacturer’s instructions;2.5 Fresh PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and resuspended in the medium; the labeled cells were cultured with rHBeAg, rHBcAg or rHCV for 7 days, or with medium only as a negative control. At the end of culture, cells were harvested and stained. The proliferation rate of CXCR5+CD4+T cells was expressed as the percentage of cells that diluted the CFSE intensity at least once at the time of harvest.3. IL-21 modulate the function of CD8+T cell in HBV infection3.1 Peripheral blood CD8+T cells were sorted by magnetic beads from patients with chronic HBV infection, HCs, and CHB patients with telbivudine therapy at baseline, week 12, and week 24. IL-21R mRNA levels of peripheral blood CD8+T cells were detected by quantitative PCR;3.2 PBMC from CHB patients was stimulated with different concentrations of IL-21 in vitro. The frequency of CD8+T cells, surface markers, such as CD69, CD45RO, and CCR7, and the expression levels of granzyme B, perforin, and IFN-γ were detected using FACS;3.3 PBMC or CFSE labeled PBMC from HLA-A2 positivity CHB patients were stimulated with HBV peptide and different concentrations of IL-21 in vitro, proliferation of CD8+T cells were measured. The frequency of HBV-specific CD8+T cells were detected by the method of MHC-I Streptamer; the surface markers (CD69, CD45RO, and CCR7) of HBV-specific CD8+T cells were detected using FACS, and the expression levels of granzyme B, perforin, and IFN-γ from HBV-specific CD8+T cells were detected using ICS.4. IL-21 modulate the function of NK cells in HBV infection4.1 Phenotypes of peripheral blood NK cells (CD16, NKG2A, NKG2D, and NKp46) were detected; ICS was used to detect IFN-y secretion from NK cells with stimulation of recombinant (r) IL-12, IL-15, and IL-18; For ADCC detection, PBMCs were co-cultured with targeted cells, HBV core 18-27 pulsed T2 cells with HLA-A2 positivity, then IFN-γ secretion was conducted by ICS with stimulation of IL-12, IL-15, and IL-18;4.2 Purified NK cells from CHB patients was stimulated with IL-21 alone or with the combination of IL-12, IL-15, and IL-18 for 72 hours, different phenotypes of NK cells (CD 16, NKG2A, NKG2D, and NKp46) and IFN-γ secretion were detected using FACS; To explore the role of IL-21 on ADCC effect, purified NK cells were co-cultured with targeted T2 cells, then IFN-γ secretion was detected by ICS with stimulation of IL-12, IL-15, and IL-18 or with the combination of IL-21;4.3 Fresh purified NK cells were labeled with CFSE and resuspended in the medium; the labeled cells were cultured with IL-21 or with combination of IL-12, IL-15, or IL-18 for 7 days. At the end of culture, cells were harvested and detected;4.4 Purified NK cells were stimulated with IL-21 or with combination of IL-12, IL-15, and IL-18, ICS was conducted for the detection of expression of phosphorylated STAT3 and STAT4.5. The study of IL-21 in HBV expression plasmid carrying mouse models5.1 Plasmid pSM2 (10μg) was hydro-dynamically injected by tail vein into wild type C57BL/6 mice and IL-21 R gene knockout mice, and plasmid pUC19 (10μg) was also injected to the wild type C57BL/6 mice as controls. Peripheral blood was collected by retroorbital puncture at each timepoint after injection and serum was collected for detection of HBsAg, HBsAb, and IL-21. Peripheral blood and intrahepatic lymphocytes were separated for FACS analysis; In addition, liver tissues were collected for HE staining, immunohistochemical analysis, and detection of mRNA levels of IL-21;5.2 rAAV8-1.3HBV was injected by tail vein into wild type C57BL/6 mice. Blood was collected by retroorbital puncture at each time point after injection and serum was collected for the detection of HBsAg. After 12 weeks, mice were classified according to HBsAg level. The experimental group was treated with injection of murine IL-21-GFP recombinant adenovirus by tail vein, while mice administrated with GFP recombinant adenovirus or PBS as control groups. Peripheral blood was collected by retroorbital puncture at day 1,4,7,10, and 13 after injection and serum was stored for detection of HBsAg, HBsAb, and IL-21.6. Statistical analysisData are expressed as mean+standard deviation (mean±SD) or interquartile range. The Mann-Whitney U test, the Wilcoxcon signed-rank test and the Chi-square test were used when 2 groups were compared. The Kruskal-Wallis H test was used when more than 2 groups were compared. Correlations between variables were assessed with the Spearman’s rank order correlation coefficient. All statistical analyses were based on a two-tailed hypothesis tests with a significance level of P< 0.05.Results1. The characteristics of IL-21 in HBV infection1.1 Compared with HCs, serum levels and mRNA levels of IL-21 in PBMC were significantly higher in chronic HBV infection patients (P<0.001; P<0.001, respectively); IHC staining reflected an accumulation of IL-21 expressing cells in livers of patients with chronic HBV infection. Although IL-21 can be secreted by Thl, Th2, Th17, and Tfh subsets, it was predominantly secreted from Thl and Tfh subsets.1.2 Serum levels of TNF-a, IL-2, IL-6, IL-10, IL17A, and IL-21 in 48 HBeAg positive CHB patients were progressively decreased during the course of antiviral treatment. Compared with baseline, serum levels of aforementioned cytokine at week 52 were significantly lower (P<0.001; P=0.002; P<0.001; P<0.001; P=0.006; P=0.007; respectively), while no significant changes can be observed for IL-4 and IFN-y. The serum levels of IL-21 at week 12 in CR patients were significantly higher than that of NCR patients (P=0.022).2. The effect of CXCR5+CD4+T cells, IL-21, and B cells on HBeAg serocon\ersion2.1 A significantly higher frequency of CXCR5+CD4+T cells was observed in patients with chronic HBV infection relative to the HC group (P<0.001). To illustrate the presence of HBV-specific cells in the overall increased CXCR5+CD4+T population in chronic HBV infection, we examined IL-21 production by these cells in response to HBV peptide stimulation. Although only a small fraction of CXCR5+CD4+T cells could secret IL-21, their representation was significantly higher in the chronic HBV infection than that in the HC group (P<0.001). Likewise, a small but definite fraction of CXCR5+CD4+T cells from the chronic HBV infection patients proliferated in the presence of either rHBeAg or rHBcAg relative to negative controls (P<0.001);2.2 Cross-sectional data showed that the frequency of CXCR5+CD4+T cells in the IC group who had achieved HBeAg seroconversion was significantly higher than the IT (P<0.001) and CHB (P<0.001) groups. Longitudinal data showed that the frequency of CXCR5+CD4+T cells in the CR group was significantly higher than the NCR group (P=0.009) during 52 weeks of telbivudine therapy. An increase in the frequency of CXCR5+CD4+T cells at week 12 relative to week 0, which was defined as "increasing pattern", was found in the majority (14 of 16) of CR patients but in only half (13 of 26) of the NCR patients. The difference was statistically significant (P=0.014); In addition, the change in frequency of CXCR5+CD4+T cells between week 12 and week 0 was negatively correlated with the change in concentration of serum HBeAg between week 12 and week 0 (r=-0.358, P=0.020);2.3 Intracellular cytokine staining was performed to examine the profile of cytokine production by CXCR5+CD4+T cells from patients with chronic HBV infection or HCs following stimulation with PMA/ionomycin or HBV peptides. Among CXCR5+CD4+T cells, PMA/ionomycin stimulation generated more IL-21-producing cells than IL-17-, IL-4-or IFN-y-secreting cells (P<0.001). This is in contrast to the CXCR5-CD4+T cell population which predominantly contained IFN-y secreting cells. In the cross-sectional study, there was a higher frequency of IL-21+CXCR5+CD4+T cells in the IC than IT or CHB groups after stimulation with either PMA/ionomycin or HBV peptides. More importantly, a significantly higher frequency of HBV-peptide-stimulated IL-21+CXCR5+CD4+T cells was detected in the CR group than NCR group after 24 weeks (P=0.005) or 52 weeks (P=0.002) of antiviral treatment;2.4 The capacity of CXCR5+CD4+T cells to promote antibody production by autologous B cells in response to HBV-specific antigens was investigated by ELISPOT assay in both CR and NCR groups. Given that the frequency of HBV-specific antibodies producing B cells was rather low, pokeweed mitogen was included in the final stage to boost antibody production after 5 days incubation with HBV antigens alone. Data showed that co-culture of autologous B cells with CXCR5+CD4+T cells resulted in significantly higher frequencies of both HBeAb-and HBcAb-secreting B cells than co-culture with CXCR5"CD4+T cells in most settings. Most remarkably, the frequency of HBeAb-secreting B cells in co-culture of CXCR5+CD4+T cells and B cells from CR patients was significantly higher than that from NCR patients (P=0.011). The concentration of IL-21 in the supernatant of the co-culture was quantitated by ELISA. There was a significantly higher level of IL-21 in the co-culture with CXCR5+CD4+T cells than in the co-culture with CXCR5-CD4+T cells after stimulation with rHBeAg in both CR (P=0.007) and NCR (P=0.013) groups. There was also a trend of elevated levels of IL-21 in co-culture of CXCR5+CD4+T cells and B cells from subjects in the CR relative to the NCR group (P=0.075). Blockade of IL-21 activity in the co-culture with soluble rIL-21R-Fc resulted in suppression of HBeAb production (P=0.027). In contrast, addition of rIL-21 to the co-culture led to an enhancement of HBeAb production (P=0.043);2.5 Although in vitro naive CD4+T cells from either patients with chronic HBV infection or HCs can differentiation into CXCR5+CD4+T cells in proper settings, the ability of the two groups showed no significant difference, and it can also be observed in CR and NCR groups;2.6 To investigate how closely the circulating CXCR5+CD4+T cells resemble Tfh cells present in lymphoid tissues, the expression of additional markers typically associated with Tfh cells were measured in circulating CXCR5+CD4+and CXCR5’CD4+T cells. Significantly higher percentages of ICOS-, PD-1-and IL-21-expressing cells were detected in the CXCR5+CD4+T cell population relative to the CXCR5"CD4+T cell population in patients with CHB (P<0.001). Next, the phenotypes of circulating and spleen-derived CXCR5+CD4+T cells from patients who underwent splenectomy due to HBV-related liver cirrhosis induced hypersplenism were directly compared. While they both expressed high levels of CD45RO, the circulating CXCR5+CD4+T cells had relatively lower expression of ICO S and PD-1. Moreover, the two populations showed differential expression of CCR7 and CD69, consistent with their different anatomical positioning. Nevertheless, the frequency of circulating CXCR5+CD4+T cells was found to be positively correlated with that of splenic ICOS+PD-1+CXCR5+CD4+T cells (r=0.732, P=0.018).3. IL-21 modulate the function of CD8T cell in HBV infection3.1 IL-21 R mRNA levels of peripheral blood CD8+T cells in patients with chronic HBV infection was significantly higher than that of HCs (P<0.001). IL-21R mRNA levels of peripheral blood CD8+T cells at week 12 in CHB patients of VR group were significantly lower than that of baseline (P=0.017). In NVR group, IL-21R mRNA levels at week 12 showed an increased trend, but no statistical difference was achieved when compared with baseline. However, it was significantly higher than that of VR group (P=0.007);3.2 IL-21 in vitro can significantly enhance the frequency of either total CD8+T cells or HBV core specific CD8+T cells in PBMC from CHB patients without antiviral treatment (P=0.008; P=0.028, respectively);3.3 IL-21 in vitro can significantly promote the expression of CD69 in CD8+T cells from CHB patients (P=0.035), and also enhance the secretion of granzyme B and perforin from total CD8+T cells of CHB patients (P<0.001). However, IL-21 (50ng/mL) only promotes the secretion of granzyme B from HBV specific CD8+T cells of CHB patients.4. IL-21 modulate the function of NK cells in HBV infection4.1 Compared with HCs, the frequency of NKG2A or NKp46 expressing NK cells was significantly increased in patients with CHB (P<0.001; P=0.001, respectively), while the frequency of CD 16 and NKG2D expressing NK cells was significantly reduced. The ability of IFN-y secretion and ADCC mediated by NK cells were significantly impaired in CHB patients (P=0.017; P<0.001, respectively);4.2 The frequency of CD 16, NKp46 or NKG2D expressing NK cells was significantly increased in patients with CHB when stimulated with rIL-21 in vitro (P<0.001). With combined stimulation of IL-12, IL-15, and IL-18, IL-21 can significantly increase IFN-y secretion and this effect was in a time dependent manner. Although IL-21 alone stimulation can enhance IFN-y expression, the effect was quite weak. However, in the context of combined stimulation of IL-12, IL-15, and IL-18, addition of IL-21 can significantly increase IFN-y secretion;4.3 In vitro IL-21 can significantly enhance the ability of NK cell mediated ADCC function and promote NK cell proliferation. Additionally; IL-21 predominantly promotes high levels of phosphorylated STAT3 and STAT4.5. The study of IL-21 in HBV expression plasmid carrying mouse models5.1 Wild type C57BL/6 mice with hydro-dynamic injection of plasmid pSM2 have significant higher level of IL-21 in peripheral blood and liver than that of control group (P<0.05), and higher frequency of CXCR5+CD4+T cells and CD19+B cells can also be detected in pSM2 wild type mice (P<0.05);5.2 Serum HBsAg of IL-21R gene knockout mice injected with plasmid pSM2 remained positivity at day 10,13, and 16 after administration. HBsAg levels were significantly higher than that of wild type mice treated with plasmid pSM2 (P=0.003; P=0.002; P=0.01, respectively), while HBsAb was undetectable in all the IL-21R gene knockout mice;5.3 Injection of murine IL-21-GFP recombinant adenovirus by tail vein can accelerate HBsAg clearance and promote HBcAb production in HBV chronic infection mice established by administration of rAAV8-1.3HBV.Conclusions1. Serum levels of IL-21 at week 12 in HBeAg positive patients with CHB with telbivudine treatment can predict HBeAg seroconversion at week 52, suggesting that serum levels of IL-21 may act as an immunological marker for evaluating treatment response;2. High frequency of circulating CXCR5+CD4+T cells may promote HBeAg seroconversion in patients with chronic HBV infection and IL-21 produced by CXCR5+CD4+T cells may represent an important mediator of this effect. Therapy that targets expansion of CXCR5+CD4+T cells or IL-21 release may be beneficial for the treatment of chronic HBV infection;3. IL-21 in vitro can promote the activation of CD8+T cells from CHB patients, and also enhance the cytotoxic activity of non-HBV specific CD8+T cells, implying the effect of IL-21 on liver inflammation and injury;4. NK cells function was impaired in chronic HBV infection. IL-21 in vitro could significantly promote IFN-y secretion and ADCC function of NK cells in patients with CHB, thus enhancing NK cell mediated HBV clearance. Immunotherapeutic strategy that targeting restoration of NK cells capacities maybe contribute to treatment in CHB patients;5. IL-21 may play an important role in HBsAg clearance and HBV specific antibody production in HBV expression plasmid carrying mouse model.