Yongyin Li

High serum IL-21 levels after 12 weeks of antiviral therapy predict HBeAg seroconversion in chronic hepatitis B

BACKGROUND & AIMS Interleukin-21 (IL-21) stimulates T cell and B cell responses and plays a role in control of chronic viral infections. The role of IL-21 in chronic hepatitis B virus (HBV) infection is not understood. METHODS Serum IL-21 levels were measured by enzyme immunoassay in 75 HBeAg-positive chronic hepatitis B (CHB) patients undergoing telbivudine treatment. The findings were validated in 103 patients from a separate clinical trial of telbivudine. A complete response to telbivudine was defined as having both HBeAg seroconversion and serum HBV-DNA level <300 copies/ml by treatment week 52. The proportions of T-cells producing IL-21 and/or expressing programmed death 1 (PD-1) in peripheral blood mononuclear cells were assessed longitudinally during treatment by intracellular cytokine staining and flow cytometry. RESULTS Median serum IL-21 levels at treatment week 12 were significantly higher in patients who did achieve vs. patients who did not achieve a complete response in both the initial (128.4 vs. 69.2 pg/ml, p=0.003) and the validation (142.2 vs. 89.9 pg/ml, p=0.004) trials. Serum levels of IL-21 (p=0.005) or HBV-DNA (p=0.003) levels at treatment week 12 independently predicted HBeAg seroconversion in the first year of treatment. The decrease in PD-1 expression on CD4(+) and CD8(+) T cells during the first 12 weeks on telbivudine treatment was not correlated with changes in IL-21 concentrations. CONCLUSIONS Serum IL-21 levels may be a biomarker for HBeAg seroconversion, and may contribute to individualization of antiviral therapy in HBeAg-positive CHB. IL-21 may also have a role in immunotherapy for CHB.

Circulating chemokine (C‐X‐C Motif) receptor 5+CD4+ T cells benefit hepatitis B e antigen seroconversion through IL‐21 in patients with chronic hepatitis B virus infection

Given the clinical significance of hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B virus (HBV) infection, it is critical to elucidate the mechanisms regulating this process. In the present study, we found that the frequency of circulating chemokine (C‐X‐C motif) receptor 5 (CXCR5)+CD4+ T cells was higher in patients who had achieved HBeAg seroconversion in both cross‐sectional (P < 0.001) and longitudinal (P = 0.009) studies. These cells were able to produce a significantly higher level of intracellular interleukin 21 (IL‐21) after stimulation with HBV peptides in patients with telbivudine‐induced HBeAg seroconversion (P = 0.007). Furthermore, sorted CXCR5+CD4+ T cells from HBeAg seroconverters boosted a higher frequency of antibody against hepatitis B e antigen (anti‐HBe)‐secreting B cells in coculture assay (P = 0.011). Of note, the increase in frequency of anti‐HBe‐secreting B cells was abrogated by soluble recombinant IL‐21 receptor‐Fc chimera (P = 0.027), whereas exogenous recombinant IL‐21 enhanced this effect (P = 0.043). Additionally, circulating CXCR5+CD4+ T cells shared similar phenotypic markers, and were positively correlated in frequency with, splenic follicular T helper cells. Conclusion: Circulating CXCR5+CD4+ T cells, by producing IL‐21, may have a significant role in facilitating HBeAg seroconversion in patients with chronic HBV infection. (Hepatology 2013;58:1277–1286)

Restored Circulating Invariant NKT Cells Are Associated with Viral Control in Patients with Chronic Hepatitis B

Invariant NKT (iNKT) cells are involved in the pathogenesis of various infectious diseases. However, their role in hepatitis B virus (HBV) infection is not fully understood, especially in human species. In this study, 35 chronic hepatitis B (CHB) patients, 25 inactive carriers (IC) and 36 healthy controls (HC) were enrolled and the proportions of circulating iNKT cells in fresh isolated peripheral blood mononuclear cells (PBMC) were detected by flow cytometry. A longitudinal analysis was also conducted in 19 CHB patients who received antiviral therapy with telbivudine. Thereafter, the immune functions of iNKT cells were evaluated by cytokine secretion and a two-chamber technique. The median frequency of circulating iNKT cells in CHB patients (0.13%) was lower than that in HC (0.24%, P = 0.01) and IC (0.19%, P = 0.02), and increased significantly during antiviral therapy with telbivudine (P = 0.0176). The expressions of CC chemokine receptor 5 (CCR5) and CCR6 were dramatically higher on iNKT cells (82.83%±9.87%, 67.67%±16.83% respectively) than on conventional T cells (30.5%±5.65%, 14.02%±5.92%, both P<0.001) in CHB patients. Furthermore, iNKT cells could migrate toward the CC chemokine ligand 5. Patients with a high ratio (≥1.0) of CD4−/CD4+ iNKT cells at baseline had a higher rate (58.33%) of HBeAg seroconversion than those with a low ratio (<1.0, 0%, P = 0.0174). In conclusion, there is a low frequency of peripheral iNKT cells in CHB patients, which increases to normal levels with viral control. The ratio of CD4−/CD4+ iNKT cells at baseline may be a useful predictor for HBeAg seroconversion in CHB patients on telbivudine therapy.

Chemokine (C‐X‐C motif) ligand 13 promotes intrahepatic chemokine (C‐X‐C motif) receptor 5+ lymphocyte homing and aberrant B‐cell immune responses in primary biliary cirrhosis

The serologic hallmark of primary biliary cirrhosis (PBC) is the presence of high titer, and specific anti-mitochondrial antibodies (AMA). Interestingly, although there is no global immune defect in patients with PBC, there is widespread dysregulated B cell function, including increased sera levels of IgM and enhanced B cell responses to CpG stimulation. The mechanisms involved in this B cell dysfunction have remained unknown. To address this issue, we focused on identifying the frequencies of B cell subsets in patients with PBC and the mechanisms that lead to B cell dysregulation, including the relationships with CXCR5+CD4+T cells. Herein we report that elevations of both serum and intrahepatic IL-21 were found in patients with PBC and, in particular, promoted B cell proliferation, STAT3 phosphorylation and AMA production in vitro. More importantly, upon stimulation with rPDC-E2, CXCR5+CD4+T cells in PBC produced higher levels of IL-21 than healthy controls. Additionally, sorted CXCR5+CD4+T cells increased production of AMA by autologous CD19+B cells. Indeed, elevated expression of intrahepatic CXCL13, a key chemokine of CXCR5+ cells, was uniquely found within the portal tracts in PBC, accompanied by infiltrates of CD4+, CXCR5+, CD19+, and CD38+ cells. In conclusion, CXCL13 promotes aggregation of CD19+B cells and CXCR5+CD4+T cells, which directs the aberrant AMA response via IL-21. These data have implications for potential immunotherapy and also reflect the unique lymphoid biology in the liver of PBC.The serological hallmark of primary biliary cirrhosis (PBC) is the presence of high titer and specific antimitochondrial antibodies (AMAs). Although there is no global immune defect in patients with PBC, there is widespread dysregulated B‐cell function, including increased sera levels of immunoglobulin M and enhanced B‐cell responses to cytosine‐phosphate‐guanine stimulation. The mechanisms involved in this B‐cell dysfunction have remained unknown. To address this issue, we focused on identifying the frequencies of B‐cell subsets in patients with PBC and the mechanisms that lead to B‐cell dysregulation, including the relationships with chemokine (C‐X‐C motif) receptor 5 (CXCR5)+CD4+T cells. Herein, we report that elevations of both serum and intrahepatic interleukin‐21 (IL‐21) were found in patients with PBC and, in particular, promoted B‐cell proliferation, signal transducer and activator of transcription 3 phosphorylation and AMA production in vitro. More important, upon stimulation with recombinant E2 subunit of pyruvate dehydrogenase complex, CXCR5+CD4+T cells in PBC produced higher levels of IL‐21 than healthy controls. Additionally, sorted CXCR5+CD4+T cells increased production of AMAs by autologous CD19+B cells. Indeed, elevated expression of intrahepatic chemokine (C‐X‐C motif) ligand 13 (CXCL13), a key chemokine of CXCR5+ cells, was uniquely found within the portal tracts in PBC, accompanied by infiltrates of CD4+, CXCR5+, CD19+, and CD38+ cells. Conclusion: CXCL13 promotes aggregation of CD19+B cells and CXCR5+CD4+T cells, which directs the aberrant AMA response by IL‐21. These data have implications for potential immunotherapy and also reflect the unique lymphoid biology in liver of PBC. (Hepatology 2015;61:1998‐2007)

Complementarity-Determining Region 3 Size Spectratypes of T Cell Receptor β Chains in CD8+ T Cells following Antiviral Treatment of Chronic Hepatitis B

ABSTRACT An increased CD8+ T cell response to hepatitis B virus (HBV) peptides occurs between 12 and 24 weeks after starting antiviral therapy for chronic hepatitis B. It is not known whether these cells have antiviral function. The aim of this study was to determine whether clonal expansions of CD8+ T cells at these time points predict the virological response to therapy. Peripheral blood CD8+ T cells were obtained from 20 patients treated with lamivudine or telbivudine for chronic hepatitis B at baseline, 12 weeks, and 24 weeks. The CDR3 spectratype of each T cell receptor (TCR) β chain variable region (Vβ) gene family was analyzed, and the changes in the numbers of Vβ families with clonal expansions were compared in subjects with (n = 12) and without (n = 8) a virological response (52 week HBV DNA < 300 copies/ml). The number of CD8+ TCR Vβ families with clonal expansions at 12 weeks relative to baseline (median [10th to 90th percentile], +2.5 [0 to +7] versus +1 [0 to +2], P = 0.03) and at 24 weeks relative to 12 weeks (+1 [0 to +2] versus −1 [−3 to +4], P = 0.006) was higher in subjects with a virological response versus subjects without a virological response, as were interleukin-2 (IL-2) but not IL-21 mRNA levels in peripheral blood mononuclear cells. The duration of new expansions at 12 weeks was higher (P < 0.0001) in responders. Increased numbers of CD8+ T cell expansions after antiviral therapy are associated with a virological response to treatment. These CD8+ T cells are a potential target for a therapeutic vaccine for chronic hepatitis B.

Role of interleukin-21 in HBV infection: friend or foe?

Interleukin (IL)-21, a cytokine produced by activated CD4+ T cells, has broad pleiotropic actions that affect the functions of a variety of lymphoid cells. The roles of IL-21 in modulating immunity to infections are currently being defined. Notably, IL-21-mediated cellular and humoral immune responses play an important role in determining the outcome of viral infection. This article reviews the current knowledge on the critical role of IL-21 in hepatitis B virus (HBV) infection. As a competent intermediary, IL-21 derived from virus-specific CD4+ T cells plays key roles in sustaining CD8+ T cells and promoting B-cell responses that are essential for effective viral control. However, as a mediator of inflammation, IL-21 is also involved in the development of HBV-induced liver cirrhosis and exacerbating liver injury. Overall, the current data point to IL-21 as an immunomodulatory cytokine in HBV infection. Immunotherapeutic strategies aimed at optimizing the beneficial effects of IL-21 in HBV infection may prove to be a rigorous challenge in the future, as they should foster the strengths of IL-21 while circumventing potential drawbacks.Cellular & Molecular Immunology advance online publication, 3 November 2014; doi:10.1038/cmi.2014.109

Presence of valine at position 27 of the hepatitis B virus core gene is associated with severe liver inflammation in Chinese patients.

Although it is widely believed that cytotoxic T lymphocytes (CTL) are responsible for severe flares of chronic hepatitis B that lead to liver failure, the published evidence to support this hypothesis is weak. The frequency of the I27V mutation in the HBV core gene, which produces a core 18–27 peptide capable of binding HLA‐A*02, was compared in Chinese patients with severe liver inflammation (n = 77, including 39 with acute‐on‐chronic liver failure), moderate liver inflammation (n = 44) and inactive disease (n = 45). The frequency with which V27 reverted to the wild‐type I27 was compared in severe liver inflammation patients who were either HLA‐A*02 positive (n = 5) or negative (n = 5). The frequency of patients with a V27 positive HBV was higher in severe than in moderate liver inflammation (23.4% vs. 6.8%, P = 0.02) or inactive disease (23.4% vs. 4.7%, P = 0.006). After a minimum of 3 months follow‐up, the frequency of reversion of V27 to the wild‐type I27 was higher in HLA‐A*02 positive than negative patients (5/5 vs. 1/5, P = 0.05). In summary, this is the first data showing an association between a specific amino acid mutation (I27V) and severe liver inflammation in patients with chronic hepatitis B. This mutation would produce a peptide that is known to bind HLA‐A*02 and stimulate CTL. The high frequency of reversion to wild‐type I27 in HLA‐A*02 positive subjects suggests that CTL recognizing this peptide exist, and is consistent with the possibility that they contribute to the pathophysiology of severe liver inflammation in chronic hepatitis B. J. Med. Virol. 83:218–224, 2011.

Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy

C–X–C-chemokine ligand 13 (CXCL13), the ligand for C–X–C chemokine receptor type 5 (CXCR5), is a major regulator of B-cell trafficking and plays an integral role in age-dependent clearance of hepatitis B virus (HBV) in the mouse model. However, the expression and function of CXCL13 in patients with chronic hepatitis B (CHB) remain unknown. By use of liver cell subpopulations isolated from CHB patients, we found that CXCL13 mRNA was abundantly expressed in Kupffer cells (KCs), but not in primary hepatocytes, liver sinusoidal endothelial cells, and hepatic stellate cells. Interestingly, KC isolated from HBV-positive liver had much higher level of CXCL13 expression than non-HBV-infected controls. And its expression was induced by toll-like receptor 3 ligand poly I:C stimulation. Moreover, intense expression of CXCL13 protein and accumulation of CD4+ T and B cells were evident in follicular-like structures in the liver tissue of CHB patients, which indicated its chemotactic effect on CXCR5+ CD4+ cells and B cells. Consistently, the levels of serum CXCL13 were significantly higher in the CHB patients than in healthy controls. Furthermore, CXCL13 concentration was increased in the complete response (CR) group during weeks 0–12 and did not change significantly during the course of telbivudine treatment, compared with the patients who didn’t achieve CR. In conclusion, the HBV-related increase of CXCL13 production in KC and serum CXCL13 level during telbivudine treatment might be associated with immune control of chronic HBV infection.

TCRγδ+CD4−CD8− T Cells Suppress the CD8+ T-Cell Response to Hepatitis B Virus Peptides, and Are Associated with Viral Control in Chronic Hepatitis B

The immune mechanisms underlying failure to achieve hepatitis B e antigen (HBeAg) seroconversion associated with viral control in chronic hepatitis B (CHB) remain unclear. Here we investigated the role of CD4−CD8− T (double-negative T; DNT) cells including TCRαβ+ DNT (αβ DNT) and TCRγδ+ DNT (γδ DNT) cells. Frequencies of circulating DNT cell subsets were measured by flow cytometry in a retrospective cohort of 51 telbivudine-treated HBeAg-positive CHB patients, 25 immune tolerant carriers (IT), 33 inactive carriers (IC), and 37 healthy controls (HC). We found that γδ DNT cell frequencies did not significantly change during treatment, being lower at baseline (P = 0.019) in patients with HBeAg seroconversion after 52 weeks of antiviral therapy (n = 20) than in those without (n = 31), and higher in the total CHB and IT than IC and HC groups (P<0.001). αβ DNT cell frequencies were similar for all groups. In vitro, γδ DNT cells suppressed HBV core peptide-stimulated interferon-γ and tumor necrosis factor-α production in TCRαβ+CD8+ T cells, which may require cell–cell contact, and could be partially reversed by anti-NKG2A. These findings suggest that γδ DNT cells limit CD8+ T cell response to HBV, and may impede HBeAg seroconversion in CHB.

Interleukin 21 Reinvigorates the Antiviral Activity of Hepatitis B Virus (HBV)–Specific CD8+ T Cells in Chronic HBV Infection

BACKGROUND Strategies that target functional recovery of exhausted hepatitis B virus (HBV)-specific CD8+ T cells are beneficial for viral control, but the potential for interleukin 21 (IL-21) to rescue CD8+ T-cell function is not well understood. METHODS We investigated the effect of IL-21 on CD8+ T-cell responses by phenotypic and functional analysis of samples from patients with chronic HBV infection and a mouse model with HBV expression. RESULTS IL-21 promoted the proliferative capacity of HBV-specific CD8+ T cells and down-regulated expression of the inhibitory receptors programmed death 1 and T-cell immunoglobulin domain and mucin domain 3. Additionally, IL-21 boosted the production of interferon-γ, granzyme B, and CD107a in HBV-specific CD8+ T cells and enhanced the cytolytic activity of CD8+ T cells against HepG2.2.15 cells. Notably, an HBV mouse model established from IL-21 receptor knockout mice showed significantly decreased frequency of HBV-specific CD8+ T cells and increased levels of serum hepatitis B surface antigen (HBsAg). Meanwhile, administration of recombinant mouse IL-21 in an HBV mouse model established from wild-type mice resulted in enhanced functionality of HBV-specific CD8+ T cells and accelerated HBsAg clearance. CONCLUSIONS IL-21 enhances the antiviral effect of HBV-specific CD8+ T cells, suggesting that it may contribute to viral clearance in chronic HBV infection.