S. Chokshi

Hepatitis C virus–specific T-cell reactivity during interferon and ribavirin treatment in chronic hepatitis C

BACKGROUND & AIMS The role of virus-specific T-helper lymphocyte reactivity in determining the therapeutic response in chronic hepatitis C virus (HCV) infection is not fully understood. METHODS We studied CD4(+) T lymphocyte proliferation together with interferon (IFN)-gamma and interleukin (IL)-10 production from peripheral blood mononuclear cells in response to 4 HCV antigens (core, NS3, NS4, and NS5) in 25 patients with chronic hepatitis C undergoing antiviral therapy with IFN alone or in combination with ribavirin, prospectively, before, during, and after treatment. RESULTS HCV-specific T-cell reactivity was uncommon at baseline but increased markedly during antiviral therapy, peaking around treatment weeks 4-8. Resolution of hepatitis C viremia was significantly more likely in patients who developed HCV-specific T-cell proliferation with increased IFN-gamma production. The main difference in T-cell reactivity of patients treated with IFN plus ribavirin was a significantly lower production of IL-10, whereas lymphocyte proliferation was similar to that in patients receiving IFN monotherapy. CONCLUSIONS Treatment-induced control of hepatitis C viremia is associated with the development of HCV-specific T-cell responses with enhanced IFN-gamma and low IL-10 production. The greater efficacy of combination therapy with IFN-alpha plus ribavirin may be related to its ability to suppress HCV-specific IL-10 production.

Hepatitis C virus (HCV) specific immune responses in anti-HCV positive patients without hepatitis C viraemia.

BACKGROUND/AIMS Most patients infected with hepatitis C virus (HCV) develop chronic infection and persistent viraemia. The immune mechanisms responsible for resolution of viraemia remain poorly understood. HCV specific humoral and cellular immune responses in patients with and without viraemia were investigated. METHODS In vitro T helper (TH) lymphocyte responses to structural and non-structural HCV proteins were determined by means of proliferative response and cytokine production in 35 anti-HCV positive/HCV RNA negative patients and in 31 patients with chronic HCV infection and persistent viraemia. Humoral responses were determined by measuring HCV specific antibody quantity and specificity. RESULTS A TH response to two or more HCV proteins was present in 18 of 35 patients with serological viral clearance compared with just one of 31 viraemic patients (p = 0.00001). HCV specific interferon-γ production was increased only in the former group. In contrast, the antibody levels were significantly lower and directed at fewer HCV antigens in patients with undetectable HCV RNA. CONCLUSIONS Patients without viraemia after HCV infection frequently have strong TH lymphocyte responses of the TH1 type to multiple HCV antigens many years after the onset of infection, whereas antibody responses are less marked. These results suggest that control of HCV replication may depend on effective TH lymphocyte activation.

CD8+ T Cell Control of Hepatitis B Virus Replication: Direct Comparison between Cytolytic and Noncytolytic Functions

Resolution of hepatitis B virus (HBV) infection was believed to be attributed to the cytotoxic T cell–mediated killing of infected hepatocytes. However, studies in HBV transgenic mice and HBV-infected chimpanzees revealed that T cell control of HBV replication also involves cytokine-mediated noncytolytic mechanisms. The relative role of cytolytic and noncytolytic functions of virus-specific CD8+ T cells during interaction with HBV-producing hepatocytes is not well understood. By using HLA-A2 matched effector cells (CD8+ T cell line or clone) and target cells supporting full HBV replication, we demonstrate that virus-specific CD8+ T cells can inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. Although CD8+ T cells kill a fraction of infected cells, this effect is minimal, and most of the viral inhibition is mediated by noncytolytic mechanisms. CD8+ T cells produce an array of cytokines, among which IFN-γ and TNF-α are responsible for HBV inactivation in the target cells. Blockade of IFN-γ and TNF-α abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines mediate the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN-γ and rTNF-α resulted in an efficient suppression of viral replication without cytotoxicity. In contrast, coculture of the same target cells with activated HLA-mismatched mitogen-activated lymphomononuclear cells caused a marked cytolytic effect and was less effective in HBV control. These results provide direct evidence that virus-specific CD8+ T cells efficiently control HBV replication by noncytolytic mechanisms, and this effect is mediated by IFN-γ and TNF-α.

Programmed death 1 expression during antiviral treatment of chronic hepatitis B: Impact of hepatitis B e‐antigen seroconversion

Hyperexpression of the programmed death 1 (PD‐1) molecule is a hallmark of exhausted T‐cells, having a negative impact on T‐cell activation and function. We studied longitudinally 18 hepatitis B e antigen (HBeAg)–positive patients undergoing treatment with direct antivirals (telbivudine or lamivudine) to determine the relationship between treatment‐induced viremia reduction and HBeAg seroconversion with respect to PD‐1 levels and T‐cell reactivity. PD‐1 expression was assessed by (1) flow cytometry and (2) quantitative real‐time polymerase chain reaction; hepatitis B virus (HBV)–specific CD8+ T‐cells were quantitated by pentamer staining; T‐cell reactivity to HBV antigens was determined by interferon gamma (IFNγ) and interleukin 10 (IL‐10) enzyme‐linked immunosorbent spot (ELISPOT) assays; and central/effector memory phenotypes were defined by phenotypic markers. PD‐1 expression correlated closely with viremia levels. On therapy, PD‐1 decreased significantly on total CD8+ T‐cells, HBV‐specific CD8+ T‐cells, and CD3+/CD8− T‐cells both as the percentage of positive cells (P < 0.01) and as the mean fluorescent intensity (P < 0.05), and this was paralleled by a marked reduction of PD‐1 messenger RNA levels (P = 0.001). HBeAg serocoversion (in 6/18 patients) resulted in a further PD‐1 decrease with a 50% reduction in the frequency of PD‐1+/CD8+ T‐cells, which was not observed in patients remaining HBeAg‐positive. The decrease in PD‐1 expression was associated with increased frequencies of IFNγ‐producing T‐cells and decreased frequencies of IL‐10 producing T‐cells. At baseline, PD‐1 expression correlated directly with the frequency of hepatitis B core antigen (HBcAg) central and effector memory phenotypes, whereas an inverse correlation was observed between PD‐1 expression and HBcAg‐specific effector phenotypes. Conclusion: These results demonstrate that in chronic HBV infection, both viremia levels and HBeAg drive PD‐1 expression and resulting T‐cell impairment. Treatment‐induced suppression of HBV replication reduces PD‐1 expression; however, additional immunotherapeutic interventions are needed for restoration of T‐cell functions. (HEPATOLOGY 2008.)

Lamivudine plus interleukin‐12 combination therapy in chronic hepatitis B: Antiviral and immunological activity

Interleukin‐12 (IL‐12) is an immunomodulatory cytokine that promotes cellular immunity. Pre‐clinical data suggest that IL‐12 inhibits hepatitis B virus (HBV) replication by stimulating interferon‐gamma (IFN‐γ) production. We investigated whether a combination treatment with lamivudine plus recombinant human interleukin‐12 (rhIL‐12) will result in a greater and prolonged suppression of HBV replication in comparison with lamivudine monotherapy. Fifteen patients with HBeAg‐positive chronic hepatitis B were randomized to receive either lamivudine alone for 24 weeks (group 1); combination of lamivudine for 16 weeks and rhIL‐12 (200 ng/kg twice weekly), starting 4 weeks after initiation of lamivudine, for 20 weeks (group 2), or the same schedule as for group 2, with lamivudine and a higher dose of rhIL‐12 (500 ng/kg, group 3). Serum HBV DNA levels, T‐cell proliferation, frequency of virus‐specific T‐cells, and IFN‐γ production were evaluated serially during and 24 weeks posttreatment. Lamivudine plus rhIL‐12/500 showed greater antiviral activity than lamivudine monotherapy. However, after stopping lamivudine in groups 2 and 3, serum HBV DNA increased significantly despite continuing rhIL‐12 administration. Lamivudine plus rhIL‐12 treatment was associated with a greater increase in virus‐specific T‐cell reactivity, IFN‐γ production, and an inverse correlation between the frequency of IFN‐γ–producing CD4+ T‐cells and viremia. The T‐cell proliferative response to HBcAg did not differ between the three groups. In conclusion, the addition of IL‐12 to lamivudine enhances T‐cell reactivity to HBV and IFN‐γ production. However, IL‐12 does not abolish HBV replication in HBeAg‐positive patients and does not maintain inhibition of HBV replication after lamivudine withdrawal. (HEPATOLOGY 2005.)

Blockade of PD1 and TIM3 Restores Innate and Adaptive Immunity in Patients with Acute Alcoholic Hepatitis

BACKGROUND & AIMS Susceptibility to bacterial infection is a feature of alcohol-related liver disease. Programmed cell death 1 (PD1), the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3, also known as hepatitis A virus cellular receptor 2), and their respective ligands-CD274 (also known as PD ligand 1 [PDL1]) and galectin-9-are inhibitory receptors that regulate the balance between protective immunity and host immune-mediated damage. However, their sustained hyperexpression promotes immune exhaustion and paralysis. We investigated the role of these immune inhibitory receptors in driving immune impairments in patients with alcoholic liver disease. METHODS In a prospective study, we collected blood samples from 20 patients with acute alcoholic hepatitis (AAH), 16 patients with stable advanced alcohol-related cirrhosis, and 12 healthy individuals (controls). Whole blood or peripheral blood mononuclear cells were assessed for expression of PD1, PDL1, TIM3, galectin-9, and Toll-like receptors on subsets of innate and adaptive immune effector cells. We measured antibacterial immune responses to lipopolysaccharide (endotoxin) using ELISpot assays, and used flow cytometry to quantify cytokine production, phagocytosis, and oxidative burst in the presence or absence of blocking antibodies against PD1 or TIM3. RESULTS Antibacterial innate and adaptive immune responses were greatly reduced in patients with AAH, compared with controls, and patients with alcohol-related cirrhosis had less severe dysfunctions in innate immune effector cells and preserved functional T-cell responses. Fewer T cells from patients with AAH produced interferon gamma in response to lipopolysaccharide, compared with controls. In addition, patients with AAH had greater numbers of interleukin 10-producing T cells, and reduced levels of neutrophil phagocytosis and oxidative burst in response to Escherichia coli stimulation, compared with controls. T cells from patients with AAH, but not alcohol-related cirrhosis, expressed higher levels of PD1 and PDL1, or TIM3 and galectin-9, than T cells from controls. Antibodies against PD1 and TIM3 restored T-cell production of interferon gamma, reduced the numbers of interleukin 10-producing T cells, and increased neutrophil antimicrobial activities. Circulating levels of endotoxin in plasma from patients with AAH caused over expression of immune inhibitory receptors on T cells via Toll-like receptor 4 binding to CD14(+) monocytes. CONCLUSIONS Antibacterial immune responses are impaired in patients with AAH. Lymphocytes from these patients express high levels of immune inhibitory receptors, produce lower levels of interferon gamma, and have increased IL10 production due to chronic endotoxin exposure. These effects can be reversed by blocking PD1 and TIM3, which increase the antimicrobial activities of T cells and neutrophils.

Genomic variations in the hepatitis B core gene : a possible factor influencing response to interferon alfa treatment

BACKGROUND/AIMS Interferon treatment causes a sustained loss of virus replication only in a proportion of patients with chronic hepatitis B. This study investigated whether genomic variations in the precore/core gene of hepatitis B virus affect the response to interferon alfa. METHODS The precore/core region was sequenced in 46 serum samples obtained before, during, and after interferon treatment of 12 patients. RESULTS In 23 samples from 7 responders (group A), there were 24 missense mutations, whereas in 23 samples from 5 patients who did not respond or relapsed after treatment (group B), there were 141 missense mutations (P < 0.001). All group B patients had cirrhosis, but only 2 of 7 patients in group A had cirrhosis (P = 0.026). Substitutions in amino acids 21-27 of the core protein, known to diminish HLA-A2-restricted cytotoxic T-cell function, were found in all nonresponders but in none of the responders. No significant changes occurred in the precore/core region in responders after seroconversion to antibody to hepatitis B e antigen, but multiple variations persisted in group B during treatment and new mutations appeared with the relapse of hepatitis. CONCLUSIONS Specific mutations in the core protein that can interfere with T-cell function occur frequently in patients with advanced chronic hepatitis B and may affect the response to interferon.

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice

Background Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. Methods Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. Results OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-β signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. Conclusions OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.

Hepatitis C virus infection in the development of hepatocellular carcinoma in cirrhosis

BACKGROUND/AIMS The role of hepatitis C virus replication and different genotypes in the progression of cirrhosis to hepatocellular carcinoma is examined on the basis of a prospective follow-up of 1438 patients with histologically proven cirrhosis. METHODS The presence of HCV RNA, anti-HCV and characterisation of virus genotypes were determined in 72 cases who developed hepatocellular carcinoma after a median follow-up of 5.3 years (range 1 to 16) and compared to 72 controls who had cirrhosis only, after a median follow-up of 4.8 years (range 1 to 16). Patients in the hepatocellular carcinoma group and controls were matched, one to one, for age, sex, nationality, HBsAg seropositivity, duration of follow-up and aetiology of cirrhosis. RESULTS HCV RNA was detected in 31 of 72 (44%) patients who developed hepatocellular carcinoma, significantly more frequently than in 17 of 72 (23%) controls with cirrhosis (odds ratio 2.4, 95% confidence interval 1.2 to 5.0; p = 0.013). When cirrhosis of different aetiologies was analysed, hepatitis C virus replication was more frequently detected in patients developing hepatocellular carcinoma in association with cryptogenic cirrhosis (p = 0.007), alcoholic cirrhosis (p = 0.043) and hepatitis B virus seronegative cirrhosis (p = 0.05). Hepatitis C virus genotypes 1b and 4 were the most prevalent; they were found in 53% and 25%, respectively, of the patients studied, but were equally distributed between cirrhosis progressing to hepatocellular carcinoma and controls. CONCLUSIONS Persistent hepatitis C virus replication is closely associated with hepatocellular carcinoma development in cirrhosis, and there is no preferential role of individual hepatitis C virus genotypes.

Hepatitis B Virus e Antigen Loss during Adefovir Dipivoxil Therapy Is Associated with Enhanced Virus-Specific CD4+ T-Cell Reactivity

ABSTRACT Weak T-cell reactivity to hepatitis B virus (HBV) is thought to be the dominant cause for chronic HBV infection. Treatment with adefovir dipivoxil (ADV) increases the rate of HBV e antigen (HBeAg) loss; however, the immune mechanisms associated with this treatment response are not understood. Serial analysis of HBV-specific CD4+ T-cell reactivity was performed during 48 weeks of therapy with ADV and correlated with treatment outcome for 19 HBeAg-positive patients receiving ADV (n = 13) or the placebo (n = 6). We tested T-cell reactivity to HBV at seven protocol time points by proliferation, cytokine production, and enzyme-linked immunospot assays. A panel of serum cytokines was quantitated by cytokine bead array. ADV-treated patients showed increased CD4+ T-cell responses to HBV and lower serum levels of cytokines compared to those of placebo-treated patients. Enhanced CD4+ T-cell reactivity to HBV, which peaked at treatment week 16, was confined to a subgroup of ADV-treated patients who achieved greater viral suppression (5.3 ± 0.3 log10 copies/ml [mean ± standard error of the mean {SEM}] serum HBV DNA reduction from baseline) and HBeAg loss, but not to ADV-treated patients with moderate (3.4 ± 0.2 log10 copies/ml [mean ± SEM]) viremia reduction who remained HBeAg positive or to patients receiving the placebo. In conclusion, T-cell reactivity to HBV increases in a proportion of ADV-treated patients and is associated with greater suppression of HBV replication and HBeAg loss.