Assessment of the effect of ribavirin on myeloid and plasmacytoid dendritic cells during interferon-based therapy of chronic hepatitis B patients.

The combination of ribavirin and peginterferon is the current standard of anti-viral treatment for chronic HCV patients. However, little is known on the mode of action of ribavirin in the anti-viral treatment of HCV patients. To investigate the immunomodulatory mechanism of ribavirin, we studied peginterferon alone versus peginterferon and ribavirin in chronic HBV patients. The addition of ribavirin did not affect the number of myeloid dendritic cells (mDC) or plasmacytoid dendritic cells (pDC), nor did it enhance T-helper-1 cell activity or T-cell proliferation. In contrast, it increased upregulation of activation markers on mDC and pDC, which was sustained throughout treatment. However, the addition of ribavirin had no effect on IFNα production by pDC. Our findings demonstrate that, although ribavirin does not lead to a viral load decline, in vivo treatment with ribavirin affects the activation of pDC and mDC in chronic HBV patients.

PI3K-PKB hyperactivation augments human plasmacytoid dendritic cell development and function.

Plasmacytoid dendritic cells (pDCs) are considered potential tools or targets for immunotherapy. However, current knowledge concerning methodologies to manipulate their development or function remains limited. Here, we investigated the role of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)-mammalian target of rapamycin (mTOR) axis in human pDC development, survival, and function. In vitro pDC generation from human cord blood-derived CD34(+) hematopoietic progenitors was reduced by pharmacologic inhibition of PI3K, PKB, or mTOR activity, and peripheral blood pDCs required PI3K-PKB-mTOR signaling to survive. Accordingly, activity of this pathway in circulating pDCs correlated with their abundance in peripheral blood. Importantly, introduction of constitutively active PKB or pharmacologic inhibition of negative regulator phosphatase and tensin homolog (PTEN) resulted in increased pDC numbers in vitro and in vivo. Furthermore, MHC class II and costimulatory molecule expression, and production of IFN-α and TNF-α, were augmented, which could be explained by enhanced IRF7 and NF-κB activation. Finally, the numerically and functionally impaired pDCs of chronic hepatitis B patients demonstrated reduced PI3K-PKB-mTOR activity. In conclusion, intact PI3K-PKB-mTOR signaling regulates development, survival, and function of human pDCs, and pDC development and functionality can be promoted by PI3K-PKB hyperactivation. Manipulation of this pathway or its downstream targets could be used to improve the generation and function of pDCs to augment immunity.

Alpha-galactosylceramide in chronic hepatitis B infection: results from a randomized placebo-controlled Phase I/II trial.

BACKGROUND: The glycosphingolipid alpha-galactosylceramide (alpha-GalCer) is known to stimulate invariant natural killer T-cells (iNKTs) and is able to induce powerful antiviral immune responses. The present dose-escalating randomized placebo-controlled Phase I/II trial aimed to investigate antiviral activity and safety of alpha-GalCer as a novel class of treatment for chronic hepatitis B patients. METHODS: Patients were randomly assigned to 0.1 microg/kg (n=8), 1 microg/kg (n=6) or 10 microg/kg (n=6) alpha-GalCer or placebo (n=7) treatment. RESULTS: Almost all alpha-GalCer-treated patients showed a rapid and strong decrease in natural killer T-cell (NKT) numbers. Patients with high baseline NKT numbers showed immune activation, including natural killer cell activation, increased serum tumour necrosis factor-alpha and interleukin-6 levels, and development of fever. Three patients demonstrated a transient decrease in hepatitis B virus (HBV) DNA. Only one alpha-GalCer-treated patient had a sustained decrease in HBV DNA at the end of follow-up. Four patients discontinued therapy because of fever shortly after drug administration. No significant side effects were observed. CONCLUSIONS: alpha-GalCer (0.1-10 microg/kg) used as monotherapy for chronic hepatitis B infection resulted in a strong decrease of NKTs, but did not clearly affect HBV DNA and alanine aminotransferase levels. alpha-GalCer was poorly tolerated and is unlikely to be suitable as an alternative monotherapy to the current treatment regimen.

The mannose receptor acts as hepatitis B virus surface antigen receptor mediating interaction with intrahepatic dendritic cells.

Dendritic cells (DC) play a key role in anti-viral immunity. Direct interactions between DC and hepatitis B virus (HBV) may explain the impaired DC function and the ineffective anti-viral response of chronic HBV patients resulting in HBV persistence. Here, the interaction between HBV surface antigens (HBsAg) and DC and the receptor involved were examined by flow cytometry in blood and liver tissue of HBV patients. The in vitro data showed that the mannose receptor (MR) is involved in HBsAg recognition and uptake by DC. The presence of HBsAg-positive DC was demonstrated sporadically in blood, but frequently in the liver of HBV patients. Interestingly, a positive correlation was found between HBsAg positivity and MR expression level in both liver- and blood-derived DC. These data suggest that in HBV infected patients, MR-mediated interaction between HBsAg and DC and subsequent impairment of DC predominantly occurs at the main site of infection, the liver.

Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is the result of an inadequate immune response towards the virus. Myeloid dendritic cells (mDC) of patients with chronic HBV are impaired in their maturation and function, resulting in more tolerogenic rather than immunogenic responses, which may contribute to viral persistence. The mechanism responsible for altered mDC function remains unclear. The HBV-infected patients display large amounts of HBV particles and viral proteins in their circulation, especially the surface antigen HBsAg, which allows multiple interactions between the virus, its viral proteins and DC. To assess whether HBV directly influences mDC function, the effects of HBV and HBsAg on human mDC maturation and function were investigated in vitro. As already described for internalization of HBV by DC, the present study shows that peripheral blood-derived mDC of healthy controls also actively take up HBsAg in a time-dependent manner. Cytokine-induced maturation in the presence of HBV or HBsAg resulted in a significantly more tolerogenic mDC phenotype as demonstrated by a diminished up-regulation of costimulatory molecules and a decreased T-cell stimulatory capacity, as assessed by T-cell proliferation and interferon-gamma production. In addition, the presence of HBV significantly reduced interleukin-12 production by mDC. These results show that both HBV particles and purified HBsAg have an immune modulatory capacity and may directly contribute to the dysfunction of mDC in patients with chronic HBV. The direct immune regulatory effect of HBV and circulating HBsAg particles on the function of DC can be considered as part of the mechanism by which HBV escapes immunity.

Intrahepatic regulatory T cells are phenotypically distinct from their peripheral counterparts in chronic HBV patients.

Peripheral blood CD4+CD25+ regulatory T cells (Treg) prevent the development of strong HBV-specific T cell responses in vitro. In this study, we examined the phenotype of FoxP3+ regulatory T cells in the liver of patients with a chronic HBV infection. We showed that the liver contained a population of CD4+FoxP3+ cells that did not express CD25, while these cells were absent from peripheral blood. Interestingly, intrahepatic CD25-FoxP3+CD4+ T cells demonstrated lower expression of HLA-DR and CTLA-4 as compared to their CD25+ counterparts. Patients with a high viral load have a higher proportion of regulatory T cells in the liver, but not in blood, compared to patients with a low viral load. In conclusion, the intrahepatic Treg are phenotypically distinct from peripheral blood Treg. Our data suggest that the higher proportion of intrahepatic Treg observed in patients with a high viral load may explain the lack of control of viral replication.

Induction of regulatory T-cells and interleukin-10-producing cells in non-responders to pegylated interferon-alpha therapy for chronic hepatitis B.

BACKGROUND: Treatment with interferon-alpha (IFN-alpha) leads to a response in only a minority of patients with chronic hepatitis B virus (HBV) infection, but the reasons for this are poorly understood. It was recently shown that in patients with chronic HBV infection, CD4+CD25+ regulatory T-cells (Treg) can suppress the HBV-specific immune response. We aimed to investigate whether in non-responders to IFN-alpha therapy Treg contribute to treatment failure by downregulating the HBV-specific T-cell responses. PATIENTS AND METHODS: Fourteen patients positive for hepatitis B e antigen received pegylated IFN-alpha monotherapy for 52 weeks and were followed for 26 weeks. RESULTS: Compared with non-responders, responders displayed an increased HBV-specific T-helper cell proliferation. At the start of treatment there was no difference in the frequencies of CD4+CD25+ Treg between responders and non-responders. During therapy, the frequency of CD4+CD25+ Treg increased in non-responders, but not in responders. In contrast to the responders, the non-responders showed a significant increase in the frequency of interleukin-10-producing cells. Treg depletion resulted in increased proliferation capacity, but did not affect the frequency of interleukin-10-producing cells measured during the course of the treatment. CONCLUSION: This study indicates that Treg might have an important role in HBV persistence during and after pegylated IFN-alpha therapy.

Aberrant composition of the dendritic cell population in hepatic lymph nodes of patients with hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) are characterized by a weak T-cell response to their tumor, and chronic carriers of hepatitis B virus or hepatitis C virus have a poor T-cell response against the virus. These inadequate T-cell responses may be due to insufficient activation of the T cells by dendritic cells (DCs). Because lymph nodes (LNs) are the primary site of antigen-specific T-cell activation, we hypothesized that hepatic LNs of patients with HCC and/or chronic viral hepatitis might have aberrant compositions of their DC populations. To address this hypothesis, we enumerated mature myeloid DCs (MDCs) and plasmacytoid DCs (PDCs) in hepatic LNs by quantitative immunohistochemistry. Patients with HCC and chronic viral hepatitis and patients with chronic viral hepatitis without HCC were compared with patients with liver inflammation of nonviral etiology and with organ donors with healthy livers. The numbers of PDCs and mature MDCs in hepatic LNs of patients with chronic viral hepatitis did not differ from those of patients with liver inflammation of nonviral etiology nor from individuals with healthy livers. However, hepatic LNs of patients with HBV or HCV infection complicated by HCC showed a 1.5-fold reduction in numbers of mature MDCs and a 4-fold increase in numbers of PDCs in their T-cell areas compared with those of patients with viral hepatitis only (P <.01). In conclusion, patients with HCC have an aberrant composition of the DC population in their hepatic LNs. This may be one of the causes of the inadequate T-cell response against HCC in these patients.

Intrahepatic CD8+ T-lymphocyte response is important for therapy-induced viral clearance in chronic hepatitis B infection.

BACKGROUND/AIMS: To determine which immune cells contribute to HBV-clearance during antiviral therapy, we performed a longitudinal analysis of intrahepatic immune cells during interferon-alpha therapy of chronic HBV-patients using the FNAB technique. METHODS: Twenty chronic HBeAg+-patients were treated with pegylated alpha-interferon combined with lamivudine or placebo for 52 weeks. FNAB and blood specimens were obtained at week 0, 2, 8 and 52. CD4+- and CD8+ T-lymphocytes, CD56+ cells, IFNgamma and granzyme B (GrB) were immunocytochemically quantified. RESULTS: The relative numbers of CD56+ cells and CD8+ T-lymphocytes were significantly higher in FNAB compared to blood at all time-points. Responders (n=9) exhibited significant increases in intrahepatic CD8+ and CD8+GrB+ lymphocytes, a small elevation in CD8+IFNgamma+ T-lymphocytes, no change in CD4+ T-lymphocytes, and a decrease in intrahepatic CD56+ cells during the first weeks of therapy. In non-responders (n=11) no significant changes in CD4+- and CD8+ T-lymphocytes and an increase in intrahepatic and CD56+ cells were observed during therapy. CONCLUSIONS: The intrahepatic CD8+ T-lymphocyte, but not the CD4+ T-lymphocyte or NK/NKT-cell response, is important for HBV clearance during interferon-alpha therapy, and the antiviral effect may be mediated by both cytolytic and non-cytolytic mechanisms.

Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B.

Dendritic cells (DC) play an important role in the induction of T-cell responses. We hypothesize that the hampered antiviral T-cell response in chronic hepatitis B patients is a result of impaired dendritic cell function. In this study, we compared the number, phenotype and functionality of two important blood precursor DC, myeloid DC (mDC) and plasmacytoid DC (pDC), of chronic hepatitis B patients with healthy volunteers. No differences in percentages of mDC and pDC in peripheral blood mononuclear cells were observed between chronic hepatitis B patients and healthy controls. The allostimulatory capacity of isolated and in vitro matured mDC, but not of pDC, was significantly decreased in patients compared to controls. Accordingly, a decreased percentage of mDC expressing CD80 and CD86 was observed after maturation, compared to controls. In addition, mDC of patients showed a reduced capacity to produce tumor necrosis factor alpha after a stimulus with synthetic double-stranded RNA and interferon gamma. Purified pDC from patients produced less interferon alpha, an important antiviral cytokine, in response to stimulation with Staphylococcus aureus Cowan strain I than pDC isolated from controls. In conclusion, mDC and pDC are functionally impaired in patients with chronic hepatitis B. This might be an important way by which hepatitis B virus evades an adequate immune response, leading to viral persistence and disease chronicity.