HBsAg preferentially binds to CpG-activated pDC.

<p><b>A</b>: pDC were cultured with or without Lox or CpG and with or without HBsAg. After 4h, cells were harvested and surface bound HBsAg was detected by flow cytometry. Data are representative for 5 independent experiments. Open: HBsAg detection in cultures without HBsAg; Filled: HBsAg detection in cultures with HBsAg. <b>B</b>: PBMC were cultured in the presence of CpG, Lox or HSV-1 either with or without anti-BDCA-2 or BDCA-4. IFNα production in pDC was detected by flow cytometry. Data show mean±SD relative IFNα production compared to cultures without BDCA-2/4 crosslinking for 2 independent experiments. <b>C</b>: pDC were cultured with or without CpG or Lox for 24h. Data show mean±SEM BDCA-2 expression as assessed by flow cytometry of 5 independent experiments. NS: not significant. <b>D</b>: PBMC were cultured with or without CpG and with or without HBsAg. After 4h, cells were harvested and surface bound HBsAg was detected on pDC by flow cytometry. Data show mean±SEM HBsAg positive pDC of 5 independent experiments.</p

HBV inhibits cytokine production and pDC-induced NK cell activation.

<p><b>ABCD</b>: Purified pDC were cultured with CpG in the presence or absence HepG2.215-derived HBV. Supernatants were analysed for TNFα (A), IP-10 (B), IL-6 (C) and IL-8 (D). Data demonstrate mean±SEM of 8 independent experiments. <b>EFG</b>: Purified pDC were stimulated with CpG in the presence or absence of patient serum-derived HBV. Healthy control serum was treated in a similar way and added in the same volume to pDC. After 24h, supernatants were harvested and tested for the presence of IFNα (E), TNFα (F) and IL-6 (G) by ELISA. Shown is the mean±SD of triplicate cultures from one out of 2 experiments with different donors. <b>HI</b>: NK cells were cultured with or without pDC and with or without HepG2.215-derived HBV in medium containing IL-3 and CpG. Data show mean±SEM CD25 expression on CD56+ cells (E) and IFNγ production (F) of 7 independent experiments. *p<0.05, Wilcoxon signed rank test.</p

Effect of HBV on pDC maturation.

<p>To investigate possible pDC activation by HepG2.215-derived HBV in comparison with other pDC stimuli, PBMC were cultured in the presence or absence of HBV (100 geq/cell), CpG, HSV-1, Lox or Influenza (see ‘ctr’ lane). To investigate the immune regulatory effect of HBV on pDC maturation induced by other known pDC activating ligands, PBMC were cultured with or without CpG, HSV-1, Lox or Influenza in the presence (‘HBV’ lane) or absence (‘ctr’ lane) of HBV (100 geq/cell). After 24h, cells were harvested and the expression of CD40, CD80, CD86 and HLA-DR on pDC was determined by flow cytometry. Data are presented as mean±SEM fluorescent intensity of 3 independent experiments with different donors. Similar data were observed for purified pDC (not shown).</p><p>*p<0.05, paired t-test; nd = not done.</p

The Effect of Chronic Hepatitis B Virus Infection on BDCA3+ Dendritic Cell Frequency and Function

<div><p>Chronic hepatitis B virus (HBV) infection results from inadequate HBV-specific immunity. BDCA3⁺ dendritic cells (DCs) are professional antigen presenting cells considered to be important for antiviral responses because of specific characteristics, including high interferon-λ production. BDCA3⁺ DCs may thus also have a role in the immune response against HBV, and immunotherapeutic strategies aiming to activate DCs, including BDCA3⁺ DCs, in patient livers may represent an interesting treatment option for chronic HBV. However, neither the effect of chronic hepatitis B (CHB) infection on the frequency and function of BDCA3⁺ DCs in liver and blood, nor the effect of the viral surface protein (HBsAg) that is abundantly present in blood of infected individuals are known. Here, we provide an overview of BDCA3⁺ DC frequency and functional capacity in CHB patients. We find that intrahepatic BDCA3⁺ DC numbers are increased in CHB patients. BDCA3⁺ DCs from patient blood are not more mature at steady state, but display an impaired capacity to mature and to produce interferon-λ upon polyI:C stimulation. Furthermore, in vitro experiments exposing blood and intrahepatic BDCA3⁺ DCs to the viral envelope protein HBsAg demonstrate that HBsAg does not directly induce phenotypical maturation of BDCA3⁺ DCs, but may reduce IFN-λ production via an indirect unknown mechanism. These results suggest that BDCA3⁺ DCs are available in the blood and on site in HBV infected livers, but measures may need to be taken to revive their function for DC-targeted therapy.</p></div

HBsAg does not have a direct effect on BDCA3⁺ DC function.

<p>(A) Isolated BDCA3⁺ DCs from healthy subjects were stimulated for 6 hours with or without polyI:C or TNFα and IL-1β in the presence or absence of rHBsAg or pHBsAg. Mean±SEM percentages of maturation marker-expressing BDCA3⁺ DCs are shown (n = 2–3). (B) Isolated BDCA3⁺ DCs from healthy subjects were stimulated for 24 hours with polyI:C in the presence or absence of rHBsAg. Data are shown as mean±SEM cytokine levels determined by CBA (n = 5). (C) Isolated blood BDCA3⁺ DCs from healthy subjects were stimulated for 24 hours with polyI:C in the presence (n = 6) or absence (n = 7) of PMBC and/or rHBsAg, and cytokine levels in the culture supernatant were determined by ELISA (mean±SEM). *<i>p <</i> 0.05 by paired Student’s <i>t</i>-test.</p

Characteristics of donors used for intrahepatic DC quantification.

<p>Characteristics of donors used for intrahepatic DC quantification.</p

HBV does not activate pDC.

<p><b>A</b>: PBMC were stimulated with CpG and analysed for IFNα production by pDC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015324#s4" target="_blank"><i>Materials and Methods</i></a>. Shown is a representative FACS plot of PBMC stained for CD123 and BDCA-4 to identify pDC and the detection of IFNα positive cells within this pDC-gate. <b>B</b>: IFNα and TNFα producing pDC within PBMC cultured in the presence or absence of HepG2.215-derived HBV, CpG, HSV-1, Lox or Influenza are presented as mean±SEM of 10 independent experiments.</p

HBsAg diminishes IFN-λ1 production by BDCA3⁺ DCs.

<p>(A) PBMC from healthy subjects were incubated with or without fluorescently-labeled HBsAg for 2 hours at indicated temparatures and HBsAg binding/uptake by BDCA3⁺ DCs was measured by flow cytometry. Representative plots of 3 independent experiments and donors are shown. (B) PBMC from healthy subjects were stimulated for 6 hours with or without rHBsAg, pHBsAg or polyI:C and maturation marker-expression on BDCA3⁺ DCs was analyzed by flow cytometry (n = 3; mean±SEM; * p<0.05 by paired Student’s <i>t</i>-test). (C-D) PBMC from healthy subjects were stimulated for 7 hours with polyI:C in the presence or absence of rHBsAg and the production of IFN-λ1 by BDCA3⁺ DCs was measured by ICS. Representative flow cytometry plots (C) and the summarized percentage of IFN-λ1-producing BDCA3⁺ DCs (D; n = 7; mean±SEM) are shown. To determine the percentage of IFN-λ-producing BDCA3⁺ DCs in blood, a minimum threshold of 70 BDCA3⁺ DCs was used. **<i>p <</i> 0.01 by paired Student’s <i>t</i>-test. (E) Liver cells from peri-tumor liver tissue were stimulated for 5 hours with or without polyI:C in the presence or absence of rHBsAg and the production of IFN-λ1 by BDCA3⁺ DCs was measured by ICS. The percentage of IFN-λ1-producing BDCA3⁺ DCs is shown (n = 3; mean±SEM). **<i>p <</i> 0.01 by paired Student’s <i>t</i>-test.</p

Negative Regulation of Hepatitis C Virus Specific Immunity Is Highly Heterogeneous and Modulated by Pegylated Interferon-Alpha/Ribavirin Therapy

<div><p>Specific inhibitory mechanisms suppress the T-cell response against the hepatitis C virus (HCV) in chronically infected patients. However, the relative importance of suppression by IL-10, TGF-β and regulatory T-cells and the impact of pegylated interferon-alpha and ribavirin (PegIFN-α/ribavirin) therapy on these inhibitory mechanisms are still unclear. We revealed that coregulation of the HCV-specific T-cell responses in blood of 43 chronic HCV patients showed a highly heterogeneous pattern before, during and after PegIFN-α/ribavirin. Prior to treatment, IL-10 mediated suppression of HCV-specific IFN-γ production in therapy-naive chronic HCV patients was associated with higher HCV-RNA loads, which suggests that protective antiviral immunity is controlled by IL-10. In addition, as a consequence of PegIFN-α/ribavirin therapy, negative regulation of especially HCV-specific IFN-γ production by TGF-β and IL-10 changed dramatically. Our findings emphasize the importance of negative regulation for the dysfunctional HCV-specific immunity, which should be considered in the design of future immunomodulatory therapies.</p> </div

Quantification of intrahepatic BDCA3⁺ DCs from HBV patients and controls.

<p>(A-B) Liver cells were isolated from HBV patients and controls. The DC population was identified as CD45⁺Lineage^-HLA-DR⁺ mononuclear cells, within which BDCA3⁺CLEC9A⁺ DCs were detected. (A) Representative flow cytometry plots and (B) the percentage CD45⁺ cells of total cells (control n = 4, HBV n = 9), percentage DCs of total cells (control n = 4, HBV n = 9) and percentage BDCA3⁺ DCs of total cells (control n = 5, HBV n = 11) in livers of controls and HBV patients. Indicated are the mean percentage and SEM. Open dots represent cells from donor livers and filled dots represent cells from peri-tumor liver tissue. **<i>p <</i> 0.01 by Mann-Whitney test. (C-D) FFPE sections of HBV-infected and control livers were stained with anti-CLEC9A Abs or non-specific sheep polyclonal Abs (green) and quantified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161235#sec002" target="_blank">methods</a>). Nuclei were visualized using DAPI (blue). Magnification 200x. (C) Representative pictures of an HBV-infected liver with high ALT (defined as > 60 IU L^-1) and high viral load (>10,000 IU ml^-1) and a control liver (healthy donor liver accepted for transplantation). White arrows indicate CLEC9A⁺ DCs. (D) Number of CLEC9A⁺ DCs per microscopic field in control livers (n = 6) and total HBV-infected livers with different levels of viral load (low n = 8, high n = 6), ALT (low n = 8, high n = 6), and fibrosis (F0–F0-1 n = 6, F1–F4 n = 7) (mean±SEM). *<i>p <</i> 0.05, **<i>p</i> <0.01 by Mann-Whitney test.</p