Early viraemia clearance during antiviral therapy of chronic hepatitis C improves dendritic cell functions

Clinical Immunology (2009) 131, 415–425

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

Clinical Immunology w w w. e l s e v i e r. c o m / l o c a t e / y c l i m

Early viraemia clearance during antiviral therapy of chronic hepatitis C improves dendritic cell functions Ioannis Pachiadakis a,⁎,1 , Shilpa Chokshi a , Helen Cooksley a , Dimitrios Farmakiotis b , Christoph Sarrazin c , Stefan Zeuzem c , Tomasz I. Michalak d , Nikolai V. Naoumov a,2 a

Institute of Hepatology, University College London, London, WC1E 6HX, UK Infectious Diseases Hospital of Thessaloniki, Thessaloniki, Greece c Medizinische Klinik I, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany d Molecular Virology and Hepatology Research, Division of Basic Medical Science, Faculty of Medicine, Memorial University, St. John's, Newfoundland, Canada b

Received 8 October 2008; accepted with revision 2 February 2009 Available online 20 March 2009 KEYWORDS Hepatitis C; Dendritic cells; Antiviral treatment

Abstract Plasma and cellular HCV RNA and core antigen were tested in monocyte-derived DC (MDDC) from chronic hepatitis C patients undergoing treatment with peg-interferon α2b/ ribavirin. DC allostimulatory capacity, HCV-specific T-cell reactivity and IL-12 production were measured at baseline and treatment week (TW)12. Using DC and autologous CD4+T-cells, obtained at baseline and TW12, we performed cross-over experiments to determine the relative role of DC and/or T-cells for impaired immune reactivity to HCV. HCV RNA and HCV core plasma levels had an impact on DC phenotype and allostimulatory capacity. In contrast, HCV genome/core protein, although detectable in DC from some patients had no effect on DC function. Antiviral immunity at TW12 was not improved in patients who remained HCV RNA positive, while early viraemia clearance (TW12) improved antiviral responses. The cross-over experiment revealed that changes in DC, rather than CD4+T cells have a major role for enhanced anti-HCV responses. © 2009 Elsevier Inc. All rights reserved.

Introduction

⁎ Corresponding author. Department of Gastroenterology and Hepatology, 424 Army General Teaching Hospital of Thessaloniki, Ring Road, 564 29, Thessaloniki, Greece. Fax: +302310381010. E-mail address: [email protected] (I. Pachiadakis). 1 Currently in the Department of Gastroenterology and Hepatology, 424 Army General Teaching Hospital of Thessaloniki. 2 Current address is Immunology and Infectious Diseases, Novartis Pharma AG, CH-4002 Basel, Switzerland.

It is estimated that 170 million people worldwide are chronically infected with the hepatitis C virus (HCV), one of the principal causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma [1,2]. Some individuals are able to clear HCV spontaneously during acute HCV infection by mounting vigorous cellular immune responses directed to multiple HCV antigens [1,3–6]. However in the great majority of patients viraemia persists, associated with impaired HCVspecific T-cell reactivity [6,7]. The mechanisms by which HCV establishes chronic infection are not fully understood.

1521-6616/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2009.02.001

416

I. Pachiadakis et al.

Table 1 Patients' characteristics for all patients included in the study (both treated and un-treated). N = 35 Gender (males/females) Age (years) Race (Caucasian/non-Caucasian ⁎) Genotype (1/3) Pre-treatment plasma HCV RNA (log10 IU/ml) Pre-treatment ALT levels (IU/L) Inflammation (grade ⁎⁎) Fibrosis (stage ⁎⁎)

24/11 45.5 ± 10.2 34/1 16/19 −6.07 ± 6.14 83 ± 52.7 3.4 ± 1.4 2.03 ± 1.07 Stages 0–3: n = 18, stages 4–6: n = 17

Scale data are presented as mean ± standard deviation. ⁎ Asian. ⁎⁎ Modified Ishak scale.

Dendritic cells (DC), the most potent antigen presenting cells, have a critical role in the initiation of adaptive immune responses [8–10]. Consequently, DC functions are targeted by numerous viruses in order to disrupt the generation of efficient antiviral cellular immune responses [11–17]. In HCV infection, several studies, using monocyte-derived DC (MDDC), have suggested that dysfunctional DC are responsible for the impaired Th1 response [18–24]. However, other authors found no significant defects in the phenotype and function of MDDC generated from patients with chronic hepatitis C (CHC) [25–28]. Similarly, controversial findings have been reported when using circulating myeloid or plasmacytoid dendritic cells, directly isolated from peripheral blood [27,29–32]. In the present study we investigated viraemia levels and MDDC functions and phenotype before treatment and early during antiviral therapy, trying to understand the role of DC in CHC. Our working hypothesis was that ‘early’ (by week 12 of treatment, TW12) clearance of viraemia and subsequent

Table 2

viral antigen loss leads to improvement of dendritic cell functions, promoting HCV-specific Th1 responses in the host. We further investigated whether improved adaptive immune response after ‘early’ viraemia clearance is due to restored DC function, or to improvement of CD4+ T-cell reactivity. In addition, we tested the relation between the presence of HCV genomic sequences and proteins (in particular HCV core) in patients' plasma and in DC lysates, and DC functions and phenotype.

Materials and methods Patients Thirty-five consecutive (n = 35), treatment-naïve patients with chronic hepatitis C, monitored at the Hepatitis Clinic, University College Hospital, London, were enrolled in the study (Table 1) after giving informed consent. All patients were positive for anti-HCV and HCV RNA (Amplicor HCV v2.0; Roche Molecular Systems, Pleasanton, CA). The HCV genotype was determined by a restriction–fragment–length– polymorphism method [33]. All patients were tested negative for HBsAg and antibodies against HIV1,2 by commercially available assays (Abbott Diagnostics, Maidenhead, UK). Patients underwent a liver biopsy with evaluation of the grade of liver inflammation and stage of fibrosis according to established criteria [34]. Twenty-two (n = 22) (Table 2) of the thirty-five enrolled patients received antiviral treatment with pegylated Interferon-α2b (1.5 μg/kg) once weekly and daily Ribavirin (Viraferon-Peg® and Rebetol®, both from Schering-Plough, Welwyn Garden City, UK) for 24 to 48 weeks, according to HCV genotype. The study was approved by the University College Hospital Ethics Committee.

Generation of monocyte-derived DC (MDDC) Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation with Lymphoprep 1077

Patients' characteristics. N = 22

Gender (males/females) Age (years) Race (Caucasian/non-Caucasian ⁎) Genotype (1/3) Pre-treatment plasma HCV RNA (log10 IU/ml) Pre-treatment ALT levels (IU/L) Inflammation (grade ⁎⁎) Viral load decline at TW12 (‘log10 drop’), ‘good responders’ ⁎⁎⁎ (n = 17) Viral load decline at TW12 (‘log10 drop’), ‘poor responders’ ⁎⁎⁎⁎ (n = 5) Viral load at TW12, ‘poor responders’ ⁎⁎⁎⁎ (log10 IU/ml) Fibrosis (stage ⁎⁎)

13/9 47.3 ± 9.7 21/1 8/14 −5.95 ± 5.98 83 ± 49.6 4.3 ± 1.2 5.48 ± 0.59 1.12 ± 0.94 −5.07 ± 0.79 2.7 ± 1.7 Stages 0–3: n = 17, stages 4–6: n = 5

Patients who received antiviral treatment with Pegylated-IFNα2b and Ribavirin. Scale data are presented as mean ± standard deviation. ⁎ Asian. ⁎⁎ Modified Ishak scale. ⁎⁎⁎ ‘good responders’: undetectable HCV RNA by TaqMan real-time PCR at TW12. ⁎⁎⁎⁎ ‘poor responders’: still detectable HCV RNA by TaqMan real-time PCR at TW12.

Early viraemia clearance during hepatitis C therapy improves dendritic cell functions (Nycomed Pharma, Oslo, Norway). Subsequently, CD14+ monocytes were separated using anti-CD14 Microbeads (Miltenyi Biotec Ltd, Bisley UK) according to the manufacturer's instructions (purity ≥ 95% by flow-cytometry). CD14− cells were cryo-preserved [35]. CD14+ monocytes were subsequently cultured in RPMI 1640 supplemented with: 2 mM L-glutamine, HEPES buffer (5 mM), 100 U/ml penicillin and 100 μg/ml streptomycin, supplemented with 10% foetal bovine serum, plus 100 ng/ml recombinant human GM-CSF and 100 ng/ml recombinant human IL-4 (both from R&D Systems, Abingdon, UK), for 6 days at 37 °C and 5% CO2. Highly viable (N 95% by Trypan Blue staining) MDDC (90% –97% pure-CD1a+, CD40+, CD83+, CD86+ and HLA-DR+ on flow cytometry) were harvested at the end of this process.

Enumeration of HCV-specific CD4+ T-cells by Elispot assays MDDC (104 cells/well, in 96-well plates, triplicate wells) were incubated overnight (16–20 h), at 37 °C/5% CO2, with commercially available recombinant HCV antigens: HCV core [aminoacids (aa) 1–115] or HCV NS3 (aa1007–1534) (Mikrogen, Munich, Germany)], at a concentration of 1 mg/ml each, and also lipopolysaccharide (LPS) (Salmonella Abortus Equi, 0.1 μg/ml, Sigma-Aldrich, Gillingham, UK) as maturation factor. Successful maturation was confirmed by significant increases in CD80, 83 and 86 expressions on DC. Tetanus toxoid (TT) (0.5 μg/ml) (Connaught Laboratories, Ontario, Canada) and phytohaemaglutinin (PHA) (1 mg/ml) (Sigma, Gillingham, UK) were used as control antigens. Bulk (memory and naive) autologous CD4+ T cells, isolated from the CD14− PBMC fraction using ‘negative’ immuno-magnetic bead separation (Miltenyi Biotec Ltd, Bisley UK) (purity ≥ 95% by flow-cytometry), were added to the culture (105 cells/ well), for 20 h at 37 °C/5% CO2. In parallel, Immobilon-P microtitre plates (Millipore, Bedford, UK) were coated with anti-IFN-γ (5 μg/ml) (BD Biosciences, Oxford, UK) for 16 h at 4 °C. The MDDC-CD4+ T cell cultures were transferred to the antibody-coated plates, incubated for another 20 h and subsequently IFN-γ-producing cells were detected, according to the manufacturer's instructions (BD Biosciences) and counted with an automated reader (AID Diagnostika GmbH, Strassberg, Germany). Response to HCV antigens was calculated and expressed as spot forming cells (SFC) per million CD4+ T cells. An IFN-γ response was judged as positive if the SFC, after antigenic stimulation, was above 10 per 106 CD4+ T-cells, which was the average SFC plus 2 standard deviations, determined with CD4+ T cells from 20 anti-HCV negative controls studied similarly [36].

417

above, and tested in parallel using the following combinations for each patient: i) MDDC (BL) with CD4+ T-cells (BL); ii) MDDC (BL) with CD4+ T-cells (TW12); iii) MDDC (TW12) with CD4+ T-cells (BL); and iv) MDDC (TW12) with CD4+ T-cells (TW12). MDDC were pulsed with HCV core and NS3 proteins, matured with LPS, and the frequency of IFN-γ-producing, HCV-specific, CD4+T-cells was determined with an Elispot assay.

Mixed lymphocyte reaction (MLR) Testing the capacity of MDDC to stimulate, naïve to HCV antigens, CD4+ T-cells we used MLR. Briefly, MDDC were incubated with CD4+ T-cells isolated from PBMC of a single healthy donor (allogeneic CD4+ T cells), at variable ratios (1/10–1/40), both with and without the addition of LPS (0.1 μg/ml) as a maturation factor, in triplicate wells, for 6 days at 37 °C/5% CO2. The cultures were subsequently pulsed with 1 μCi/well of [methyl-3H] thymidine (Amersham Pharmacia, Buckinghamshire, UK) during the last 12–16 h of the culture. The incorporation of [methyl-3H] thymidine in proliferating CD4+ T-cells was measured using a 1450 Microbeta counter (Wallac, Turku, Finland) and expressed as Counts Per Minute (CPM).

Detection of interleukin-12 p70 in DC culture supernatants MDDC secretion of interleukin-12p70 (IL-12p70) was assessed with a commercially available ELISA kit (R&D Systems, Abingdon, UK) according to the manufacturer's instructions. MDDC (1.5 × 105/well, in triplicate wells), were incubated overnight with LPS (1 μg/ml) and human recombinant IFN-γ (2 ng/ml; R&D) at 37 °C and 5% CO2. Culture supernatants

Analysis of autologous MDDC and CD4+ T-cells in a ‘cross-over’ experiment PBMCs were obtained from a group of 8 patients (part of the initial group of the 22 treated patients) with early viraemia clearance (all ‘good’ responders to antiviral treatment, with undetectable HCV RNA at TW12), both at baseline (BL) and at treatment week 12 (TW12), and were cryo-preserved [35]. MDDC and bulk (memory and naive) CD4+ T-cells were generated/isolated from PBMC samples, as described

Figure 1 Allostimulatory capacity of LPS-matured MDDC from 30 patients with chronic HCV infection (Patients) and LPS-matured DC from 12 non-infected healthy individuals (Controls), prior to the initiation of antiviral treatment.

418 were subsequently harvested and tested with IL-12p70 ELISA (lower limit of detection, 5 pg/ml).

I. Pachiadakis et al. (Scripps Institute, La Jolla, CA) software for data analysis. The efficiencies of immuno-magnetic cell separations were also assessed by flow cytometry on the day of preparation.

Flow cytometry Detection and quantitation of HCV RNA in MDDC We used mouse monoclonal anti-human antibodies: antiCD1α-FITC, anti-CD14-PE, anti-CD40-FITC, anti-CD80-FITC, anti-CD83-FITC, anti-CD86-PE, anti-HLA DR-FITC and antiCD4-FITC (all from Beckton Dickinson), and the corresponding isotype controls. MDDC, 5 × 104, were incubated for 30 min with the specific antibodies at 4 °C, in PBS supplemented with human AB serum and the analyses were performed on a FACSCalibur flow-cytometer with CellQuest software (both from BD) for data acquisition and WinMDI

We also investigated whether HCV RNA within DC has an impact on their functional characteristics. Total RNA was extracted from different numbers of MDDC (2.5 × 105 to 106) using RNeasy® Minikit, (Qiagen, Crawley, UK) according to the manufacturer's instructions and was subsequently used as a template in ‘real-time’, reverse-transcription, polymerase chain reaction (RT-PCR) with a lower limit of detection 20 IU/ml as described previously [36]. Briefly, HCV RNA was

Figure 2.1 (A) HCV core plasma levels and effect on CD83 expression on LPS-matured MDDC. (B) HCV core plasma levels and effect on CD86 expression on immature MDDC. (C) HCV RNA plasma levels and effect on CD40 expression on immature MDDC. (D) HCV core plasma levels and effect on CD80 expression on immature MDDC. Scatterplot and boxplot extending from the 25th to the 75th percentile and whiskers to the largest and smallest observed values within 1.5 box lengths; the solid line is the median.

Early viraemia clearance during hepatitis C therapy improves dendritic cell functions reverse transcribed and amplified in a single-tube, one-step process using the QuantiTect® RT-PCR Kit (Qiagen) and quantitated with the TaqMan® fluorigenic detection system (ABI Prism® 7700 sequence detector; Applied Biosystems, Warrington, UK). All samples and controls were tested in duplicates and the quantitation values were determined from an internal standard, which has been validated against the WHO International Standard for HCV (National Institute of Biological Standards and Control, Potters Bar, UK). The presence of HCV genome in MDDC was also tested with a highly sensitive [≤10 viral genomes(copies)/ml] assay, combining PCR and hybridisation detection of the amplicons (RT-PCR nucleic acid hybridisation — RT-PCR NAH), as previously described [37]. Briefly, MDDC aliquots (2 × 105– 5 × 105) were lysed using Trizol by an overnight chloroform/ isopropanol precipitation protocol and the extracted total RNA was transcribed with Moloney murine leukaemia virus reverse transcriptase (Invitrogen Life Technologies, Burlington, Ontario, Canada) and nested-PCR amplification of HCV 5′-untranslated region (UTR) followed in a PTC-200 thermocycler (MJR research, Watertown, MA). Detection of HCV RNA negative strand was performed with cDNA synthesis using

419

rTth DNA polymerase (Promega Corp., Madison, WI) as previously described [38].

Detection of HCV core antigen in plasma and MDDC Aliquots of two concentrations of MDDC-105 and 5 × 105 cells, and total PBMC-106 cells, as controls, were lysed with repeated passages through a 25 G needle and a subsequent incubation for 30 min with lysis buffer [50 mM Tris (hydroxymethyl) aminomethane, 150 mM NaCl, 1 mM EDTA, 1% NP40 (Nonidet P40)] at 4 °C. Plasma samples (500 μl), collected at the same time as the cell aliquots, were also tested in parallel. The concentration of HCV core protein in the cell lysates and plasma samples was determined with Ortho trak-C assay (Ortho Clinical Diagnostics, Raritan, NJ) (range of detection 1.5 to 300 pg/ml), as previously described [39–41].

Statistical analyses Non-parametric tests, in particular Mann–Whitney (for comparisons between two different groups) and Wilcoxon signed rank (for comparisons before and after treatment)

Figure 2.2 (A–D) Scatterplots of raw data, including correlation coefficients and P values [Kendall's τ (tau) correlation], corresponding to data presented in Figs. 2.1 A–D respectively.

420

I. Pachiadakis et al.

Results MDDC from CHC patients and healthy controls were tested both as ‘immature’ DC and as ‘mature’ DC following stimulation with LPS. ‘Immature’ DC from healthy controls showed a trend for higher allostimulatory capacity than MDDC from CHC patients (P = 0.063) (data not shown).The difference between the two groups was significant when testing ‘mature’ DC from the same subjects (P = 0.015) (Fig. 1). Using ultra-sensitive PCR methodology (RT-PCR NAH), the positive strand of HCV RNA was detected in MDDC lysates in 13 out of 33 patients with CHC. No detectable HCV sequences were found in the same samples by TaqMan® real-time RT PCR. The presence of negative strand HCV RNA in DC lysates was also detected in 5 out of 33 patients tested. All patients that had detectable levels of negative strand HCV RNA in their DC lysates also had detectable positive strand HCV RNA in the same cell lysates.

Figure 3.1 Effect of HCV core plasma levels on DC allostimulatoty capacity (MLR with LPS-matured MDDC). Scatterplot and boxplot extending from the 25th to the 75th percentile and whiskers to the largest and smallest observed values within 1.5 box lengths; the solid line is the median.

tests, were used as appropriate. In the ‘cross-over’ experiments of our study, a mixed model-based ANOVA was used, with ‘treatment’ (‘treated’ vs ‘untreated’) and ‘cell type’ (DC or CD4+ T-cells) as the within-group factors. Correlations between continuous, non-normally distributed data were assessed with Kendall's τ (tau) correlation coefficient. P values less than 0.05 were considered statistically significant. P values greater than 0.05 but less than 0.1 were considered indicative of a trend. Results were analysed using SPSS for Windows v.13.0 (SPSS Inc, Chicago, IL).

Figure 3.2 Scatterplot of raw data, including correlation coefficient and P value [Kendall's τ (tau) correlation], corresponding to data presented in Fig. 3.1.

Relation between patient characteristics and DC phenotype and functions We investigated whether viral or host parameters bear an impact on the expression of DC phenotypic markers and functions. The parameters studied and patients' stratification were as follows: HCV viraemia (‘low’: b 6 × 105 IU/ml or ‘high’: ≥6 × 105 IU/ml); presence/absence of HCV RNA sequences (‘positive’ and ‘negative’ strand HCV RNA) in DC lysates; concentration of HCV core antigen in DC lysates [‘low’: b1.1 pg/ml or ‘high’ ≥1.1 pg/ml (the 1.1 pg/ml cut-off limit was based on the median of the measured titres)]; HCV core protein levels in patients' plasma [‘low’: b184 pg/ml and ‘high’: ≥184 pg/ml (cut-off based on the median of the measured titres)]; ethanol consumption (‘low’: b 20 units/ week and ‘high’: ≥20 units/week) and severity of liver disease

Figure 4 Effect of alcohol consumption on IL-12p70 secretion from LPS-stimulated MDDC, generated from patients chronically infected with HCV, prior to the initiation of antiviral treatment.

Early viraemia clearance during hepatitis C therapy improves dendritic cell functions [‘mild fibrosis, stages 0–2, or ‘moderate/severe’ fibrosis, stages 3–6, modified Ishak score [34]]. Also raw data (i.e. without stratification) of the mentioned viral and host parameters were correlated to data regarding DC phenotypic surface markers and functions. CD83 expression on LPS-matured DC, as well as CD86 expression on immature DC was significantly higher in patients with high plasma levels of HCV core protein (Figs. 2.1A and B, P = 0.05). Also HCV RNA plasma levels were found to influence significantly the surface marker

421

expression on DC. In particular, CD40 and CD80 expression on immature DC was significantly higher at high viraemia levels (P = 0.04 and 0.007, respectively) (Figs. 2.1C and D). Raw data and correlations corresponding to Figs. 2.1(A to D) are presented separately in Figs. 2.2(A to D respectively). The expression of DC phenotypic markers (CD40, CD80, CD83, CD86, HLA-DR) was not affected by the presence of HCV RNA or the concentration of HCV core protein in DC lysates neither was it affected by ethanol consumption and fibrosis stage (data not shown).

Figure 5 (A) Autologous CD4+ T-cell response, tested with IFN-γ Elispot assay, for ‘good’ and ‘poor’ responders to antiviral treatment, with MDDC pulsed with recombinant HCV core protein. (⁎) Significant increase in IFN-γ production at TW12 as compared to BL values in ‘good’ responders to treatment (P = 0.036); (†) Significant difference (P = 0.038) in Δ(Delta) (TW12-preRx) values between ‘good’ responders and ‘poor’ responders (non-responders) (ΔIFN-γ). (B) Allostimulatory capacity in ‘good’ and in ‘poor’ responders to antiviral treatment, tested with MLR assay. (‡) Significant difference (P = 0.048) in Δ(Delta) (TW12-preRx) values between responders and non-responders (ΔMLR). Boxplot extending from the 25th to the 75th percentile and whiskers to the largest and smallest observed values within 1.5 box lengths; the solid line is the median. Numbers of data-points appear inside the boxplots.

422 Plasma levels of HCV core protein affected significantly the DC allostimulatory capacity (P = 0.001; Fig. 3.1). Raw data and correlations appear in Fig. 3.2. A significant inverse correlation between ethanol consumption and IL-12p70 secretion (P = 0.02) (Fig. 4) was also observed. The presence of HCV genome (either positive or negative strand HCV RNA) or HCV core protein in DC lysates had no statistically significant effect on MDDC function or surface marker expression (data not shown).

Effect of antiviral treatment on DC functions MDDC from 22 of the 35 studied patients were tested both before the initiation of antiviral treatment and at treatment week 12 (TW12). Patients were divided into: ‘good responders’ to treatment (n = 17) — undetectable HCV RNA in

I. Pachiadakis et al. plasma at TW12 and ‘poor responders’ to treatment (n = 5) — still detectable HCV RNA at TW12. A significant increase in the frequency of IFN-γ-producing T-cells at TW12 was observed in ‘good responders’ when DC were pulsed with recombinant HCV core, compared to pretreatment (P = 0.036), while there were no significant changes in the ‘poor-responder’ group (Fig. 5A). Furthermore a significant difference was observed between ‘good responders’ and ‘poor responders’ in Δ(Delta) (TW12 — pretreatment) values for the number of IFN-γ-producing T-cells after HCV core pulsing (P = 0.038) (Fig. 5A). Early viraemia clearance was also associated with improved allostimulatory capacity of DC. Allogeneic CD4+ T cell proliferation at TW12 was increased, non-significantly though, in ‘good responders’, while ‘poor responders’ also showed a non-significant decrease, as compared to pretreatment. The difference between ‘good’ and ‘poor-

Figure 6 IL-12p70 production prior to the initiation of antiviral treatment (baseline, BL) and at week 12 of antiviral treatment (TW12), in ‘good’ (undetectable HCV RNA at TW12) and ‘poor’ (still detectable HCV RNA at TW12) responders to antiviral treatment. (⁎) Significant difference in ΔIL-12 (TW12-BL) values between ‘good’ and ‘poor’ responders. Boxplot extending from the 25th to the 75th percentile and whiskers to the largest and smallest observed values within 1.5 box lengths; the solid line is the median.

Early viraemia clearance during hepatitis C therapy improves dendritic cell functions

423

responders’ in Δ(Delta) (TW12 — pre-treatment) values for ‘naïve’ CD4+ T cell proliferation (MLR) was statistically significant (P = 0.048) (Fig. 5B). Both ‘good responders’ and ‘poor responders’ demonstrated a decrease in IL-12p70 secretion at TW12 when compared to baseline, with the reduction (ΔIL12) being significantly more obvious in the ‘good’ responder group (P = 0.041) (Fig. 6).

Analysis of DC functions in a ‘cross-over’ experiment In order to define whether the enhanced adaptive responses that we observed in patients with early, on treatment, HCV clearance were due to improvement of the DC function or of the CD4+ T-cell reactivity, we performed a ‘cross-over’ experiment (Fig. 7). PBMC were isolated both before treatment and at TW12 from eight patients who were ‘good responders’ to antiviral treatment (had undetectable HCV RNA at TW12) and were part of the initial group of the 22 treated patients. When MDDC generated from PBMC harvested at baseline were tested against autologous CD4+ T-cells isolated both before the initiation of treatment (baseline, BL) and at TW12 (pulsed with either recombinant HCV core or NS3), in an IFN-γ Elispot assay, no significant difference was observed with regard to the frequency of IFNγ-producing CD4+ T-cells, between ‘treated’ (TW12) and ‘untreated’ (BL) CD4+ T-cells. In contrast, when MDDC generated at TW12 (‘treated’) were tested against the same two subsets of CD4+ T-cells (isolated at baseline visit and at TW12 and pulsed with recombinant HCV core), a trend (P = 0.084) towards an increased frequency of IFN-γ-producing CD4+ T-cells was observed (Fig. 7A) and a similar trend (P = 0.062) was also seen when the response to HCV NS3 was assessed (Fig. 7B).

Discussion The mechanisms that HCV uses to down-regulate antiviral immunity, thus promoting HCV persistence, are still not well defined. To gain a better understanding, we investigated the impact of MDDC phenotypic and functional changes in relation to early on-treatment viraemia clearance on CD4+ T-cell reactivity, extending previous findings that rapid viraemia clearance is associated with enhanced HCV-specific, T-cell responses [36]. We demonstrated that in patients who cleared the virus by TW12, the enhanced adaptive immunity observed is related to restoration of DC functions, which was not observed in patients who remained viraemic during treatment with peg-interferon/ribavirin (Figs. 5A and B). We also observed overall defective DC function in the setting of chronic HCV infection (patients vs non-infected controls) (Fig. 1). A novel aspect in the present study was to quantitate HCV core antigen levels in patients' sera and in MDDC lysates, in correlation to DC function and phenotype. Most previous studies used transfection of naïve DC with either adenovirus vectors expressing HCV proteins [23,24] and liposomal transfection agents [42], or by pulsing cultures of DC or PBMC (‘naïve’ or CHC) with recombinant HCV core protein [31]. Our observations indicate that HCV core present inside DC, does not influence DC function and phenotype while high

Figure 7 (A) ‘Cross-over’ experiment with recombinant HCV core pulsing (DC and CD4+ T-cells both from BL-pre-treatment, and from TW12-week 12 of antiviral treatment). A trend towards an increased contribution of ‘treated’ MDDC to the improved IFN-γ production from autologous CD4+ T-cells (P = 0.084), is observed. (B) ‘Cross-over’ experiment with recombinant HCV NS3 pulsing (DC and CD4+ T-cells both from BL, and from TW12). Again a trend towards an increased contribution of ‘treated’ MDDC to the improved IFN-γ production from autologous CD4+ Tcells (P = 0.062), is observed.

HCV core plasma levels were shown to be related to increased maturation (CD83, Fig. 2.1A; P= 0.05), and co-stimulatory (CD86, Fig. 2.1B; P= 0.05) marker expression on DC surface as well as to enhanced DC allostimulatory capacity (Fig. 3.1; P = 0.001). Thus, contrary to previous reports [20,23,24] and in line with other studies [42,43] our experiments show a ‘stimulatory’ effect of circulating HCV viral products (plasma core protein) on DC phenotype and function. In a similar manner, HCV viraemia levels appear directly related to DC activation and co-stimulation phenotypic marker expression (CD40 and CD80 on immature DC with P values 0.04 and 0.007 respectively, Figs. 2.1C and D) but not to any other functional output of DC. The observed increase in MLR activity in samples from subjects with high HCV core plasma levels combined with the observed defect in allostimulatory capacity of MDDC derived from HCV subjects as a whole (compared to healthy controls) is suggestive of other factors, in the setting of chronic HCV infection, that ‘outdo’ the stimulatory effect of core antigen.

424 Using an ultra-sensitive PCR methodology [37], the positive strand HCV RNA was detected in MDDC lysates in 13 of 33 patients. Our observations also demonstrate the presence of a low level replication of HCV in monocyte-derived dendritic cells, supporting previous studies [19,44]. The detection of the replicative intermediate, negative strand HCV RNA, in MDDC lysates (in 5 of the 13 patients with detectable positive strand HCV RNA) argues against the presence of HCV sequences and antigens in DC being only the product of non-specific scavenging of virions by dendritic cells. Although both HCV RNA and HCV core proteins are detectable in DC, the present study suggests that they do not affect directly the DC functions. The presence and replication of HCV in DC do not appear to affect the functional output (at least the functional parameters that we tested) and the phenotype of the latter, but is probably a mechanism that HCV implements to evade immunological control. It was shown previously [45] that HCV is internalized via binding to DC-SIGN receptors in non-lysosomal compartments in DC, escapes degradation and probably ‘hides’ from the immune system, facilitating viral dissemination. As an ‘immunologically privileged’ reservoir of the virus, DC may contribute to viral persistence, even after successful control of viraemia, through antiviral treatment (SVR), or spontaneous clearance [37]. With regard to patients' characteristics affecting DC function in CHC, we observed a suppressive effect of alcohol on DC IL-12p70 secretion (P = 0.02) (Fig. 4), potentially leading to impaired induction of Th1 responses. An interesting observation regarding IL-12p70 production from DC, both in ‘good responders’ and ‘poor responders’, is that it appears decreased at TW12 compared to baseline levels (Fig. 6), probably as an effect of the antiviral treatment itself, as also demonstrated elsewhere [46]. In the present study we used a novel strategy to dissect whether the impaired immune response to HCV is primarily due to defect of DC or the effector T-cells. We conducted “cross-over” experiments with DC and CD4+ T-cells isolated from patients that cleared the virus early on treatment (DC and T-cells hypothesised with improved functions because of early on-treatment viraemia clearance) to interact respectively with DC and T-cells functionally impaired by the chronic infection. An increased frequency of IFN-γ-producing CD4+ Tcells was associated with ‘treatment’ of DC, but not of CD4+ T-cells (Fig. 7). Therefore, our “cross-over” observations, for the first time demonstrate the importance of dendritic cells in the development of an effective adaptive response against HCV and render DC a potential primary target for immunemodulatory therapeutic approaches to chronic hepatitis C. In conclusion, dendritic cell function is impaired in patients with chronic HCV infection and the dysfunction is associated with viral load. This observation is supported by the finding that viraemia clearance, early on-treatment, leads to the improvement of DC functions which was not observed in patients that remain viraemic on-therapy. The present study also demonstrates that improvement of antiviral immunity is due to improved DC function as a result of viraemia clearance.

Acknowledgment This study was funded in part by a research grant from Schering-Plough, Welwyn Garden City, United Kingdom.

I. Pachiadakis et al.

References [1] F. Lechner, D.K. Wong, P.R. Dunbar, R. Chapman, R.T. Chung, P. Dohrenwend, G. Robbins, R. Phillips, P. Klenerman, B.D. Walker, Analysis of successful immune responses in persons infected with hepatitis C virus, J. Exp. Med. 191 (2000) 1499–1512. [2] T. Poynard, M.F. Yuen, V. Ratziu, C.L. Lai, Viral hepatitis C, Lancet 362 (2003) 2095–2100. [3] F. Lechner, N.H. Gruener, S. Urbani, J. Uggeri, T. Santantonio, A.R. Kammer, A. Cerny, R. Phillips, C. Ferrari, G.R. Pape, P. Klenerman, CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained, Eur. J. Immunol. 30 (2000) 2479–2487. [4] R. Thimme, D. Oldach, K.M. Chang, C. Steiger, S.C. Ray, F.V. Chisari, Determinants of viral clearance and persistence during acute hepatitis C virus infection, J. Exp. Med. 194 (2001) 1395–1406. [5] R. Thimme, J. Bukh, H.C. Spangenberg, S. Wieland, J. Pemberton, C. Steiger, S. Govindarajan, R.H. Purcell, F.V. Chisari, Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 15661–15668. [6] M.E. Cramp, P. Carucci, S. Rossol, S. Chokshi, G. Maertens, R. Williams, N.V. Naoumov, Hepatitis C virus (HCV) specific immune responses in anti-HCV positive patients without hepatitis C viraemia, Gut 44 (1999) 424–429. [7] P. Botarelli, M.R. Brunetto, M.A. Minutello, P. Calvo, D. Unutmaz, A.J. Weiner, Q.L. Choo, J.R. Shuster, G. Kuo, F. Bonino, et al., Tlymphocyte response to hepatitis C virus in different clinical courses of infection, Gastroenterology 104 (1993) 580–587. [8] R.M. Steinman, M.D. Witmer, Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice, Proc. Natl. Acad. Sci. U. S. A 75 (1978) 5132–5136. [9] R.M. Steinman, D. Hawiger, M.C. Nussenzweig, Tolerogenic dendritic cells, Annu. Rev. Immunol. 21 (2003) 685–711. [10] P. Guermonprez, J. Valladeau, L. Zitvogel, C. Thery, S. Amigorena, Antigen presentation and T cell stimulation by dendritic cells, Annu. Rev. Immunol. 20 (2002) 621–667. [11] J. Engelmayer, M. Larsson, M. Subklewe, A. Chahroudi, W.I. Cox, R.M. Steinman, N. Bhardwaj, Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion, J. Immunol. 163 (1999) 6762–6768. [12] I. Fugier-Vivier, C. Servet-Delprat, P. Rivailler, M.C. Rissoan, Y.J. Liu, C. Rabourdin-Combe, Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells, J. Exp. Med. 186 (1997) 813–823. [13] I. Grosjean, C. Caux, C. Bella, I. Berger, F. Wild, J. Banchereau, D. Kaiserlian, Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells, J. Exp. Med. 186 (1997) 801–812. [14] M. Moutaftsi, A.M. Mehl, L.K. Borysiewicz, Z. Tabi, Human cytomegalovirus inhibits maturation and impairs function of monocyte-derived dendritic cells, Blood 99 (2002) 2913–2921. [15] G. Pollara, K. Speidel, L. Samady, M. Rajpopat, Y. McGrath, J. Ledermann, R.S. Coffin, D.R. Katz, B. Chain, Herpes simplex virus infection of dendritic cells: balance among activation, inhibition, and immunity, J. Infect. Dis. 187 (2003) 165–178. [16] M.J. Raftery, M. Schwab, S.M. Eibert, Y. Samstag, H. Walczak, G. Schonrich, Targeting the function of mature dendritic cells by human cytomegalovirus: a multilayered viral defense strategy, Immunity 15 (2001) 997–1009. [17] C. Servet-Delprat, P.O. Vidalain, H. Bausinger, S. Manie, F. Le Deist, O. Azocar, D. Hanau, A. Fischer, C. Rabourdin-Combe, Measles virus induces abnormal differentiation of CD40 ligand-activated human dendritic cells, J. Immunol. 164 (2000) 1753–1760. [18] S. Auffermann-Gretzinger, E.B. Keeffe, S. Levy, Impaired dendritic cell maturation in patients with chronic, but not resolved, hepatitis C virus infection, Blood 97 (2001) 3171–3176.

Early viraemia clearance during hepatitis C therapy improves dendritic cell functions [19] C. Bain, A. Fatmi, F. Zoulim, J.P. Zarski, C. Trepo, G. Inchauspe, Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection, Gastroenterology 120 (2001) 512–524. [20] A. Dolganiuc, K. Kodys, A. Kopasz, C. Marshall, T. Do, L. Romics Jr., P. Mandrekar, M. Zapp, G. Szabo, Hepatitis C virus core and nonstructural protein 3 proteins induce pro- and antiinflammatory cytokines and inhibit dendritic cell differentiation, J. Immunol. 170 (2003) 5615–5624. [21] T. Kanto, N. Hayashi, T. Takehara, T. Tatsumi, N. Kuzushita, A. Ito, Y. Sasaki, A. Kasahara, M. Hori, Impaired allostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis C virus-infected individuals, J. Immunol. 162 (1999) 5584–5591. [22] C. Rollier, J.A. Drexhage, B.E. Verstrepen, E.J. Verschoor, R.E. Bontrop, G. Koopman, J.L. Heeney, Chronic hepatitis C virus infection established and maintained in chimpanzees independent of dendritic cell impairment, Hepatology 38 (2003) 851–858. [23] P. Sarobe, J.J. Lasarte, A. Zabaleta, L. Arribillaga, A. Arina, I. Melero, F. Borras-Cuesta, J. Prieto, Hepatitis C virus structural proteins impair dendritic cell maturation and inhibit in vivo induction of cellular immune responses, J. Virol. 77 (2003) 10862–10871. [24] P. Sarobe, J.J. Lasarte, N. Casares, d.C. Lopez-Diaz, E. Baixeras, P. Labarga, N. Garcia, F. Borras-Cuesta, J. Prieto, Abnormal priming of CD4(+) T cells by dendritic cells expressing hepatitis C virus core and E1 proteins, J. Virol. 76 (2002) 5062–5070. [25] R.S. Longman, A.H. Talal, I.M. Jacobson, M.L. Albert, C.M. Rice, Patients chronically infected with hepatitis C virus have functional dendritic cells, Blood (2003). [26] R.S. Longman, A.H. Talal, I.M. Jacobson, M.L. Albert, C.M. Rice, Presence of functional dendritic cells in patients chronically infected with hepatitis C virus, Blood 103 (2004) 1026–1029. [27] D. Piccioli, S. Tavarini, S. Nuti, P. Colombatto, M. Brunetto, F. Bonino, P. Ciccorossi, F. Zorat, G. Pozzato, C. Comar, S. Abrignani, A. Wack, Comparable functions of plasmacytoid and monocyte-derived dendritic cells in chronic hepatitis C patients and healthy donors, J. Hepatol. 42 (2005) 61–67. [28] E. Barnes, M. Salio, V. Cerundolo, L. Francesco, D. Pardoll, P. Klenerman, A. Cox, Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus, J. Viral Hepatitis 15 (2008) 219–228. [29] M. Zimmermann, C. Flechsig, N. La Monica, M. Tripodi, G. Adler, N. Dikopoulos, Hepatitis C virus core protein impairs in vitro priming of specific T cell responses by dendritic cells and hepatocytes, J. Hepatol. 48 (2008) 51–60. [30] R.S. Longman, A.H. Talal, I.M. Jacobson, C.M. Rice, M.L. Albert, Normal functional capacity in circulating myeloid and plasmacytoid dendritic cells in patients with chronic hepatitis C, J. Infect. Dis. 192 (2005) 497–503. [31] A. Dolganiuc, S. Chang, K. Kodys, P. Mandrekar, G. Bakis, M. Cormier, G. Szabo, Hepatitis C virus (HCV) core proteininduced, monocyte-mediated mechanisms of reduced IFNalpha and plasmacytoid dendritic cell loss in chronic HCV infection, J. Immunol. 177 (2006) 6758–6768. [32] T. Kanto, M. Inoue, H. Miyatake, A. Sato, M. Sakakibara, T. Yakushijin, C. Oki, I. Itose, N. Hiramatsu, T. Takehara, A. Kasahara, N. Hayashi, Reduced numbers and impaired ability of myeloid and plasmacytoid dendritic cells to polarize T helper

[33]

[34]

[35] [36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

425

cells in chronic hepatitis C virus infection, J. Infect. Dis. 190 (2004) 1919–1926. J. Mellor, A. Hawkins, P. Simmonds, Genotype dependence of hepatitis C virus load measurement in commercially available quantitative assays, J. Clin. Microbiol. 37 (1999) 2525–2532. K. Ishak, A. Baptista, L. Bianchi, F. Callea, J. De Groote, F. Gudat, H. Denk, V. Desmet, G. Korb, R.N. MacSween, et al., Histological grading and staging of chronic hepatitis, J. Hepatol. 22 (1995) 696–699. B. Rehermann, N.V. Naoumov, Immunological techniques in viral hepatitis, J. Hepatol. 46 (2007) 508–520. K.H. Tang, E. Herrmann, H. Cooksley, N. Tatman, S. Chokshi, R. Williams, S. Zeuzem, N.V. Naoumov, Relationship between early HCV kinetics and T-cell reactivity in chronic hepatitis C genotype 1 during peginterferon and ribavirin therapy, J. Hepatol. 43 (2005) 776–782. T.N. Pham, S.A. MacParland, P.M. Mulrooney, H. Cooksley, N.V. Naoumov, T.I. Michalak, Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C, J. Virol. 78 (2004) 5867–5874. R.E. Lanford, D. Chavez, F.V. Chisari, C. Sureau, Lack of detection of negative-strand hepatitis C virus RNA in peripheral blood mononuclear cells and other extrahepatic tissues by the highly strand-specific rTth reverse transcriptase PCR, J. Virol. 69 (1995) 8079–8083. S. Laperche, N. Le Marrec, N. Simon, F. Bouchardeau, C. Defer, M. Maniez-Montreuil, T. Levayer, J.P. Zappitelli, J.J. Lefrere, A new HCV core antigen assay based on disassociation of immune complexes: an alternative to molecular biology in the diagnosis of early HCV infection, Transfusion 43 (2003) 958–962. L.H. Tobler, S.L. Stramer, S.R. Lee, D. Baggett, D. Wright, D. Hirschkorn, I. Walsh, M.P. Busch, Performance of ORTHO HCV core antigen and trak-C assays for detection of viraemia in preseroconversion plasma and whole blood donors, Vox Sang. 89 (2005) 201–207. H.L. Tillmann, J. Wiegand, I. Glomb, A. Jelineck, G. Picchio, H. Wedemeyer, M.P. Manns, Diagnostic algorithm for chronic hepatitis C virus infection: role of the new HCV-core antigen assay, Z. Gastroenterol. 43 (2005) 11–16. W. Li, J. Li, D.L. Tyrrell, B. Agrawal, Expression of hepatitis C virus-derived core or NS3 antigens in human dendritic cells leads to induction of pro-inflammatory cytokines and normal T-cell stimulation capabilities, J. Gen. Virol. 87 (2006) 61–72. W. Li, D.K. Krishnadas, J. Li, D.L. Tyrrell, B. Agrawal, Induction of primary human T cell responses against hepatitis C virusderived antigens NS3 or core by autologous dendritic cells expressing hepatitis C virus antigens: potential for vaccine and immunotherapy, J. Immunol. 176 (2006) 6065–6075. N. Goutagny, A. Fatmi, V.D. Ledinghen, F. Penin, P. Couzigou, G. Inchauspe, C. Bain, Evidence of viral replication in circulating dendritic cells during hepatitis C virus infection, J. Infect. Dis. 187 (2003) 1951–1958. I.S. Ludwig, A.N. Lekkerkerker, E. Depla, F. Bosman, R.J. Musters, S. Depraetere, Y. van Kooyk, T.B. Geijtenbeek, Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation, J. Virol. 78 (2004) 8322–8332. E. Barnes, M. Salio, V. Cerundolo, J. Medlin, S. Murphy, G. Dusheiko, P. Klenerman, Impact of alpha interferon and ribavirin on the function of maturing dendritic cells, Antimicrob. Agents Chemother. 48 (2004) 3382–3389.