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Digestive and Liver Disease 43 (2011) 807– 813

Contents lists available at ScienceDirect

Digestive and Liver Disease

j our nal ho me page: www.elsev ier .com/ locate /d ld

iver, Pancreas and Biliary Tract

edistribution of regulatory T-cells across the evolvingtages of chronic hepatitis C

ilvia Ferri a,∗, Claudine Lalannea, Giulia Lanzonia, Mirna Bassib, Sofia Asioli c,alentina Ciprianoa, Georgios Pappasa, Paolo Muratori a, Marco Lenzia, Luigi Muratori a

Department of Clinical Medicine, University of Bologna, Bologna, ItalyDepartment of Clinical Pathology, S. Orsola-Malpighi Hospital, Bologna, ItalyDepartment of Biomedical Sciences and Human Oncology, University of Turin, Turin, Italy

r t i c l e i n f o

rticle history:eceived 14 January 2011ccepted 28 April 2011

eywords:hronic HCV-related liver diseaseecompensated cirrhosisELD score

egulatory T-cells

a b s t r a c t

Background: Hepatitis C virus infection frequently leads to chronic hepatitis, possibly evolving to end-stage liver disease and hepatocellular carcinoma. Regulatory T cells can affect antiviral immune responsethus influencing the outcome of the disease.Aim: To determine numeric and functional distribution of regulatory T cells expressingCD4+CD25hiFoxp3+ (T-regs) during the different stages of hepatitis C virus-related liver disease.Methods: 90 hepatitis C viraemic patients and 50 healthy controls were included. Surface and intracellular(Foxp3) T-reg markers were evaluated by flow cytometry. Target cell proliferation and interferon-gammaproduction were evaluated in 37 HCV patients. In 16 cases intrahepatic distribution of Foxp3 by immuno-histochemistry was assessed.Results: T-regs were increased in hepatitis C virus infected patients and correlated inversely withaminotransferases and directly with MELD score and disease duration. A preserved inhibitory ability

of interferon-gamma production was distinctive of patients with normal aminotransferases. CirculatingT-regs did not correlate with intrahepatic distribution of Foxp3.Conclusions: In chronic hepatitis C, selective expansion of peripheral T-regs in patients with normalaminotransferases and advanced disease suggests that, though a continual low level inflammation doesnot prevent liver disease progression, once cirrhosis has developed it may represent an attempt to preventimmuno-mediated decompensation.

Gast

© 2011 Editrice

. Introduction

Hepatitis C virus (HCV) infection affects some 180 million indi-iduals worldwide and in the vast majority of cases it leads tohronic liver disease; a proportion of these patients are expectedo develop, over 2–4 decades, progressive hepatic fibrosis possiblyvolving in cirrhosis and liver failure, or hepatocellular carcinoma1,2].

Different host factors such as older age, male gender, African-merican ethnicity, alcoholism, smoking, co-infection with human

mmunodeficiency virus (HIV), hepatitis B virus (HBV) and co-

orbidity with steatosis and metabolic syndrome are responsible

or a quicker evolution of the disease; whereas viral features, suchs HCV viral load and genotypes, are not universally associated with

∗ Corresponding author at: Department of Clinical Medicine, University ofologna, Via Massarenti 9, 40138 Bologna, Italy. Tel.: +39 051 6363865;

ax: +39 051 6363631.E-mail address: silvia.ferri9@unibo.it (S. Ferri).

590-8658/$36.00 © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevieroi:10.1016/j.dld.2011.04.020

roenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

liver disease progression [3]. Immunological factors play a promi-nent role in controlling HCV infection, whose eradication requiresa coordinated inter-play between innate and adaptive immunity. Avigorous and multi-specific immune response, involving CD4 andCD8 T lymphocytes, NK cells, B lymphocytes and dendritic cells,is associated with pathogen clearance and disease resolution. Incontrast, a narrowly focused and/or delayed response of T andB lymphocytes seems to favour the development of chronic HCVinfection [4], but, limiting liver damage, may ultimately guaranteehost survival [5].

In the last decade, new classes of T lymphocytes with regula-tory properties capable of suppressing effector T cells have beendescribed. Amongst them, the best known are the so-called T-regs, naturally occurring CD4+CD25+T cells expressing CD25 athigh levels (CD25hi), surface markers such as CD45RO and CD62Land the forkhead/winged helix transcription factor Foxp3 [6–9].

CD4+CD25hiFoxp3+ T cells suppress the activities of effector Tcells both directly and indirectly via down-regulation of antigen-presenting cells by reducing their histo-compatibility moleculeclass II (MHC-II) and costimulatory protein expression, alter-

Ltd. All rights reserved.

808 S. Ferri et al. / Digestive and Liver Disease 43 (2011) 807– 813

Table 1Clinical, biochemical and virological features of hepatitis C virus positive patients as a whole and according to the stage of liver disease.

HCV (n = 90) CHC (n = 51) HCC (n = 23) ESLD (n = 16)

Age (years)* 60 (23–92) 61 (23–92) 60 (45–84) 60 (43–72)Sex (M/F) 54/36 23/28 21/22 10/6AST (×unl) 2.0 ± 1.1 1.9 ± 1.2 2.2 ± 1.2 1.8 ± 0.7ALT (×unl) 2.0 ± 1.5 2.3 ± 1.5 1.8 ± 1.4 1.0 ± 0.42,3

Gamma-GT (×unl) 1.1 ± 1.0 1.1 ± 0.9 1.6 ± 1.4 0.5 ± 0.41,4

Viral load (×103 UI)* 361 (7–7692) 597 (24–7692) 257 (37–1388) 54 (7–327) 2

Genotype (%)1 70 67 69 842 15 19 12 83 12 14 12 04 3 0 7 8

Disease (months) 205 ± 144 163 ± 105 305 ± 192 384 ± 1651

Bilirubin (mg/dl)* 1.2 (0.3–55.1) 0.6 (0.3–6.1) 1.7 (0.6–55.1) 2 6.9 (1.2–37.0)2,4

INR 1.45 ± 0.57 1.10 ± 0.12 1.52 ± 0.432 2.18 ± 0.692,4

Total proteins (g/dl) 7.3 ± 1.0 7.7 ± 0.6 7.0 ± 1.21 6.7 ± 1.01

Gamma-globulins (g/dl) 1.7 ± 0.7 1.5 ± 0.5 2.1 ± 0.91 1.9 ± 0.9Alpha fetoprotein (ng/ml)* 8 (1–1954) 8 (2–41) 23 (4–1954)1 2 (1–33)1,4

Lymphocytes (/�l) 1528 ± 1014 2124 ± 979 899 ± 4812 743 ± 4602

MELD score 14 ± 8 8 ± 2 15 ± 61 24 ± 61,4

HCV: hepatitis C virus positive patients, CHC: chronic hepatitis C, HCC: hepatocelluar carcinoma, ESLD: end-stage liver disease, AST: aspartate transaminase, ALT: alaninetransaminase, gamma-GT: gamma-glutamil transpeptidase, INR: International Normalized Ratio, MELD: Mayo End-stage Liver Disease score.unl: upper normal level.D

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ata are expressed as mean ± standard deviation.* Median with range in parentheses:p < 0.01 vs. CHC; 2 p < 0.001 vs. CHC; 3 p < 0.05 vs. HCC; 4 p < 0.01 vs. HCC.

ng their cytokine secretive abilities and modulating tryptophanatabolism [10]. Several mechanisms of suppression have been pro-osed, including the secretion of immunosuppressive cytokinesnd cell-to-cell-contact leading to functional modification or directilling of target cells [6,11]. Peripheral T-regs are expanded dur-ng acute HCV infection [12–15], maintained at high levels duringhe chronic phase [16–19], when they also represent a key com-onent of the liver-infiltrating lymphocytes [20,21], and loweredo the same level as control subjects upon spontaneous recoveryr treatment-induced HCV eradication [13,16]. The further expan-ion of T-regs observed in patients with hepatocellular carcinomaas been associated with tumour recurrence and poor prognosis22–27], but only scarce information is available on T-regs duringhe advanced stages of HCV-related liver disease [27].

In this paper we analyse T-reg frequency and function during thevolving stages of liver disease in HCV-infected patients demon-trating a selective and progressive expansion of T-regs in advancediver disease.

. Patients and methods

.1. Patients

Ninety consecutive patients with HCV-related chronic liver dis-ase not receiving antiviral therapy (median age 60 years [range3–92], 57% males) were enrolled between January 2009 and Jan-ary 2010. Fifty blood donors served as healthy controls (HC,edian age 42 years [range 25–66], 54% males). The local ethi-

al committee approved the study and all patients gave writtennformed consent to participate in the study.

All patients were HCV RNA-positive by bDNA or TMA testing.nown viral, metabolic, genetic and autoimmune causes of liverisease were excluded.

Patients were divided into 3 groups according to the stagef their liver disease, namely: chronic hepatitis C (CHC, n = 51),epatocellular carcinoma (HCC, n = 23) and decompensated cir-

hosis with end-stage liver disease (ESLD, n = 16) on the waitingist for liver transplantation. We also enrolled 5 patients withompensated cirrhosis which were included in the CHC group,aving comparable biochemical and immunological parameters.

HCC was diagnosed by ultrasonography and confirmed by contrast-enhanced ultrasonography, computed tomography or magneticresonance.

Liver disease was presumed to have begun from the first deter-mination of increased levels of serum aminotransferases togetherwith the detection of anti-HCV antibodies and HCV-RNA. Prior to1989, only increased aminotransferases were used to date diseaseinitiation.

Each subgroup of patients was subdivided into 3 subsets accord-ing to the degree of hepatocyte necrosis expressed as alanineaminotransferase (ALT) levels in at least two consecutive tests: [N]persistently normal ALT, [A] ALT levels ≥2.5 times the upper nor-mal levels and [I] intermediate ALT levels. ALT was chosen as theserological marker of liver damage because of its selective increasefollowing hepatocyte necrosis. Clinical and laboratory features ofpatients are reported in Table 1.

2.2. Flow cytometry

Six-colour flow-cytometry analysis was performed on freshwhole blood. Fifty microliters of peripheral blood were stained withthe following combinations of anti-surface antigens monoclonalantibodies: phycoerythrin-cychrome (PE-Cy7)-conjugated anti-CD4 and phycoerythrin (PE)-conjugated anti-CD25; fluorescein(FITC)-conjugated anti-CD25/PE-CD45RO; FITC-CD25/PE-CD62L(BD Biosciences, San Jose, CA, USA). To better characterise T-regulatory cells, samples were also fixed, permealized (Fix andPerm, Caltag Medsystems, Buckingham, UK) and stained with FITC-conjugated anti-FOXP3 (eBioscience, Inc., San Diego, CA). Foxp3production was evaluated both as percentage and as mean fluo-rescence intensity (MFI) of positive cells (Fig. 1).

Flow-cytometry was performed on a Becton Dickinson flow-cytometer (FACScanto II, BD Biosciences, San Jose, CA, USA) andFACSDiva Software was used for analysis. A mean of 20,000 lym-phocyte gated events per sample were collected.

2.3. Cell separation and purification

Peripheral blood mononuclear cells (PBMCs) were separatedusing Ficoll Histopaque (Sigma–Aldrich s.r.l. Milan, Italy). Cell via-

S. Ferri et al. / Digestive and Liver Disease 43 (2011) 807– 813 809

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ig. 1. A representative example of flow cytometry on peripheral blood mononuclells; PBMC are included in the square. (B) Expression of CD4 and CD25 amongst Pigh intensity. (C) Amongst CD4+CD25hi cells, T-regs express Foxp3 and represent

ility, determined by Trypan blue exclusion, always exceeded8%. CD4+CD25+ T cells were isolated using immuno-magneticeads (CD4+CD25+ Regulatory T Cell Isolation Kit, Miltenyi Biotec,ergisch Gladbach, Germany). The resulting CD4+CD25− T lympho-ytes were used as targets in function experiments. The purity ofhe CD4+CD25+ T-regs and of the CD4+CD25− subset exceeded 95%nd 92%, respectively; Foxp3 was expressed by more than 90% ofsolated CD4+CD25+ T lymphocytes.

.4. Functional assays

Purified CD4+CD25+ T cells were added at a ratio of 1:4 toutologous CD4+CD25− cells. Control cultures with CD4+CD25−

cells alone were performed under identical conditions. Cellsere cultured for 5 days in RPMI 1640, supplemented with 2 mM

-glutamine, 25 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml strep-omycin, 2.5 �g/ml amphotericin B, and 10% inactivated FCS at7 ◦C and 5% CO2, in the presence of a T cell expander (CD3/CD28ynabeads, Dynal Invitrogen, Oslo, Norway). Recombinant IL-2

Chiron) was added at a concentration of 30 U/ml on day 1. Allhe proliferation and cytokine production experiments were per-ormed in triplicate.

Cell proliferation was tested using Click-iT EdU Alexa Fluor47 Flow Cytometry Assay (Invitrogen Life Technologies, Carls-ad, CA, USA), following the manufacturer’s instructions. Briefly,ells were labelled with 5-ethynil-2′-deoxyuridine (EdU), a nucle-side analogue to thymidine that is incorporated into DNA duringctive DNA synthesis. After 16 h of incubation, cells were harvested,ashed and labelled with FITC-conjugated anti-CD4 antibody (BDiosciences, San Jose, CA, USA). After fixation and permealization,dU incorporation was detected by a click reaction and standardow cytometry was used to determine the percentage of cells in-phase.

On the fifth day of culture, cells from different wells cul-ured under the same conditions were exposed, in the presence ofrefeldin A (4 �g/ml), to phorbol 12-myristate 13-acetate (PMA)20 ng/ml) and Ionomycin (500 ng/ml) and incubated for 5 h at7 ◦C and 5% CO2; after washing they were stained with anti-D4 FITC monoclonal antibodies, fixed, permeabilized and finallytained with PE-Cy7 conjugated anti-interferon-gamma (IFN-�)onoclonal antibody (BD Biosciences, San Jose, CA, USA). Cells wereashed once and analysed by flow cytometry.

Percentage inhibition was calculated using the formula: − (percentage of proliferating or IFN-� producing cells in the pres-

nce of CD4+CD25+ T cells/percentage of proliferating or IFN-�roducing cells in the absence of CD4+CD25+ T cells) × 100.

IFN-� production was evaluated both as percentage and as meanuorescence intensity (MFI) of positive cells.

lls (PBMC) of a healthy control. (A) Physical parameters of peripheral blood white T-regs are located in the square embracing cells that co-express CD4 and CD25 at.08% of PBMC.

2.5. Immuno-histochemistry

Immunohistochemical staining for Foxp3 was performed onparaffin-embedded sections of diagnostic liver biopsy specimensobtained from 16 patients with chronic hepatitis (6 HCV[N] and8 HCV[A]). Mean grading and staging were 6 and 2 respectively,according to Ishak scoring system, with no differences betweenHCV[N] and HCV[A].

Immunohistochemical staining was applied to 4-�m-thickparaffin-embedded sections of diagnostic liver biopsy specimens.Sections were incubated at room temperature with the pri-mary antibody FOXP3 (mAbcam 22510, dilution 1:200, Abcam,Cambridge, MA), stained using a standard indirect avidin-biotinhorseradish peroxidase method and diaminobenzidine colourdevelopment, and counterstained with haematoxylin [28]. Onlynuclei with a strongly positive signal were counted. The number oflymphocytes per field ranged from 10 to 400 cells per high powerfield (40× magnification). A mean of 1000 lymphocytes per case(range, 400–1800) was counted.

2.6. Statistical analysis

Mann–Whitney test or t-test were used where appropriate forcontinuous variables, Fisher’s exact test was used for categori-cal variables. Correlation analysis was determined by Pearson’s orSpearman’s correlation coefficient. A value of p < 0.05 was consid-ered significant. Statistical analysis was performed using GraphPadInStat version 3.0a for Macintosh (GraphPad Software Inc., SanDiego, CA, USA).

3. Results

3.1. Clinical and laboratory features

Age and sex were comparable amongst the different groups,with the exception of HCC patients, who were more often men. InESLD, ALT and gamma-glutamiltranspeptidase (�-GT) levels werelower than in patients with CHC and HCC, and the estimatedduration of liver disease was longer in patients with cirrho-sis (HCC and ESLD). As expected, subjects with HCC and ESLDhad higher serum bilirubin levels and International NormalizedRatio (INR), besides a lower lymphocyte count, whereas signifi-cant increase of alpha fetoprotein was distinctive of patients withHCC (Table 1). No differences in HCV genotype distribution were

appreciated, but lower viral load was detected in ESLD. Comparedto patients with increased aminotransferases, those with persis-tently normal and intermediate liver enzyme levels also had lower�-GT levels, both when analysed together (HCV[N]: 34 ± 28 mg/dl,

810 S. Ferri et al. / Digestive and Liver Disease 43 (2011) 807– 813

Table 2T-reg quantification in hepatitis C virus positive patients as a whole, according to the stage of liver disease, transaminase levels.

CD4+CD25+% PBMC CD4+CD25hiFoxp3+% PBMC CD4+CD25hiFoxp3+% CD4+ CD4+CD25hiFoxp3 MFI

HC (n = 50) 13.7 ± 6.1 0.09 ± 0.07 0.28 ± 0.24 2783 ± 804

HCV (n = 90) 14.9 ± 6.5 0.27 ± 0.302 * ,#,§ 0.63 ± 0.732 * ,#,§ 3212 ± 1401 *,#

HCV[N] (n = 34) 13.9 ± 5.5 0.32 ± 0.381 0.78 ± 0.931 3576 ± 16891

HCV[I] (n = 27) 16.5 ± 7.0 0.33 ± 0.291 0.63 ± 0.54 2 3002 ± 962HCV[A] (n = 29) 14.0 ± 6.8 0.14 ± 0.105,6 0.39 ± 0.355,6 2847 ± 12085

CHC (n = 51) 14.7 ± 6.2 0.14 ± 0.14 0.36 ± 0.33 2992 ± 1365CHC[N] (n = 15) 14.4 ± 5.4 0.11 ± 0.10 0.29 ± 0.31 3298 ± 1853CHC[I] (n = 15) 17.6 ± 6.8 0.20 ± 0.19 0.42 ± 0.51 2635 ± 693CHC[A] (n = 21) 12.9 ± 5.9 0.13 ± 0.14 0.38 ± 0.55 2994 ± 1271

HCC (n = 23) 17.2 ± 7.6 0.34 ± 0.272,3 0.71 ± 0.512,3 3296 ± 1604HCC[N] (n = 9) 17.9 ± 6.4 0.42 ± 0.311,7 0.80 ± 0.642,7 4043 ± 2005HCC[I] (n = 9) 18.2 ± 9.2 0.41 ± 0.251,8 0.83 ± 0.441 3038 ± 1064HCC[A] (n = 5) 15.9 ± 8.2 0.14 ± 0.129 0.36 ± 0.159 2188 ± 298

ESLD (n = 16) 12.1 ± 4.34 0.55 ± 0.45 2, 3 1.35 ± 1.102,4,3 3858 ± 10912,3

ESLD[N] (n = 11) 11.1 ± 3.8 0.60 ± 0.511,7 1.53 ± 1.251,7 3862 ± 11372

ESLD[I] (n = 5) 14.3 ± 4.8 0.44 ± 0.30 0.94 ± 0.59 3849 ± 11331,8

HC: healthy controls, HCV: hepatitis C virus positive patients, CHC: chronic hepatitis C, HCC: hepatocellular carcinoma, ESLD: end-stage liver disease, PBMC: peripheral bloodmononuclear cells.[N]: Patients with persistently normal transaminases, [I]: Patients with intermediate levels of transaminases, [A]: Patients with persistently abnormal transaminases.Data are expressed as mean ± standard deviation1 p < 0.05 vs. HC; 2 p < 0.005 vs. HC; 3 p < 0.05 vs. CHC; 4 p < 0.05 vs. HCC; 5 p < 0.05 vs. HCV[N]; 6 p < 0.05 vs. HCV[I]; 7 p < 0.05 vs. CHC[N]; 8 p < 0.05 vs. CHC[I]; 9 p < 0.05 vs.HCC[I].

* Inverse correlation with alanine transaminase (ALT) levels:r = −0.29, p = 0.007 for CD4+CD25hiFoxp3+ ‰ PBMC;r = −0.21, p = 0.05 for CD4+CD25hiFoxp3+ ‰ CD4+;r = −0.30, p = 0.008 for CD4+CD25hiFoxp3 mean fluorescence intensity (MFI).

# Direct correlation with Mayo End-stage Liver Disease (MELD) score:r = 0.50, p < 0.0001 for CD4+CD25hiFoxp3+ ‰ PBMC;r = 0.46, p = 0.0002 for CD4+CD25hiFoxp3+ ‰ CD4+;r = 0.42, p = 0.001 for CD4+CD25hiFoxp3 MFI.§ Direct correlation with duration of disease:

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The ability of regulatory T cells to suppress proliferation andIFN-� production by CD4+CD25− target cells was investigated in16 HC and in 37 HCV patients (11 CHC, 15 HCC, 11 ESLD), of whom

= 0.37, p = 0.01 for CD4+CD25hiFoxp3+ ‰ PBMC; = 0.32, p = 0.03 for CD4+CD25hiFoxp3+ ‰ CD4+; = 0.26, p = 0.07 for CD4+CD25hiFoxp3 MFI.

CV[I]: 41 ± 34 mg/dl, HCV[A]: 122 ± 127 mg/dl, p < 0.0001 and.002 respectively) and as patient subgroups (data not shown).

.2. Frequency and phenotype of regulatory T-cells

The proportion of CD4+CD25hiFoxp3+ in PBMC and in CD4+ells was significantly increased in HCV-positive patients takens a whole compared to HC, whereas CD4+CD25+ T cell fre-uency and Foxp3 mean fluorescence intensity (MFI) were similarTable 2 and data not shown). Amongst HCV patients, HCC andSLD showed significantly more circulating CD4+CD25hiFoxp3+-regs (Fig. 2) that also expressed Foxp3 at higher intensity com-ared to HC, the highest MFI values being found in patientsith ESLD (Table 2). However, T-reg absolute number was notifferent amongst patient subgroups (data not shown). Whenhe patients were stratified according to aminotransferase lev-ls, an ascending climax in the percentage of Foxp3 positive cellsas observed: HCV[A] showed the lowest proportion of posi-

ive cells, HCV[I] an intermediate figure, and HCV[N] reachedhe highest T-reg peak. This climax was observed in all HCVatients as a whole, in HCC and in ESLD, but not in CHC (Table 2,ig. 2).

CD4+CD25hiFoxp3+ frequency and Foxp3 MFI were correlatednversely to ALT levels, but directly to estimated disease durationnd MELD score (Table 2).

CD45RO and CD62L expression, indicative of a central mem-ry phenotype, did not differ between patients and controls,eing present on about 85% of CD4+CD25hi T cells (data nothown).

3.3. Functional assays

Fig. 2. CD4+CD25hiFoxp3+ % peripheral blood mononuclear cells (PBMC) in healthycontrols (HC), hepatitis C virus-positive patients as a whole (HCV) and accord-ing to the stage of liver disease (chronic hepatitis: CHC, hepatocellular carcinoma:HCC, end-stage liver disease: ESLD) or transaminase levels (normal [N]/intermediate[I]/abnormal [A]).

S. Ferri et al. / Digestive and Liver Disease 43 (2011) 807– 813 811

Table 3T-reg functional assays: inhibition of target cell proliferation and interferon-gamma (IFN-�) production in hepatitis C virus positive patients as a whole, according to thestage of liver disease and transaminase levels.

% CD4+CD25− single cultureproliferation

Inhibition of target cellproliferation by T-regs (%)

% CD4+CD25− single cultureIFN-� production

Inhibition of target cell IFN-�production by T-regs (%)

HC (n = 18) 59.3 ± 20.4 51.8 ± 22.2 24.3 ± 14.0 31.8 ± 13.7

HCV (n = 37) 51.7 ± 28.2#,§ 45.9 ± 29.3* 28.0 ± 12.4#,§ 14.0 ± 21.71

HCV[N] (n = 17) 36.6 ± 27.21 67.2 ± 19.91 22.2 ± 13.1 18.5 ± 24.0HCV[I] (n = 10) 54.4 ± 27.3 28.9 ± 23.21,3 30.5 ± 10.9 3.5 ± 6.81

HCV[A] (n = 10) 74.7 ± 10.01,3,4 20.8 ± 17.92,3 34.6 ± 8.91,3 15.0 ± 17.41

CHC (n = 11) 57.5 ± 22.5 44.7 ± 30.9 28.5 ± 10.3 23.5 ± 33.7CHC[N] (n = 4) 45.9 ± 27.2 76.1 ± 10.71 29.5 ± 16.5 61.1 ± 18.51

CHC[I] (n = 3) 50.4 ± 21.9 52.0 ± 29.6 25.9 ± 8.9 30.4 ± 20.0CHC[A] (n = 4) 74.4 ± 4.9 17.6 ± 12.41,5 29.5 ± 4.5 12.0 ± 10.42,5

HCC (n = 15) 45.1 ± 31.3 46.6 ± 31.9 32.1 ± 12.1 24.3 ± 17.9HCC[N] (n = 6) 16.7 ± 16.72 73.4 ± 12.11 21.7 ± 14.6 28.6 ± 17.05

HCC[I] (n = 4) 55.3 ± 21.5 11.0 ± 4.61,6 39.7 ± 6.9 5.2 ± 12.7HCC[A] (n = 5) 71.3 ± 10.86 24.0 ± 23.26 38.6 ± 10.9 22.2 ± 20.0

ESLD (n = 11) 54.9 ± 29.7 46.1 ± 27.3 21.8 ± 10.08 4.8 ± 6.41,7,8

ESLD[N] (n = 7) 46.5 ± 29.5 56.5 ± 26.2 17.3 ± 7.8 5.0 ± 7.31,5,6

ESLD[I] (n = 4) 69.7 ± 27.2 25.2 ± 16.91 29.6 ± 8.99 4.3 ± 6.12

HC: healthy controls, HCV: hepatitis C virus positive patients, CHC: chronic hepatitis C, HCC: hepatocellular carcinoma, ESLD: end-stage liver disease.[N]: Patients with persistently normal transaminases, [I]: patients with intermediate levels of transaminases, [A]: patients with persistently abnormal transaminases.Data are expressed as mean ± standard deviation.1 p < 0.05 vs. HC; 2 p < 0.005 vs. HC; 3 p < 0.05 vs. HCV[N]; 4 p < 0.05 vs. HCV[I]; 5p < 0.05 vs. CHC[N]; 6p < 0.05 vs. HCC[N]; 7p < 0.05 vs. CHC; 8p < 0.05 vs. HCC; 9p < 0.05 vs.ESLD[N].

# Direct correlation with alanine transaminase (ALT) levels: r = 0.52, p = 0.001 for % CD4+CD25− proliferation; r = 0.40, p = 0.013 for % CD4+CD25− IFN-� production.* Inverse correlation with ALT levels: r = −0.67, p < 0.0001.

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7 HCV[N] (mean ALT: 0.8 ± 0.2 times upper normal level [×unl]),0 HCV[I] (mean ALT: 1.5 ± 0.3 × unl) and 10 HCV[A] (mean ALT:.8 ± 1.2 × unl).

After 5-day culture, the percentage of CD4+CD25− target cellsroliferating in single culture experiments was similar in HC andCV patients taken as a whole and in the different stages of the dis-ase; furthermore, the presence of T-regs significantly reduced theroliferation in a comparable degree in all the different subgroups.owever, the spontaneous proliferation rate considerably variedccording to ALT levels: HCV[N] showed a significantly lower tar-et cell proliferation compared to HC and HCV[A] (36.6% vs. 59.3%nd 74.7%, p = 0.011 and p < 0.001 respectively). Similar observa-ions were made in the subgroups, in particular in CHC and HCC.o-culture with T-regs determined a very strong inhibition of base-

ine proliferation in HCV[N], but only partial in HCV[I] and [A] (67.2%s. 28.9% and 20.8%, p < 0.001 for both). The association betweenower ALT levels and high T-reg inhibitory abilities was maintainedlso when we analysed patient subgroups (Table 3, Fig. 3A and B).

An analogous proportion of IFN-� producing target cellsas present in HC and in HCV patients as a whole, but

n contrast with HC, HCV-derived T-regs were poorly effec-ive in inhibiting IFN-� secretion, particularly in ESLD. Thoughn HCV[N] patients the frequency of baseline IFN-� produc-ng target cells was reduced in comparison with HCV[A] (22.2%s. 34.6%, p = 0.015), T-reg presence determined only a weaknhibition of IFN-� production in HCV patients (Fig. 3B) indepen-ently from aminotransferase levels. Moving to patient subgroups,e found a selective impairment of ESLD regulatory cells in inhibit-

ng IFN-� production (p vs. CHC = 0.031, p vs. HCC 0.007), whereasnly T-regs from CHC[N] were able to determine a vigorous IFN-�eduction (61.1%) (Table 3, Fig. 3C and D).

No differences were noticed in the IFN-� MFI between patients

nd controls in any culture setting (data not shown).

In HCV patients as a whole, ALT levels correlated directly withingle culture target cell proliferation and IFN-� production andnversely with the T-reg-mediated inhibition of target cell prolif-

0.05 for % CD4+CD25− proliferation; r = −0.45, p = 0.009 for % CD4+CD25− IFN-�

eration; at variance, MELD score was inversely correlated to targetcell proliferation and IFN-� production (Table 3).

No correlations were observed between CD4+CD25hiFoxp3+frequency, Foxp3 MFI and T-reg effects on target cell proliferationor IFN-� production.

Control experiments were performed under the same condi-tions with T-reg single cultures (no substantial proliferation orIFN-� production) and using CD4+CD25− T cells in place of T-regs in co-culture experiments (no appreciable differences fromCD4+CD25− single cultures) (data not shown).

3.4. Immuno-histochemistry

Foxp3-positive lymphocytes were detected amongst the inflam-matory infiltrate, especially in the portal tracts, in 8 out of 16biopsies (4 HCV[N] and 4 HCV[A]) whereas, in the remaining 8 cases,no lymphocytes expressing Foxp3 were identified (Fig. 4).

On the whole, Foxp3-positive cells represented 8.0% of lym-phocytes infiltrating the liver (range 0–22.5%), with no correlationwith any biochemical, virological or histological parameters (inparticular ALT, HCV viral load, histological stage). In addition, nocorrelation was observed between absolute number and frequencyof intra-hepatic and circulating T-regs (data not shown).

No differences in peripheral T-reg frequency or function werenoted according to histological grade and stage.

4. Discussion

Chronic HCV infection causes progressive liver damage that mayevolve over decades, through sequential phases of the disease, fromchronic hepatitis to compensated cirrhosis, and its long-term com-plications such as HCC and ESLD [1]. The mechanisms leading to

viral persistence in most HCV-infected patients are only partiallyunderstood, however induction of T-reg priming and expansionmay be one of the strategies exploited by the virus to escapeimmune control [5].

812 S. Ferri et al. / Digestive and Liver Disease 43 (2011) 807– 813

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ig. 3. Functional assays in hepatitis C virus-positive patients as a whole (HCV), aCC, end-stage liver disease: ESLD), transaminase levels (normal [N]/intermediate

arget cell proliferation by T-regs. (C) % of interferon-gamma (IFN-�) production by

In this study for the first time we investigate T-reg re-istribution and behaviour in a cross-sectional population ofatients with HCV-related liver disease at different stages of its nat-ral history, through the analysis of their frequency and functionmongst peripheral lymphocytes and in liver biopsies of patientsith chronic HCV-related disease.

An overall increased frequency of CD4+CD25hiFoxp3+ T-regsnd Foxp3 fluorescence intensity was observed in HCV-positiveatients as a whole with respect to healthy controls; the increase of-reg activity was conspicuous in patients with persistently normalLT values, in keeping with previous observations [17–19,27,29],uggesting that enhanced T-reg function may be instrumental inimiting the immuno-mediated liver damage.

As far as the different chronic stages of hepatitis C are con-erned, we observed that T-regs were distributed following a sortf ascending gradient over time, which was directly correlated notnly to the estimated duration of disease, but also to disease pro-ression: in chronic hepatitis, T-reg proportion was higher thann healthy controls; it was increased further in cirrhosis compli-ated by HCC, and eventually reached its peak during the end-stagehase of the disease, as similarly reported in patients with chronicepatitis B [30].

Liver cirrhosis is associated with a decrease in white cell countue to various mechanisms, such as portal hypertension-inducedequestration, alterations in bone marrow stimulating factors,iral- and toxin-induced bone marrow suppression and consump-ion [31] but our data demonstrate that T-regs were selectivelypared, or even expanded, in advanced liver disease, possibly con-ributing to the low liver enzymes commonly found in patients with

CV-related ESLD.

Notwithstanding the increasing frequency, the inhibitory prop-rties of T-regs on the proliferative capacity of target cells werereserved all over the different stages of the disease, being sig-

ng to the stage of liver disease (chronic hepatitis: CHC, hepatocellular carcinoma:ormal [A]). (A) % of target cell proliferation in single cultures. (B) % of inhibition of

t cell single cultures. (D) % of inhibition of target cell IFN-� production by T-regs.

nificantly more effective in patients with persistently normal ALTvalues.

The observation that T-reg frequency and function wereinversely correlated to the biochemical expression of hepato-cyte damage supports the notion that their activity may limitthe immuno-mediated liver damage, down-regulating not onlythe HCV-specific cytotoxic reactivity targeting HCV-infected hep-atocytes [4,32], but also limiting the bystander killing effectinduced by the increased intrahepatic levels of pro-inflammatorycytokines [33,34]. On the other hand, the direct correlation of T-reg frequency and Foxp3 expression with disease duration andprogression points to a sort of continuous adaptation of theimmune system to the viral presence, allowing the developmentof progressive viral tolerance. To further support this assumption,an in vitro model has recently demonstrated that HCV-infectedhepatocytes, through the increased production of transforminggrowth factor beta (TGF-�), are capable of directly inducing T-regsdevelopment, thus contributing to impaired host T cell responses[35].

Lymphocytes expressing Foxp3, identified as T-regs [20], weredetected into liver portal tracts of half of the patients examined, butthey appeared not to associate to any clinical or laboratory parame-ter, nor to mirror frequency and composition of circulating T-regs,an observation indicating that T-reg turnover within the liver isquite elevated.

HCV patients, especially those with persistently elevated liverenzymes, displayed an increased number of IFN-� secreting cellsamongst the bulk of targets that was not significantly reduced bythe addition of T-regs. However, this high amount of IFN-� did not

emerge to be effective in eradicating the virus, possibly due to bothan HCV-induced disturbance of crucial IFN-� dependent pathways[36] and a selective impairment of HCV-specific immune response[16,37,38].

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[39] Patzwahl R, Meier V, Ramadori G, et al. Enhanced expression of interferon-

S. Ferri et al. / Digestive and

An overall up-regulated expression of IFN-responsive genes isorrelated to the extent of liver damage [39] and the interferon-amma transgenic mouse, which strongly expresses the IFN-�ene in the liver, is a well known model of chronic hepatitis [40];ccordingly, patients with chronic hepatitis and a preserved T-regnhibition on IFN-� secreting target cells displayed normal amino-ransferase levels.

On the contrary, T-regs from ESLD patients were not able toontrol IFN-� secretion, independently from ALT levels. The selec-ive expansion of peripheral T-regs in these patients may reflect anttempt of the immune system to determine a sort of “quantitative”ompensation aimed to avoid immuno-mediated liver decompen-ation in subjects with an already delicate equilibrium.

Further investigations on T-reg mechanisms and functionogether with longitudinal studies through the evolving stages ofiver disease will pave the way to immunotherapeutic interventionsor chronic hepatitis C and in particular its long-term complications.

onflict of interest statement

one declared.

cknowledgements

This work was supported in part by a grant from theegione Emilia Romagna-University research project 2007–2009:Immunological, virological and oncological follow-up of the liverecipients”.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.dld.2011.04.020.

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