Increased Expression of Regulatory Tr1 Cells in Recurrent Hepatitis C after Liver Transplantation

American Journal of Transplantation 2009; 9: 2102–2112Wiley Periodicals Inc.

C© 2009 The AuthorsJournal compilation C© 2009 The American Society of

Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2009.02743.x

Increased Expression of Regulatory Tr1 Cells inRecurrent Hepatitis C after Liver Transplantation

A. Carpentiera,b, F. Contia,c,*, F. Stenarda,c,

L. Aoudjehanea, C. Mirouxb, P. Podevina,

O. Moralesb, S. Chouzenouxa, O. Scattonc,

H. Grouxd, C. Auriaultd, Y. Calmusa,c,

V. Pancreb,† and N. Delhemb,†

aLaboratoire de Biologie Cellulaire, Universite ReneDescartes, Paris, FrancebUMR 8527—Institut de Biologie de Lille, Lille,FrancecUnite de Transplantation, Hopital Cochin, Paris,FrancedInstitut de Pharmacologie Moleculaire et Cellulaire,Valbonne, Sophia Antipolis, France*Corresponding author: Filomena Conti,[email protected]†These authors contributed equally.

Immune response failure during HCV infection hasbeen associated with the activity of regulatory T cells.Hepatitis C-related cirrhosis is the main reason forliver transplantation. However, 80% of transplantedpatients present an accelerated recurrence of the dis-ease. This study assessed the involvement of reg-ulatory T-cell subsets (CD4+CD25+ cells: ‘Treg’ andCD49b+CD18+ cells: ‘T regulatory-1’ cells), in the re-currence of HCV after liver transplantation, using tran-scriptomic analysis, ELISA assays on serum samplesand immunohistochemistry on liver biopsies from liverrecipients 1 and 5 years after transplantation. Threegroups of patients were included: stable HCV-negativerecipients and those with mild and severe hepatitisC recurrence. At 5 years, Treg markers were overex-pressed in all HCV+ recipients. By contrast, Tr1 markerswere only overexpressed in patients with severe recur-rence. At 1 year, a trend toward the overexpression ofTr1 was noted in patients evolving toward severe recur-rence. IL-10 production, a characteristic of the Tr1 sub-set, was enhanced in severe recurrence at both 1 and5 years. These results suggest that Tr1 are enhancedduring severe HCV recurrence after liver transplanta-tion and could be predictive of HCV recurrence. Highlevels of IL-10 at 1 year could be predictive of severe re-currence, and high IL-10 producers might warrant moreintensive management.

Key words: Hepatitis C, liver transplantation, regula-tory T cells, Tr1

Abbreviations: HCV, Hepatitis C Virus; LT, liver trans-plantation; Th1, T Helper 1; Treg, regulatory T cell;Tr1, Type 1 regulatory T cell

Received 18 December 2008, revised 19 May 2009 andaccepted for publication 19 May 2009

Introduction

Cirrhosis due to hepatitis C virus (HCV) is a common indica-tion for liver transplantation (LT). HCV infection of the graftis universal, and graft damage is often accelerated, leadingto cirrhosis in 30% of patients within 5 years (1) and re-duced patient survival compared to other indications (2,3).The mechanisms of accelerated HCV-induced liver damageafter LT are poorly understood.

Viral clearance seems to be associated with persistentCD4+ and CD8+ antiviral responses (4). Strong T helper1cell activity, specific for Core, NS3, NS4 and NS5 proteins,is associated with spontaneous recovery (5), whereas aweak or nonpersistent CD4 response is associated with apoor outcome (6). The role of CD8+ T cells during acuteinfection has been clearly analyzed (7). Much attention hasrecently focused on regulatory T cells (Tregs) and theircontribution to HCV disease. The Treg population, which ac-counts for 5–10% of peripheral CD4+ T cells, constitutivelyexpresses CD25 (8) and can suppress host immune re-sponses in the setting of autoimmune diseases, transplan-tation and antitumor immunity (9,10). Treg also constitu-tively express surface markers such as GITR (CD133) (11),CTLA4 (CD152) (12) and the transcription factor Foxp3,which is characteristic of this subpopulation of Tregs inthe mouse (13), although its specificity in humans is lessclear (14). Other subpopulations of regulatory T-cell sub-sets, such as IL-10-secreting Tr-1 cells that express CD18and CD49b (15,16) and TGF-b-secreting Th3 cells (17), typ-ify induced T-cell regulatory subpopulations as they aregenerated in the periphery and exert cytokine-mediatedsuppression.

An increased frequency of CD4+CD25+T cells has recentlybeen described in the blood of patients with persistentHCV infection, compared with those who cleared HCV(18,19), as well as an in vitro depletion of CD25+ T cellsresulting in increased HCV-specific T-cell responsiveness.

2102

Regulatory T Cells in Recurrent Hepatitis C

It has been proposed that CD4+CD25+ cells contributeto HCV persistence by suppressing HCV-specific T-cell re-sponses (20,21). Some studies have shown a correlationbetween a reduced HCV-specific T-cell response and thesecretion of TGF-b by liver-infiltrating CD4+CD25+ T cells(18), and that regulatory T cells are able to inhibit HCV-specific T-cell activity, independently of IL-10 and TGF-b(18,19). However, it has also been shown that functionalFoxp3+CD4+CD25+ Tregs are detectable during both per-sistent HCV infection and after recovery, suggesting thatthey are part of the normal immune response to thispathogen (21).

Previous studies have demonstrated the involvement of IL-10 in suppression of the anti-HCV immune response (22).In chronically infected patients, IL-10-secreting Tr1 cells cir-culate concomitantly with IFN-c -secreting Th1 cells, andthese two subtypes of CD4 T cells recognize the sameepitopes on HCV core protein (23). PBMCs that secreteIL-10 in response to HCV NS4 protein inhibit the activa-tion of dendritic cells inducing a Th1 response (24), andin vitro HCV itself is able to induce a secretion of IL-10(22). During treatment for HCV, a downregulation of IL-10 production is observed, coinciding with an upregula-tion of IFNc production by CD4+ T cells (25). These find-ings suggest that Tr1 cells may also be implicated in HCVpathogenesis.

In the postorgan transplant setting, numerous experimen-tal studies have demonstrated that Tregs induce allografttolerance (26,27). Tregs are influenced by immunosuppres-sive therapy; in particular, calcineurine inhibitors reducethe Treg function in vitro (28). It has also been shownthat Treg levels decrease significantly after LT, especiallyduring allograft rejection (29), and that the reduction in cir-culating Tregs is counterbalanced by an intragraft increase(30).

No evaluations have ever been made of the expression andactivity of regulatory T cells, and their potential involvementin the accelerated progression of recurrent hepatitis C afterLT. During the present study, the expression of regulatoryT cells was analyzed in transplanted HCV-negative patientsand in patients with mild or severe recurrent hepatitis C.Our results suggest, for the first time, that Tregs are signif-icantly enhanced in recurrent hepatitis C, and that Tr1 cellsare specifically enhanced in severe recurrent hepatitis C.We also show that serum IL-10 levels, characteristic ofthe Tr1 subset, are specifically and significantly enhancedin patients prior to a severe recurrence, when comparedwith patients experiencing a mild recurrence of HCV. Thusat 1 year post-LT, IL-10 levels could be predictive of severerecurrence. These findings may have therapeutic implica-tions, insofar as at 1 year post-LT, high IL-10 producersmight represent a subset of liver transplant recipients whowarrant more intensive management.

Patients and Methods

Patients

The study population consisted of patients who had undergone LT be-tween 1994 and 1999 at Cochin Hospital, Paris, France. Thirty transplantedpatients were included in this study. Inclusion was performed after the5th year of follow-up when liver biopsy results became available. Chronichepatitis C was defined by biochemical abnormalities, the RT-PCR detec-tion of serum HCV-RNA (Cobas Amplicor HCV test 2.0, Roche Diagnos-tics) and consistent histological findings. All biopsy specimens were as-sessed by the same pathologist, and only biopsy samples with >10 portaltracts were retained for the study. A histological diagnosis of recurrenthepatitis C was based on combined evidence of hepatocellular necrosis,chronic inflammatory infiltrate and portal and periportal fibrosis. Biliary le-sions were required to be focal and mild, and the absence of endothe-lialitis was necessary. Mild to moderate steatosis could be present. Hep-atitis C was scored using scales for activity (A0–A3) and fibrosis (F0–F4),according to the METAVIR scoring system (31). Recurrent hepatitis C, 5years after LT, was considered to be severe when histological examina-tion produced a METAVIR score of A1F2 or higher and mild when thescore was lower than A1F2. Patients with cirrhosis (F4) and with fibros-ing cholestatic hepatitis were excluded from this study. Patients with his-tological evidence of acute or chronic rejection were also excluded. Thehistological diagnosis of acute rejection was based on the following com-bination of lesions: portal inflammatory infiltrate containing eosinophils,presence of biliary epithelial lesions and endothelialitis, absence of promi-nent piecemeal necrosis, possible presence of centrilobular lesions, includ-ing central vein endothelialitis and pericentrilobular inflammatory infiltrates.Chronic rejection was identified by dystrophic changes of the biliary ep-ithelium affecting a majority of the bile ducts, with or without bile ductloss.

Thirty transplanted patients were thus included; 10 were HCV-negative andhad normal histological findings, while the other 20 suffered from recurrentchronic hepatitis C (mild in 10 cases and severe in 10). Only HCV-positivepatients with genotype 1 were included in this study. All patients whohad undergone a systematic evaluation about 5 years after their LT wereincluded consecutively, once histological findings of the liver biopsy had be-come available. When a liver biopsy was performed, clinical variables wereassessed and patients with a recent episode of rejection (6 months), biliarycomplications or severe complications other than hepatitis C recurrencewere ruled out of the study. A serum sample from the 1-year posttransplantworkup was also available from 24 of the patients (8 in each group). Frozenportions of the liver biopsy from four patients in each group were availablefor PCR and immunohistological analysis.

Following LT, the patients had been treated with prednisolone (5 mg/kgdaily, tapered to 20 mg from day 7) and cyclosporine (5 mg/kg daily) ortacrolimus (0.15 mg/kg daily). Cyclosporine and tacrolimus dosages wereadjusted to maintain blood levels of between 100 and 200 ng/mL and 5and 12 ng/mL, respectively. The immunosuppressive regimen was similarwith respect to cyclosporine and tacrolimus (dose and dosage) and cor-ticosteroids in the three patient groups (data not shown). The incidenceof a past history of acute rejection was similar in the three groups. Noneof the patients were receiving antiviral therapy at the time of our study.Patient characteristics are summarized in Table 1. The three groups werecomparable for age, sex, albumin, bilirubin and viral load at both 1 and 5years post-LT. ALT levels were statistically higher in patients with severerecurrent hepatitis C at 1 and 5 years, and the METAVIR score was onlysignificantly higher in patients with severe recurrent hepatitis C at 5 yearsafter LT.

American Journal of Transplantation 2009; 9: 2102–2112 2103

Carpentier et al.

Table 1: Patient characteristics

Transplanted patients HCV-negative stable transplants Mild recurrent hepatitis C Severe recurrent hepatitis C pb

1 year post-LT, na = 24 8 8 8Sex: male/female 3/5 3/5 5/3 nsALT (IU/L) 20.6 ± 3.2 30.5 ± 7.3 57.4 ± 9.9 0.02Bilirubin (mg/dL) 20.4 ± 4.1 24.5 ± 3 26.2 ± 2.9 nsAlbumin (g/L) 44 ± 1.6 42.6 ± 1.9 39.2 ± 1.2 nsProthrombin rate (%) 96 ± 7 91 ± 3 88 ± 4 nsMETAVIR activity 0 0.6 1.2 nsMETAVIR fibrosis 0 0.7 1.8 nsViral load (IU/mL) 0.98 10−6 ± 0.6 10−6 2.5 10−6 ± 0.8 10−6 ns

5-year post-LT, na = 30 10 10 10Sex: male/ female 4/6 4/6 6/4 nsMean age (years) 43 ± 9 54 ± 8 56 ± 5 nsALT (IU/L) 24 ± 6 24.7 ± 5.7 73.2 ± 15.4 0.01Bilirubin (mg/dL) 22.1 ± 5.2 25.5 ± 12 28.7 ± 6.9 nsAlbumin (g/L) 43.9 ± 3.6 42.9 ± 2.2 36.2 ± 2 nsProthrombin rate (%) 95 ± 5 87 ± 9 74 ± 16 nsMETAVIR activity 0.2 0.4 1.4 0.05METAVIR fibrosis 0.4 0.7 2.8 <0.001Viral load (IU/mL) 1.2 10−6 ± 0.4 10−6 2.9 10−6 ± 0.6 10−6 ns

aNumber; bstatistical evaluation: p—severe versus other groups; cmean age (range).

Transcriptomic analysis

Transcriptome analysis was performed on both liver specimens and serumsamples, because when using this technique on a large panel of genesin the setting of chronic HCV infection, we had found a strong correlationregarding the transcription of genes between liver and serum (32).

RNA extraction

The extraction of total RNA from frozen liver biopsy samples was performedusing TRIzolTM reagent (GIBCO BRL, Invitrogen, Paisley, Scotland, UK),as described by the manufacturer. Total RNA was then immunoprecipi-tated with isopropanol. The RNA pellet was finally washed and dissolvedin DNAse and Rnase-free water. Before reverse transcription, a DNAsetreatment was performed using the Message clean kit (GenHunter Corp.,France), as described by the manufacturer.

Serum mRNA extraction was achieved using the QIAampR©

Viral RNA kit(QIAGEN, Courtaboeuf, France), as described by the manufacturer. Briefly,a serum sample was incubated with the AVL buffer supplemented withcarrier RNA. After the addition of ethanol, the sample was applied to theQIAmp spin column and centrifuged. The column was then cleaned, andmRNA were eluted and then stored at −80◦C prior to analysis.

Reverse transcription

RNA were supplemented with the following mixture [oligo dT (Roche, Mey-lan, France) + RNAsin (PROMEGA, Charbonnieres; France) + H2O], andthen incubated at 70◦C for 5 min. After a further 5 min at room tempera-ture, the following mixture [buffer 5× (Invitrogen, Cergy Pontoise, France)+ DTT (Invitrogen) + d NTPs 10 mM + RNAsin (PROMEGA) + Superscript(Invitrogen] was added. This reaction was followed by an initial incubationstep at 45◦C for 60 min and a second incubation at 95◦C for 5 min. Finally,H2O was added to obtain a concentration of 10 ng total RNA/1 lL.

ABI PRISMR 7000 sequence detection systemThe quantification of transcripts from liver and serum samples was per-formed using real-time quantitative RT-PCR with the ABI PRISM

R 7000 se-quence detection system (Applied Biosystems, CA, USA). The mix was

optimized for real-time PCR analysis using SYBR Green 1 Dye, AmplitaqGold

R©DNA polymerase, dNTPs with UTP, Passive Reference 1 required

for signal normalization and optimized buffer components, in a total vol-ume of 50 lL. Each sample was run in a 96-well plate containing 94primers by performing initial denaturation at 95◦C for 5 min, after whichPCR reactions were cycled 40 times as follows: 15 s at 95◦C and 1 min at60◦C. Fluorescence intensity was measured at the end of each elongationphase. Melting curve analysis was performed immediately after amplifica-tion, in accordance with the manufacturer’s instructions. The amplificationof liver cDNA was successfully repeated twice with cDNA from the sameextraction.

Light cycler-based PCR assay

cDNA was synthesized from total RNA at a concentration of 100 ng/lL usingoligo dT primers and Superscript reverse transcriptase (GIBCO BRL). Thequantification of transcripts from samples was confirmed by real-time quan-titative RT-PCR using the Light Cycler system (Roche Diagnostics, Meylan,France). The PCR mixture contained the following: Taq polymerase, 1× ofLightCycler-DNA master SYBRGreen I (Roche Diagnostics), 3 mM of MgCl2,0.5 lmol/L of each primer and 1 lL of cDNA preparation (patient cDNA sam-ples) in a total volume of 20 lL. Thirty-two samples were run in parallel byperforming an initial denaturation at 95◦C for 8 min, and then the PCRreactions were cycled 35–40 times as follows: 15 s at 95◦C, 7 s at theappropriate annealing temperature (Table 2) and 18–64 s at 72◦C accordingto the length of the target sequence annealing (40 s at 58◦C). Fluorescenceintensity was measured at the end of each elongation phase. The meltingcurve analysis was carried out immediately after amplification, following themanufacturer’s instructions.

Primers

All primers were designed for real-time PCR use and were purchased fromMWG-Biotech (Germany; Table 2). The housekeeping genes b-actin andG3PDH were used as controls.

Immunohistochemical analysis

Markers of regulatory T cells were also evaluated by immunohistochem-istry, and TGF-b and IL-10 levels were also determined. Liver biopsies were

2104 American Journal of Transplantation 2009; 9: 2102–2112


Table 2: Primer sequences

HybridizationGene Primer sequences T◦c

CD4 5′-GGGAAATCAGGGCTCCTTCTTA 59◦5′-TGGTCCCAAAGGCTTCTTCTT

CD25 5′-GGGACTGCTCACGTTCATCA 59◦5′-TTCAACATGGTTCCTTCCTTGTAG

GITR 5′-TGTGTCCAGCCTGAATTCCA 59◦5′-CCGAGGCACAGTCGATACACT

CTLA4 5′-TTCTTCTCTTCATCCCTGTCTTCT 61◦5′-GAGATGCATACTCACACACAAAGCT

FoxP3 5′-TCACCTACGCCACGGTCA 59◦5′-CACAAAGCACTTGTGCAG

CD18 5′-ATGCTTGATGACCTCAGGAATGT 59◦5′-ACGGTCTTGTCCACGAAGGA

CD49b 5′-CAACGGGTGTGTGTTCTGACA 59◦5′-TCATCACACACAACCACAACATCT

IL-10 5′-GCCTAACATGCTTCGAGATC 59◦5′-TGATGTCTGGGTCTTCGTTC

TGF-b 5′-CGAGCCTGAGGCCGACTAC 62◦5′-CGGAGCTCTGATGTGTTGAAGA

b Actin 5′-TGCTATCCAGGCTGTGCTA 58◦5′-ATGGAGTTGAAGGTAGTTT

GAPDH 5′-GCCAAGGTCATCCATGACAACTTTGG 58◦5′-GCCTGCTTCACCACCTTCTTGATGTC

snap-frozen in liquid nitrogen for subsequent immunohistochemical analy-sis. The DAKO LSABR2 System, HRP (Dako, Carpinteria, CA), was used,based on a modified labeled avidin–biotin technique. Briefly, the frozen sec-tions were fixed in acetone and blocked with 0.1% H2O2 and horse serum,and were then incubated with primary Ab (anti-IL-10: 1/10, other antibod-ies: 1/40), after which biotinylated rabbit anti-mouse IgG was added. Theavidin–biotin complex was added, followed by 3-amino-9-ethylcarbazole,and the slides were finally counterstained. Anti-human mAbs CD4, CD25,IL-10, Integrin-a2 (CD49b) and Integrin-b2 (CD18) were purchased fromSanta Cruz (Tebu-Bio, France). Slides were examined by two of the authors,who were blinded to the diagnosis. Positive cells exhibiting a characteristicstaining were counted in three different portal tracts and three separatelobular fields of each sample (magnification ×200), and the mean numberof positive cells was calculated for each patient group.

ELISA method

Serum levels of TGF-b and IL-10 were evaluated. 96-well plates (NUNC,Denmark) were coated overnight with 50 lL of a primary antibody solutionat a concentration of 2 lg/mL. After washes, the plates were saturatedwith PBS-BSA (Sigma-Aldrich, USA) and serum diluted to half in PBS-BSA,were applied in duplicate and left overnight. A recombinant cytokine range(Pharmingen, Becton Dickinson, USA) was then applied to each plate atrates ranging from 10 ng/mL to 10 pg/mL. After that, the biotin-coupledsecondary antibody (1 lg/mL) was added, and an amplification phase wasperformed using streptavidin-peroxidase. Plates were revealed and the rev-elation reaction was stopped by the addition of HCl 2N. The plates werethen read at 492 nm.

Data interpretation and statistics

PCR results were analyzed using the ‘Relative Gene Expression Method’as described elsewhere (33). Briefly, individual CT values were normalizedusing the average CT values for housekeeping genes (�CT = CT − CTHKG).Average �CT values for each group were then compared with the �CTvalues of the control group (S group) (��CT = �CT − �CTSgroup). The

evaluation of 2−��CT then indicated the fold change in gene expressionrelative to the control group.

The relevance of PCR and ELISA results was validated using the statisticalrank sum Mann–Whitney test with Sigma Stats software.

Results

Peripheral expression of regulatory T-cell markers

5 years after LT

Peripheral transcriptome analysis on 5-year samples re-vealed a significant increase in markers associated withCD4+CD25+ Treg expression (Figure 1A). CD4 expres-sion was increased in patients with severe recurrencewhen compared to HCV-negative patients and those withmild recurrence (p < 0.01 in both cases). When com-pared with the values in HCV-negative patients, CD25expression was significantly increased (p < 0.05) in pa-tients with both types of recurrence. A significant increasein CTLA4 and GITR expression was observed in groupswith HCV recurrence when compared to the HCV-negativegroup, but no significant difference was found betweenthe two HCV+ groups. Foxp3 transcription factor expres-sion did not differ between the three groups. Taken to-gether, these data showed that CD4+CD25+ Treg markerswere overexpressed in HCV-positive versus HCV-negativerecipients, without any difference between mild or severerecurrence.

The expression of Tr1 markers (CD49b and CD18) was eval-uated in the serum of the same patients. Levels of thesemarkers were significantly elevated in patients with a se-vere hepatitis C recurrence when compared to patientswith a mild recurrence and with HCV-negative patients(p < 0.05 in both cases) (Figure 1B).

Expression of regulatory T cells in liver biopsies

5 years after LT

mRNA levels: The expression of CD4+CD25+ mRNA inliver biopsies was increased in both patient groups expe-riencing recurrence (Figure 2A). CD4 expression was 30-fold higher in severe recurrence compared to HCV-negativerecipients. CD25 mRNA was 6-fold higher in severe re-currence and 14-fold higher in mild recurrence comparedto HCV-negative recipients. The expression of GITR andFoxp3 were also increased in patients with HCV recur-rence, when compared to HCV-negative recipients. Theintrahepatic expression of CD18 and CD49b mRNA wasincreased with HCV recurrence, and more markedly in theevent of severe recurrence (Figure 2B).

Protein levels: Intrahepatic RT-PCR results were con-firmed by the immunohistochemical evaluation of proteinparameters (Table 3). For Treg markers (Figure 3), the meanCD4-positive cell count in lobular and portal fields was16.8 ± 3.7 in severe recurrence (Figure 3C), 4 ± 0.8 in mildrecurrence (Figure 3B) and 3 ± 1.5 in HCV-negative patients


Carpentier et al.

Figure 1: Peripheral mRNA expression of regulatory T-cell markers 5 years after LT. (A) CD4+ CD25+ regulatory T cells; (B) Type 1regulatory T cells (Tr1) mRNA expression was evaluated on sera from HCV-negative recipients and recipients with a mild or severe HCVrecurrence 5 years after LT. Results were calculated using the 2−��CT method, as described by Livak et al. ∗p < 0.05 versus HCV-negativegroup; ∗∗p < 0.01 versus HCV- negative group; ◦p < 0.05 versus mild recurrence group.

(Figure 3A). The mean CD25-positive cell count was alsohigher in severe recurrence (13 ± 3.8, Figure 3G) than thatin mild (4 ± 0.6, Figure 3F) or HCV-negative patients (4 ±0.3, Figure 3E). Immunohistochemical analysis revealed an

overexpression of Tr1-specific markers in the liver with se-vere recurrence when compared with mild recurrence orHCV-negative findings (Figure 4). The mean CD4-positivecell count was 16.8 ± 3.7 in severe recurrence (Figure 4C),

Figure 2: Intrahepatic mRNA expression of regulatory T-cell markers 5 years after LT. (A) CD4+ CD25+ regulatory T cells; (B) Type1 regulatory T cells (Tr1) mRNA expression was evaluated on liver biopsies from HCV-negative recipients and recipients with a mild orsevere HCV recurrence 5 years after LT. Results were calculated using the 2−��CT method, as described by Livak.



Table 3: Intrahepatic expression of Treg and Tr1 cell markers (im-munohistochemical evaluation)

HCV-negative Mild Severepatients recurrence recurrence

CD4a 3 ± 1.5 4 ± 0.8 16.8 ± 3.7CD25a 4 ± 0.3 4 ± 0.6 13 ± 3.8CD49ba 3 ± 1.5 8 ± 2.2 13.5 ± 3.5CD18a 2 ± 1.4 7.3 ± 2 14.4 ± 1.4aMean of positive cell counts in lobular and portal fields.

4 ± 0.8 in mild recurrence (Figure 4B) and 3 ± 1.5 in HCV-negative patients (Figure 4A). The mean CD49b-positivecell count was 3 ± 1.5, 8 ± 2.2 and 13.5 ± 3.5, respec-tively, in the HCV-negative, mild and severe recurrencegroups (Figure 4G, E and F) and that of CD18 cells was2 ± 4.4, 7.3 ± 2 and 14.4 ± 1.4, respectively (Figure 4K,I and J). Taken together, these results suggest that Tregmarkers were overexpressed in the liver of patients withHCV recurrence when compared to HCV-negative trans-planted patients, whereas intrahepatic Tr1 cells were onlyincreased in severe recurrent hepatitis C and could thusbe implicated in the increased severity of the course ofrecurrence after LT.

Peripheral expression of regulatory T-cell markers

1 year after LT

The gene expression of Tregs was analyzed in sera thathad been obtained 1 year after LT from some of thepatients. All CD4+CD25+Treg markers were significantlyoverexpressed in HCV-positive recipients when compared

to HCV-negative recipients (p < 0.05 for HVC-positive vs.HCV-negative patients), except for FoxP3, levels of whichwere similar in all three groups (Figure 5A). No differenceswere detected regarding CD4, CD25, GITR and CTLA4 be-tween patients who would develop a severe or a mild re-currence of HCV 4 years later.

As for Tr1 cell markers, significant increases in CD4 (p <

0.05 vs. the HCV-negative group), CD18 (p < 0.05 vs.the HCV-negative group) and CD49b (p < 0.05 vs. theHCV-negative group) were observed in the serum of HCV-positive recipients compared to HCV-negative patients (Fig-ure 5B), with a trend toward the overexpression of Tr1markers in patients who would progress to severe recur-rence compared to those with a milder course. However,the difference between these two groups was not signif-icant, probably because of the small number of samplesavailable.

Tr1-associated immunosuppressive cytokines: TGF-band IL-10

Five years after LT, TGF-bmRNA was overexpressed in theserum and liver of recipients with recurrent hepatitis C,when compared to HCV-negative patients (9-fold in themild group and 16-fold in the severe recurrence group; p <

0.01 in both cases) (Figure 6A). One year after LT, TGF-bwas significantly overexpressed in patients with mild (3-fold, p < 0.05) and severe recurrence (9-fold, p < 0.01)compared with HCV-negative patients. There was a trendtoward an increased expression of TGF-b in patients with

Figure 3: Intrahepatic expression of regulatory T cells determined by the immunohistochemistry assay. CD4 (A–D) and CD25(E–H) protein expression was assessed on frozen sections of liver biopsies from HCV-negative recipients and recipients with a mild orsevere HCV recurrence 5 years after LT. The mean CD4-positive cell count in lobular and portal fields was higher in severe recurrence (C)than that in mild recurrence (B) and in HCV-negative patients (A). The mean CD25-positive cell count was also higher in severe recurrence(G) than that in mild forms (F) or in HCV-negative patients (E).


Carpentier et al.

Figure 4: Intrahepatic expression of Tr1 cell markers. CD4 (A–D), CD49b (E–H) and CD18 (I–L) protein expressions were assessed byimmunohistochemistry on frozen sections of liver biopsies from HCV-negative recipients and recipients with a mild or severe recurrenceof hepatitis C after LT. The mean CD49b-positive cell count was higher in severe recurrence (G) than that in mild recurrence (F) andin HCV-negative patients (E). The CD18-positive cell count was also higher in severe recurrence (K) than that in mild forms (J) or inHCV-negative patients (I).

severe recurrence compared with mild recurrence, but thisdifference did not attain significance.

At 5 years, IL-10 mRNA expression was significantly in-creased in the serum and liver of patients with HCV re-currence (Figure 6B), and the difference was significantbetween severe recurrence and HCV-negative recipients(8-fold increase, p < 0.05, in serum and liver) and betweensevere and mild recurrence (4- and 2-fold increase in liverand serum, respectively, p < 0.05). IL-10 levels determinedin sera using ELISA (Figure 6C) were significantly higher inpatients with mild or severe recurrence compared to HCV-negative patients (3.4- and 8-fold, respectively, p < 0.05),without there being a significant difference between themild and severe recurrence groups. IL-10 protein levels, as-

sessed in the liver by immunohistochemistry, confirmedthese findings (Figure 6D). The mean number of IL-10-positive cells was higher in severe recurrence (18.6 ± 3.5)than that in mild recurrence (3 ± 0.2) or in HCV-negativepatients (0).

Finally, IL-10 mRNA (Figure 6B, upper graph) and protein(Figure 6C, upper graph) levels were compared in sera andliver samples that had been obtained 1 year after LT withthe future pattern of recurrence at 5 years. IL-10 mRNA ex-pression (Figure 6B) was higher in patients who wouldgo on to develop severe recurrence, compared to theHCV-negative and mild recurrence groups (5.5-fold, p <

0.05, and 3-fold, p < 0.05, respectively). Moreover, IL-10serum levels (Figure 6C) were higher in patients who would



Figure 5: Peripheral mRNA expression of Treg markers 1 year post-LT. (a) CD4+ CD25+ regulatory T cells; (b) Type 1 regulatory Tcells (Tr1) mRNA expression was evaluated on sera from HCV-negative recipients and recipients with a mild or severe HCV recurrence1 year after LT. Results were calculated using the 2−��CT method, as described by Livak. ∗p < 0.05 versus HCV-negative group ∗∗p <

0.01 versus HCV-negative group.

experience severe recurrence compared to HCV-negativepatients or those with a mild recurrence (6.4- and 13.2-fold, respectively, p < 0.05).

Discussion

An increased frequency of CD4+CD25+ Tregs has beendescribed in patients with persistent HCV infection, whichcould contribute to HCV persistence by suppressing HCV-specific T-cell responses (19,20). The accelerated progres-sion of recurrent hepatitis C prompted us to propose thepotential role of Tregs after LT. During the present study,we found that 5 years after LT, Treg markers were over-expressed in sera from HCV-positive patients, comparedto HCV-negative transplant patients, without there beingany significant differences between mild and severe recur-rence. By contrast, we showed for the first time that Tr1markers (CD18, CD49b) were significantly overexpressedin patients with severe recurrence compared to those withmild recurrence (or to HCV-negative recipients). The over-expression of these markers was confirmed in liver biop-sies obtained 5 years after LT. The data obtained 1 yearafter LT concerning some of the patients included in the5-year study were analyzed in the function of their futurepattern of recurrence at 5 years. An overexpression of Tregmarkers was observed in patients with HCV recurrence,

and a trend toward an overexpression of Tr1 markers wasobserved in patients who would experience severe recur-rence, when compared to patients with a persistent mildcourse at 5 years. However, this difference was not signif-icant, probably because of the small number of samples(eight in each group).

Tr1 subpopulation activity was also assessed in terms ofTGF-b and IL-10 production. We thus demonstrated for thefirst time, to our knowledge, that these two cytokines areoverexpressed in sera and liver biopsies from patients withHCV recurrence when compared to HCV-negative patients,5 years post-LT. More importantly, we detected an overex-pression of these cytokines at 1 year after LT in patientswho would subsequently develop a recurrence. IL-10 lev-els were significantly higher in patients experiencing se-vere recurrence than those in patients with persistent mildrecurrence. These results were confirmed in peripheralblood using ELISA, and by immunohistochemistry on liverbiopsies.

The first impressive finding was the significant increasein Treg and Tr1 cell markers alongside that of TGF-b andIL-10, but not of FoxP3, in serum and liver from HCV-positive recipients when compared to HCV-negative re-cipients. These data could explain that HCV-positive liverrecipients have a risk of acute rejection, lower than or


Carpentier et al.

Figure 6: TGF-b and interleukin-10 expression in HCV-negative recipients with a mild or severe HCV recurrence after LT. (A) TGF-bmRNA and (B) IL-10 mRNA expression were assessed in serum 1 and 5 years post-LT, and intrahepatic mRNA expression 5 years post-LT.(C) The peripheral cytokine secretion of IL-10 was measured by ELISA in sera from patients at 1 and 5 years post-LT. ( D) Intrahepatic IL-10expression was assessed by immunohistochemistry on frozen sections of liver biopsies 5 years post-LT. ∗p < 0.05 versus HCV− group;∗∗p < 0.01 versus HCV− group; ◦p < 0.05 versus mild recurrence group.

similar to that of other transplanted patients (34), whichis enhanced under antiviral therapy (35). The low expres-sion of FoxP3 in these patients may reflect the low andinconsistent expression of FoxP3 by Tr1 cells.

Little has been reported to date concerning the role ofTregs in recurrent hepatitis C after LT. Our results showthat CD4+CD25+ Tregs are overexpressed, both periph-erally and in the liver, in HCV-positive patients after LT,when compared to HCV-negative patients. This could beexplained by the specific activation of this subpopulationin infected liver and by a sequestration of CD4+CD25+

Tregs in the graft. Similar results had previously been ob-served in a context of acute rejection (30). One could spec-ulate that in the same way as in nontransplanted patients,CD4+CD25+ Tregs contribute to HCV persistence (19), de-spite the use of immunosuppressive drugs that reduce thenumber of CD4+CD25+ T cells in peripheral blood (36,37).However, our data did not allow us to determine whetherthese findings were a result, or a cause, of severe HCVrecurrence.

Our results show that Tr1 cell markers and activity (TGF-b,IL-10) were increased in patients with severe HCV recur-rence compared to mild recurrence. One major finding ofthis study is that IL-10 may predict the severity of HCV re-currence in the long term. Indeed, high IL-10 levels couldalready be detected 1 year after LT, and this could be in-terpreted as a specific activation of Tr1 cells. Interactionsbetween Tr1 cells and immunosuppressive treatments areunknown; Tr1 cell proliferation is dependent on IL-15, a cy-tokine which is not inhibited by immunosuppressive ther-apy (38,39). Serum levels of IL-15 and IL-10 are elevatedin a context of chronic hepatitis C (40), proportionally todisease severity in nontransplanted patients. Tr1 cells maythus proliferate and play a role in the course and severityof HCV recurrence. The assessment of samples obtained1 year post-LT gave us the opportunity to investigate thishypothesis retrospectively, in the light of the subsequentcourse of HCV recurrence. IL-10 was significantly overex-pressed in patients who would experience severe recur-rence in the future, compared with those suffering froma persistent mild course and with HCV-negative patients.



IL-10 is characteristic of Tr1 immunosuppressive activityand these cells could thus be implicated at the very begin-ning of the recurrence. Peripheral IL-10 expression couldprovide an important insight into the future course of hep-atitis C recurrence, and in our hands, elevated IL-10 levelswere predictive of severe recurrent hepatitis C.

Although these data are encouraging, we acknowledgeseveral limitations. The study was retrospective, no func-tional assays were performed, the number of patients wassmall and we did not have documented serum and liverdata at 1 year for all the recipients assessed at 5 years. In-deed, frozen samples of serum and liver stored at −80◦Cunder optimal conditions constitute unique and rare mate-rials. It will be a lengthy (more than 5 years) and difficultprocess to design a prospective trial in order to confirmthese findings. A prospective trial is currently under wayto confirm these data and to add functional assays, but theresults will not be available for several years.

In conclusion, we have shown that regulatory T cells, andmore particularly the Tr1 subpopulation, are increased insevere forms of recurrent hepatitis C, 5 years after LT. Moreimportantly, at 1 year post-LT, high serum levels of IL-10 (themain Tr1 immunosuppressive cytokine) could be predictiveof a severe course of hepatitis C recurrence. These findingsmay have therapeutic implications, insofar as such patientscould benefit from reduced immunosuppressive therapy orfrom the earlier introduction of antiviral treatment in orderto inhibit an accelerated course of HCV recurrence.

Acknowledgements

This work and A.C. received support from the ‘Agence Nationale de laRecherche sur le Sida et les Hepatites’ (ANRS) and the ‘Comite du Nord dela Ligue contre le cancer’.

References

1. Curry MP. Hepatitis B and hepatitis C viruses in liver transplanta-tion. Transplantation 2004; 78: 955–963.

2. Velidedeoglu E, Mange KC, Frank A et al. Factors differentiallycorrelated with the outcome of liver transplantation on HCV+ andHCV− recipients. Transplantation 2004; 77: 1834–1842.

3. Berenguer M, Prieto M, San Juan F et al. Contribution of donorage to the recent decrease in patient survival among HCV-infectedliver transplant recipients. Hepatology 2002; 36: 202–210. Erratumin Hepatology 2003; 37: 489.

4. Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV.Determinants of viral clearance and persistence during acute hep-atitis C virus infection. J Exp Med 2001; 194: 1395–1406.

5. Gerlach JT, Diepolder HM, Jung MC et al. Recurrence of hepatitisC virus after loss of virus-specific CD4(+) T-cell response in acutehepatitis C. Gastroenterology 1999; 117: 933–941.

6. Tsai SL, Liaw YF, Chen MH, Huang CY, Kuo GC. Detection of type2-like T-helper cells in hepatitis C virus infection: Implications forhepatitis C virus chronicity. Hepatology 1997; 25: 449–458.

7. Murata M, Nabeshima S, Maeda N, Nakashima H, Kashiwagi S,Hayashi J. Increased frequency of IFN-gamma-producing periph-eral CD8+ T cells with memory-phenotype in patients with chronichepatitis C. J Med Virol 2002; 67: 162–170.

8. O’Garra A, Vieira P. Regulatory T cells and mechanisms of immunesystem control. Nat Med 2004; 10: 801–805.

9. Sakaguchi S, Sakagushi N, Shimizu J et al. Immunologic tolerancemaintained by CD4+CD25+ regulatory T cells: Their common rolein controlling autoimmunity, tumor immunity and transplantationtolerance. Immunol Rev 2001; 182: 18–32.

10. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immuno-logic self-tolerance maintained by activated T cells expressing IL-2receptor a-chains (CD25). J Immunol 1995; 155: 1151–1164.

11. Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakagushi S. Stim-ulation of CD25+CD4 +regulatory T cells through GITR breaksimmunological self-tolerance Nat Immunol 2002; 3: 135–142.

12. Birebent B, Lorho R, Lechartier H et al. Suppressive properties ofhuman CD4+CD25+ regulatory T cells are dependent on CTLA-4expression Eur J Immunol 2004; 34: 3485–3496.

13. Hori S, Nomura T, Sakagushi S. Control of regulatory T cells de-velopment by the transcription factor FoxP3 Science 2003; 299:1057–1061.

14. Morgan ME, van Bilsen JHM, Bakker AM et al. Expression of FoxP3 mRNA is not confined to CD4+CD25+ T regulatory cells inhumans Hum Immunol 2005; 66: 13–20.

15. Groux H, O’Garra A, Bigler M et al. A CD4 +T-cell subset inhibitsantigen-specific T-cell responses and prevent colitis Nature 1997;389: 737–742.

16. Rahmoun M, Foussat A, Groux H, Pene J, Yssel H, Chanez P. En-hanced frequency of CD18- and CD49b-expressing T cells in pe-ripheral blood of asthmatic patients correlates with disease sever-ity. Int Arch Allergy Immunol 2006; 140: 139–149.

17. Weiner HL. Induction and mechanism of action of transforminggrowth factor-beta-secreting Th3 regulatory cells. Immunol Rev2001; 182: 207–214.

18. Cabrera R, Tu Z, Xu Y et al. An immunomodulatory role for CD4 (+)CD25 (+) regulatory T lymphocytes in hepatitis C virus infection.Hepatology 2004; 40: 1062–1071.

19. Rushbrook SM, Ward SM, Unitt E et al. Regulatory T cells sup-press in vitro proliferation of virus-specific CD8+ T cells duringpersistent hepatitis C virus infection. J Virol 2005; 79: 7852–7859.

20. Boettler T, Spangenberg HC, Neumann-Haefelin C et al. T cellswith a CD4+CD25+ regulatory phenotype suppress in vitro pro-liferation of virus-specific CD8+ T cells during chronic hepatitis Cvirus infection. J Virol 2005; 79: 7860–7867.

21. Manigold T, Shin EC, Mizukoshi E et al. Foxp3+CD4+CD25 +Tcells control virus-specific memory T cells in chimpanzees thatrecovered from hepatitis C. Blood 2006; 107: 4424–4432.

22. Delpuech O, Buffello-Le Guillou DB, Rubinstein E, Feray C, PetitMA. The hepatitis C virus (HCV) induces a long-term increase ininterleukin-10 production by human CD4+ T cells (H9). Eur Cy-tokine Netw 2001; 12: 69–77.

23. MacDonald AJ, Duffy M, Brady MT, McKiernan S, Hall W, HegartyJ et al. CD4 T helper type 1 regulatory T cells induced againstthe same epitopes on the core protein in hepatitis C virus-infectedpersons J Infect Dis 2002; 185: 720–727.

24. Brady MT, MacDonald AJ, Rowan AG, Mills KH. Hepatitis C virusnon-structural protein 4 suppresses Th1 responses by stimulatingIL-10 production from monocytes. Eur J Immunol 2003; 33: 3448–3457.

25. Cramp ME, Rossol S, Chokshi S, Carucci P, Williams R, NaoumovNV. Hepatitis C virus-specific T-cell reactivity during interferon and


Carpentier et al.

ribavirin treatment in chronic hepatitis C. Gastroenterology 2000;118: 346–355.

26. Velasquez-Lopera MM, Eaton VL, Lerret NM et al. Induc-tion of transplantation tolerance by allogeneic donor-derivedCD4+CD25+Foxp3+ regulatory T cells. Transpl Immunol 2008; 19:127–135.

27. Joffre O, Santolaria T, Calise D et al. Prevention of acute andchronic allograft rejection with CD4+CD25+Foxp3 +regulatory Tlymphocytes. Nat Med 2008; 14: 88–92.

28. Zeiser R, Nguyen VH, Beilhack A et al. Inhibition of CD4+CD25+regulatory T-cell function by calcineurin-dependent interleukin-2production. Blood 2006; 108: 390–399.

29. Demirkiran A, Kok A, Kwekkeboom J et al. Low circulating regula-tory T-cell levels after acute rejection in liver transplantation. LiverTransplant 2006; 12: 277–284.

30. Stenard F, Nguyen C, Cox K et al. Decreases in circulat-ing CD4(+)CD25(hi)FOXP3(+) cells and increases in intragraftFOXP3(+) cells accompany allograft rejection in pediatric liver al-lograft recipients. Pediatr Transplant 2009; 13: 70–80.

31. Bedossa P, Poynard T. An algorithm for the grading of activity inchronic hepatitis C. The METAVIR cooperative study group. Hepa-tology 1996; 24: 289–293.

32. Carpentier A, Conti F, Carriere M et al. Analysis of gene transcrip-tion in sera during chronic hepatitis C infection. J Med Virol 2009;473–480.

33. Livak KJ, Schmittgen TD. Analysis of relative gene expression datausing real-time quantitative PCR and the 2−��CT method. Methods2001; 25: 402–408.

34. Burton JR and Rosen HR. Acute rejection in HCV-infected livertransplant recipients: The great conundrum. Liver Transpl 2006;12: S38–S47.

35. Walter T, Dumortier J, Guillaud O, Hervieu B, Paillard,Scoazec JY,Boillot O. Rejection under alpha interferon therapy in livertransplantrecipients. Am J Transplant 2007; 7: 177–1.

36. Demirkiran A, Kok A, Kwekkeboom J, Metselaar HJ, Tilanus HW,Van Der Laan LJ. Decrease of CD4(+)CD25(+) T cells in peripheralblood after liver transplantation: Association with immunosuppres-sion. Transplant Proc 2005; 37: 1194–1196.

37. Furtado GC, Curotto de Lafaille MA, Kutchukhidze N, Lafaille JJ.Interleukin 2 signaling is required for CD4+ regulatory T cell func-tion. J Exp Med 2002; 196: 851–857.

38. Roncarolo MG, Bachetta R, Bordignon C, Narula S, Levings MK.Type 1 T regulatory cells. Immunol Rev 2001; 182: 68–79.

39. Conti F, Frappier J, Dharancy S et al. Interleukin-15 productionduring liver allograft rejection in humans. Transplantation 2003;76: 210–216.

40. Kakumu S, Okumura A, Ishikawa T et al. Serum levels of IL-10, IL-15 and soluble tumour necrosis factor-alpha (TNF alpha) receptorsin type C chronic liver disease. Clin Exp Immunol 1997; 109: 458–463.


Increased Expression of Regulatory Tr1 Cells in Recurrent Hepatitis C after Liver Transplantation

Documents

Transcript of Increased Expression of Regulatory Tr1 Cells in Recurrent Hepatitis C after Liver Transplantation