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

Authors


* Corresponding author: Filomena Conti, filomena.conti@cch.aphp.fr

Abstract

Immune response failure during HCV infection has been associated with the activity of regulatory T cells. Hepatitis C-related cirrhosis is the main reason for liver transplantation. However, 80% of transplanted patients present an accelerated recurrence of the disease. This study assessed the involvement of regulatory T-cell subsets (CD4+CD25+ cells: ‘Treg’ and CD49b+CD18+ cells: ‘T regulatory-1’ cells), in the recurrence of HCV after liver transplantation, using transcriptomic analysis, ELISA assays on serum samples and immunohistochemistry on liver biopsies from liver recipients 1 and 5 years after transplantation. Three groups of patients were included: stable HCV-negative recipients and those with mild and severe hepatitis C recurrence. At 5 years, Treg markers were overexpressed in all HCV+ recipients. By contrast, Tr1 markers were only overexpressed in patients with severe recurrence. At 1 year, a trend toward the overexpression of Tr1 was noted in patients evolving toward severe recurrence. IL-10 production, a characteristic of the Tr1 subset, was enhanced in severe recurrence at both 1 and 5 years. These results suggest that Tr1 are enhanced during severe HCV recurrence after liver transplantation and could be predictive of HCV recurrence. High levels of IL-10 at 1 year could be predictive of severe recurrence, and high IL-10 producers might warrant more intensive management.

Abbreviations: 
HCV

Hepatitis C Virus

LT

liver transplantation

Th1

T Helper 1

Treg

regulatory T cell

Tr1

Type 1 regulatory T cell

Introduction

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

Viral clearance seems to be associated with persistent CD4+ and CD8+ antiviral responses (4). Strong T helper1 cell activity, specific for Core, NS3, NS4 and NS5 proteins, is associated with spontaneous recovery (5), whereas a weak or nonpersistent CD4 response is associated with a poor outcome (6). The role of CD8+ T cells during acute infection has been clearly analyzed (7). Much attention has recently focused on regulatory T cells (Tregs) and their contribution to HCV disease. The Treg population, which accounts for 5–10% of peripheral CD4+ T cells, constitutively expresses CD25 (8) and can suppress host immune responses in the setting of autoimmune diseases, transplantation and antitumor immunity (9,10). Treg also constitutively express surface markers such as GITR (CD133) (11), CTLA4 (CD152) (12) and the transcription factor Foxp3, which is characteristic of this subpopulation of Tregs in the mouse (13), although its specificity in humans is less clear (14). Other subpopulations of regulatory T-cell subsets, such as IL-10-secreting Tr-1 cells that express CD18 and CD49b (15,16) and TGF-β-secreting Th3 cells (17), typify induced T-cell regulatory subpopulations as they are generated in the periphery and exert cytokine-mediated suppression.

An increased frequency of CD4+CD25+T cells has recently been described in the blood of patients with persistent HCV infection, compared with those who cleared HCV (18,19), as well as an in vitro depletion of CD25+ T cells resulting in increased HCV-specific T-cell responsiveness. It has been proposed that CD4+CD25+ cells contribute to HCV persistence by suppressing HCV-specific T-cell responses (20,21). Some studies have shown a correlation between a reduced HCV-specific T-cell response and the secretion of TGF-β 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-β (18,19). However, it has also been shown that functional Foxp3+CD4+CD25+ Tregs are detectable during both persistent HCV infection and after recovery, suggesting that they are part of the normal immune response to this pathogen (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 circulate concomitantly with IFN-γ-secreting Th1 cells, and these two subtypes of CD4 T cells recognize the same epitopes on HCV core protein (23). PBMCs that secrete IL-10 in response to HCV NS4 protein inhibit the activation of dendritic cells inducing a Th1 response (24), and in 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 upregulation of IFNγ production by CD4+ T cells (25). These findings suggest that Tr1 cells may also be implicated in HCV pathogenesis.

In the postorgan transplant setting, numerous experimental studies have demonstrated that Tregs induce allograft tolerance (26,27). Tregs are influenced by immunosuppressive therapy; in particular, calcineurine inhibitors reduce the Treg function in vitro (28). It has also been shown that Treg levels decrease significantly after LT, especially during allograft rejection (29), and that the reduction in circulating Tregs is counterbalanced by an intragraft increase (30).

No evaluations have ever been made of the expression and activity of regulatory T cells, and their potential involvement in the accelerated progression of recurrent hepatitis C after LT. During the present study, the expression of regulatory T cells was analyzed in transplanted HCV-negative patients and in patients with mild or severe recurrent hepatitis C. Our results suggest, for the first time, that Tregs are significantly enhanced in recurrent hepatitis C, and that Tr1 cells are specifically enhanced in severe recurrent hepatitis C. We also show that serum IL-10 levels, characteristic of the Tr1 subset, are specifically and significantly enhanced in patients prior to a severe recurrence, when compared with patients experiencing a mild recurrence of HCV. Thus at 1 year post-LT, IL-10 levels could be predictive of severe recurrence. These findings may have therapeutic implications, insofar as at 1 year post-LT, high IL-10 producers might represent a subset of liver transplant recipients who warrant more intensive management.

Patients and Methods

Patients

The study population consisted of patients who had undergone LT between 1994 and 1999 at Cochin Hospital, Paris, France. Thirty transplanted patients were included in this study. Inclusion was performed after the 5th year of follow-up when liver biopsy results became available. Chronic hepatitis C was defined by biochemical abnormalities, the RT-PCR detection of serum HCV-RNA (Cobas Amplicor HCV test 2.0, Roche Diagnostics) and consistent histological findings. All biopsy specimens were assessed by the same pathologist, and only biopsy samples with >10 portal tracts were retained for the study. A histological diagnosis of recurrent hepatitis C was based on combined evidence of hepatocellular necrosis, chronic inflammatory infiltrate and portal and periportal fibrosis. Biliary lesions were required to be focal and mild, and the absence of endothelialitis was necessary. Mild to moderate steatosis could be present. Hepatitis C was scored using scales for activity (A0–A3) and fibrosis (F0–F4), according to the METAVIR scoring system (31). Recurrent hepatitis C, 5 years after LT, was considered to be severe when histological examination produced a METAVIR score of A1F2 or higher and mild when the score was lower than A1F2. Patients with cirrhosis (F4) and with fibrosing cholestatic hepatitis were excluded from this study. Patients with histological evidence of acute or chronic rejection were also excluded. The histological diagnosis of acute rejection was based on the following combination of lesions: portal inflammatory infiltrate containing eosinophils, presence of biliary epithelial lesions and endothelialitis, absence of prominent piecemeal necrosis, possible presence of centrilobular lesions, including central vein endothelialitis and pericentrilobular inflammatory infiltrates. Chronic rejection was identified by dystrophic changes of the biliary epithelium affecting a majority of the bile ducts, with or without bile duct loss.

Thirty transplanted patients were thus included; 10 were HCV-negative and had normal histological findings, while the other 20 suffered from recurrent chronic hepatitis C (mild in 10 cases and severe in 10). Only HCV-positive patients with genotype 1 were included in this study. All patients who had undergone a systematic evaluation about 5 years after their LT were included consecutively, once histological findings of the liver biopsy had become available. When a liver biopsy was performed, clinical variables were assessed and patients with a recent episode of rejection (6 months), biliary complications or severe complications other than hepatitis C recurrence were ruled out of the study. A serum sample from the 1-year posttransplant workup was also available from 24 of the patients (8 in each group). Frozen portions of the liver biopsy from four patients in each group were available for PCR and immunohistological analysis.

Following LT, the patients had been treated with prednisolone (5 mg/kg daily, tapered to 20 mg from day 7) and cyclosporine (5 mg/kg daily) or tacrolimus (0.15 mg/kg daily). Cyclosporine and tacrolimus dosages were adjusted to maintain blood levels of between 100 and 200 ng/mL and 5 and 12 ng/mL, respectively. The immunosuppressive regimen was similar with respect to cyclosporine and tacrolimus (dose and dosage) and corticosteroids in the three patient groups (data not shown). The incidence of a past history of acute rejection was similar in the three groups. None of the patients were receiving antiviral therapy at the time of our study. Patient characteristics are summarized in Table 1. The three groups were comparable for age, sex, albumin, bilirubin and viral load at both 1 and 5 years post-LT. ALT levels were statistically higher in patients with severe recurrent hepatitis C at 1 and 5 years, and the METAVIR score was only significantly higher in patients with severe recurrent hepatitis C at 5 years after LT.

Table 1.  Patient characteristics
Transplanted patientsHCV-negative stable transplantsMild recurrent hepatitis CSevere recurrent hepatitis Cpb
  1. aNumber; bstatistical evaluation: p—severe versus other groups; cmean age (range).

1 year post-LT, na= 24888 
 Sex: male/female3/53/55/3ns
 ALT (IU/L)20.6 ± 3.230.5 ± 7.357.4 ± 9.90.02
 Bilirubin (mg/dL)20.4 ± 4.124.5 ± 3  26.2 ± 2.9ns
 Albumin (g/L)   44 ± 1.642.6 ± 1.939.2 ± 1.2ns
 Prothrombin rate (%)96 ± 791 ± 388 ± 4ns
 METAVIR activity00.61.2ns
 METAVIR fibrosis00.71.8ns
 Viral load (IU/mL) 0.98 10−6± 0.6 10−6 2.5 10−6± 0.8 10−6ns
5-year post-LT, na= 30101010 
 Sex: male/ female4/64/66/4ns
 Mean age (years)43 ± 954 ± 856 ± 5 ns
 ALT (IU/L)24 ± 624.7 ± 5.773.2 ± 15.40.01
 Bilirubin (mg/dL)22.1 ± 5.225.5 ± 12 28.7 ± 6.9 ns
 Albumin (g/L)43.9 ± 3.6 42.9 ± 2.236.2 ± 2  ns
 Prothrombin rate (%)95 ± 587 ± 9  74 ± 16ns
 METAVIR activity0.20.41.40.05
 METAVIR fibrosis0.40.72.8<0.001
 Viral load (IU/mL)  1.2 10−6± 0.4 10−6  2.9 10−6± 0.6 10−6ns

Transcriptomic analysis

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

RNA extraction

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

Serum mRNA extraction was achieved using the QIAamp® Viral RNA kit (QIAGEN, Courtaboeuf, France), as described by the manufacturer. Briefly, a serum sample was incubated with the AVL buffer supplemented with carrier RNA. After the addition of ethanol, the sample was applied to the QIAmp spin column and centrifuged. The column was then cleaned, and mRNA were eluted and then stored at −80°C prior to analysis.

Reverse transcription

RNA were supplemented with the following mixture [oligo dT (Roche, Meylan, France) + RNAsin (PROMEGA, Charbonnières; France) + H2O], and then incubated at 70°C for 5 min. After a further 5 min at room temperature, 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 incubation step 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 μL.

ABI PrismR 7000 sequence detection system

The quantification of transcripts from liver and serum samples was performed using real-time quantitative RT-PCR with the ABI PrismR 7000 sequence detection system (Applied Biosystems, CA, USA). The mix was optimized for real-time PCR analysis using SYBR Green 1 Dye, Amplitaq Gold® DNA polymerase, dNTPs with UTP, Passive Reference 1 required for signal normalization and optimized buffer components, in a total volume of 50 μL. Each sample was run in a 96-well plate containing 94 primers by performing initial denaturation at 95°C for 5 min, after which PCR reactions were cycled 40 times as follows: 15 s at 95°C and 1 min at 60°C. Fluorescence intensity was measured at the end of each elongation phase. Melting curve analysis was performed immediately after amplification, in accordance with the manufacturer's instructions. The amplification of liver cDNA was successfully repeated twice with cDNA from the same extraction.

Light cycler-based PCR assay

cDNA was synthesized from total RNA at a concentration of 100 ng/μL using oligo dT primers and Superscript reverse transcriptase (GIBCO BRL). The quantification of transcripts from samples was confirmed by real-time quantitative RT-PCR using the Light Cycler system (Roche Diagnostics, Meylan, France). The PCR mixture contained the following: Taq polymerase, 1× of LightCycler-DNA master SYBRGreen I (Roche Diagnostics), 3 mM of MgCl2, 0.5 μmol/L of each primer and 1 μL of cDNA preparation (patient cDNA samples) in a total volume of 20 μL. Thirty-two samples were run in parallel by performing an initial denaturation at 95°C for 8 min, and then the PCR reactions were cycled 35–40 times as follows: 15 s at 95°C, 7 s at the appropriate annealing temperature (Table 2) and 18–64 s at 72°C according to the length of the target sequence annealing (40 s at 58°C). Fluorescence intensity was measured at the end of each elongation phase. The melting curve analysis was carried out immediately after amplification, following the manufacturer's instructions.

Table 2.  Primer sequences
GenePrimer sequencesHybridization T°c
CD45′-GGGAAATCAGGGCTCCTTCTTA59°
5′-TGGTCCCAAAGGCTTCTTCTT 
CD255′-GGGACTGCTCACGTTCATCA59°
5′-TTCAACATGGTTCCTTCCTTGTAG 
GITR5′-TGTGTCCAGCCTGAATTCCA59°
5′-CCGAGGCACAGTCGATACACT 
CTLA45′-TTCTTCTCTTCATCCCTGTCTTCT61°
5′-GAGATGCATACTCACACACAAAGCT 
FoxP35′-TCACCTACGCCACGGTCA59°
5′-CACAAAGCACTTGTGCAG 
CD185′-ATGCTTGATGACCTCAGGAATGT59°
5′-ACGGTCTTGTCCACGAAGGA 
CD49b5′-CAACGGGTGTGTGTTCTGACA59°
5′-TCATCACACACAACCACAACATCT 
IL-105′-GCCTAACATGCTTCGAGATC59°
5′-TGATGTCTGGGTCTTCGTTC 
TGF-β5′-CGAGCCTGAGGCCGACTAC62°
5′-CGGAGCTCTGATGTGTTGAAGA 
β Actin5′-TGCTATCCAGGCTGTGCTA58°
5′-ATGGAGTTGAAGGTAGTTT 
GAPDH5′-GCCAAGGTCATCCATGACAACTTTGG58°
5′-GCCTGCTTCACCACCTTCTTGATGTC 

Primers

All primers were designed for real-time PCR use and were purchased from MWG-Biotech (Germany; Table 2). The housekeeping genes β-actin and G3PDH were used as controls.

Immunohistochemical analysis

Markers of regulatory T cells were also evaluated by immunohistochemistry, and TGF-β and IL-10 levels were also determined. Liver biopsies were snap-frozen in liquid nitrogen for subsequent immunohistochemical analysis. The DAKO LSABR2 System, HRP (Dako, Carpinteria, CA), was used, based on a modified labeled avidin–biotin technique. Briefly, the frozen sections 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 antibodies: 1/40), after which biotinylated rabbit anti-mouse IgG was added. The avidin–biotin complex was added, followed by 3-amino-9-ethylcarbazole, and the slides were finally counterstained. Anti-human mAbs CD4, CD25, IL-10, Integrin-α2 (CD49b) and Integrin-β2 (CD18) were purchased from Santa Cruz (Tebu-Bio, France). Slides were examined by two of the authors, who were blinded to the diagnosis. Positive cells exhibiting a characteristic staining were counted in three different portal tracts and three separate lobular fields of each sample (magnification ×200), and the mean number of positive cells was calculated for each patient group.

ELISA method

Serum levels of TGF-β and IL-10 were evaluated. 96-well plates (NUNC, Denmark) were coated overnight with 50 μL of a primary antibody solution at a concentration of 2 μg/mL. After washes, the plates were saturated with 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 at rates ranging from 10 ng/mL to 10 pg/mL. After that, the biotin-coupled secondary antibody (1 μg/mL) was added, and an amplification phase was performed using streptavidin-peroxidase. Plates were revealed and the revelation reaction was stopped by the addition of HCl 2N. The plates were then 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 normalized using the average CT values for housekeeping genes (ΔCT = CT − CTHKG). Average ΔCT values for each group were then compared with the ΔCT values of the control group (S group) (ΔΔCT =ΔCT −ΔCTS group). The evaluation of 2−ΔΔCT then indicated the fold change in gene expression relative to the control group.

The relevance of PCR and ELISA results was validated using the statistical rank 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 revealed a significant increase in markers associated with CD4+CD25+ Treg expression (Figure 1A). CD4 expression was increased in patients with severe recurrence when compared to HCV-negative patients and those with mild recurrence (p < 0.01 in both cases). When compared with the values in HCV-negative patients, CD25 expression was significantly increased (p < 0.05) in patients with both types of recurrence. A significant increase in CTLA4 and GITR expression was observed in groups with HCV recurrence when compared to the HCV-negative group, but no significant difference was found between the two HCV+ groups. Foxp3 transcription factor expression did not differ between the three groups. Taken together, these data showed that CD4+CD25+ Treg markers were overexpressed in HCV-positive versus HCV-negative recipients, without any difference between mild or severe recurrence.

Figure 1.

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

The expression of Tr1 markers (CD49b and CD18) was evaluated in the serum of the same patients. Levels of these markers were significantly elevated in patients with a severe hepatitis C recurrence when compared to patients with 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 in liver biopsies was increased in both patient groups experiencing recurrence (Figure 2A). CD4 expression was 30-fold higher in severe recurrence compared to HCV-negative recipients. CD25 mRNA was 6-fold higher in severe recurrence and 14-fold higher in mild recurrence compared to HCV-negative recipients. The expression of GITR and Foxp3 were also increased in patients with HCV recurrence, when compared to HCV-negative recipients. The intrahepatic expression of CD18 and CD49b mRNA was increased with HCV recurrence, and more markedly in the event of severe recurrence (Figure 2B).

Figure 2.

Intrahepatic mRNA expression of regulatory T-cell markers 5 years after LT. (A) CD4+ CD25+ regulatory T cells; (B) Type 1 regulatory T cells (Tr1) mRNA expression was evaluated on liver biopsies from HCV-negative recipients and recipients with a mild or severe HCV recurrence 5 years after LT. Results were calculated using the 2−ΔΔCT method, as described by Livak.

Protein levels:  Intrahepatic RT-PCR results were confirmed by the immunohistochemical evaluation of protein parameters (Table 3). For Treg markers (Figure 3), the mean CD4-positive cell count in lobular and portal fields was 16.8 ± 3.7 in severe recurrence (Figure 3C), 4 ± 0.8 in mild recurrence (Figure 3B) and 3 ± 1.5 in HCV-negative patients (Figure 3A). The mean CD25-positive cell count was also higher in severe recurrence (13 ± 3.8, Figure 3G) than that in 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 severe recurrence when compared with mild recurrence or HCV-negative findings (Figure 4). The mean CD4-positive cell count was 16.8 ± 3.7 in severe recurrence (Figure 4C), 4 ± 0.8 in mild recurrence (Figure 4B) and 3 ± 1.5 in HCV-negative patients (Figure 4A). The mean CD49b-positive cell count was 3 ± 1.5, 8 ± 2.2 and 13.5 ± 3.5, respectively, in the HCV-negative, mild and severe recurrence groups (Figure 4G, E and F) and that of CD18 cells was 2 ± 4.4, 7.3 ± 2 and 14.4 ± 1.4, respectively (Figure 4K, I and J). Taken together, these results suggest that Treg markers were overexpressed in the liver of patients with HCV recurrence when compared to HCV-negative transplanted patients, whereas intrahepatic Tr1 cells were only increased in severe recurrent hepatitis C and could thus be implicated in the increased severity of the course of recurrence after LT.

Table 3.  Intrahepatic expression of Treg and Tr1 cell markers (immunohistochemical evaluation)
 HCV-negative patientsMild recurrenceSevere recurrence
  1. aMean of positive cell counts in lobular and portal fields.

CD4a3 ± 1.54 ± 0.816.8 ± 3.7
CD25a4 ± 0.34 ± 0.6   13 ± 3.8
CD49ba3 ± 1.58 ± 2.213.5 ± 3.5
CD18a2 ± 1.47.3 ± 214.4 ± 1.4
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 or severe 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).

Figure 4.

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

Peripheral expression of regulatory T-cell markers 1 year after LT

The gene expression of Tregs was analyzed in sera that had been obtained 1 year after LT from some of the patients. All CD4+CD25+Treg markers were significantly overexpressed 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 which were similar in all three groups (Figure 5A). No differences were detected regarding CD4, CD25, GITR and CTLA4 between patients who would develop a severe or a mild recurrence of HCV 4 years later.

Figure 5.

Peripheral mRNA expression of Treg markers 1 year post-LT. (a) CD4+ CD25+ regulatory T cells; (b) Type 1 regulatory T cells (Tr1) mRNA expression was evaluated on sera from HCV-negative recipients and recipients with a mild or severe HCV recurrence 1 year after LT. Results were calculated using the 2−ΔΔCT method, as described by Livak. *p < 0.05 versus HCV-negative group **p < 0.01 versus HCV-negative group.

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. the HCV-negative group) were observed in the serum of HCV-positive recipients compared to HCV-negative patients (Figure 5B), with a trend toward the overexpression of Tr1 markers in patients who would progress to severe recurrence compared to those with a milder course. However, the difference between these two groups was not significant, probably because of the small number of samples available.

Tr1-associated immunosuppressive cytokines: TGF-β and IL-10

Five years after LT, TGF-βmRNA was overexpressed in the serum and liver of recipients with recurrent hepatitis C, when compared to HCV-negative patients (9-fold in the mild group and 16-fold in the severe recurrence group; p < 0.01 in both cases) (Figure 6A). One year after LT, TGF-β was 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 trend toward an increased expression of TGF-β in patients with severe recurrence compared with mild recurrence, but this difference did not attain significance.

Figure 6.

TGF-βand interleukin-10 expression in HCV-negative recipients with a mild or severe HCV recurrence after LT. (A) TGF-β mRNA 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-10 expression 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.

At 5 years, IL-10 mRNA expression was significantly increased in the serum and liver of patients with HCV recurrence (Figure 6B), and the difference was significant between severe recurrence and HCV-negative recipients (8-fold increase, p < 0.05, in serum and liver) and between severe and mild recurrence (4- and 2-fold increase in liver and serum, respectively, p < 0.05). IL-10 levels determined in sera using ELISA (Figure 6C) were significantly higher in patients 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 the mild and severe recurrence groups. IL-10 protein levels, assessed in the liver by immunohistochemistry, confirmed these 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-negative patients (0).

Finally, IL-10 mRNA (Figure 6B, upper graph) and protein (Figure 6C, upper graph) levels were compared in sera and liver samples that had been obtained 1 year after LT with the future pattern of recurrence at 5 years. IL-10 mRNA expression (Figure 6B) was higher in patients who would go on to develop severe recurrence, compared to the HCV-negative and mild recurrence groups (5.5-fold, p < 0.05, and 3-fold, p < 0.05, respectively). Moreover, IL-10 serum levels (Figure 6C) were higher in patients who would experience severe recurrence compared to HCV-negative patients 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 been described in patients with persistent HCV infection, which could contribute to HCV persistence by suppressing HCV-specific T-cell responses (19,20). The accelerated progression of recurrent hepatitis C prompted us to propose the potential role of Tregs after LT. During the present study, we found that 5 years after LT, Treg markers were overexpressed in sera from HCV-positive patients, compared to HCV-negative transplant patients, without there being any significant differences between mild and severe recurrence. By contrast, we showed for the first time that Tr1 markers (CD18, CD49b) were significantly overexpressed in patients with severe recurrence compared to those with mild recurrence (or to HCV-negative recipients). The overexpression of these markers was confirmed in liver biopsies obtained 5 years after LT. The data obtained 1 year after LT concerning some of the patients included in the 5-year study were analyzed in the function of their future pattern of recurrence at 5 years. An overexpression of Treg markers was observed in patients with HCV recurrence, and a trend toward an overexpression of Tr1 markers was observed in patients who would experience severe recurrence, when compared to patients with a persistent mild course at 5 years. However, this difference was not significant, probably because of the small number of samples (eight in each group).

Tr1 subpopulation activity was also assessed in terms of TGF-β and IL-10 production. We thus demonstrated for the first time, to our knowledge, that these two cytokines are overexpressed in sera and liver biopsies from patients with HCV recurrence when compared to HCV-negative patients, 5 years post-LT. More importantly, we detected an overexpression of these cytokines at 1 year after LT in patients who would subsequently develop a recurrence. IL-10 levels were significantly higher in patients experiencing severe recurrence than those in patients with persistent mild recurrence. These results were confirmed in peripheral blood using ELISA, and by immunohistochemistry on liver biopsies.

The first impressive finding was the significant increase in Treg and Tr1 cell markers alongside that of TGF-β and IL-10, but not of FoxP3, in serum and liver from HCV-positive recipients when compared to HCV-negative recipients. These data could explain that HCV-positive liver recipients have a risk of acute rejection, lower than or similar to that of other transplanted patients (34), which is enhanced under antiviral therapy (35). The low expression of FoxP3 in these patients may reflect the low and inconsistent expression of FoxP3 by Tr1 cells.

Little has been reported to date concerning the role of Tregs in recurrent hepatitis C after LT. Our results show that CD4+CD25+ Tregs are overexpressed, both peripherally and in the liver, in HCV-positive patients after LT, when compared to HCV-negative patients. This could be explained by the specific activation of this subpopulation in infected liver and by a sequestration of CD4+CD25+ Tregs in the graft. Similar results had previously been observed in a context of acute rejection (30). One could speculate that in the same way as in nontransplanted patients, CD4+CD25+ Tregs contribute to HCV persistence (19), despite the use of immunosuppressive drugs that reduce the number of CD4+CD25+ T cells in peripheral blood (36,37). However, our data did not allow us to determine whether these findings were a result, or a cause, of severe HCV recurrence.

Our results show that Tr1 cell markers and activity (TGF-β, IL-10) were increased in patients with severe HCV recurrence compared to mild recurrence. One major finding of this study is that IL-10 may predict the severity of HCV recurrence in the long term. Indeed, high IL-10 levels could already be detected 1 year after LT, and this could be interpreted as a specific activation of Tr1 cells. Interactions between Tr1 cells and immunosuppressive treatments are unknown; Tr1 cell proliferation is dependent on IL-15, a cytokine which is not inhibited by immunosuppressive therapy (38,39). Serum levels of IL-15 and IL-10 are elevated in a context of chronic hepatitis C (40), proportionally to disease severity in nontransplanted patients. Tr1 cells may thus proliferate and play a role in the course and severity of HCV recurrence. The assessment of samples obtained 1 year post-LT gave us the opportunity to investigate this hypothesis retrospectively, in the light of the subsequent course of HCV recurrence. IL-10 was significantly overexpressed in patients who would experience severe recurrence in the future, compared with those suffering from a persistent mild course and with HCV-negative patients. IL-10 is characteristic of Tr1 immunosuppressive activity and these cells could thus be implicated at the very beginning of the recurrence. Peripheral IL-10 expression could provide an important insight into the future course of hepatitis C recurrence, and in our hands, elevated IL-10 levels were predictive of severe recurrent hepatitis C.

Although these data are encouraging, we acknowledge several limitations. The study was retrospective, no functional assays were performed, the number of patients was small and we did not have documented serum and liver data at 1 year for all the recipients assessed at 5 years. Indeed, frozen samples of serum and liver stored at −80°C under optimal conditions constitute unique and rare materials. It will be a lengthy (more than 5 years) and difficult process to design a prospective trial in order to confirm these findings. A prospective trial is currently under way to confirm these data and to add functional assays, but the results will not be available for several years.

In conclusion, we have shown that regulatory T cells, and more particularly the Tr1 subpopulation, are increased in severe forms of recurrent hepatitis C, 5 years after LT. More importantly, at 1 year post-LT, high serum levels of IL-10 (the main Tr1 immunosuppressive cytokine) could be predictive of a severe course of hepatitis C recurrence. These findings may have therapeutic implications, insofar as such patients could benefit from reduced immunosuppressive therapy or from the earlier introduction of antiviral treatment in order to inhibit an accelerated course of HCV recurrence.

Acknowledgements

This work and A.C. received support from the ‘Agence Nationale de la Recherche sur le Sida et les Hépatites’ (ANRS) and the ‘Comité du Nord de la Ligue contre le cancer’.

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