hepatitis c virus ( hcv ) is a parentally transmitted hepatotropic rna virus that causes chronic hepatitis , which may lead to cirrhosis and hepatocellular carcinoma ( hcc ) . evidence suggests that clearance and control of hcv infection during acute phase is dependent on vigorous and multispecific cd4 and cd8 t lymphocyte responses [ 2 , 3 ] . on the contrary , the development and maintenance of chronic infection is linked to weak or absence of hcv - specific th1 response and to the presence of th2 cytokines ( il-4 and il-10 ) [ 4 , 5 ] . so , some studies have implicated il-10 in hcv pathogenesis [ 4 , 6 ] . regulatory t cell 1 ( tr1 ) was first described in 1996 [ 7 , 8 ] as a further subtype of cd4 t cells , without specific markers but with cytokine profile distinct from that of th1 or th2 cells . indeed , they were shown as secreting high level of il-10 , moderate levels of ifn- and il-5 , quite undetectable il-4 , and low amounts of transforming growth factor ( tgf- ) . recent studies have suggested that tr1 could be induced against bacterial , viral and parasite antigens in vivo and might prevent infection - induced immunopathology or prolong pathogen persistence by suppressing protective th1 response [ 9 , 10 ] but has never been directly implicated in hepatitis c viral pathogenesis . the dysfunction of the immune response against hcv could be explained by immunosuppressive mechanisms supported by tr1 via their high production of il-10 . the recent identification of combined expression of cd4 , cd18 ( integrin 2 ) , and cd49b ( integrin 2 ) as specific markers for tr1 cells should facilitate their characterization in vivo . an increased frequency of tregs was recently described in the blood of patients with persistent hcv infection when compared with those who had cleared hcv [ 12 , 13 ] . it has been proposed that tregs contribute to hcv persistence by suppressing hcv - specific t - cell responses [ 14 , 15 ] . some studies have shown a correlation between a reduced hcv - specific t - cell response and the secretion of il-10 and tgf- by liver - infiltrating tregs   and that tregs may inhibit hcv - specific t - cell activity in a cell - cell contact manner [ 12 , 13 ] . it has also been shown that treg levels are significantly enhanced in recurrent hepatitis c and that treg type 1 cell ( tr1 ) levels are specifically higher in severe recurrent hepatitis c . moreover , the opportunity of studying three histologically well - defined liver biopsies of an hcv genotype 1b infected patient followed from the chronic infection to the hcc allowed us to investigate the tr1 cells infiltration within these liver biospies . the first biopsy was performed during the chronic phase ( date not specified in the file ) and appeared normal without any liver injury , the second one was performed during the cirrhotic phase ( march 1990 ) , and the third one was performed during hcc ( 1998 ) . thus , we examined the intrahepatic t cell response in terms of cytokines , cytokine receptors , and adhesion molecules in a patient under ifn-/ribavirin treatment and who remained persistently infected > 18 years . the hcv infected patient was followed up in the hepatogastroenterology department of the necker university hospital . information concerning the patient included age alat > three times the normal and negative anti - hav , hbv , and hev igm , and positive serology for hcv ( third generation microparticle eia ( abbott axsym , abbott park ) ) confirmed by riba strip immunoblot assay ( chiron corporation , emeryville , ca ) . genotyping of hcv was done using a multiplex pcr method with genotype - specific primers , and the concentration of hcv rna was determined by rt - pcr with the cobas amplicor hcv monitor test ( roche diagnostics , branchburg , nj ) . the patient presented no human immunodeficiency virus ( hiv ) coinfection , immunosuppressive therapy , chronic liver disease due to hepatitis b virus , autoimmune hepatitis , or primary biliary cirrhosis and other causes of chronic liver disease . three liver biopsies were performed for one patient who underwent several biopsies for a longitudinal study . the three biopsies were performed as part of the routine medical followup and were obtained by the standard menghini procedure ( needle diameter : 1.6  mm ) with a biopsy sample length of approximately 1  cm . based on histologic fibrosis ( f ) and necroinflammatory ( ni ) scores , liver biopsies were divided into biopsies with small hepatitis and without liver lesion ( f < or = 1/6 ; n < or = 1/18 ) , biopsies with fibrosis grade and liver lesions corresponding to histologically proved cirrhosis ( f = 6/6 ) , and biopsies with hcc histologically proved by anatomopathologic expertise . the study was approved by the institut de biologie de lille ( cnrs ) institutional review boards and informed consent was obtained in writing from the donor . extraction of total rna from frozen liver biopsy samples was performed using trizol reagent ( gibco brl , invitrogen , scotland , uk ) as described by the manufacturer . homogenized samples were incubated for 5 minutes at room temperature and resuspended in 60  l of chloroform ( prolabo , merck eurolab , france ) . total rna was then immunoprecipitated with 100  l of isopropanol ( acros organics , usa ) . the rna pellet was finally washed twice with 75% ethanol ( carlo erba reagent , france ) and dissolved in dnase , rnase - free distilled water ( gibco brl , invitrogen , scotland , uk ) . before reverse transcription , we performed dnase treatment using the kit message clean ( genhunter corp . , france ) . cdna was synthetized from total rna at a concentration of 100  ng/l using random hexamers and superscript reverse transcriptase ( gibco brl , invitrogen , scotland , uk ) . the quantification of transcripts from liver samples was performed by real - time quantitative rt - pcr using the light cycler system ( roche diagnostics , mannheim , germany ) . the pcr mixture contained the following : taq polymerase , 1x of light - cycler dna master sybr green i ( roche diagnostics , meylan , france ) , 3  mm of mgcl2 , 0.5 mol / l of each primer , and 1  l of cdna preparation ( patient cdna samples ) in a total volume of 20  l . thirty - two samples were run in parallel by performing an initial denaturation at 95c for 8 minutes ; the pcr reactions were cycled 35 to 40 times as follows : 15 seconds at 95c , 7 seconds at the appropriate annealing temperature ( table 1 ) , and 18 to 64 seconds at 72c according to the length of the target sequence , followed by annealing ( 40  s at 58c ) . the melting curve analysis was carried out immediately after amplification , following the manufacturer 's instructions . amplification of liver cdna was successfully repeated three times with cdna from the same extraction . all primers were designed for real - time pcr use and were purchased from mwg - biotech ( germany ) ( cf . samples are quantified using relative standard curves for each amplification reaction , and results were normalized to the internal controls -actin and g3pdh . after each light cycler run , agarose gel electrophoresis with tbe ( tris - borate - edta ) 1.5% agarose ( sigma - aldrich , germany ) gel , followed by dna staining with ethidium bromide , was performed to have an independent validation check of the presence of an amplicon . in order to control the length of the amplicon generated , a 100 bp dna ladder ( gibco brl , invitrogen , scotland , uk ) was used . as the lymphotropism of hepatitis c virus ( hcv ) has already been ascertained , and in the light of the fact that the immune defense system is an organized network composed of functionally immune cells , this study was carried out to verify the possible involvement of tr1 cells in the progression of hcv - related chronic hepatitis . in this study , we first analyzed the quantitative expression of the different intrahepatic cell populations by real - time pcr using light cycler system ( figure 1 ) , and we performed after each rt - pcr an agarose gel electrophoresis to have an independent validation check ( figure 2 ) . we observed a significant increase of cd19 expression in cirrhotic and hcc liver biopsies in comparison with the first biopsy without liver lesions . this observation confirmed data of the literature showing disturbances of b lymphocyte activation and function associated with hcv chronic infection [ 17 , 18 ] . the cd8 but not cd8 gene expression was also increased in the cirrhotic and hcc biopsies . this observation was in correlation with recent data evidencing by a similar approach of real - time quantitative assays a significant increase and infiltration of cd8 during chronic hcv infection . but the major observation of our work was the significant increase of cd4 expression in the course of time and proportionally with the severity of the fibrosis . however , we did not observe a variation of ifn- or il-2 expression , suggesting that the cd4 marker detected in cirrhosis and hcc liver biopsies was probably not associated with the th1 protective phenotype . due to the absence of il-4 expression , the cd4 cells with th2 phenotype were also excluded in accordance with the work of bergamini et al . . a further subtype of cd4 t cells , with immunosuppressive function and cytokine profiles distinct from either th1 or th2 t cells , termed regulatory t cells has been described . they include tr1 cells which secrete high level of il-10 and low to moderate levels of tgf- , th3 cells which primarily secrete tgf- , and cd4cd25 cells , which inhibit immune response through cell - cell contact ( 9 ) . although the level of il-10 and tgf- produced in vitro may not be significantly higher than that produced by classical th2 cells , what is important is that tr1 cells make these cytokines in the absence of significant levels of il-4 . we confirmed within cirrhotic and hcc biopsies a significant increase of expression of il-10 , tgf-1 and their respective receptors il-10r , il-10r , and tgf-r2 whereas we did not detect an increase of il-4 . so , the production of il-10 and tgf- in the absence of il-4 argued in favor of the presence of tr1 in the cirrhotic and hcc biopsies . although liver dendritic cells also secrete high levels of il-10 , in the present study we could not detect cd11c expression , confirming a tr1 cells origin for il-10 production . in this sense , functional alteration of hcv - specific cd4 t cells or failure to develop a long - lasting t helper response was recently correlated to a loss of ifn- secretion and the presence of a significant antigen - specific il-10 and tgf- , which could contribute to chronic hepatitis c persistence . moreover , tgf- plays a pivotal role in inducing fibrosis and has been proposed as its surrogate marker . indeed , a very interesting study has shown that serum tgf-1 could be used to assess therapeutic outcome and short - term prognosis of hcv - related chronic hepatitis . moreover , results obtained for tr1 specific markers cd49b and cd18 confirmed the tr1 phenotype and showed that tr1 cells increased during the progression of the pathology . despite their low proliferative capacity , cloned tr1 express normal levels of t cell activation markers such as cd25 following tcr - mediated activation . they also expressed ccr5 and t1-st2 , an il-1-r - like molecule , markers previously expressed preferentially on th1 and th2 cells , respectively . we clearly confirmed here within cirrhotic and hcc biopsies the expression of cd25 , ccr5 , and t1-st2 in relation to the presence of tr1 cells . in accordance with this tr1 phenotype , the expression of tgf--r2 was also observed . however , we can not exclude the implication of other regulatory t cell subsets , insofar as we observed a significant increase of cd25 , pselectin , and icam i expression , which were known as specific markers for cd4cd25 regulatory t cells . these observations were also conforted by other experiments we performed on liver biopsies of three well - defined cohorts of 45 hcv genotype 1b infected patients , including patients without liver lesions , patients with cirrhosis , and patients with histologically proved hcc that confirmed an increase of tr1 proportional to the aggravation of the pathology . a first implication of regulatory t cells during hcv infection was examined in peripheral blood of patients infected with hcv genotype 1b , showing a secretion of ifn- or il-10 but not il-4 by hcv core - specific cd4 t cell clones . this work demonstrated that helper type 1 and regulatory t cells were induced probably against the same epitopes on the core protein . moreover , sugimoto et al .   have suggested for the first time that hcv persistence was associated with a reversible cd4-mediated suppression of hcv - specific cd8 t cells and with higher frequency of cd4cd25 regulatory t cells that could directly suppress hcv - specific type 1 cd8 t cells ex vivo . indeed , it is possible that the induction of regulatory t cells during hcv infection may suppress antiviral th1 and consequently cd8 ctl responses in vivo , and this may explain the persistence of infection and prevalence of cirrhosis . evidence of regulatory t cells installation in the liver of a chronically infected patient with cirrhosis and hepatocellular carcinoma ( hcc ) suggests a key role for these cells in the aggravation of the liver pathology and could potentially represent a predictive factor of liver damage aggravation . as previously described   high serum il-10 levels may be related to a poor response to ifn treatment in patients with chronic hepatitis c. in this sense , it would have been interesting to investigate other markers such as spleen markers or beta2-microglobulin , although it has been described that neither spleen measurements nor serum beta2-microglobulin levels were able to predict therapeutic response to antiviral therapy . in conclusion of our study , the increase of tr1 cells , which suppressed protective th1 responses via their high production of il-10 , could explain the failure of the ifn treatment observed for this patient . in a more general way , the implication of tr1 in viral persistence and associated hepatic pathologies could have significant implications for our understanding of the role of t cells in immunity to infectious diseases and for the development of new therapies for the control of immune - mediated disorders .
hepatitis c virus ( hcv ) is an important causative agent of liver disease , but factors that determine the resolution or progression of infection are poorly understood . in this study , we suggested that existence of immunosuppressive mechanisms , supported by regulatory t cells and especially the regulatory t cell 1 subset ( tr1 ) , may explain the impaired immune response during infection and thus the fibrosis aggravation to hepatocellular carcinoma ( hcc ) . using quantitative real - time pcr , we investigated the intra - hepatic presence of tr1 cells in biopsies from a genotype 1b infected patient followed for an 18-year period from cirrhosis to hcc . 
 we described a significant increase of gene expression in particular for the cytokines il-10 , tgf- , and their receptors that were perfectly correlated with an increased expression of the tr1 specific markers ( combined expression of cd4 , cd18 , and cd49b ) . 
 this was strongly marked since the patient evolved in the pathology and could explain the failure of the treatment . in conclusion , evidence of regulatory t cell installation in the liver of chronically infected patient with cirrhosis and 
 hcc suggests for the first time a key role for these cells in the course of hcv infection .