|Year : 2012 | Volume
| Issue : 2 | Page : 116-122
Identification of FoxP3 expression in peripheral blood and liver tissues in Egyptian patients with hepatitis C virus infection
Samy B.M. El-Hady1, Eman Almasry2, Mahmoud Abdu A. Ashour3, Isam Sabe4
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Biochemistry, Faculty of Medicine, El_Minia University, El_Minia, Egypt
3 Department of Internal Medicine, Faculty of Medicine, Zagazig University, Zagazig, Egypt
4 Department of Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||23-Jan-2012|
|Date of Acceptance||20-Feb-2012|
|Date of Web Publication||23-Jun-2014|
Samy B.M. El-Hady
Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig
Source of Support: None, Conflict of Interest: None
FoxP3 constitutive expression is necessary for the suppressive function of regulatory T cells (Tregs). The majority of infected patients with hepatitis C virus (HCV) develop only a weak, narrow, or nonpersistent adaptive response to acute infection. The aim of this study was to investigate the frequency of CD4+FoxP3+ Tregs in peripheral blood and liver biopsy tissues in patients with acute and chronic hepatitis C and their potential association with viral load.
Patients and methods
The study participants were divided into three groups. Group I comprised 15 recently diagnosed patients with acute hepatitis C. Group II comprised 46 patients with chronic hepatitis C, of whom 21 patients had chronic hepatitis C without hepatic cirrhosis (group IIa) and 25 patients had chronic hepatitis C with hepatic cirrhosis (group IIb). Group III comprised 22 apparently normal individuals and they constituted the control group. Double immunohistochemical analyses were performed on liver biopsy samples for CD4/FoxP3 and CD8/FoxP3. Further, flow cytometric analysis of peripheral blood for CD4, CD8, and FoxP3-positive lymphocytes was carried out.
Most FoxP3+ cells in the peripheral blood and liver tissues were CD4+ cells, and CD8+FoxP3+ cells were very scare. A considerable number of FoxP3+ cells were observed in the portal tracts and fibrous septa, particularly when lymphoid aggregates were present. They were also observed in parenchymatous areas, where they preferentially localized in necrotic areas. In group I patients there were significant positive correlations between CD4+FoxP3+ cells in peripheral blood and alanine aminotransferase, aspartate aminotransferase, total bilirubin, and viral copies. In chronic hepatitis cases, no correlations were found between CD4+FoxP3+ cells in peripheral blood and liver tissues and other laboratory parameters.
FoxP3− Tregs are upregulated in HCV patients, suggesting an important role for Tregs in establishing and/or maintaining HCV persistence. Further studies are needed to examine the role of Tregs in HCV disease pathogenesis and to develop therapeutic approaches to control the balance between Tregs and effector T cells to enhance viral clearance.
Keywords: FoxP3, hepatitis C virus, regulatory T cells
|How to cite this article:|
El-Hady SB, Almasry E, Ashour MA, Sabe I. Identification of FoxP3 expression in peripheral blood and liver tissues in Egyptian patients with hepatitis C virus infection. Egypt J Haematol 2012;37:116-22
|How to cite this URL:|
El-Hady SB, Almasry E, Ashour MA, Sabe I. Identification of FoxP3 expression in peripheral blood and liver tissues in Egyptian patients with hepatitis C virus infection. Egypt J Haematol [serial online] 2012 [cited 2019 Dec 12];37:116-22. Available from: http://www.ehj.eg.net/text.asp?2012/37/2/116/135065
| Introduction|| |
Regulatory T cells (Tregs) constitute a specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens 1. Tregs are characterized by a number of markers including CD4, CD25, and the transcription factor FoxP3 2,3. Other surface markers shown to be expressed by Tregs include CD127, CD39, and CTLA-4 4–8.
FoxP3, a member of the forkhead/winged-helix family of transcription factors, acts as a ‘master’ regulator for the development of Tregs. Further, its constitutive expression is necessary for the suppressive function of Tregs 2. In the peripheral lymphoid organs, the large majority of FoxP3-expressing Tregs are CD4+ and express high levels of the interleukin (IL)-2 receptor α chain (CD25). In addition to the FoxP3-expressing CD4+CD25+ Tregs, there appears to be a minor population of CD8+FoxP3-expressing Tregs. Because CD25 is also expressed on activated T cells, the Treg population is more accurately defined by FoxP3 expression 9. Some FoxP3+ Tregs acquire the ability to produce IL-17; hence, they can potentially contribute to the antimicrobial innate immune defense while controlling inflammation and autoimmunity at the same time, particularly at mucosal sites 10. Multiple mechanisms of action of Tregs have been proposed, including cell-contact-dependent and cytokine-dependent mechanisms 11.
In human disease, alterations in the numbers of Tregs are found in a number of disease states. For example, patients with tumors have a local relative excess of FoxP3-positive T cells, which inhibits the body’s ability to suppress the formation of cancerous cells 12. In contrast, patients with an autoimmune disease such as systemic lupus erythematosus have a relative dysfunction of FoxP3-positive cells 13.
Hepatitis C virus (HCV) is a highly persistent human pathogen that causes chronic necroinflammatory liver disease with progression to liver failure and cancer 14,15. The adaptive T-cell immune response is important in mediating HCV clearance 16–21; however, the reason that the majority of infected patients develop only a weak, narrow, or nonpersistent adaptive response to acute infection is not well understood. Studies on some patients acutely infected with HCV have shown impairment of cytokine production during this phase of infection 21. Impaired cell responses of HCV-specific CD8+ T cells during the earliest phases of acute HCV infection have been observed 19. During the chronic phase of HCV infection, HCV-specific CD8+ T cells also display significant functional deficits, including impaired cytokine production and proliferative capacity 22,23. Further, it is known that HCV-infected patients have an IL-2 deficiency 24. This study was undertaken to study the frequency of CD4+FoxP3+ Tregs in peripheral blood and liver biopsy tissues in patients with acute and chronic hepatitis C and their potential association with viral load.
| Patients and methods|| |
This study included 83 individuals, all of whom were unaware of current or past immunomodulatory or antiviral treatment, were negative for HBsAg, other causes of hepatitis, anti-HIV antibodies and schistosoma infection, had no other causes of liver disease with no focal hepatic masses, and no history of alcohol consumption. These patients did not suffer from ascites, bleeding tendency, or esophageal varices. Chronic hepatitis is defined as the persistence of HCV RNA for at least 6 months after initial infection. Duration of infection describes the time from the estimated date of HCV acquisition to enrollment. These patients were classified into three groups:
Group I (patients with acute hepatitis C): Group I comprised 15 patients (nine males and six females; mean age 27.5±6.23 years) with acute hepatitis C who were diagnosed recently. They were healthcare workers infected by needle sticks with HCV antibody seroconversion. Patients were enrolled within 6 months of infection. The diagnosis was based on history taking, clinical features, and laboratory investigations.
Group II (patients with chronic hepatitis C): Group II comprised patients with chronic hepatitis C who were infected. These patients get infected more than 1 year ago. The diagnosis was based on history taking, clinical features, laboratory investigations, abdominal ultrasound, and liver biopsy. Samples were taken during follow-up. Outpatients were admitted for liver biopsy. For liver biopsy, a reticulin (Wilder’s) stain was used for outlining nodules of regeneration and septa surrounding cirrhotic nodules. The term cirrhosis was reserved for those with diffuse nodules surrounded by septa. Group II patients were classified into:
Group IIa (chronic hepatitis C without cirrhosis): This subgroup comprised 21 patients (12 males and nine females; mean age 40.3±6.66 years) with chronic hepatitis C without cirrhosis.
Group IIb (chronic hepatitis C with hepatic cirrhosis): This subgroup comprised 25 patients (14 males and 11 females; mean age 7±7.27 years) with chronic hepatitis C with hepatic cirrhosis.
Group III: Group III comprised 22 apparently healthy individuals (12 males and 10 females; mean age 36±7.04 years), matched for age and sex, who served as the control group.
All the participant were subjected to the following:
- Full history taking.
- Complete physical examination including abdominal ultrasound.
- Routine hematological and biochemical investigations including
- complete blood count using Sysmex S.F.3000 (Kobe, Japan), with examination of Leishman-stained films;
- liver and kidney function tests using a Dimension Autoanalyzer (Dade Behring, Illinois, USA);
- liver biopsy on fresh needle biopsy specimens obtained using 1.5 mm-diameter disposable needles, which was performed for chronic cases only. The intercostal technique guided by ultrasound (directed biopsy) was implemented by a radiologist in the presence of the treating clinician. Rest in bed for 24 h with careful observation was essential after the biopsy. Samples were fixed in 10% neutral formalin. Sections of paraffin-embedded tissues were used for hematoxylin and eosin, reticulin (Wilder’s), and immunohistochemical staining.
- Quantitative RT-PCR for HCV: HCV viremia was quantified by the Cobas Amplicor HCV Monitor test (Roche Diagnostic, Mannheim, Germany).
- Specific laboratory investigations.
Double immunohistochemical analysis
Double immunohistochemical labeling was performed manually on formalin-fixed, paraffin-embedded liver biopsy specimens. Endogenous peroxidase was blocked before staining. Two methods of immunodetection were applied: the streptavidin–biotin–peroxidase (SBP) method with diaminobenzidine (DAB) as chromogen and the streptavidin–biotin–alkaline phosphatase (SBAP) method with fast blue (FB) as chromogen.
For CD8/FoxP3 double staining, primary mouse anti-CD8 antibody (CD8, clone C8/144B, 1/100; Dako, Carpinteria, California, USA) and secondary biotinylated goat anti-mouse antibody (1/500; Jackson ImmunoResearch, West Grove, Pennsylvania, USA) were used, followed by detection with SBAP complex (1/50; Dako) and FB chromogen staining. For FoxP3 staining, slides were incubated at 4πC overnight with an mAb to FoxP3 (FoxP3, 1/20; Abcam, Cambridge, UK) and a secondary biotinylated goat anti-mouse antibody (1/500; Jackson ImmunoResearch); detection was performed with the SBP complex (1/200; Dako) and DAB chromogen staining. CD8+FoxP3+ costained cells were characterized by brown nuclear staining with the anti-FoxP3 antibody and circled by blue cytoplasmic membrane staining with the anti-CD8 antibody.
For CD4/FoxP3 double staining, primary mouse antibody (CD4, clone 4B12, 1/75; Novocastra, Newcastle, UK) was used, followed by incubation with secondary biotinylated goat anti-mouse antibody; detection was performed with SBP complex and DAB chromogen staining. The second immunostaining with FoxP3 was performed as above with the SBAP method and FB staining. Staining with an unrelated primary antibody (mouse IgG, 2 μg/ml, Jackson ImmunoResearch) provided a negative control for each reaction. CD4+FoxP3+ costained cells were identified by a blue nuclear staining with anti-FoxP3 antibody and were circled by a brown cytoplasmic membrane staining with the anti-CD4 antibody.
Cell percentages were ascertained on serial sections obtained from paraffin blocks. Recorded parameters included percentages of CD8+, CD4+, and FoxP3+ cells and distribution of FoxP3+ cells.
Flow cytometric analysis of peripheral blood
Whole blood was collected by venipuncture into vacutainer tubes containing ethylenediaminetetraacetic acid.
The whole blood staining method was used. A volume of 100 µl of fresh blood was incubated with anti-CD4 FITC and anti-CD8 PerCP monoclonal antibodies (Becton Dickinson, San Jose, California, USA) for 30 min at 4πC in one tube. Blood was then washed twice. This tube became ready to be analyzed by flow cytometry. In the same tube, the intracellular costaining of FoxP3 was carried out using the FoxP3 staining protocol. Cells were fixed and permeabilized with FoxP3 staining buffer (eBioscience, San Diego, California, USA). The resultant cells were stained with PE-conjugated antibodies to FoxP3 (20 μl/106 cells, clone PCH101 Set; eBioscience). Intracellular costaining of FoxP3 was performed on gated T cells using a flow cytometer (Becton Dickinson). Isotype-matched control antibodies were used to determine the background levels of staining.
Flow cytometry gating strategy
Total lymphocytes were gated on the basis of CD3+ expression. First, the proportion of each CD4+ or CD8+ population was calculated. Finally, the proportion of Tregs was determined by evaluating FoxP3-positive cells coexpressed with CD4+ cells. Average percentages of each subpopulation of CD4+, CD4+FoxP3+, CD8+, and CD8+FoxP3+ cells were calculated.
| Results|| |
Some laboratory data pertaining to patients with acute hepatitis C (group I), with chronic hepatitis C without cirrhosis (group IIa), with chronic hepatitis C with cirrhosis (group IIb), and the control group (group III) are illustrated in [Table 1].
Most FoxP3+ cells in the peripheral blood and liver tissues were CD4+ cells, and CD8+FoxP3+ cells were very scarce (ranging from 0 to 0.1%) [Table 2] and [Table 3], [Figure 1] and [Figure 2]. A considerable number of FoxP3+ cells were observed in the portal tracts and fibrous septa, particularly when lymphoid aggregates were present. They were also observed in parenchymatous areas, where they preferentially localized in necrotic areas. CD4+ T cells were the most common cells in portal tracts and piecemeal necrosis, whereas CD8+ T cells were the predominant cells in lobules.
|Table 2: Percentage of FoxP3 expression in peripheral blood in the studied groups|
Click here to view
|Table 3: Percentage of intrahepatic FoxP3 expression in the studied groups|
Click here to view
[Table 4] and [Table 5] and [Figure 3] and [Figure 4] show that in group I there were significant positive correlations between CD4+FoxP3+ cells in peripheral blood and alanine aminotransferase, aspartate aminotransferase, total bilirubin, and viral copies. Interestingly, in chronic hepatitis cases, no correlations were found between CD4+FoxP3+cells in peripheral blood and liver tissues and other laboratory parameters.
|Figure 3: A significant positive correlation found between the absolute count of CD4+FoxP3+ cells in PBMCs and alanine transaminase (ALT) in group I (r=0.49, P<0.05).|
Click here to view
|Figure 4: A significant positive correlation found between the absolute count of CD4+FoxP3+ cells in PBMCs and viral copies in group I (r=0.56, <0.05).|
Click here to view
|Table 4: Correlations between absolute counts of CD4+FoxP3+ lymphocytes in PBMCs and some laboratory findings in the studied groups|
Click here to view
|Table 5: Correlations between intrahepatic CD4+FoxP3+/CD4 expression and some laboratory findings in the studied groups|
Click here to view
| Discussion|| |
A member of the Fox protein family, FoxP3 appears to function as a master regulator in the development and function of Tregs 2.
Persistent HCV infection is associated with a weak, narrow, cell-mediated immune response that is characterized by a low frequency of HCV-specific interferon (IFN)-γ-producing T cells 17, 22, 25 and an even lower frequency of IL-2-producing cells 24. In HCV infection, most of the studies related to the role of FoxP3− Tregs are focused on peripheral Tregs, which probably do not reflect the precise composition and functions of these cells in the liver. The purpose of this study was to evaluate the frequency of CD4+FoxP3+ Tregs in peripheral blood and liver tissues of HCV patients in relation to viral load.
In this study, there were increased numbers of CD4+FoxP3+ cells in peripheral blood of all patient groups compared with normal controls. This result agrees with that found in the study by Smyk Pearson et al. 26 who found that acute HCV groups had significantly higher frequencies of circulating FoxP3+ Tregs compared with healthy controls, irrespective of whether they ultimately developed spontaneous resolution or persistence, suggesting that expansion of Tregs occurs during the earliest phases of acute HCV infection. Their data provide insight into the long-standing observation that HCV-specific cytotoxic T lymphocytes function poorly during acute HCV infection irrespective of the final outcome of disease.
Further, Claassen et al. 27 and Shevach 28 have shown that HCV patients have a higher number of Tregs in peripheral blood compared with healthy individuals. Similarly, Ebinuma et al. 29 found that among HCV-infected patients, HCV-specific FoxP3+ Tregs were detected in the blood and had the capacity to suppress HCV-specific T cells.
As regards chronic HCV infection, several studies have revealed an increased frequency of Tregs in the peripheral blood of these patients compared with persons whose HCV infection spontaneously resolved or in healthy controls 30,31. Further, Langhans et al. 32 found that FoxP3+CD25+CD4+ Tregs were detected more frequently in patients with chronic hepatitis C than in those with self-limited HCV infection, which responded to HCV core stimulation and inhibited proliferation of reporter cells. Furthermore, Speletas et al. 33 observed significant increase in FoxP3 in all disease groups compared with controls, which was positively correlated with the intensity of inflammation.
In our study an increase in CD4+FoxP3+ cells was seen on liver biopsy in chronic HCV cases (with and without cirrhosis); this finding agrees with that of Sturm et al. 34 who found that intrahepatic Tregs in chronic hepatitis C patients are mainly CD4+FoxP3+ cells, whereas CD8+FoxP3+ cells are very scarce. This point is supported by their complementary flow cytometry analysis, which does not detect CD8+FoxP3+Treg in liver samples. Further, Ward et al. 35 found a much higher percentage of CD4 FoxP3+ Tregs within the infected liver than in the blood. However, Speletas et al. 33 found that increased FoxP3 expression, and therefore Tregs intrahepatic accumulation, characterizes not only viral hepatitis but also, to an equal degree, nonalcoholic fatty liver disease as well as autoimmune hepatitis, primary biliary cirrhosis, and methotrexate-related hepatotoxicity.
There are several possibilities that may explain the increased frequency of CD4+FoxP3+ Tregs in HCV patients. Treg induction may be enhanced by HCV gene products with immune regulatory capacities 36,37. Further, a report indicates that HCV proteins can induce FoxP3+ Tregs 38. However, a more likely mechanism is related to certain cytokines that are characteristically overproduced in HCV-infected patients. It is now established that IL-10 is mainly produced by T cells with regulatory or suppressive functions 39,40. Secretion of transforming growth factor β, also produced by Tregs, is considered an important factor for the local survival and function of Tregs 30,41 and also contributes to induction of fibrosis 42. In-vitro studies have demonstrated that both transforming growth factor-β and IL-10 inhibit the cytokine secretion and cytotoxicity of CD8+ T lymphocytes 30,43.
Our study showed that FoxP3+ cells are predominantly localized in piecemeal and lobular necrosis. We found that CD4+FoxP3+ cells infiltrate the hepatic lymphoid aggregates, portal or septal tracts, and parenchymatous lobules or nodules; these findings are in agreement with those of Sturm et al. 34. This would mean that Tregs, within HCV-infected livers, have direct access to interact with cytotoxic T cells at the site of action. As the effector T cells home to the site of antigen expression, activation of antigen-specific Tregs (e.g. by HCV) in close proximity to the effector T cells will provide local rather than global immune regulation to limit immune-mediated damage while promoting viral persistence. Furthermore, activated lymphocytes, either by viral or other antigens, that enter the liver are likely to encounter Tregs as these cells are localized in portal tracts.
Several studies suggest an important role for Tregs in establishing and/or maintaining HCV persistence. The CD4+FoxP3+ T-cell fraction isolated from peripheral blood mononuclear cells of infected patients suppressed virus-specific CD8+ T-cell proliferation and IFN-γ production, and depletion of these cells resulted in increased IFN-γ production by the remaining cells in response to HCV proteins 33, 36, 44. Ebinuma et al. 29 also showed that HCV can prime virus-specific FoxP3− Tregs with antigen-specific expansion and suppression of HCV-specific CD8 T cells. They also showed that FoxP3 extends to non-HCV-specific CD4 T cells, suggesting a more global role for antigen-specific Tregs in immune regulation beyond that for HCV infection.
However, Langhans et al. 32 exclusively demonstrated that Tregs inhibit reporter T cells through secretion of IL-10 and IL-35 rather than through cell-contact-dependent mechanisms. The HCV-specific Treg clones lost their functional capacity, along with FoxP3 expression, if kept in culture without HCV core exposure.
In contrast to the results described above, Treg deficiency was reported in patients with persistent HCV infection. These patients often develop mixed cryoglobulinemia, an autoimmune B-cell proliferative disorder 45.
Interestingly, in patients with acute HCV, significant positive correlations were found between peripheral blood and serum liver transaminase levels and viral load, whereas at the chronic stage of the disease no such correlations were found. These findings are in agreement with Ebinuma et al. 29 who found that the ratio between CD25+FoxP3 T cells correlated positively with viremia in HCV-infected patients, suggesting that the balance between Tregs and effector T cells determines the level of viremia. In their study, a similar correlation was not observed for serum liver transaminase levels, perhaps because of generally well-preserved liver function among their study patients.
In conclusion, FoxP3− Tregs are upregulated in HCV patients, which may suppress HCV-specific CD8 T cells leading to viral persistence. Further studies are needed to examine the role of Tregs in HCV disease pathogenesis and to develop therapeutic approaches to control the balance between Tregs and effector T cells to enhance viral clearance while limiting liver inflammation.
| References|| |
|1.||Feuerer M, Hill JA, Mathis D, Benoist C. Foxp3+ regulatory T-cells: differentiation, specification, subphenotypes. Nat Immunol. 2009;10:689–695 |
|2.||Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T-cells. Nat Immunol. 2003;4:330–336 |
|3.||Sakaguchi S. Naturally arising Foxp3-expressing CD25+ CD4+ regulatory T-cells in immunological tolerance to self and non-self. Nat Immunol. 2005;6:345–352 |
|4.||Liu W, Putnam AL, Xu Yu Z, Szot GL, Lee MR, Zhu S, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ Treg-cells. J Exp Med. 2006;103:1701–1711 |
|5.||Borsellino G, Kleinewietfeld M, Di Mitri D, Sternjak A, Diamantini A, Giometto R, et al. Expression of ectonucleotidase CD39 by Foxp3+ Treg-cells: hydrolysis of extracellular ATP and immune suppression. Blood. 2007;110:1225–1232 |
|6.||Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T-cells mediates immune suppression. J Exp Med. 2007;204:1257–1265 |
|7.||Sansom DM, Walker LSK. The role of CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA-4) in regulatory T-cell biology. Immunol Rev. 2006;212:131–148 |
|8.||Tang Q, Boden EK, Henriksen KJ, Bour Jordan H, Bi M, Bluestone JA. Distinct roles of CTLA-4 and TGF-β in CD4+CD25+ regulatory T-cell function. Eur J Immunol. 2004;34:2996–3005 |
|9.||Pandiyan P, Lenardo MJ. The control of CD4+CD25+Foxp3+ regulatory T-cell survival. Biol Direct. 2008;3:6–10 |
|10.||Voo KS, Wang YH, Santori FR, Boggiano C, Wang YH, Arima K, et al. Identification of IL-17-producing FOXP3+ regulatory T-cells in humans. Proc Natl Acad Sci USA. 2009;106:4793–4798 |
|11.||Haque R, Lei F, Xiong X, Song J. The regulation of FoxP3-expressing regulatory T-cells. Endocr Metab Immune Disord Drug Targets. 2011;11:334–346 |
|12.||Beyer M, Schultze JL. Regulatory T-cells in cancer. Blood. 2006;108:804–811 |
|13.||Alvarado Sánchez B, Hernández Castro B, Portales Pérez D, Baranda L, Layseca Espinosa E, Abud Mendoza C, et al. Regulatory T-cells in patients with systemic lupus erythematosus. J Autoimmun. 2006;27:110–118 |
|14.||Hoofnagle JH. Hepatitis C: the clinical spectrum of disease. Hepatology. 1997;26(Suppl. 3):15S–20S |
|15.||Liang TJ, Rehermann B, Seeff LB, Hoofnagle JH. Pathogenesis, natural history, treatment and prevention of hepatitis C. Ann Intern Med. 2000;132:296–305 |
|16.||Cooper S, Erickson AL, Adams EJ, Kansopon J, Weiner AJ, Chien DY, et al. Analysis of a successful immune response against hepatitis C virus. Immunity. 1999;10:439–449 |
|17.||Diepolder HM, Zachoval R, Hoffmann RM, Wierenga EA, Santantonio T, Jung MC, et al. Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection. Lancet. 1995;346:1006–1007 |
|18.||Gerlach JT, Diepolder HM, Jung MC, Gruener NH, Schraut WW, Zachoval R, et al. Recurrence of hepatitis C virus after loss of virus-specific CD4+ T-cell response in acute hepatitis C. Gastroenterology. 1999;117:933–941 |
|19.||Lechner F, Wong DKH, Dunbar PR, Chapman R, Chung RT, Dohrenwend P, et al. Analysis of successful immune responses in persons infected with hepatitis C virus. J Exp Med. 2000;191:1499–1512 |
|20.||Missale G, Bertoni R, Lamonaca V, Valli A, Massari M, Mori C, et al. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response. J Clin Invest. 1996;98:706–714 |
|21.||Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med. 2001;194:1395–1406 |
|22.||Gruener NH, Lechner F, Jung MC, Diepolder H, Gerlach T, Lauer G, et al. Sustained dysfunction of antiviral CD8+ T-lymphocytes after infection with hepatitis C virus. J Virol. 2001;75:5550–5558 |
|23.||Wedemeyer H, He XS, Nascimbeni M, Davis AR, Greenberg HB, Hoofnagle JH, et al. Impaired effector function of hepatitis C virus-specific CD8+ T-cells in chronic hepatitis C virus infection. J Immunol. 2002;169:3447–3458 |
|24.||Semmo N, Day CL, Ward SM, Lucas M, Harcourt G, Loughry A, et al. Preferential loss of IL-2-secreting CD4+ T-helper cells in chronic HCV infection. Hepatology. 2005;41:1019–1028 |
|25.||Chisari FV. Unscrambling hepatitis C virus-host interactions. Nature. 2005;436:930–932 |
|26.||Smyk Pearson S, Golden Mason L, Klarquist J, Burton JR Jr., Tester IA, Wang CC, et al. Functional suppression by FoxP3+CD4+CD25 high regulatory T-cells during acute hepatitis C virus infection. J Infect Dis. 2008;197:46–57 |
|27.||Claassen M, de Knegt R, Turgut D, Groothuismink A, Janssen H. Role of multiple regulatory T-cell populations in controlling peripheral blood and liver immunity to human hepatitis C virus infections. Cytokine. 2009;48:98 |
|28.||Shevach EM. CD4+CD25+ suppressor T-cells: more questions than answers. Nat Rev Immunol. 2002;2:389–400 |
|29.||Ebinuma H, Nakamoto N, Li Y, Price DA, Gostick E, Levine BL, et al. Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T-cells in hepatitis C virus infection. J Virol. 2008;82:5043–5053 |
|30.||Cabrera R, Tu Z, Xu Y, Firpi RJ, Rosen HR, Liu C, et al. An immunomodulatory role for CD4+CD25+ regulatory T-lymphocytes in hepatitis C virus infection. Hepatology. 2004;40:1062–1071 |
|31.||Sugimoto K, Ikeda F, Stadanlick J, Nunes FA, Alter HJ, Chang KM. Suppression of HCV-specific T-cells without differential hierarchy demonstrated ex vivo in persistent HCV infection. Hepatology. 2003;38:1437–1448 |
|32.||Langhans B, Braunschweiger I, Arndt S, Schulte W, Satoguina J, Layland LE, et al. Core-specific adaptive regulatory T-cells in different outcomes of hepatitis C. Clin Sci. 2010;119:97–109 |
|33.||Speletas M, Argentou N, Germanidis G, Vasiliadis T, Mantzoukis K, Patsiaoura K, et al. Foxp3 expression in liver correlates with the degree but not the cause of inflammation. Mediators Inflamm. 2011;9:323–328 |
|34.||Sturm N, Thélu MA, Camous X, Dimitrov G, Ramzan M, Dufeu Duchesne T, et al. Characterization and role of intra-hepatic regulatory T-cells in chronic hepatitis C pathogenesis. J Hepatol. 2010;53:25–35 |
|35.||Ward SM, Fox BC, Brown PJ, Worthington J, Fox SB, Chapman RW, et al. Quantification and localisation of FOXP3+ T-lymphocytes and relation to hepatic inflammation during chronic HCV infection. J Hepatol. 2007;47:316–324 |
|36.||Boettler T, Spangenberg HC, Neumann Haefelin C, Panther E, Urbani S, Ferrari C, et al. T-cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T-cells during chronic hepatitis C virus infection. J Virol. 2005;79:7860–7867 |
|37.||Alatrakchi N, Graham CS, Van Der Vliet HJJ, Sherman KE, Exley MA, Koziel MJ. Hepatitis C Virus (HCV)-specific CD8+ cells produce transforming growth factor β that can suppress HCV-specific T-cell responses. J Virol. 2007;81:5882–5892 |
|38.||Li S, Jones KL, Woollard DJ, Dromey J, Paukovics G, Plebanski M, et al. Defining target antigens for CD25+FOXP3+IFN-γ-regulatory T-cells in chronic hepatitis C virus infection. Immunol Cell Biol. 2007;85:197–204 |
|39.||Abel M, Séne D, Pol S, Bourlière M, Poynard T, Charlotte F, et al. Intrahepatic virus-specific IL-10-producing CD8 T-cells prevent liver damage during chronic hepatitis C virus infection. Hepatology. 2006;44:1607–1616 |
|40.||Billerbeck E, Thimme R. CD8+ regulatory T-cells in persistent human viral infections. Hum Immunol. 2008;69:771–775 |
|41.||Bolacchi F, Sinistro A, Ciaprini C, Demin F, Capozzi M, Carducci FC, et al. Increased Hepatitis C Virus (HCV)-specific CD4+CD25+ regulatory T-lymphocytes and reduced HCV-specific CD4+ T-cell response in HCV-infected patients with normal versus abnormal alanine aminotransferase levels. Clin Exp Immunol. 2006;144:188–196 |
|42.||Schuppan D, Krebs A, Bauer M, Hahn EG. Hepatitis C and liver fibrosis. Cell Death Differ. 2003;10(Suppl. 1):S59–S67 |
|43.||Uraushihara K, Kanai T, Ko K, Totsuka T, Makita S, Iiyama R, et al. Regulation of murine inflammatory bowel disease by CD25+ and CD25- CD4+ glucocorticoid-induced TNF-receptor family-related gene+ regulatory T-cells. J Immunol. 2003;171:708–716 |
|44.||Rushbrook SM, Ward SM, Unitt E, Vowler SL, Lucas M, Klenerman P, et al. Regulatory T-cells suppress in vitro proliferation of virus-specific CD8+ T-cells during persistent hepatitis C virus infection. J Virol. 2005;79:7852–7859 |
|45.||Boyer O, Saadoun D, Abriol J, Dodille M, Piette JC, Cacoub P, et al. CD4+CD25+ regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis. Blood. 2004;103:3428–3430 |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]