- Email: [email protected]
TC1 response, in hepatitis C virus (HCV) infection and human immunodeficiency virus (HIV)-HCV co-infection
POSTERS
S 160
munity. Reduced number and/or functional impairment of these cells may contribute to the immune dysregulation associated with HCV infection.
]•]
DYNAMICS OF CD81-EXPRESSION ON LYMPHOCYTE SUBSETS DURING ANTIVIRAL TREATMENT OF PATIENTS WITH CHRONIC HEPATITIS C
B. Kronenberger1, E. Herrmann 1, T. Ghaliai 1, H. Wedemeyel 2, W.R Hofmann 1, U. Mihm 1, S. Zeuzem 1, C. Sarra~zin1. 1Klinikfiir
Innere Medizin IL Universitiitsklinikum des Saarlandes, Homburg/Saar, Germany," 2Abt. fiir Gastroenterologie, Hepatologie und Endokrinologie, Medizinisehe Hoehsehule Hannover, Germany Aim: CD81 is a hepatitis C virus (HCV) co-receptor with important functions on lymphocytes. During treatment CD81 expression may be affected directly the antiviral therapy or indirectly by reduction of HCV-RNA levels. The dynamics of CD81 on lymphocyte subtypes have not yet been investigated and may be relevant for viral replication and treatment response. Methods: CD81 was flow cytometrically quantified on CD8(+), CD4(+), CD19(+) and CD56(+) lymphocyte subtypes from patients with chronic hepatitis C before, during and after antiviral treatment with pegylated interferon-alpha and ribavirin. Dynamics of CD81 were investigated in correlation with HCV-RNA dynamics and the viral kinetic parameters efficacy of blocking virus production and infected cell loss. Results: Patients with chronic HCV showed a higher baseline CD81expression (n 20) than healthy controls (n 8). Baseline CD81-expression on CD4(+) and CD8(+) cells inversely correlated with HCV-RNA serum levels. During treatment, the following typical patterns of CD81-regulation were observed: 1. Downregulation on CD8(+) T-cells (Figure A) and most significantly on CD56(+) natural killer cells (Figure B), 2. Transient upregulation on CD19(+) B-cells and 3. No change on CD4(+) T-cells. During treatment, CD81 on CD8(+) and CD19(+) cells correlated with antiviral efficacy and infected cell loss, respectively, but not with HCVRNA serum levels. After end of treatment, CD56(+) cells and CD8(+) cells from sustained responders maintained lower CD81-expression compared with baseline. Conclusion: Lymphocyte subsets show different patterns of CD81response before and during antiviral treatment which are associated with antiviral response, HCV-RNA serum levels, and administration of pegylated interferon-alpha and ribavirin. 7OO
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CHARACTERISATION OF HCV CORE-SPECIFIC ADAPTIVE REGULATORY T CELL CLONES FROM PATIENTS WITH CHRONIC HEPATITIS C INFECTION
B. Langhans, I. Braunschweiger, H.D. Nieschalke, T. Sauerbruch, U. Spengler. Department of Internal Medicine I, Universitdtsklinikum
Bonn, Bonn, Germany Background and Alms: Circumstantial evidence indicates a potential role of regulatory CD4+ T cells in the control of virus-specific T cell responses during acute and chronic hepatitis C virus (HCV) infection. However, to date only so called natural regulatory CD4+ CD25+ T cells with HCV specificity have been analysed. Here, we report on T cell clones representing adaptive HCV-core specific regulatory on CD4+ cells.
Methods: For generation ofT cell lines, isolated peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C infection were stimulated in vitro with HCV core protein in the presence of human rIL-2 and autologous feeder cells over 24 days. For cloning, T cells were seeded under limiting dilution conditions. T cell clones (TCC) were picked after 10 days from plates with <10% proliferative wells and were re-stimulated with PHA at 10- to 14-day intervals. Specificity of TCC was analysed with respect to proliferation (3H thymidin incorporation) and cytokine production (IL-2, IL-4, IL-10, IFN-T, TGF-[J) after stimulation with HCV core protein and anti-CD3/CD28, respectively. Candidates of HCV-specific regulatory CD4+ T cells were characterised phenotypically (typical markers of regulatory CD4+ T cells: CD25, GITR, CTLA-4) and functionally (inhibition of autologous IFN-g producing TH1 reporter clones). Results: Among the remaining clones, clone H7_3 and H7_16 were outstanding, because they produced substantial amounts of IL-10 and TGF-[J upon restimulation with HCV core protein and anti-CD3/CD28, respectively, but did not proliferate in response to HCV core or antiCD3/CD28 stimulation nor did they produce IL-2, IL-4 and IFN-T. Importantly, both clones inhibited IFN-g production of autologous TH1 reporter clones. Both clones expressed CTLA-4 and GITR. Moreover, clone H7_16 belonged to the CD4+CD25high T cell subpopulation, while clone H7_3 was CD4+CD25negative. Conclusion: Our results demonstrate that HCV-specific T cells exist in chronic hepatitis C, which inhibit anti-viral TH1 effector cells. Moreover, such regulatory CD4+ cells can also be found in T cell subpopulations other than CD4+CD25high T cells.
~ T
CELL CHEMOKINE RECEPTOR EXPRESSION AND CHEMOTAXIS, ASSOCIATED TO TH1/TC1 RESPONSE, IN HEPATITIS C VIRUS (HCV) INFECTION AND HUMAN IMMUNODEFICIENCY VIRUS (HIV)-HCV CO-INFECTION
M. Calvino 1, J.R. Larmbia 1'2, E. Sanz 2, J. P6rez 1, S. Benito 1, F. Gonzfilez 2, C. Perna4, M. Montes 3, M. Rodriguez-Zapata 3, T. Parra 1, A. Bienvenido 2. 1Liver Research Unit, 2Gastroenterology Department,
3Internal Medicine Department, 4pathology Department Hospital Universitario, Guadalajara, Spain Background: Interaction between CCR5, CXCR3 receptors and its ligands favours T cells migration into the liver. Intrahepatic inflammatory infiltrate is responsible of liver damage in chronic HCV infection. HIV HCV infection is characterized by a more aggressive natural history than isolated HCV infection. This fact could be related to different potential of migration in T cells. Objectives: (1) To evaluate the ratio of CXCR3 and CCR5 expression between CD4+ and CD8+ cells in HCV infection (HCV/HIV), HCV HIV co-infection (HCV/HIV+) and healthy controls (HC). (2) To evaluate invitro migration capacity of CD4+ and CD8+ cells after MIP-la, RANTES and IP-10 stimulation. Methods: Peripheral blood mononuclear cells (PBMC) from 27 HCV/HIV , 9 HCV/HIV+ and 10 HC were obtained. PBMC were stained with anti-CD8, anti-CD4, anti-CCR5 and anti-CXCR3 monoclonal antibodies and analysed by flow-cytometry. PBMC from 5 patients of each group were used for in-vitro analysis of migration in response to chemokines. Migration index was calculated as: (cells migrating toward chemokine gradient)/(cells migrating toward medium alone). Data are shown as median plus interquantile range (IQR). Kmskall Wallis and Mann Whitney-U tests were used for statistical comparisons. Results: In HCV/HIV the ratio TCD8+CCR5+/TCD4+CCR5+ (1.4; IQR 1) decreased with respect to HC (2.6; IQR 1.14) (p<0.001) and this was not observed in HCV/HIV+. The ratio TCD8+CXCR3+/ TCD4+CXCR3+ decreased in HCV/HIV (1.4; IQR 0.3) and in HCV/HIV+ (1.6; IQR 0.9) in relation to HC (2.9; IQR 2.3) (p <0.001). Migration index after stimulation with IP-10 was higher in HCV/HIV+ (CD8+ 2.5; IQR 2.5/CD4+ 3.4; IQR 2.7) than in HCV/HIV
05C. Viral Hepatitis'
(e) Hepatitis' C Experimental (immunology)
(CD8+ 1.6; IQR 0.7/CD4+ 1.7; IQR 1.1) (p <0.05). No difference was found after stimulation with RANTES and MIP-la. Conclusions: (1) HCV HIV co-infection and isolated HCV infection display a relative decrease of CXCR3 expression on CD8+ cells in relation to CD4+ cells. (2) Isolated HCV infection shows a relative decrease of CCR5 expression on CD8+ cells with respect to CD4+, not observed in HCV/HIV+. (3) HCV/HIV+ group presents a migration potential in response to IP-10 higher than HCV/HIV group. (4) These last two differences could favour a more aggressive intrahepatic inflammatory infiltrate in HIV HCV co-infection.
•
C H E M O K I N E R E C E P T O R E X P R E S S I O N ON T CELLS A C C O R D I N G TO I N F L A M M A T O R Y ACTIVITY A N D LIVER FIBROSIS IN C H R O N I C HEPATITIS C VIRUS (HCV) INFECTION
M. Calvino 1, J.R. Larrubia 1'2, E. Sanz 2, C. Perna 3, J. P~rez 1, S. Benito 1, F. Gonzfilez 2, T. Parra 1, A. Bienvenido 2. 1Research
Unit, 2Gastroenterology Department, 3Pathology Department, Hospital Universitario, Guadalajara, Spain Background: Chemokine receptors play an important role in specific T cells migration into liver to control HCV during primoinfection. Nevertheless, during chronic infection these receptors attract a non-specific mononuclear infiltrate into the liver responsible for hepatic damage. The study of chemokine receptor profiles on T cells has an undoubted therapeutic interest. Objective: To correlate in chronic hepatitis C (CHC) CXCR3, CCR5 and CCR3 expression on CD4+ T cells from peripheral blood (PB) and on CD8+ T cells from liver and PB, with hepatic inflammatory activity and fibrosis according to Scheuer index. Methods: Peripheral blood (PBMC) and intrahepatic (IHMC) mononuclear cells from 27 CHC patients were obtained. PBMC were separated through a Ficoll-Hypaque gradient and IHMC through collagenase-I and DNAase digestion. Mononuclear cells were marked with anti-CD8, antiCD4, anti-CCR5, anti-CCR3 and anti-CXCR3 monoclonal antibodies and analysed by flow-cytometry. Hepatic inflammatory activity and fibrosis grade were evaluated by Scheuer index after hemtoxilin-eosin staining. Spearman correlation coefficient was used for statistical analysis. Results: It was observed a positive correlation between intrahepatic CD8+/CXCR3+ T cells and porto-periportal (PPA) (r 0.7; p < 0.01) and lobular (LA) (r 0.6; P<0.05) activity. Also, it was found a positive correlation between PB CD4+/CCR5+ T cells and LA (r 0.6; p <0.05) and between PB CD4/CXCR3 T cells and PPA (r 0.4; p < 0.05). There seemed to exist a positive correlation between CD8+/CCR5+ T cells from liver and PB and intrahepatic inflammatory activity, although it did not reach statistical significance in our study. There was not any correlation between CCR5 and CXCR3 expression and liver fibrosis. CCR3 expression did not correlate with any histological variable. Conclusions: (1) Chemokine receptor expression associated to Tcl/Thl response on T cells correlates with intrahepatic inflammation but not with liver fibrosis. (2) Treatments focus on CXCR3 and CCR5 regulation on T cells may modify liver inflammation in chronic hepatitis C.
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A S S O C I A T I O N OF I M M U N E CELL INFILTRATION A N D HEPATIC I N T E R F E R O N - S T I M U L A T E D GENE E X P R E S S I O N IN THE A B S E N C E OF IRF-3 ACTIVATION IN C H R O N I C HEPATITIS C
D. Lau 1, P. Fish2, S.M. Lemon 3, M. Gale Jr. 2. 1Dept Internal Medicine,
UTMB, Galveston, TX, USA," 2Dept of Microbiology, UT Southwestern, Dallas, TX, USA," 3Dept of Microbiology, UTMB, Galveston, TX, USA a/[J Interferons (IFNs) play important role in viral infections. HCV encodes proteins that block IFN production and antagonize IFN actions to support
S161
viral persistence. The NS3/4A protease of HCV disrupts viral activation of IRF-3, thereby attenuating IFN production and IFN-stimulated gene (ISG) expression. [Foy et al, Science 2003; 300:1145 48]. In this study, we compared the transcriptional profiles of ISGs among liver biopsy samples from normal controls, HCV genotype-1 patients and patients with non-alcoholic fatty liver disease (NAFLD) using Affymetrix HG-U95A Human GeneChips. Liver biopsy slides were also examined by immunofluorescence staining and confocal microscopy analysis to define the subcellular distribution of IRF-3, the extent of viral protein expression in hepatocytes, and the composition of hepatic infiltrating immune cells. HCV samples had significantly higher transcriptional levels of ISGs associated with IFN such as OAS2, MXl,ISG56 compared to controls and NAFLD (p<0.005). The level of IRF-3, however, was similar in HCV and normal controls. HCV NS3 or NS5A proteins were observed within focal areas of biopsy specimen and distributed in a perinuclear context within hepatocytes. IRF-3, though abundant, was typically found in an inactive state as defined by its cytoplasmic-bound distribution in liver cells. Furthermore, HCV infection was associated with specific CD3positive T cell and plasmacytoid dendritic cell (PDC) infiltrates in biopsy specimens. Our results indicated that in chronic HCV, IRF-3 activation within infected hepatocytes is limited, which lends further evidence of NS3/4A disruption of IRF-3 activation. Despite the lack of active IRF-3, increased hepatic ISG expression in HCV infection was observed and was correlated with the presence of immune cell infiltrates in liver samples. Infiltrating immune cells could be an important source of alpha/beta IFNs that potentially influence hepatic ISG expression and the outcome of HCV infection.
[•
C O - I N H I B I T O R Y M O L E C U L E B7-H1 MAY PLAY A N I M P O R T A N T ROLE F O R C H R O N I C I T Y OF HEPATITIS C
Y.J. Lee 1'3, S.J. Park 1'3, S.R. Jee 1, S.H. Lee 1, S.Y. Seol 1, J.M. Chung 1, H.Y. Jeong 2'3, S.K. Seo 2'3, S.G. Park 2'3, I.H. Choi 2'3. 1Department
of Internal Medicine, 2Department of Microbiology, 3Center for Viral Disease Researeh, Inje University School of Medicine, Busan, South Korea Background and Aims: The establishment of a chronic hepatitis C (CHC) infection is believed to be associated with the failure of an effective HCVspecific T cell response. Little is known regarding the role of negative T cell regulators such as B7-H1 (CD274) in the impaired HCV-specific T cell response. B7-H1 is a member of the B7 co-signaling family and has been shown to suppress both CD4+ and CD8+ effector T cell responses. We therefore examined the expression B7-H1 on immune cells from CHC patients. Methods: Twenty-one age-matched healthy volunteers and twenty-four CHC patients who were enrolled in the Busan Paik Hospital of Inje University (Busan, Korea) participated in this study. We did flow cytometry for analysis of B7-H1 on PBMC, T cell proliferation assay using co-culture system involving purified T cells and monocytes, ELISA for measurement of IL-2, IL-4, IL-10 and IFN-y in the culture supematants, and ELISPOT for IFN-y production from CD4+ or CD8+ T cells in response to HCV NS4 protein and NS3 and NS4 peptides. Results: B7-H1 was significantly up-regulated on CD14+ monocytes and B cells isolated from CHC patients compared to normal subjects. In a T cell proliferation assay, we found that B7-HI+ CD14+ monocytes significantly reduced T cell proliferation in response to anti-CD3 compared to B7H1- CD14+ monocytes. The involvement of B7-H1 in an impaired HCVspecific T cell response was further supported by the finding that blockade of monocyte-associated B7-H1 significantly augmented the generation of IFN-producing HCV-specific CD4+ and CD8+ effector T cells. In addition, monocyte-B7-H1 blockade greatly enhanced the production of type 1 cytokines including IFN-y and IL-2, but not type 2 cytokines such as IL-4 and IL-10. Furthermore, blocking of monocyte-B7-H1 not only significantly increased perforin production but also decreased apoptosis of HCV-specific CD8+ T cells.
S 160
munity. Reduced number and/or functional impairment of these cells may contribute to the immune dysregulation associated with HCV infection.
]•]
DYNAMICS OF CD81-EXPRESSION ON LYMPHOCYTE SUBSETS DURING ANTIVIRAL TREATMENT OF PATIENTS WITH CHRONIC HEPATITIS C
B. Kronenberger1, E. Herrmann 1, T. Ghaliai 1, H. Wedemeyel 2, W.R Hofmann 1, U. Mihm 1, S. Zeuzem 1, C. Sarra~zin1. 1Klinikfiir
Innere Medizin IL Universitiitsklinikum des Saarlandes, Homburg/Saar, Germany," 2Abt. fiir Gastroenterologie, Hepatologie und Endokrinologie, Medizinisehe Hoehsehule Hannover, Germany Aim: CD81 is a hepatitis C virus (HCV) co-receptor with important functions on lymphocytes. During treatment CD81 expression may be affected directly the antiviral therapy or indirectly by reduction of HCV-RNA levels. The dynamics of CD81 on lymphocyte subtypes have not yet been investigated and may be relevant for viral replication and treatment response. Methods: CD81 was flow cytometrically quantified on CD8(+), CD4(+), CD19(+) and CD56(+) lymphocyte subtypes from patients with chronic hepatitis C before, during and after antiviral treatment with pegylated interferon-alpha and ribavirin. Dynamics of CD81 were investigated in correlation with HCV-RNA dynamics and the viral kinetic parameters efficacy of blocking virus production and infected cell loss. Results: Patients with chronic HCV showed a higher baseline CD81expression (n 20) than healthy controls (n 8). Baseline CD81-expression on CD4(+) and CD8(+) cells inversely correlated with HCV-RNA serum levels. During treatment, the following typical patterns of CD81-regulation were observed: 1. Downregulation on CD8(+) T-cells (Figure A) and most significantly on CD56(+) natural killer cells (Figure B), 2. Transient upregulation on CD19(+) B-cells and 3. No change on CD4(+) T-cells. During treatment, CD81 on CD8(+) and CD19(+) cells correlated with antiviral efficacy and infected cell loss, respectively, but not with HCVRNA serum levels. After end of treatment, CD56(+) cells and CD8(+) cells from sustained responders maintained lower CD81-expression compared with baseline. Conclusion: Lymphocyte subsets show different patterns of CD81response before and during antiviral treatment which are associated with antiviral response, HCV-RNA serum levels, and administration of pegylated interferon-alpha and ribavirin. 7OO
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CHARACTERISATION OF HCV CORE-SPECIFIC ADAPTIVE REGULATORY T CELL CLONES FROM PATIENTS WITH CHRONIC HEPATITIS C INFECTION
B. Langhans, I. Braunschweiger, H.D. Nieschalke, T. Sauerbruch, U. Spengler. Department of Internal Medicine I, Universitdtsklinikum
Bonn, Bonn, Germany Background and Alms: Circumstantial evidence indicates a potential role of regulatory CD4+ T cells in the control of virus-specific T cell responses during acute and chronic hepatitis C virus (HCV) infection. However, to date only so called natural regulatory CD4+ CD25+ T cells with HCV specificity have been analysed. Here, we report on T cell clones representing adaptive HCV-core specific regulatory on CD4+ cells.
Methods: For generation ofT cell lines, isolated peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C infection were stimulated in vitro with HCV core protein in the presence of human rIL-2 and autologous feeder cells over 24 days. For cloning, T cells were seeded under limiting dilution conditions. T cell clones (TCC) were picked after 10 days from plates with <10% proliferative wells and were re-stimulated with PHA at 10- to 14-day intervals. Specificity of TCC was analysed with respect to proliferation (3H thymidin incorporation) and cytokine production (IL-2, IL-4, IL-10, IFN-T, TGF-[J) after stimulation with HCV core protein and anti-CD3/CD28, respectively. Candidates of HCV-specific regulatory CD4+ T cells were characterised phenotypically (typical markers of regulatory CD4+ T cells: CD25, GITR, CTLA-4) and functionally (inhibition of autologous IFN-g producing TH1 reporter clones). Results: Among the remaining clones, clone H7_3 and H7_16 were outstanding, because they produced substantial amounts of IL-10 and TGF-[J upon restimulation with HCV core protein and anti-CD3/CD28, respectively, but did not proliferate in response to HCV core or antiCD3/CD28 stimulation nor did they produce IL-2, IL-4 and IFN-T. Importantly, both clones inhibited IFN-g production of autologous TH1 reporter clones. Both clones expressed CTLA-4 and GITR. Moreover, clone H7_16 belonged to the CD4+CD25high T cell subpopulation, while clone H7_3 was CD4+CD25negative. Conclusion: Our results demonstrate that HCV-specific T cells exist in chronic hepatitis C, which inhibit anti-viral TH1 effector cells. Moreover, such regulatory CD4+ cells can also be found in T cell subpopulations other than CD4+CD25high T cells.
~ T
CELL CHEMOKINE RECEPTOR EXPRESSION AND CHEMOTAXIS, ASSOCIATED TO TH1/TC1 RESPONSE, IN HEPATITIS C VIRUS (HCV) INFECTION AND HUMAN IMMUNODEFICIENCY VIRUS (HIV)-HCV CO-INFECTION
M. Calvino 1, J.R. Larmbia 1'2, E. Sanz 2, J. P6rez 1, S. Benito 1, F. Gonzfilez 2, C. Perna4, M. Montes 3, M. Rodriguez-Zapata 3, T. Parra 1, A. Bienvenido 2. 1Liver Research Unit, 2Gastroenterology Department,
3Internal Medicine Department, 4pathology Department Hospital Universitario, Guadalajara, Spain Background: Interaction between CCR5, CXCR3 receptors and its ligands favours T cells migration into the liver. Intrahepatic inflammatory infiltrate is responsible of liver damage in chronic HCV infection. HIV HCV infection is characterized by a more aggressive natural history than isolated HCV infection. This fact could be related to different potential of migration in T cells. Objectives: (1) To evaluate the ratio of CXCR3 and CCR5 expression between CD4+ and CD8+ cells in HCV infection (HCV/HIV), HCV HIV co-infection (HCV/HIV+) and healthy controls (HC). (2) To evaluate invitro migration capacity of CD4+ and CD8+ cells after MIP-la, RANTES and IP-10 stimulation. Methods: Peripheral blood mononuclear cells (PBMC) from 27 HCV/HIV , 9 HCV/HIV+ and 10 HC were obtained. PBMC were stained with anti-CD8, anti-CD4, anti-CCR5 and anti-CXCR3 monoclonal antibodies and analysed by flow-cytometry. PBMC from 5 patients of each group were used for in-vitro analysis of migration in response to chemokines. Migration index was calculated as: (cells migrating toward chemokine gradient)/(cells migrating toward medium alone). Data are shown as median plus interquantile range (IQR). Kmskall Wallis and Mann Whitney-U tests were used for statistical comparisons. Results: In HCV/HIV the ratio TCD8+CCR5+/TCD4+CCR5+ (1.4; IQR 1) decreased with respect to HC (2.6; IQR 1.14) (p<0.001) and this was not observed in HCV/HIV+. The ratio TCD8+CXCR3+/ TCD4+CXCR3+ decreased in HCV/HIV (1.4; IQR 0.3) and in HCV/HIV+ (1.6; IQR 0.9) in relation to HC (2.9; IQR 2.3) (p <0.001). Migration index after stimulation with IP-10 was higher in HCV/HIV+ (CD8+ 2.5; IQR 2.5/CD4+ 3.4; IQR 2.7) than in HCV/HIV
05C. Viral Hepatitis'
(e) Hepatitis' C Experimental (immunology)
(CD8+ 1.6; IQR 0.7/CD4+ 1.7; IQR 1.1) (p <0.05). No difference was found after stimulation with RANTES and MIP-la. Conclusions: (1) HCV HIV co-infection and isolated HCV infection display a relative decrease of CXCR3 expression on CD8+ cells in relation to CD4+ cells. (2) Isolated HCV infection shows a relative decrease of CCR5 expression on CD8+ cells with respect to CD4+, not observed in HCV/HIV+. (3) HCV/HIV+ group presents a migration potential in response to IP-10 higher than HCV/HIV group. (4) These last two differences could favour a more aggressive intrahepatic inflammatory infiltrate in HIV HCV co-infection.
•
C H E M O K I N E R E C E P T O R E X P R E S S I O N ON T CELLS A C C O R D I N G TO I N F L A M M A T O R Y ACTIVITY A N D LIVER FIBROSIS IN C H R O N I C HEPATITIS C VIRUS (HCV) INFECTION
M. Calvino 1, J.R. Larrubia 1'2, E. Sanz 2, C. Perna 3, J. P~rez 1, S. Benito 1, F. Gonzfilez 2, T. Parra 1, A. Bienvenido 2. 1Research
Unit, 2Gastroenterology Department, 3Pathology Department, Hospital Universitario, Guadalajara, Spain Background: Chemokine receptors play an important role in specific T cells migration into liver to control HCV during primoinfection. Nevertheless, during chronic infection these receptors attract a non-specific mononuclear infiltrate into the liver responsible for hepatic damage. The study of chemokine receptor profiles on T cells has an undoubted therapeutic interest. Objective: To correlate in chronic hepatitis C (CHC) CXCR3, CCR5 and CCR3 expression on CD4+ T cells from peripheral blood (PB) and on CD8+ T cells from liver and PB, with hepatic inflammatory activity and fibrosis according to Scheuer index. Methods: Peripheral blood (PBMC) and intrahepatic (IHMC) mononuclear cells from 27 CHC patients were obtained. PBMC were separated through a Ficoll-Hypaque gradient and IHMC through collagenase-I and DNAase digestion. Mononuclear cells were marked with anti-CD8, antiCD4, anti-CCR5, anti-CCR3 and anti-CXCR3 monoclonal antibodies and analysed by flow-cytometry. Hepatic inflammatory activity and fibrosis grade were evaluated by Scheuer index after hemtoxilin-eosin staining. Spearman correlation coefficient was used for statistical analysis. Results: It was observed a positive correlation between intrahepatic CD8+/CXCR3+ T cells and porto-periportal (PPA) (r 0.7; p < 0.01) and lobular (LA) (r 0.6; P<0.05) activity. Also, it was found a positive correlation between PB CD4+/CCR5+ T cells and LA (r 0.6; p <0.05) and between PB CD4/CXCR3 T cells and PPA (r 0.4; p < 0.05). There seemed to exist a positive correlation between CD8+/CCR5+ T cells from liver and PB and intrahepatic inflammatory activity, although it did not reach statistical significance in our study. There was not any correlation between CCR5 and CXCR3 expression and liver fibrosis. CCR3 expression did not correlate with any histological variable. Conclusions: (1) Chemokine receptor expression associated to Tcl/Thl response on T cells correlates with intrahepatic inflammation but not with liver fibrosis. (2) Treatments focus on CXCR3 and CCR5 regulation on T cells may modify liver inflammation in chronic hepatitis C.
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A S S O C I A T I O N OF I M M U N E CELL INFILTRATION A N D HEPATIC I N T E R F E R O N - S T I M U L A T E D GENE E X P R E S S I O N IN THE A B S E N C E OF IRF-3 ACTIVATION IN C H R O N I C HEPATITIS C
D. Lau 1, P. Fish2, S.M. Lemon 3, M. Gale Jr. 2. 1Dept Internal Medicine,
UTMB, Galveston, TX, USA," 2Dept of Microbiology, UT Southwestern, Dallas, TX, USA," 3Dept of Microbiology, UTMB, Galveston, TX, USA a/[J Interferons (IFNs) play important role in viral infections. HCV encodes proteins that block IFN production and antagonize IFN actions to support
S161
viral persistence. The NS3/4A protease of HCV disrupts viral activation of IRF-3, thereby attenuating IFN production and IFN-stimulated gene (ISG) expression. [Foy et al, Science 2003; 300:1145 48]. In this study, we compared the transcriptional profiles of ISGs among liver biopsy samples from normal controls, HCV genotype-1 patients and patients with non-alcoholic fatty liver disease (NAFLD) using Affymetrix HG-U95A Human GeneChips. Liver biopsy slides were also examined by immunofluorescence staining and confocal microscopy analysis to define the subcellular distribution of IRF-3, the extent of viral protein expression in hepatocytes, and the composition of hepatic infiltrating immune cells. HCV samples had significantly higher transcriptional levels of ISGs associated with IFN such as OAS2, MXl,ISG56 compared to controls and NAFLD (p<0.005). The level of IRF-3, however, was similar in HCV and normal controls. HCV NS3 or NS5A proteins were observed within focal areas of biopsy specimen and distributed in a perinuclear context within hepatocytes. IRF-3, though abundant, was typically found in an inactive state as defined by its cytoplasmic-bound distribution in liver cells. Furthermore, HCV infection was associated with specific CD3positive T cell and plasmacytoid dendritic cell (PDC) infiltrates in biopsy specimens. Our results indicated that in chronic HCV, IRF-3 activation within infected hepatocytes is limited, which lends further evidence of NS3/4A disruption of IRF-3 activation. Despite the lack of active IRF-3, increased hepatic ISG expression in HCV infection was observed and was correlated with the presence of immune cell infiltrates in liver samples. Infiltrating immune cells could be an important source of alpha/beta IFNs that potentially influence hepatic ISG expression and the outcome of HCV infection.
[•
C O - I N H I B I T O R Y M O L E C U L E B7-H1 MAY PLAY A N I M P O R T A N T ROLE F O R C H R O N I C I T Y OF HEPATITIS C
Y.J. Lee 1'3, S.J. Park 1'3, S.R. Jee 1, S.H. Lee 1, S.Y. Seol 1, J.M. Chung 1, H.Y. Jeong 2'3, S.K. Seo 2'3, S.G. Park 2'3, I.H. Choi 2'3. 1Department
of Internal Medicine, 2Department of Microbiology, 3Center for Viral Disease Researeh, Inje University School of Medicine, Busan, South Korea Background and Aims: The establishment of a chronic hepatitis C (CHC) infection is believed to be associated with the failure of an effective HCVspecific T cell response. Little is known regarding the role of negative T cell regulators such as B7-H1 (CD274) in the impaired HCV-specific T cell response. B7-H1 is a member of the B7 co-signaling family and has been shown to suppress both CD4+ and CD8+ effector T cell responses. We therefore examined the expression B7-H1 on immune cells from CHC patients. Methods: Twenty-one age-matched healthy volunteers and twenty-four CHC patients who were enrolled in the Busan Paik Hospital of Inje University (Busan, Korea) participated in this study. We did flow cytometry for analysis of B7-H1 on PBMC, T cell proliferation assay using co-culture system involving purified T cells and monocytes, ELISA for measurement of IL-2, IL-4, IL-10 and IFN-y in the culture supematants, and ELISPOT for IFN-y production from CD4+ or CD8+ T cells in response to HCV NS4 protein and NS3 and NS4 peptides. Results: B7-H1 was significantly up-regulated on CD14+ monocytes and B cells isolated from CHC patients compared to normal subjects. In a T cell proliferation assay, we found that B7-HI+ CD14+ monocytes significantly reduced T cell proliferation in response to anti-CD3 compared to B7H1- CD14+ monocytes. The involvement of B7-H1 in an impaired HCVspecific T cell response was further supported by the finding that blockade of monocyte-associated B7-H1 significantly augmented the generation of IFN-producing HCV-specific CD4+ and CD8+ effector T cells. In addition, monocyte-B7-H1 blockade greatly enhanced the production of type 1 cytokines including IFN-y and IL-2, but not type 2 cytokines such as IL-4 and IL-10. Furthermore, blocking of monocyte-B7-H1 not only significantly increased perforin production but also decreased apoptosis of HCV-specific CD8+ T cells.