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Phenotype and function of monocyte-derived dendritic cells from patients with hepatitis C virus infection
434A
AASLD ABSTRACTS
HEPATOLOGY October 2001
1047
1048
GENETIC DIFFERENCES IN INTERLEUKIN 10 P R O D U C T I O N DO NOT INFLUENCE THE SEVERITY OF CHRONIC HEPATITIS C. William Ab-
IMPAIRED MIXED LEUCOCYTE REACTION A N D ALTERED PHENOTYPE OF DENDRITIC CELLS IN CHRONIC HEPATITIS C. Elizabeth A
bott, Eirini Rigopoulou, Philip Haigh, Helen Cooksley, Ivana Muflerova, Institute of Hepatology, London Uk; Marco Novelli, Alison Winstanley, University College London, London Uk; Roger Williams, Nikolai Naoumov, Institute of Hepatology, London Uk
Sanders, Lucy A Fowkes, Judith A Holloway, Corinne L Brooks, William M Rosenberg, Southampton University, Southampton Uk
The cellular and molecular mechanisms responsible for inter-individual differences in severity of chronic hepatitis C (CHC) have not been defined. Interleukin 10 (IL-10) is an anti-inflammatory cytokine that down-regulates anti-viral immune responses. Understanding the role of IL-10 in chronic hepatitis C (CHC) may be of value for designing new treatment strategies. Aim: To determine whether genetic polymorphisms in the IL-10 promoter and/or the ability to produce IL-10 influence HCV-specific T cell reactivity and the severity of CHC. Methods: 113 consecutive patients with CHC were characterised for an IL-10 promoter polymorphism (either GCC, ACC, or ATA), for concanavalin A (Con A) stimulated IL-10 production from PBMC, and for T cell proliferation to HCV core, NS3, NS4 and NS5 antigens. The grade and stage of liver histology was correlated with these parameters and also with clinical variables. Results: A mild grade of CHC was associated with high T cell proliferation to core (p=0.04), NS3 (p=0.05) and NS4 (p=0.03) antigens, young age (p=0.005) and intravenous drug use (IVDU) (p=0.05). There were no associations between the grade of CHC and either Con A stimulated IL-10 production, IL-10 promoter haplotype, sex, alcohol intake or HCV genotype. Age was independently associated with the grade of CHC on multivariate analysis (p=0.01). An early stage of CHC was associated with young age (p=0.0001), short duration of HCV infection (p=0.007) and low grade of CHC (p=0.0001), but not with IL-10 promoter haplotype, Con A stimulated IL-10 production, T cell proliferation, sex, alcohol intake or HCV genotype. On multivariate analysis, age and the grade of CHC were independently associated with the stage of CHC (p= 0.0001). There were no associations between HCV-specific T cell reactivity and either Con A stimulated IL- 10 production or IL-10 promoter haplotype. Conclusions: Inter-individual differences in Con A stimulated IL-10 production and functional genetic polymorphisms in the IL-10 promoter have no influence on the severity of chronic hepatitis C.
Introduction: In comparison with acute hepatitis C infection, chronic hepatitis C (CHC) is characterised by weak cellular immune responses with both HCVspecific C D 4 + T lymphocyte proliferative responses and CD8+ T lymphocyte cytotoxicity being weak and oligospecific. We have studied the role of antigen presentation by dendritic cells (DCs) which are the most potent antigen presenting cells. We investigated the allogeneic stimulatory capacity of cultured DCs and the phenotype of ex vivo peripheral blood DCs (PBDCs) which had not been manipulated in culture or exposed to exogenous cytokines. Materials and Methods: Mixed leucocyte reactions (MLR) were compared in 6 normal healthy donors and 7 HCV infected individuals using 7-14 day old monocyte derived DCs propagated in culture medium containing IL-4 and GM-CSF. The phenotype of ex vivo PBDCs was ascertained by 3 colour flow cytometry. Markers of antigen uptake (CD32, CD64 and mannose receptor), DC maturation (CD54, CD4 and CD8~) and costimulation (CD40, CD54, CD80 and CD86) were assessed in 13 CHC and 8 uninfected individuals. Results: MLR was impaired in CHC compared to uninfected individuals (p<0.05). The altered function was less pronounced at day 14 compared to day 7. The phenotype of ex vivo DCs was altered in patients with CHC compared to uninfected individuals. The expression of the costimulatory molecules CD40 and CD86 on DCs was reduced in CHC compared to uninfected individuals (p<0.0001 and p<0.05 respectively). Comparable levels of expression of markers of antigen uptake and DC maturation were observed between the two groups. Conclusion: Mixed leucocyte reaction of CI-IC DCs is impaired compared with uninfected individuals. Our study is the first to report an altered phenotype of ex vivo DCs which reflect the phenotype of circulating DCs in the body. The reduced levels of costimulatory markers CD40 and CD80 observed on the surface of the DCs from individuals with CHC may lead to the impaired T cell responses that are characteristic of chronic HCV infection.
1049
1050
DETERMINANTS OF VIRAL CLEARANCE A N D PERSISTENCE D U R I N G ACUTE HEPATITIS C VIRUS INFECTION. Robert Thimme, The Scripps
PHENOTYPE A N D F U N C T I O N OF MONOCYTE-DERIVED DENDRITIC CELLS FROM PATIENTS W I T H HEPATITIS C VIRUS INFECTION. Helena Daniels, Joti Hannoe, Sinead Costelloe, Vinod Patel, Farzin Farzaneh,
Research Institute, La Jofla, CA; David Oldach, Institute of Human Virology, Baltimore, MD; Carola Steiger, The Scripps Research Institute, La Jolla, CA; Stuart Ray,John Hopkins University School of Medicine, Baltimore, MD; Francis V Chisari, The Scripps Research Institute, La Jofla, CA In this study, we analyzed the virological and immunological features of acute HCV infection starting immediately after accidental needlestick exposure in 2 health care workers. Both patients were HLA-A2 positive, and they were infected by the same HCV genotype (lb), achieved comparable viral titers ( - 5 x 107 GE/ml) and similar peak sALT activity (1386 vs 1809 U/I), and they experienced asymptomatic infections with similar antibody profiles. Nevertheless, one patient cleared the virus while the other developed persistent infection. Viral clearance was preceded by a prolonged episode of acute hepatitis during which the viral titer decreased only -3-fold. The onset of the liver disease coincided precisely with the appearance of CD38+ (activated), NS3 1406 (KLVALGINAV) specific, HLA-A2 tetramer-binding CD8+ T cells. Surprisingly, these T cells'failed to produce IFNg when they were stimulated by the NS3 1406 pepdde present in the tetramer. Since the dominant viral sequence in this patient was subsequently found to be KLSGLGINAV, that peptide was synthesized and used to stimulate the patient's CD8+ T cells to produce IFNg. Unexpectedly, during the peak of acute hepatitis the patient's CD8+ T ceils did not produce IFNg in response to KLSGLGINAV. Importantly, the patient's CD8+ T cells began to produce IFNg in response to that peptide during the resolution phase of acute hepatitis, coinciding with a 5-log decrease in viremia and elimination of the virus without a surge in serum ALT activity. This was accompanied by loss of the CD38 activation marker and a rise in the CD4+ T cell response. In contrast to these findings, HCV persisted in the second patient who failed to m o u n t a significant virus-specific CD8+ and CD4+ T cell response. In sum, these results suggest that disease pathogenesis and viral clearance may be mediated by different effector mechanisms and T cell populations. Specifically, acute hepatitis appears to be mediated by CD38+, CD8+ cytolytic T ceils that don't produce IFNg and are relatively inefficient at controlling the infection. In contrast, the virus appears to be very efficiently controlled by relatively noncytolytic CD38-, CD8+ T cells that produce IFNg when they recognize viral antigen. Finally, the results indicate that primary failure of the T cell response is an important pathway to viral persistence during acute HCV infection.
Phillip Harrison, GKT School of Medicine and Dentistry, London Uk Dendriticcells (DC) are the principalinitiators of an antigen-specificT cellimmune responsedue to their abilityto acquire,processand presentantigen in associationwith high expressionof HLA class I and class II, adhesion and co-stimulator'/molecules.Previousstudies have indicated that monoeyte-derivedDC from patients with chronic hepatitis C virus infection (HCV) do not respond to maturation stimuli and have an impaired ability to stimulate allogeneicT lymphocytes comparedto DC derivedfromhealthy donors.We havestudied the abilityof monocytederivedDC frompatients with chronic HCV (n = 12 ) and matchednormal controls (n= 12) to stimulateT cell proliferation not only in mixed lymphocyte reaction (MLR) but also to both recall and neoantigens (purifiedprotein derivativeof tuberculin (PPD) and keyholelimpet bemocyanni(KLH), respectively).Dendritic cells were derived from monocytes cultured for 7 days in medium containing GM-CSF (800U/ml),IL-4 (500U/ml) and 1%autologousplasma. Theywere left unpulsed or pulsed overnightwith PPD (20/zg/ml)or KLH (80/~g/ml)before further maturation in monocyte conditionedmedium for 48 hours. In allogeneicMLR, the proliferationof normal T cells,as assessedby thymidineincorporation,was signihcandylower in responseto DC frompatients with HCV comparedto allogeneicnormal controls (mean cpm 6784 ±4854 vs 11959 -+7032, p<0.01). Proliferationof autologousT celLato DC pulsed with the recall antigen PPD was highly variable from patients with HCV overallresponseswere not significantlydifferem to those seen in normal controls (Table).However,proliferationof autologousT cells to DC pulsed with the neo-amigen KLH was significantlyhigher in patients with HCV compared to normal controls (p=0.008, Table). To investigatethe DC phenotype, the proportions of cultured cells expressingthe surface markers CDla, CD83, CD86, CD14 and MHC Class II were determined by flow cytometry.The proportion of ceils expressingthe DC marker CD83 was higher from normal controls than from HCV patients (27%vs I0%, respectively;p=0.01), CDla levels(36%vs 24%,p=0.1), CD86 (80% vs 73%,p=0.2) and MHC class I1 (90%vs 86%) were similar but expression of the macrophage marker CD14 was lower in controls (5% vs 14%,p =0.03). Followingmaturation of the cells in monocyteconditionedmedia,surfaceexpressionofCDla fellin controlsbut not in the HCV group (to 20%and 45%,respectively;p = 0.03 aftermaturation), levelsof CD83increasedsignificantlyon DC from controls but to a lesser degree on DC from HCV patients (increase to 64% and 26%, respectively;p=0.001). Levelsof CD86, MHC class II and CDld were unaffectedby exposure to monocyte conditionedmedia in both groups. These data confirmthat matured monocyte-derived DC from patients with HCV have an impaired ability to stimulate proliferation of allogeneicT lymphocytescompared to DC from normal controls. Furthermore, DC from patients with HCV have an immaturephenotype,with an increasedproportion expressinga macrophagemarker, and do not respond appropriatelyto a maturation stimulus, as reflectedby the pattern of cell surface markers.Despitethese findings,the antigen-specificstimulationof autologouslymphocytesby DC from patients with HCV is preserved.
Proliferationof aatologouslymphocytesin responseto antigenpulsed DC StimulationIndex (standarddeviation) AnUgen Normal ,HCV PPD 8.9 (7.3) 34.0 (48,5) KLH 4.4 (5,0) 16.4 (t6.9) Stimulation index = cpm with pulsed DC/cpm with unpulsed DC
p=0.13 p=0,008
AASLD ABSTRACTS
HEPATOLOGY October 2001
1047
1048
GENETIC DIFFERENCES IN INTERLEUKIN 10 P R O D U C T I O N DO NOT INFLUENCE THE SEVERITY OF CHRONIC HEPATITIS C. William Ab-
IMPAIRED MIXED LEUCOCYTE REACTION A N D ALTERED PHENOTYPE OF DENDRITIC CELLS IN CHRONIC HEPATITIS C. Elizabeth A
bott, Eirini Rigopoulou, Philip Haigh, Helen Cooksley, Ivana Muflerova, Institute of Hepatology, London Uk; Marco Novelli, Alison Winstanley, University College London, London Uk; Roger Williams, Nikolai Naoumov, Institute of Hepatology, London Uk
Sanders, Lucy A Fowkes, Judith A Holloway, Corinne L Brooks, William M Rosenberg, Southampton University, Southampton Uk
The cellular and molecular mechanisms responsible for inter-individual differences in severity of chronic hepatitis C (CHC) have not been defined. Interleukin 10 (IL-10) is an anti-inflammatory cytokine that down-regulates anti-viral immune responses. Understanding the role of IL-10 in chronic hepatitis C (CHC) may be of value for designing new treatment strategies. Aim: To determine whether genetic polymorphisms in the IL-10 promoter and/or the ability to produce IL-10 influence HCV-specific T cell reactivity and the severity of CHC. Methods: 113 consecutive patients with CHC were characterised for an IL-10 promoter polymorphism (either GCC, ACC, or ATA), for concanavalin A (Con A) stimulated IL-10 production from PBMC, and for T cell proliferation to HCV core, NS3, NS4 and NS5 antigens. The grade and stage of liver histology was correlated with these parameters and also with clinical variables. Results: A mild grade of CHC was associated with high T cell proliferation to core (p=0.04), NS3 (p=0.05) and NS4 (p=0.03) antigens, young age (p=0.005) and intravenous drug use (IVDU) (p=0.05). There were no associations between the grade of CHC and either Con A stimulated IL-10 production, IL-10 promoter haplotype, sex, alcohol intake or HCV genotype. Age was independently associated with the grade of CHC on multivariate analysis (p=0.01). An early stage of CHC was associated with young age (p=0.0001), short duration of HCV infection (p=0.007) and low grade of CHC (p=0.0001), but not with IL-10 promoter haplotype, Con A stimulated IL-10 production, T cell proliferation, sex, alcohol intake or HCV genotype. On multivariate analysis, age and the grade of CHC were independently associated with the stage of CHC (p= 0.0001). There were no associations between HCV-specific T cell reactivity and either Con A stimulated IL- 10 production or IL-10 promoter haplotype. Conclusions: Inter-individual differences in Con A stimulated IL-10 production and functional genetic polymorphisms in the IL-10 promoter have no influence on the severity of chronic hepatitis C.
Introduction: In comparison with acute hepatitis C infection, chronic hepatitis C (CHC) is characterised by weak cellular immune responses with both HCVspecific C D 4 + T lymphocyte proliferative responses and CD8+ T lymphocyte cytotoxicity being weak and oligospecific. We have studied the role of antigen presentation by dendritic cells (DCs) which are the most potent antigen presenting cells. We investigated the allogeneic stimulatory capacity of cultured DCs and the phenotype of ex vivo peripheral blood DCs (PBDCs) which had not been manipulated in culture or exposed to exogenous cytokines. Materials and Methods: Mixed leucocyte reactions (MLR) were compared in 6 normal healthy donors and 7 HCV infected individuals using 7-14 day old monocyte derived DCs propagated in culture medium containing IL-4 and GM-CSF. The phenotype of ex vivo PBDCs was ascertained by 3 colour flow cytometry. Markers of antigen uptake (CD32, CD64 and mannose receptor), DC maturation (CD54, CD4 and CD8~) and costimulation (CD40, CD54, CD80 and CD86) were assessed in 13 CHC and 8 uninfected individuals. Results: MLR was impaired in CHC compared to uninfected individuals (p<0.05). The altered function was less pronounced at day 14 compared to day 7. The phenotype of ex vivo DCs was altered in patients with CHC compared to uninfected individuals. The expression of the costimulatory molecules CD40 and CD86 on DCs was reduced in CHC compared to uninfected individuals (p<0.0001 and p<0.05 respectively). Comparable levels of expression of markers of antigen uptake and DC maturation were observed between the two groups. Conclusion: Mixed leucocyte reaction of CI-IC DCs is impaired compared with uninfected individuals. Our study is the first to report an altered phenotype of ex vivo DCs which reflect the phenotype of circulating DCs in the body. The reduced levels of costimulatory markers CD40 and CD80 observed on the surface of the DCs from individuals with CHC may lead to the impaired T cell responses that are characteristic of chronic HCV infection.
1049
1050
DETERMINANTS OF VIRAL CLEARANCE A N D PERSISTENCE D U R I N G ACUTE HEPATITIS C VIRUS INFECTION. Robert Thimme, The Scripps
PHENOTYPE A N D F U N C T I O N OF MONOCYTE-DERIVED DENDRITIC CELLS FROM PATIENTS W I T H HEPATITIS C VIRUS INFECTION. Helena Daniels, Joti Hannoe, Sinead Costelloe, Vinod Patel, Farzin Farzaneh,
Research Institute, La Jofla, CA; David Oldach, Institute of Human Virology, Baltimore, MD; Carola Steiger, The Scripps Research Institute, La Jolla, CA; Stuart Ray,John Hopkins University School of Medicine, Baltimore, MD; Francis V Chisari, The Scripps Research Institute, La Jofla, CA In this study, we analyzed the virological and immunological features of acute HCV infection starting immediately after accidental needlestick exposure in 2 health care workers. Both patients were HLA-A2 positive, and they were infected by the same HCV genotype (lb), achieved comparable viral titers ( - 5 x 107 GE/ml) and similar peak sALT activity (1386 vs 1809 U/I), and they experienced asymptomatic infections with similar antibody profiles. Nevertheless, one patient cleared the virus while the other developed persistent infection. Viral clearance was preceded by a prolonged episode of acute hepatitis during which the viral titer decreased only -3-fold. The onset of the liver disease coincided precisely with the appearance of CD38+ (activated), NS3 1406 (KLVALGINAV) specific, HLA-A2 tetramer-binding CD8+ T cells. Surprisingly, these T cells'failed to produce IFNg when they were stimulated by the NS3 1406 pepdde present in the tetramer. Since the dominant viral sequence in this patient was subsequently found to be KLSGLGINAV, that peptide was synthesized and used to stimulate the patient's CD8+ T cells to produce IFNg. Unexpectedly, during the peak of acute hepatitis the patient's CD8+ T ceils did not produce IFNg in response to KLSGLGINAV. Importantly, the patient's CD8+ T cells began to produce IFNg in response to that peptide during the resolution phase of acute hepatitis, coinciding with a 5-log decrease in viremia and elimination of the virus without a surge in serum ALT activity. This was accompanied by loss of the CD38 activation marker and a rise in the CD4+ T cell response. In contrast to these findings, HCV persisted in the second patient who failed to m o u n t a significant virus-specific CD8+ and CD4+ T cell response. In sum, these results suggest that disease pathogenesis and viral clearance may be mediated by different effector mechanisms and T cell populations. Specifically, acute hepatitis appears to be mediated by CD38+, CD8+ cytolytic T ceils that don't produce IFNg and are relatively inefficient at controlling the infection. In contrast, the virus appears to be very efficiently controlled by relatively noncytolytic CD38-, CD8+ T cells that produce IFNg when they recognize viral antigen. Finally, the results indicate that primary failure of the T cell response is an important pathway to viral persistence during acute HCV infection.
Phillip Harrison, GKT School of Medicine and Dentistry, London Uk Dendriticcells (DC) are the principalinitiators of an antigen-specificT cellimmune responsedue to their abilityto acquire,processand presentantigen in associationwith high expressionof HLA class I and class II, adhesion and co-stimulator'/molecules.Previousstudies have indicated that monoeyte-derivedDC from patients with chronic hepatitis C virus infection (HCV) do not respond to maturation stimuli and have an impaired ability to stimulate allogeneicT lymphocytes comparedto DC derivedfromhealthy donors.We havestudied the abilityof monocytederivedDC frompatients with chronic HCV (n = 12 ) and matchednormal controls (n= 12) to stimulateT cell proliferation not only in mixed lymphocyte reaction (MLR) but also to both recall and neoantigens (purifiedprotein derivativeof tuberculin (PPD) and keyholelimpet bemocyanni(KLH), respectively).Dendritic cells were derived from monocytes cultured for 7 days in medium containing GM-CSF (800U/ml),IL-4 (500U/ml) and 1%autologousplasma. Theywere left unpulsed or pulsed overnightwith PPD (20/zg/ml)or KLH (80/~g/ml)before further maturation in monocyte conditionedmedium for 48 hours. In allogeneicMLR, the proliferationof normal T cells,as assessedby thymidineincorporation,was signihcandylower in responseto DC frompatients with HCV comparedto allogeneicnormal controls (mean cpm 6784 ±4854 vs 11959 -+7032, p<0.01). Proliferationof autologousT celLato DC pulsed with the recall antigen PPD was highly variable from patients with HCV overallresponseswere not significantlydifferem to those seen in normal controls (Table).However,proliferationof autologousT cells to DC pulsed with the neo-amigen KLH was significantlyhigher in patients with HCV compared to normal controls (p=0.008, Table). To investigatethe DC phenotype, the proportions of cultured cells expressingthe surface markers CDla, CD83, CD86, CD14 and MHC Class II were determined by flow cytometry.The proportion of ceils expressingthe DC marker CD83 was higher from normal controls than from HCV patients (27%vs I0%, respectively;p=0.01), CDla levels(36%vs 24%,p=0.1), CD86 (80% vs 73%,p=0.2) and MHC class I1 (90%vs 86%) were similar but expression of the macrophage marker CD14 was lower in controls (5% vs 14%,p =0.03). Followingmaturation of the cells in monocyteconditionedmedia,surfaceexpressionofCDla fellin controlsbut not in the HCV group (to 20%and 45%,respectively;p = 0.03 aftermaturation), levelsof CD83increasedsignificantlyon DC from controls but to a lesser degree on DC from HCV patients (increase to 64% and 26%, respectively;p=0.001). Levelsof CD86, MHC class II and CDld were unaffectedby exposure to monocyte conditionedmedia in both groups. These data confirmthat matured monocyte-derived DC from patients with HCV have an impaired ability to stimulate proliferation of allogeneicT lymphocytescompared to DC from normal controls. Furthermore, DC from patients with HCV have an immaturephenotype,with an increasedproportion expressinga macrophagemarker, and do not respond appropriatelyto a maturation stimulus, as reflectedby the pattern of cell surface markers.Despitethese findings,the antigen-specificstimulationof autologouslymphocytesby DC from patients with HCV is preserved.
Proliferationof aatologouslymphocytesin responseto antigenpulsed DC StimulationIndex (standarddeviation) AnUgen Normal ,HCV PPD 8.9 (7.3) 34.0 (48,5) KLH 4.4 (5,0) 16.4 (t6.9) Stimulation index = cpm with pulsed DC/cpm with unpulsed DC
p=0.13 p=0,008