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P137 THE EXPRESSION OF NKG2D-LIGANDS BY ACTIVATED CD4 T CELLS RENDERS THEM SUSCEPTIBLE TO NK CELL REGULATION IN CHRONIC HEPATITIS B (CHB)
POSTERS on local metabolic conditions. Given that oxidative stress is a predominant feature in the treatment of malignant diseases, targeting this mechanism may improve immune-based therapies. P136 DEFICIENCY OF IL-33 SENSITIZES TO SEVERE LIVER INJURY INDUCED BY ConA BUT NOT BY CCl4 IN MICE M.I. Arshad1,2 , A. Filliol1 , V. Genet1 , C. Lucas-Clerc3,4 , J.-P. Girard5 , C. Piquet-Pellorce1,4,6 , M. Samson1,4,6 . 1 Inserm U 1085, Institut de Recherche Sant´e Environnement & Travail (IRSET), Rennes, France; 2 Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan; 3 Service de Biochimie CHU Rennes, 4 Universit´e de Rennes 1, Rennes, 5 Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique (IPBS-CNRS), Universit´e de Toulouse, Toulouse, 6 Structure F´ed´erative BioSit UMS 3480 CNRS-US 18 Inserm, Rennes, France E-mail: [email protected] Background and Aims: IL-33/ST2 axis play a protective role during acute hepatitis but little is known about the functional role of endogenous IL-33 in liver patho-physiology. We aimed to decipher the functional role of IL-33 by using IL-33 deficient mice during immune cell mediated and hepato-toxic driven liver injury. Methods: We used a genetic model of acute hepatitis by using IL-33 deficient mice in ConA (a T cell-mediated hepatitis) and CCl4 (a hepatotoxic agent-induced hepatitis) induced acute liver injury. The liver functions (AST/ALT), signature of cytokines and characterization of infiltrate cell in WT and IL-33−/− mice were carried out by biochemistry, qPCR and flow cytometry analyses. Results: Our results demonstrated that IL-33−/− mice exhibited more severe ConA liver injury than WT mice evidencing a protective effect of IL-33 in this hepatic model while no difference was observed in CCl4 -hepatitis between WT and IL-33−/− mice. The ConA-induced hepatic injury was associated with increased TNF-a, IL-1-b, IFN-g and IL-6 cytokines in WT and IL-33−/− mice. The level of TNF-a and IL-1-b but not of IFN-g and IL-6 was significantly higher in IL-33−/− mice than WT control. The intrahepatic percentage of NK, NKT cells, T cells and B cells was not altered significantly between WT and IL-33−/− mice following ConAhepatitis. Conclusions: We evidenced that the genetic ablation of IL-33 sensitized the mice to severe ConA liver injury but not CCl4mediated liver injury. IL-33 has a limited impact on proinflammatory cytokines and influx of infiltrate cells during immune cell mediated liver pathology. P137 THE EXPRESSION OF NKG2D-LIGANDS BY ACTIVATED CD4 T CELLS RENDERS THEM SUSCEPTIBLE TO NK CELL REGULATION IN CHRONIC HEPATITIS B (CHB) W.-C. Huang1 , D. Peppa1,2 , N. Easom1 , X.-Z. Tan1 , C. Chang3 , J. Trowsdale3 , U. Gill4 , P. Kennedy4 , M. Maini1 . 1 Division of Infection and Immunity, 2 Centre for Sexual Health and HIV Research, University College London (UCL), London, 3 Department of Pathology, University of Cambridge, Cambridge, 4 Barts and the London Hospital, London, United Kingdom E-mail: [email protected] Background and Aims: We have recently demonstrated rapid, contact-dependent killing of HBV-specific T cells by the large number of activated NK cells that they make contact with in the liver vasculature. This deletion was partially TRAIL-dependent but other pathways were implicated (Peppa JEM 2013). In this study we therefore explored a role for the NKG2D pathway in T/NK cell interactions in CHB.
Methods: An NKG2D-GFP reporter cell line was used for initial screening for NKG2D ligands (NKG2D-L). Global, activated and HBVspecific T cells from the circulation and liver (identified by IFN-g staining following HBV-peptide stimulation) were stained with a panel of NKG2D-L mAb. NK cell depletion and NKG2D-blocking mAb were used for functional experiments. Results: NKG2D-L (MICA/B and ULPB1) were upregulated on T cells (CD4 > CD8) in CHB patients, particularly those with liver inflammation (ALT >2× upper limit of normal). NKG2D ligands were preferentially expressed on activated (HLA−DR+) and HBV-specific T cells and strikingly enriched on T cells in the CHB liver compared to the circulation and control livers. In line with their expression of NKG2DL, there was preferential rescue of the activated fraction of CD4 T cells following NK cell depletion. Following NKG2D pathway blockade in vitro, HBV-specific CD4 T cells could be recovered from CHB patients with ongoing liver inflammation. Conclusions: Collectively, these results imply that in the setting of active CHB, NKG2D-dependent CD4 T cell/NK cell interactions may serve as a homeostatic response to liver inflammation, resulting in constraints on antiviral immunity. P138 SERONEGATIVE ACUTE LIVER FAILURE REPRESENTS A MACROPHAGE–T CELL ACTIVATION SYNDROME E. Triantafyllou1,2 , O.T. Pop1 , R.D. Abeles1 , W. Khamri2 , L. Possamai2 , M. Hussain1 , W. Bernal1 , G. Auzinger1 , M.A. Heneghan1 , N. Heaton1 , M. Thursz2 , J. Wendon1 , A. Quaglia1 , Y. Ma1 , C.G. Antoniades1,2 . 1 Institute of Liver Studies, King’s College London, King’s College Hospital, 2 Section of Hepatology, Imperial College London, St Mary’s Hospital, London, United Kingdom E-mail: [email protected] Background and Aims: Monocytes/macrophages are central mediators of inflammatory responses in acute liver failure (ALF). Seronegative ALF (SALF) bears striking similarities to macrophage activation syndrome (MAS), which is driven by the activation and excessive proliferation of T cells and macrophages. We sought to examine the underlying mechanisms of SALF that remain unexplored. Methods: Circulating and hepatic levels of key inflammatory cytokines, macrophage and T cell activation markers were evaluated in SALF patients (n = 10) and non-SALF patients (n = 5) on admission, compared to (HC) healthy controls (n = 5). TNF-a and IL-6 concentrations were determined following 6 hour incubation of PBMCs with 100 ng/ml of LPS in SALF (n = 10), acetaminophen-induced AALF (n = 10) and HC (n = 10). Immunohistochemistry (CD68/CD163/CD3/CD4/CD8/T-bet/Gata-3/ Th17/MPO/CD56/Perforin/FoxP3) was used to characterize the hepatic immune cell infiltrate in SALF (n = 10), AALF (n = 10) and pathological control (PC) liver tissue (n = 5). Results: Compared to HC, SALF patients had higher circulating levels of pro- (IFN-g, IL-1b, IL-6, IL-8), anti- (IL-10, TGF-b1) inflammatory cytokines and macrophage (sCD163) and T cell (IL-2Ra) activation markers (all p < 0.01) (Figure). Intrahepatic concentrations of IL-8, IL-12, TGF-b1, IL-2Ra and sCD163 were significantly elevated in SALF, compared to PC tissue (p < 0.05). LPS-induced secretion levels of TNF-a and IL-6 of PBMCs in SALF were comparable to HC (p > 0.05), suggesting intact proinflammatory responses. Immunohistochemical analysis revealed a macrophage- (CD68+,CD163+), Th1 lymphocytic- (CD4+,T-bet+) rich infiltrate in centrilobular and periportal areas, compared to AALF and PC (Figure). Conclusions: SALF is a Th1-cell driven disease, characterized by profound elevation in biomarkers of macrophage/T cell activation; thus, forming the rationale to assess immunotherapeutic strategies to attenuate proinflammatory responses and severity of liver failure.
Journal of Hepatology 2014 vol. 60 | S67–S214
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Methods: An NKG2D-GFP reporter cell line was used for initial screening for NKG2D ligands (NKG2D-L). Global, activated and HBVspecific T cells from the circulation and liver (identified by IFN-g staining following HBV-peptide stimulation) were stained with a panel of NKG2D-L mAb. NK cell depletion and NKG2D-blocking mAb were used for functional experiments. Results: NKG2D-L (MICA/B and ULPB1) were upregulated on T cells (CD4 > CD8) in CHB patients, particularly those with liver inflammation (ALT >2× upper limit of normal). NKG2D ligands were preferentially expressed on activated (HLA−DR+) and HBV-specific T cells and strikingly enriched on T cells in the CHB liver compared to the circulation and control livers. In line with their expression of NKG2DL, there was preferential rescue of the activated fraction of CD4 T cells following NK cell depletion. Following NKG2D pathway blockade in vitro, HBV-specific CD4 T cells could be recovered from CHB patients with ongoing liver inflammation. Conclusions: Collectively, these results imply that in the setting of active CHB, NKG2D-dependent CD4 T cell/NK cell interactions may serve as a homeostatic response to liver inflammation, resulting in constraints on antiviral immunity. P138 SERONEGATIVE ACUTE LIVER FAILURE REPRESENTS A MACROPHAGE–T CELL ACTIVATION SYNDROME E. Triantafyllou1,2 , O.T. Pop1 , R.D. Abeles1 , W. Khamri2 , L. Possamai2 , M. Hussain1 , W. Bernal1 , G. Auzinger1 , M.A. Heneghan1 , N. Heaton1 , M. Thursz2 , J. Wendon1 , A. Quaglia1 , Y. Ma1 , C.G. Antoniades1,2 . 1 Institute of Liver Studies, King’s College London, King’s College Hospital, 2 Section of Hepatology, Imperial College London, St Mary’s Hospital, London, United Kingdom E-mail: [email protected] Background and Aims: Monocytes/macrophages are central mediators of inflammatory responses in acute liver failure (ALF). Seronegative ALF (SALF) bears striking similarities to macrophage activation syndrome (MAS), which is driven by the activation and excessive proliferation of T cells and macrophages. We sought to examine the underlying mechanisms of SALF that remain unexplored. Methods: Circulating and hepatic levels of key inflammatory cytokines, macrophage and T cell activation markers were evaluated in SALF patients (n = 10) and non-SALF patients (n = 5) on admission, compared to (HC) healthy controls (n = 5). TNF-a and IL-6 concentrations were determined following 6 hour incubation of PBMCs with 100 ng/ml of LPS in SALF (n = 10), acetaminophen-induced AALF (n = 10) and HC (n = 10). Immunohistochemistry (CD68/CD163/CD3/CD4/CD8/T-bet/Gata-3/ Th17/MPO/CD56/Perforin/FoxP3) was used to characterize the hepatic immune cell infiltrate in SALF (n = 10), AALF (n = 10) and pathological control (PC) liver tissue (n = 5). Results: Compared to HC, SALF patients had higher circulating levels of pro- (IFN-g, IL-1b, IL-6, IL-8), anti- (IL-10, TGF-b1) inflammatory cytokines and macrophage (sCD163) and T cell (IL-2Ra) activation markers (all p < 0.01) (Figure). Intrahepatic concentrations of IL-8, IL-12, TGF-b1, IL-2Ra and sCD163 were significantly elevated in SALF, compared to PC tissue (p < 0.05). LPS-induced secretion levels of TNF-a and IL-6 of PBMCs in SALF were comparable to HC (p > 0.05), suggesting intact proinflammatory responses. Immunohistochemical analysis revealed a macrophage- (CD68+,CD163+), Th1 lymphocytic- (CD4+,T-bet+) rich infiltrate in centrilobular and periportal areas, compared to AALF and PC (Figure). Conclusions: SALF is a Th1-cell driven disease, characterized by profound elevation in biomarkers of macrophage/T cell activation; thus, forming the rationale to assess immunotherapeutic strategies to attenuate proinflammatory responses and severity of liver failure.
Journal of Hepatology 2014 vol. 60 | S67–S214
S111