812 HEPATITIS C VIRUS-SPECIFIC EFFECTOR AND REGULATORY CD4+ T CELLS IN PRIMARY INFECTION RESULTING IN CLEARANCE OR CHRONIC INFECTION

POSTERS 811 THE MOLECULAR MECHANISMS OF B CELL AND B CELL LYMPHOMA RECRUITMENT TO THE HUMAN LIVER S. Shetty1,2 , T. Bruns1 , C. Weston1 , Y. Oo1,2 , Z. Stamataki1 , S. Hubscher1 , S. Jalkanen3 , P. Lalor1 , D.H. Adams1,2 . 1 Centre for Liver Research, 2 NIHR Biomedical Research Unit, University of Birmingham, Birmingham, UK; 3 MediCity Research, University of Turku, Turku, Finland E-mail: [email protected] Introduction: There is gathering interest in the presence of B cells within liver tissue and their contribution to chronic inflammation and fibrosis but the recruitment signals for B cells into peripheral tissue is poorly understood. In addition a large proportion of lymphomas which infiltrate the liver are of B cell origin but again little is understood of this process. Lymphocyte recruitment to the liver occurs within the hepatic sinusoidal channels. These low shear vascular beds are lined by specialised hepatic sinusoidal endothelial cells (HSEC). Our aim was to understand the molecular mechanisms of B cell and B cell lymphoma recruitment to the liver. Methods: We used isolated human HSEC in flow assays with purified peripheral blood B cells to elucidate the molecular mechanisms of B cell recruitment via HSEC. The contribution of conventional adhesion molecules, ICAM-1 and VCAM-1 and unconventional molecules VAP-1 and CLEVER-1/stabilin-1 was assessed by using function blocking antibodies. We repeated our experiments with two B cell lymphoma cell lines. We assessed the contribution of chemokines by performing transwell assays and adding chemokines to our flow assays. We also tracked the motility of B cells and lymphoma cell lines on HSEC using tracking software. Results: B cells were captured from flow and firmly adhered to HSEC, the primary adhesion receptor on HSEC was VCAM-1. B cells also underwent transendothelial migration which was mediated by a combination of ICAM-1, VAP-1 and CLEVER-1/stabilin-1. Lymphoma cell line recruitment shared several features of primary lymphocyte homing, firm adhesion was mediated by ICAM-1 and VCAM-1 and they demonstrated shape-change and crawling behaviour which was ICAM-1 dependent. The lymphoma cell lines did not undergo transendothelial migration and this could not be initiated with the addition of SDF-1a. Conclusion: There is increasing evidence that B cells play an important role in chronic inflammatory liver diseases. The recruitment signals we have identified for B cells in this study may provide potential therapeutic targets for liver disease. Furthermore we have demonstrated preserved lymphocyte homing mechanisms in malignantly transformed B cells. These properties could be therapeutic targets to prevent lymphoma dissemination to the liver. 812 HEPATITIS C VIRUS-SPECIFIC EFFECTOR AND REGULATORY CD4+ T CELLS IN PRIMARY INFECTION RESULTING IN CLEARANCE OR CHRONIC INFECTION P.B. Sugden1 , M. Hunter1 , E. Keoshkerian1 , B. Cameron1 , A. Zekry1 , J. Zaunders2 , N. Sediki2 , T. Kelleher2 , A. Lloyd1 . 1 Infection and Inflamation Research Centre, 2 Kirby Institute, University of NSW, Sydney, NSW, Australia E-mail: [email protected] Background and Aims: In primary hepatitis C (HCV) infection, viral persistence has been associated with a weak and narrowly targeted cellular immune response. Functional CD4+ helper and CD8+ cytotoxic T lymphocytes are crucial components of this response. Regulatory CD4+ T cells can control effector functions of both CD4 and CD8 T cells. We hypothesised that the relative abundance of HCV-specific effector and regulatory CD4+ T cells influences HCV infection outcomes.

Method: Using a recently developed flow cytometric assay for antigen-specific effector (based on the stimulated expression of CD25 and CD134) [1] and regulatory CD4 T cells (Treg; based on CD25, CD134 and CD39), we studied a cross-sectional case-control series of chronically infected subjects (n = 15) and spontaneous clearers (n = 15), and a longitudinal case-control series of subjects followed during primary infection resulting in clearance (n = 10) or chronicity (n = 10) with up to five time points from pre-infection to post outcome. PBMCs were stimulated in the presence of HCV antigens, followed by staining for CD3, CD4, CD25, CD134 and CD39. Results: In the cross-sectional study, as expected subjects who cleared HCV responded more often to HCV stimuli than subjects with chronic infection (P < 0.05 for each). The mean HCV-specific effector to Treg ratio was 2.3 (SD 2.0) in subjects who cleared infection compared with 0.5 (SD 1.3) in subjects with persistent infection (p = 0.01 Mann Whitney). Preliminary analysis of the longitudinal dataset indicates that the HCV-specific effector to regulatory T-cell ratios evolve early during primary infection with subjects who clear developing more T-effectors while subjects who go on to become chronic developing more T-regs, predicting outcome. Also, subjects who clear have an HCV specific T-cell response that increases and develops later, while those who become chronically infected have a response that starts earlier and decreases over time. Conclusion: Clearance of HCV infection is associated with predominant HCV-specific T-effector responses relative to HCVspecific T-regs. Further investigation of the determinants of these divergent T cell response patterns is warranted. Reference(s) [1] Keoshkerian E, et al. A novel assay for detection of hepatitis C virusspecific effector CD4+ T cells via co-expression of CD25 and CD134. J Immunol Methods 2011.

813 PRIMING BY PROINFLAMMATORY CYTOKINES IS REQUIRED FOR TOLL-LIKE RECEPTORS ON HEPATIC ENDOTHELIUM TO RECRUIT MONOCYTES UNDER CONDITIONS OF FLOW R. Sutaria1 , S.M. Curbishley1 , C.J. Weston1 , S.C. Afford1 , D.H. Adams1 , K.-K. Li2 . 1 Centre for Liver Research and NIHR Biomedical Research Unit, University of Birmingham, 2 Centre for Liver Research and NIHR Biomedical Research Unit, Birmingham, UK E-mail: [email protected] Background and Aims: The liver is continuously exposed to pathogen associated molecular patterns (PAMPs) such as bacterial ligands that are transported from the gut via the portal circulation. In chronic liver disease bacterial translocation increases and patients are at risk of developing both gram negative and gram positive sepsis which is associated with poor clinical outcomes. We hypothesised that activation of toll-like receptors (TLR) on hepatic sinusoidal endothelium by bacterial ligands will modulate endothelial activation leading to enhanced leukocyte recruitment and exacerbation of chronic inflammation. Because monocytes recruited from the blood are pivotal in determining the balance between the persistent hepatic inflammation and resolution we investigated the effect of different TLR ligands on the recruitment of monocytes via hepatic sinusoidal endothelium. Methods: Human hepatic sinusoidal endothelial cells were isolated and grown as confluent monolayers in Ibidi flow chamber slides. Cells were stimulated with TLR ligands 1–9, tumour necrosis factor alpha (TNFa) or interferon gamma (IFNg), alone or in combination. Monocytes isolated from blood were perfused over the endothelial cells at a shear stress of 0.05Pa to emulate conditions within the hepatic sinusoids and interactions with endothelium recorded. Results: 1. Treatment of resting endothelium with TLR ligands to 1, 2, 4, 5 or 6 resulted in increased adhesion of monocytes from 27.8±16.8

Journal of Hepatology 2012 vol. 56 | S225–S388

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