A. Evans

Blockade of PD1 and TIM3 Restores Innate and Adaptive Immunity in Patients with Acute Alcoholic Hepatitis

BACKGROUND & AIMS Susceptibility to bacterial infection is a feature of alcohol-related liver disease. Programmed cell death 1 (PD1), the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3, also known as hepatitis A virus cellular receptor 2), and their respective ligands-CD274 (also known as PD ligand 1 [PDL1]) and galectin-9-are inhibitory receptors that regulate the balance between protective immunity and host immune-mediated damage. However, their sustained hyperexpression promotes immune exhaustion and paralysis. We investigated the role of these immune inhibitory receptors in driving immune impairments in patients with alcoholic liver disease. METHODS In a prospective study, we collected blood samples from 20 patients with acute alcoholic hepatitis (AAH), 16 patients with stable advanced alcohol-related cirrhosis, and 12 healthy individuals (controls). Whole blood or peripheral blood mononuclear cells were assessed for expression of PD1, PDL1, TIM3, galectin-9, and Toll-like receptors on subsets of innate and adaptive immune effector cells. We measured antibacterial immune responses to lipopolysaccharide (endotoxin) using ELISpot assays, and used flow cytometry to quantify cytokine production, phagocytosis, and oxidative burst in the presence or absence of blocking antibodies against PD1 or TIM3. RESULTS Antibacterial innate and adaptive immune responses were greatly reduced in patients with AAH, compared with controls, and patients with alcohol-related cirrhosis had less severe dysfunctions in innate immune effector cells and preserved functional T-cell responses. Fewer T cells from patients with AAH produced interferon gamma in response to lipopolysaccharide, compared with controls. In addition, patients with AAH had greater numbers of interleukin 10-producing T cells, and reduced levels of neutrophil phagocytosis and oxidative burst in response to Escherichia coli stimulation, compared with controls. T cells from patients with AAH, but not alcohol-related cirrhosis, expressed higher levels of PD1 and PDL1, or TIM3 and galectin-9, than T cells from controls. Antibodies against PD1 and TIM3 restored T-cell production of interferon gamma, reduced the numbers of interleukin 10-producing T cells, and increased neutrophil antimicrobial activities. Circulating levels of endotoxin in plasma from patients with AAH caused over expression of immune inhibitory receptors on T cells via Toll-like receptor 4 binding to CD14(+) monocytes. CONCLUSIONS Antibacterial immune responses are impaired in patients with AAH. Lymphocytes from these patients express high levels of immune inhibitory receptors, produce lower levels of interferon gamma, and have increased IL10 production due to chronic endotoxin exposure. These effects can be reversed by blocking PD1 and TIM3, which increase the antimicrobial activities of T cells and neutrophils.

Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease

Background/aims Intestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown. Methods/design We analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively. Results In ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments. Conclusions In ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the ‘leaky’ gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients.

Surveillance for hepatocellular carcinoma in a mixed-aetiology UK cohort with cirrhosis: does α-fetoprotein still have a role?

Mortality from hepatocellular carcinoma (HCC) in people with cirrhosis is increasing whereas mortality from other causes is declining. Surveillance appears to reduce mortality but the optimal strategy is uncertain. Current guidelines differ by recommending ultrasonography alone or with α-fetoprotein (αFP). Records in three UK hospitals were analysed from 2006 to 2011. Of 111 HCC cases identified, 24 (47.1%) of those eligible were under surveillance: 21 (87.5%) were under combined ultrasonography-αFP, 2 (8.3%) ultrasonography-only and 1 (4.2%) αFP-only surveillance. αFP was elevated in 19 (86.4%), and αFP alone triggered a confirmatory study in 11 (9.9%) overall and 7 (29.1%) under surveillance. Surveillance, but not αFP, correlated with smaller tumours. Survival did not differ significantly between groups. Given that αFP use is associated with identifying smaller HCCs and that several diagnoses would have been delayed without αFP in this real-life cohort, these data support ongoing αFP use. However, further work is necessary with regard to whether αFP translates into improved clinical outcome and overall cost effects. In our area, stopping αFP use would also represent a significant change in practice.

PWE-088 Frequency and function of anti-bacterial mait cells are significantly impaired in advanced alcoholic liver disease

Introduction Intestinal bacterial translocation and systemic gut-derived bacterial products play a central role in the immunopathogenesis of alcoholic liver disease (ALD), yet the mechanisms of susceptibility to infection and the link with intestinal immunity remain elusive. Mucosal associated invariant T cells (MAIT) are unconventional T cells which only respond to bacteria-derived metabolites and are found in large numbers in the blood, intestinal mucosae and liver. They represent a key sentinel system for the homeostatic control of the gut flora and for the control of bacterial infections. The aim of this study was to assess the role of MAIT cells in ALD. Method Peripheral blood mononuclear cells from subjects with acute alcoholic hepatitis (AAH, Maddrey’s discriminant function >32; n = 9), compensated alcohol-related cirrhosis (ARC, n = 9) and healthy controls (HC, n = 9) were examined by FACS. MAIT cells were identified as CD161+/Vα7.2+ CD8+T cells and MAIT-presenting cells (MPC) as MR1+ monocytes or B cells. We quantified (1) frequency, activation status (CD69/HLA-DR) and immunoinhibitory signatures (PD1/TIM3/LAG3) of MAIT cells; (2) frequency and immunoinhibitory status (PD1/PDL1/TIM3/mGal9) of MPC; (3) cytokine/cytotoxicity profiles (IFNγ/TNFα/IL17; GranzymeB (GrB)/Perforin/CD107a) of MAIT cells after in-vitro stimulation with fixed E. Coli. Plasma cytokines and endotoxin levels were assessed by ELISA. Results MAIT frequencies were reduced in ARC (p = 0.005) and dramatically lower in AAH (p < 0.001) compared to HC. MAIT cells from AAH and ARC were activated and overexpressed immunoinhibitory receptors compared to HC (p < 0.03). Frequencies of MR1+ MPC were comparable in all groups but expression of immunoinhibitory molecules was higher in AAH and ARC (p < 0.03). Membrane-bound MPC Gal9 was lower in AAH and ARC (p < 0.01) than HC whereas soluble Gal9 was higher (p = 0.004). Levels of E. Coli-stimulated IFNγ from MAIT cells were comparable in all groups, but in ARC they produced less TNFα (p = 0.024) than HC. IL17 responses were observed only in HC. The cytotoxic potential of MAIT cells (GrB) was higher in AAH than HC. Conclusion This is the first report addressing the role of MAIT cells in liver disease. We show that MAIT cells have quantitative and functional impairments in ALD, with defective TNFα and IL17 responses, increased killing potential and overexpression of immunoinhibitory receptors. MAIT cells may represent a novel immunotherapeutic target for ALD. Disclosure of interest None Declared.

PWE-115 Alpha-Fetoprotein Measurement in the Diagnosis of Hepatocellular Carcinoma in Real-Life Practice: A Multi-Centre, Retrospective Analysis

Introduction In hepatocellular carcinoma (HCC), earlier diagnosisimproves outcome but the optimum method of surveillance in high-risk groups is controversial. Recent AASLD and EASL guidelines[1.2] have recommended six- monthly ultrasound surveillance (USS) alone. British guidelines[3] currently recommend combining serial alpha-fetoprotein (aFP) measurements with six-monthly USS. This study aimed to assess the role of aFP measurement in HCC surveillance programmes. Methods This large retrospective multicentre study assessed newly diagnosed HCC over a 5-year period (2006–2011) at three centres: two general hospitals and one tertiary referral centre. Electronic and multi-disciplinary team data were reviewed. Results 111 patients with a confirmed diagnosis of HCC were identified. Of these, 91(81.9%) were male and the median age was 69 years (range 24–87). 52(46.8%) patients with newly diagnosed HCC had established liver disease prior to diagnosis. Of these, 21(40.4%) were participating in combined USS-aFP surveillance, 2(3.8%) USS alone and 1(1.9%) aFP alone. A diagnosis of HCC was confirmed by liver biopsy in 43(38.7%), CT in 41(36.9%), MRI in 25(22.5%) and USS combined with elevated aFP in 2(1.8%). At diagnosis, aFP was elevated in 81(73.0%), normal in 22(19.8%) and unmeasured in 8(7.2%) patients. Of those 21 diagnosed in an established surveillance programme of six- monthly USS and aFP, 17(81.0%) showed a rise in aFP. When assessing the trigger for confirmatory cross-sectional imaging ± biopsy across all data, a solely elevated aFP prompted further investigation in 11(9.9%); in those under surveillance, this number was 7(29.2%) with no abnormality detected on USS within the preceding three-month period in 6(85.7%) of these. Conclusion These results demonstrate that a significant number of patients would have had a delayed diagnosis of HCC if aFP measurement was removed from UK screening programmes. Potential contributing factors limiting the success of USS- based screening programmes include: small lesion size, sonographer error, patient factors limiting USS accuracy (e.g. body habitus) and irregular attendance for USS. This study supports continued serial measurement of aFP in patients with liver cirrhosis in contrast to European and American guidelines. Disclosure of Interest None Declared. References Hepatology, vol. 53(3)1020–1022, Mar. 2011. Journal of Hepatology, vol. 56(4)908–943, Apr. 2012 Gut, vol. 52(3)iii1-iii8, 2003.

PTU-204 Accuracy of visual estimation of adenoma size: a comparison with direct measurement in the pathology department

Introduction Large adenomatous colonic polyps (>10 mm) are associated with an increased risk of development of adenocarcinoma. Recent national guidelines require the ability to distinguish polyps above and below 10 mm in size to determine the optimal surveillance interval.1 There is no standardised technique to measure polyp size either in the literature that underpins current guidelines or in practice. Visual estimation at endoscopy is widely used. Small prospective studies have shown this method to be inaccurate when compared to direct measurement in the pathology department.2 This retrospective study aims to establish the accuracy of visual estimation of polyp size in usual clinical practice comparing to direct measurement. Methods A search for the word “polyp” was performed on the pathology reports for all colonoscopies and flexible sigmoidoscopies performed during a 1-year period. The pathology and endoscopy reports of the resultant cases were reviewed. Only adenomas completely removed by snare polypectomy without lifting and retrieved intact, where both endoscopic and measured sizes were recorded, and where the measured size was 5 to 15 mm were included. The direct measurement was subtracted from the visual estimate to give a size difference. The paired-sample t-test was used to test the null hypothesis that there was no difference between the mean sizes determined using the two methods for the group as a whole or for individual endoscopists. Results In a total of 4285 procedures, 79 polyps met the criteria for inclusion. In 39 cases (49%), the difference between visual estimate and direct measurement was >2 mm. In ascertaining whether a polyp was above or below the 10 mm cut-off, visual estimate and direct measurement were discordant in 21 cases (27%). Despite these disparities, there was no overall tendency to over or underestimate polyp size for the group as a whole (mean difference 0.05 mm p=0.88). Of the 15 individual endoscopists, the two with the highest procedure counts both showed significant tendencies to underestimate polyp size, while a third showed significant overestimation. Conclusion In clinical practice, visual estimation of polyp size is often inaccurate. Individual endoscopists may systematically over or underestimate polyp sizes. Direct measurement should be preferred in determining surveillance intervals.Abstract PTU-204 Figure 1 Visual estimate—direct measurement (mm).Abstract PTU-204 Table 1 Endoscopist Polyp count Mean difference (mm) p Value D 9 −1.9 0.01 E 5 3.0 0.01 G 17 −1.9 0.001 Competing interests None declared. References 1. National Institute for Health and Clinical Excellence. Colonoscopic Surveillance for Prevention of Colorectal Cancer. 2011. http://www.nice.org.uk/guidance/CG118 2. Schoen, et al. The pathologic measurement of polyp size is preferable to the endoscopic estimate. Gastrointest Endosc 1997;46:492–6.

P64 Hepatitis B virus upregulates hepatocyte expression of PD-L1 to evade hepatoxic adaptive immune responses

Introduction Hepatitis B virus employs a variety of strategies aimed at overwhelming, evading or neutralising the host immune response to infection resulting in chronicity. We have previously shown that the Programmed Cell Death Pathway (PD-1/PD-L1) is an inhibitory T-cell pathway implicated in the homeostasis of immune responses and the balance between cytolytic and non-cytolytic CD8+ T-cell effector functions. Aim The aim of this study was to investigate the impact of hepatitis B virus (HBV) infection on hepatocytic PD-L1 expression. Method A human hepatoma cell line that constitutively expresses HBV-DNA (HepG2215), its parent cell line (HepG2) were cultured. A human hepatoma cell line (Huh7) was transfected with a plasmid containing an HBV head-to-tail dimer using Fugene 6 reagent. We also cultured a further HepG2 cell line (AD38) that produces full infectious virus under the control of a tetracycline (Tet)-responsive promoter. HBV-DNA and PD-L1 were quantitated longitudinally. Intracellular and secreted HBV-DNA was quantified with qRT-PCR. PD-1/PD-L1 expression was assessed by FACS and qRT-PCR. Co-cultures between virus-specific CD8+ T-cell lines and hepatocytes producing HBV were also established and analysis of T cell functions performed. Results The hepatoma cell lines which constitutively produce HBV virions (HepG2215) had significantly higher basal levels of PD-L1 expression compared with their parent cell line (HepG2) (p=0.01). A significant increase in intracellular and secreted HBV-DNA levels confirmed successful transfection of Hepatitis B virus. Following transfection there was a significant increase in PD-L1 levels (p=0.01) on infected hepatocytes, which was not observed following transfection with an empty vector. A significant correlation was observed between PD-L1 expression and both intracellular HBV-DNA (r=0.98, p=0.01) and secreted HBV-DNA (r=0.908, p=0.046) following transfection. Following activation of HBV-DNA expression in the AD38 cell line (-Tet), PD-L1 expression increased. Moreover, subsequent fluctuations in HBV-DNA in the absence/presence of Tet was temporally associated with the expression of PD-L1 (r=0.83, p<0.001). Hyperexpression of PD-L1 on hepatocytes was associated with a predominance of non-cytolytic T cell functions. Conclusion These results demonstrate that HBV-DNA drives PD-L1 expression on infected hepatocytes. As we have previously demonstrated, upregulation of PD-L1 impairs adaptive immune responses to HBV infection, and this novel function of HBV may reflect an important strategy by which hepatitis B virus extends the life-span of target hepatocytes and escapes an effective immune response contributing to the development of chronicity.

OP13 Spontaneous resolution of acute hepatitis C virus infection correlates with the reconstitution of the circulating Cd56dim natural killer-cell pool

Introduction Efforts to identify the immune-correlates responsible for resolution of hepatitis C virus (HCV) infection are fundamental to develop new treatment strategies and an effective vaccine against HCV. We have previously shown that imbalanced natural killer (NK)-cell subsets, with hyper-expression of co-inhibitory markers, are associated with chronic HCV infection. Aim In this study we aim to assess the role of NK-cells in determining the outcome of acute HCV infection in a cohort of well characterised patients. Method We analysed 12 patients with acute HCV infection who met the following criteria: ALT>10xULN, exposure to HCV within previous 4 months and HCV-RNA(+). Viral load was determined by qPCR. Peripheral blood mononuclear cells collected at 3 time-points (baseline, BL; month 1, M1; month 6, M6) were stained with fluorochrome-labelled antibodies to NK-cells (CD3/CD56/CD16). Proportions of CD56dim(CD16bright) and CD56bright(CD16dim) subsets and expression of PD-1/PD-L1 were evaluated by 6-colour flow cytometry and correlated with HCV-RNA and ALT at each time-point and over-time. Supernatants from cell-cultures in the presence of HCV-antigens were collected for cytokine analysis and quantification. Results Six patients resolved HCV spontaneously (Resolvers), whilst six developed chronic infection (Chronics). At presentation, mean viraemia and ALT levels did not differ between Resolved and Chronic patients. In Resolvers HCV-RNA became undetectable at M3, which was then followed by ALT normalisation. Overall, Resolvers had higher proportions of total NKs than Chronics (p=0.023). Cytotoxic CD56dim NK cells were also higher in Resolvers (p=0.001), and became progressively predominant within their total NK pool, as shown by their progressive increase of the CD56dim/CD56bright ratio, which differentiated Resolvers from Chronics after M1 (p=0.01). In Chronic patients’ cytotoxic CD56dim NK cells had, however, an overall greater expression of the cytotoxicity marker CD16 (p=0.008). Chronic patients also had higher proportions of both subsets of NK cells expressing the immunoinhibitory marker PD-1 (p=0.02), and stronger per-cell expression of the immunoinhibitory ligand PD-L1 on CD56dim NK cells (p=0.001). Analysis of clinical parameters revealed that in Resolvers the progressive increase of CD56dim/CD56bright ratio correlated with the decline of HCV-RNA over-time (r=−0.997, p=0.042), due to reduction of CD56bright (r=0.999, p=0.011) and expansion of CD56dim (r=−0.997, p=0.041). At M1, Resolvers’ CD56dim were positively correlated with ALT (r=0.820, p=0.046). Conclusion In patients with acute HCV infection the proportions and evolution of the two functionally distinct NK cell subsets differ in patients that resolve the infection compared to those who become chronically infected. A favourable outcome of infection is associated with the establishment of a defined NK profile, with predominance of CD56dim (cytotoxic) NK cells with low expression of the immunoinhibitory markers PD-1 and PD-L1, which may be linked to an improved early clearance of virus-infected hepatocytes, as shown by the correlation of this subset with serum HCV-RNA and ALT decline.