Mark W. Robinson

Liver immunology and its role in inflammation and homeostasis

The human liver is usually perceived as a non-immunological organ engaged primarily in metabolic, nutrient storage and detoxification activities. However, we now know that the healthy liver is also a site of complex immunological activity mediated by a diverse immune cell repertoire as well as non-hematopoietic cell populations. In the non-diseased liver, metabolic and tissue remodeling functions require elements of inflammation. This inflammation, in combination with regular exposure to dietary and microbial products, creates the potential for excessive immune activation. In this complex microenvironment, the hepatic immune system tolerates harmless molecules while at the same time remaining alert to possible infectious agents, malignant cells or tissue damage. Upon appropriate immune activation to challenge by pathogens or tissue damage, mechanisms to resolve inflammation are essential to maintain liver homeostasis. Failure to clear ‘dangerous’ stimuli or regulate appropriately activated immune mechanisms leads to pathological inflammation and disrupted tissue homeostasis characterized by the progressive development of fibrosis, cirrhosis and eventual liver failure. Hepatic inflammatory mechanisms therefore have a spectrum of roles in the healthy adult liver; they are essential to maintain tissue and organ homeostasis and, when dysregulated, are key drivers of the liver pathology associated with chronic infection, autoimmunity and malignancy. In this review, we explore the changing perception of inflammation and inflammatory mediators in normal liver homeostasis and propose targeting of liver-specific immune regulation pathways as a therapeutic approach to treat liver disease.

Tissue-resident Eomeshi T-betlo CD56bright NK cells with reduced proinflammatory potential are enriched in the adult human liver

The adult human liver is enriched with natural killer (NK) cells, accounting for 30–50% of hepatic lymphocytes, which include tissue‐resident hepatic NK‐cell subpopulations, distinct from peripheral blood NK cells. In murine liver, a subset of liver‐resident hepatic NK cells have altered expression of the two highly related T‐box transcription factors, T‐bet and eomesodermin (Eomes). Here, we investigate the heterogeneity of T‐bet and Eomes expression in NK cells from healthy adult human liver with a view to identifying human liver‐resident populations. Hepatic NK cells were isolated from donor liver perfusates and biopsies obtained during orthotopic liver transplantation (N = 28). Hepatic CD56bright NK cells were Eomeshi T‐betlo, a phenotype virtually absent from peripheral blood. These NK cells express the chemokine receptor CXCR6 (chemokine (C‐X‐C motif) receptor 6), a marker of tissue residency, which is absent from hepatic CD56dim and blood NK cells. Compared to blood populations, these hepatic CD56bright NK cells have increased expression of activatory receptors (NKp44, NKp46, and NKG2D). They show reduced ability to produce IFN‐γ but enhanced degranulation in response to challenge with target cells. This functionally distinct population of hepatic NK cells constitutes 20–30% of the total hepatic lymphocyte repertoire and represents a tissue‐resident immune cell population adapted to the tolerogenic liver microenvironment.

Broad anti-hepatitis C virus (HCV) antibody responses are associated with improved clinical disease parameters in chronic HCV infection

ABSTRACT During hepatitis C virus (HCV) infection, broadly neutralizing antibody (bNAb) responses targeting E1E2 envelope glycoproteins are generated in many individuals. It is unclear if these antibodies play a protective or a pathogenic role during chronic infection. In this study, we investigated whether bNAb responses in individuals with chronic infection were associated with differences in clinical presentation. Patient-derived purified serum IgG was used to assess the breadth of HCV E1E2 binding and the neutralization activity of HCV pseudoparticles. The binding and neutralization activity results for two panels bearing viral envelope proteins representing either an intergenotype or an intragenotype 1 group were compared. We found that the HCV load was negatively associated with strong cross-genotypic E1E2 binding (P = 0.03). Overall, we observed only a modest correlation between total E1E2 binding and neutralization ability. The breadth of intergenotype neutralization did not correlate with any clinical parameters; however, analysis of individuals with genotype 1 (gt1) HCV infection (n = 20), using an intragenotype pseudoparticle panel, found a strong association between neutralization breadth and reduced liver fibrosis (P = 0.006). A broad bNAb response in our cohort with chronic infection was associated with a single nucleotide polymorphism (SNP) in the HLA-DQB1 gene (P = 0.038), as previously reported in a cohort with acute disease. Furthermore, the bNAbs in these individuals targeted more than one region of E2-neutralizing epitopes, as assessed through cross-competition of patient bNAbs with well-characterized E2 antibodies. We conclude that the bNAb responses in patients with chronic gt1 infection are associated with lower rates of fibrosis and host genetics may play a role in the ability to raise such responses. IMPORTANCE Globally, there are 130 million to 150 million people with chronic HCV infection. Typically, the disease is progressive and is a major cause of severe liver cirrhosis and hepatocellular carcinoma. While it is known that neutralizing antibodies have a role in spontaneous clearance during acute infection, little is known about their role in chronic infection. In the present work, we investigated the antibody response in a cohort of chronically infected individuals and found that a broadly neutralizing antibody response is protective and is associated with reduced levels of liver fibrosis and cirrhosis. We also found an association between SNPs in class II HLA genes and the presence of a broadly neutralizing response, indicating that antigen presentation may be important for the production of HCV-neutralizing antibodies.

Elevated interferon-stimulated gene transcription in peripheral blood mononuclear cells occurs in patients infected with genotype 1 but not genotype 3 hepatitis C virus

Hepatitis C virus (HCV) can be classified into seven distinct genotypes that are associated with differing pathologies and respond differently to antiviral therapy. In the UK, genotype 1 and 3 are present in approximately equal proportions. Chronic infection with HCV genotype 3 is associated with increased liver steatosis and reduced peripheral total cholesterol levels, which potentially influences peripheral immune responses. To understand these differences, we investigated host gene transcription in peripheral blood mononuclear cells by microarray and quantitative PCR in patients with genotype 1 (n = 22) or genotype 3 infection (n = 22) and matched healthy controls (n = 15). Enrichment of genes involved in immune response and inflammatory pathways were present in patients infected with HCV genotype 1; however, no differences in genes involved in lipid or cholesterol metabolism were detected. This genotype‐specific induction of genes is unrelated to IL28B genotype or previous treatment failure. Our data support the hypothesis that genotype 1 infection drives a skewed Type I interferon response and provides a foundation for future investigations into the host–pathogen interactions that underlie the genotype‐specific clinical outcomes of chronic HCV infection.

Non cell autonomous upregulation of CDKN2 transcription linked to progression of chronic hepatitis C disease.

Chronic hepatitis C virus infection (C‐HC) is associated with higher mortality arising from hepatic and extrahepatic disease. This may be due to accelerated biological aging; however, studies in C‐HC have thus far been based solely on telomere length as a biomarker of aging (BoA). In this study, we have evaluated CDKN2 locus transcripts as alternative BoAs in C‐HC. Our results suggest that C‐HC induces non‐cell‐autonomous senescence and accelerates biological aging. The CDKN2 locus may provide a link between C‐HC and increased susceptibility to age‐associated diseases and provides novel biomarkers for assessing its impact on aging processes in man.

Viral genotype correlates with distinct liver gene transcription signatures in chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection of the liver with either genotype 1 or genotype 3 gives rise to distinct pathologies, and the two viral genotypes respond differently to antiviral therapy.

Anti‐envelope antibody responses in individuals at high risk of hepatitis C virus who resist infection

Injection drug users uninfected by hepatitis C virus (HCV) despite likely repeated exposure through high‐risk behaviour are well documented. Factors preventing infection in these individuals are incompletely understood. Here, we looked for anti‐HCV‐envelope antibody responses in a cohort of repeatedly exposed but uninfected subjects. Forty‐two hepatitis C diagnostic antibody‐ and RNA‐negative injection drug users at high risk of exposure were studied and findings compared to healthy controls and cases with chronic HCV infection. Purified IgGs from sera were tested by ELISA for binding to genotype 1a and 3a envelope glycoproteins E1E2 with further testing for IgG and IgM reactivity against soluble E2. Virus‐neutralizing activity was assessed using an HCV pseudoparticle system. Uninfected subjects demonstrated significantly greater IgG and IgM reactivities to envelope glycoproteins than healthy controls with IgG from 6 individuals additionally showing significant neutralization. This study is the first to describe humoral immunological responses targeting the HCV envelope, important for viral neutralization, in exposed uninfected individuals. A subset of these cases also had evidence of viral neutralization via anti‐envelope antibodies. In addition to confirming viral exposure, the presence of specific anti‐envelope antibodies may be a factor that helps these individuals resist HCV infection.

Tracking TCRβ Sequence Clonotype Expansions during Antiviral Therapy Using High-Throughput Sequencing of the Hypervariable Region

To maintain a persistent infection viruses such as hepatitis C virus (HCV) employ a range of mechanisms that subvert protective T cell responses. The suppression of antigen-specific T cell responses by HCV hinders efforts to profile T cell responses during chronic infection and antiviral therapy. Conventional methods of detecting antigen-specific T cells utilize either antigen stimulation (e.g., ELISpot, proliferation assays, cytokine production) or antigen-loaded tetramer staining. This limits the ability to profile T cell responses during chronic infection due to suppressed effector function and the requirement for prior knowledge of antigenic viral peptide sequences. Recently, high-throughput sequencing (HTS) technologies have been developed for the analysis of T cell repertoires. In the present study, we have assessed the feasibility of HTS of the TCRβ complementarity determining region (CDR)3 to track T cell expansions in an antigen-independent manner. Using sequential blood samples from HCV-infected individuals undergoing antiviral therapy, we were able to measure the population frequencies of >35,000 TCRβ sequence clonotypes in each individual over the course of 12 weeks. TRBV/TRBJ gene segment usage varied markedly between individuals but remained relatively constant within individuals across the course of therapy. Despite this stable TRBV/TRBJ gene segment usage, a number of TCRβ sequence clonotypes showed dramatic changes in read frequency. These changes could not be linked to therapy outcomes in the present study; however, the TCRβ CDR3 sequences with the largest fold changes did include sequences with identical TRBV/TRBJ gene segment usage and high junction region homology to previously published CDR3 sequences from HCV-specific T cells targeting the HLA-B*0801-restricted 1395HSKKKCDEL1403 and HLA-A*0101-restricted 1435ATDALMTGY1443 epitopes. The pipeline developed in this proof of concept study provides a platform for the design of future experiments to accurately address the question of whether T cell responses contribute to SVR upon antiviral therapy. This pipeline represents a novel technique to analyze T cell dynamics in situations where conventional antigen-dependent methods are limited due to suppression of T cell functions and highly diverse antigenic sequences.

PTH-100 A comparative gene expression study between individuals with apparent resistance, spontaneous clearance, or chronic infection from hcv

Introduction We have identified a group of high risk injection drug users who share needles, syringes and other injecting paraphernalia over a long period of time yet who remain uninfected by hepatitis C virus (HCV). These individuals appear to be resistant to HCV infection and we have termed them exposed uninfected (EU). Defining factors that may prevent HCV infection in these cohorts could give valuable insight into the mechanisms of natural resistance to HCV. Method Individuals at high risk of recurrent exposure to HCV infection from long term IDU were recruited in Plymouth and if HCV antibody and HCV RNA negative were termed exposed uninfected (EU). We have compared the transcriptional profiles of intravenous drug users (IDU) with 3 different outcomes following HCV exposure – chronic hepatitis C infection (CHCV; n = 6), spontaneously resolution of HCV (SR; HCV Ab positive but HCV RNA negative, n = 6) and exposed but uninfected (EU; n = 6). Agilent single channel microarray was performed on RNA isolated from peripheral blood mononuclear cells and the results were analysed on Ingenuity Pathway Analysis software (Qiagen). The identified targets were subsequently validated at a protein level. Results 1465 genes between the EU and CHCV group, 4377 between the CHCV and SR group and 4510 between the SR and EU group were significantly up or down regulated. Of the differentially regulated genes, the association with resistance to HCV was strongest for IL27 and CXCL7 which were significantly upregulated in EU (IL27 log fold change EU vs SR: 1.943, p = 1.72E-04 and CXCL7 log fold change EU vs CHCV 2.359, p = 6.04E-03) relative to the other 2 groups. At a protein level, the median IL27 serum level in the EU group (n = 13), detected by sandwich ELISA, was significantly higher (p = 0.008) compared to CHCV (n = 14), healthy control (HC; n = 8) and SR (n = 14) groups. The CD28 mediated T-helper cell signalling pathway was significantly upregulated in SR (ratio 0.348, p = 2.77E-05) relative to the 2 other groups. Conclusion Significant differences in gene expression were found in injection drug users with different outcomes from exposure to HCV infection. EU and SR exhibit differences related to innate and adaptive immunity compared to CHCV that likely contribute to natural resistance and spontaneous clearance of HCV. Disclosure of interest None Declared.

PTU-110 What impact do hcv infection and subclinical cryoglobulinaemia have on the b cell repertoire?

Introduction In addition to causing liver injury, hepatitis C virus (HCV) is closely associated with clonal B-cell pathologies such as cryogloglobulinaemia.1The virus is also capable of evading control by antibody responses.2Understanding the mechanisms by which HCV resists clearance by the adaptive immune system is important in vaccine design. We hypothesised that HCV infection may restrict the B-cell repertoire, predisposing to clonal B-cell disorders and contributing to impaired ability to mount an effective anti-viral antibody response. We aimed to explore this by determining: Any differences in B-cell Receptor (BCR) characteristics between HCV-infected individuals with and without cryoglobulinaemia. Any functional differences in viral neutralising ability associated with subclinical cryoglobulinaemia. Method Patients chronically infected with either genotype 1 (G1) or genotype 3 (G3) HCV were prospectively recruited in addition to healthy controls (HC). None had a prior diagnosis of cryoglobulinaemia. Patient serum was tested for presence of cryoprecipitates. Purified IgG from patient serum was tested for ability to prevent infection of cultured cells by a panel of HCV envelope-coated ‘pseudoparticles’. For a subset of HCV-infected patients and HC, RNA was extracted from PBMCs and cDNA libraries generated. BCR genes were amplified and underwent 454 sequencing. Sequences were analysed for overall diversity, clonality and specific gene usage using an in-house programme. Results 75 HCV-infected patients (36 G1, 39 G3) were recruited. Of the HCV-infected individuals, 28% (21/75) had detectable cryoprecipitates. There was no association between presence of cryoglobulins and functional antibody responses (p = 0.77). BCR sequences were analysed for 6 cryoglobulin positive, 6 cryoglobulin negative and 6 HC. There was no difference in overall BCR diversity between the groups. Cryoglobulin positive individuals showed reduced expression of IGHV 1–8 (p = 0.004). The antigen binding CDR3 region was longer in cryoglobulinaemic individuals than HC (p = 0.028). Conclusion Our data suggest that asymptomatic cyoglobulinaemic antibodies are common in chronic HCV infection. While these are not associated with an obvious deficit in antibody function or BCR diversity, next generation sequencing has revealed potentially important differences in their B-cell receptor populations. These novel findings and their functional implications require further analysis. Disclosure of interest None Declared. References Charles ED, et al. Blood 2008;111:1344–1356 Edwards VC, et al. J Gen Virol. 2012;93:1–19