Tissue CD14+CD8+ T cells reprogrammed by myeloid cells and modulated by LPS.

The liver is bathed in bacterial products, including lipopolysaccharide transported from the intestinal portal vasculature, but maintains a state of tolerance that is exploited by persistent pathogens and tumours1-4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+ T cells compartmentalized in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins and are enriched in close proximity to CD14high myeloid cells in hepatic zone 2. CD14+CD8+ T cells preferentially accumulate within the donor pool in liver allografts, among hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+ T cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10 and IL-2 production ex vivo, and enhanced antiviral/anti-tumour effector function after TCR engagement. This CD14+CD8+ T cell profile can be recapitulated by the acquisition of membrane proteins-including the lipopolysaccharide receptor complex-from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T cells in vitro. CD14+CD8+ T cells express integrins and chemokine receptors that favour interactions with the local stroma, which can promote their induction through CXCL12. Lipopolysaccharide can also increase the frequency of CD14+CD8+ T cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+ T cells with immunomodulatory ability.

Hepatocellular Carcinoma in Mongolia Delineates Unique Molecular Traits and a Mutational Signature Associated with Environmental Agents.

PURPOSE: Mongolia has the world's highest incidence of hepatocellular carcinoma (HCC), with ∼100 cases/100,000 inhabitants, although the reasons for this have not been thoroughly delineated. EXPERIMENTAL DESIGN: We performed a molecular characterization of Mongolian (n = 192) compared with Western (n = 187) HCCs by RNA sequencing and whole-exome sequencing to unveil distinct genomic and transcriptomic features associated with environmental factors in this population. RESULTS: Mongolian patients were younger, with higher female prevalence, and with predominantly HBV-HDV coinfection etiology. Mongolian HCCs presented significantly higher rates of protein-coding mutations (121 vs. 70 mutations per tumor in Western), and in specific driver HCC genes (i.e., APOB and TSC2). Four mutational signatures characterized Mongolian samples, one of which was novel (SBS Mongolia) and present in 25% of Mongolian HCC cases. This signature showed a distinct substitution profile with a high proportion of T>G substitutions and was significantly associated with a signature of exposure to the environmental agent dimethyl sulfate (71%), a 2A carcinogenic associated with coal combustion. Transcriptomic-based analysis delineated three molecular clusters, two not present in Western HCC; one with a highly inflamed profile and the other significantly associated with younger female patients. CONCLUSIONS: Mongolian HCC has unique molecular traits with a high mutational burden and a novel mutational signature associated with genotoxic environmental factors present in this country.

Control of occult hepatitis B virus infection.

Background: The diagnosis of hepatitis B virus (HBV) infection requires HBV DNA testing and serologic testing for detection of the surface antigen (HBsAg) and the hepatitis B core antibody (anti-HBc). There is a population of patients with occult HBV infection (OBI), which is not detected by HBsAg or HBV DNA quantification in blood, despite the presence of active replication in the liver. Scope: This document provides a definition of OBI and describes the diagnostic techniques currently used. It also addresses the detection of patients with risk factors and the need for screening for OBI in these patients. Summary: Correct diagnosis of OBI prevents HBV reactivation and transmission. Diagnosis of OBI is based on the detection of HBV DNA in patients with undetectable HBsAg in blood. Perspectives: A high number of patients with OBI may remain undiagnosed; therefore, screening for OBI in patients with factor risks is essential. For a correct diagnosis of OBI, it is necessary that new markers such as ultrasensitive HBsAg are incorporated, and a more comprehensive marker study is performed by including markers such as cccDNA.

Epigenetic priming in chronic liver disease impacts the transcriptional and genetic landscapes of hepatocellular carcinoma.

Hepatocellular carcinomas (HCCs) usually arise from chronic liver disease (CLD). Precancerous cells in chronically inflamed environments may be 'epigenetically primed', sensitising them to oncogenic transformation. We investigated whether epigenetic priming in CLD may affect HCC outcomes by influencing the genomic and transcriptomic landscapes of HCC. Analysis of DNA methylation arrays from 10 paired CLD-HCC identified 339 shared dysregulated CpG sites and 18 shared differentially methylated regions compared with healthy livers. These regions were associated with dysregulated expression of genes with relevance in HCC, including ubiquitin D (UBD), cytochrome P450 family 2 subfamily C member 19 (CYP2C19) and O-6-methylguanine-DNA methyltransferase (MGMT). Methylation changes were recapitulated in an independent cohort of nine paired CLD-HCC. High CLD methylation score, defined using the 124 dysregulated CpGs in CLD and HCC in both cohorts, was associated with poor survival, increased somatic genetic alterations and TP53 mutations in two independent HCC cohorts. Oncogenic transcriptional and methylation dysregulation is evident in CLD and compounded in HCC. Epigenetic priming in CLD sculpts the transcriptional landscape of HCC and creates an environment favouring the acquisition of genetic alterations, suggesting that the extent of epigenetic priming in CLD could influence disease outcome.

Modeling hepatitis C virus kinetics during liver transplantation reveals the role of the liver in virus clearance.

While the liver, specifically hepatocytes, are widely accepted as the main source of hepatitis C virus (HCV) production, the role of the liver/hepatocytes in clearance of circulating HCV remains unknown. Frequent HCV kinetic data were recorded and mathematically modeled from five liver transplant patients throughout the anhepatic (absence of liver) phase and for 4 hr post-reperfusion. During the anhepatic phase, HCV remained at pre-anhepatic levels (n = 3) or declined (n = 2) with t1/2~1 hr. Immediately post-reperfusion, virus declined in a biphasic manner in four patients consisting of a rapid decline (t1/2 = 5 min) followed by a slower decline (t1/2 = 67 min). Consistent with the majority of patients in the anhepatic phase, when we monitored HCV clearance at 37°C from culture medium in the absence/presence of chronically infected hepatoma cells that were inhibited from secreting HCV, the HCV t1/2 in cell culture was longer in the absence of chronically HCV-infected cells. The results suggest that the liver plays a major role in the clearance of circulating HCV and that hepatocytes may be involved.

Aberrant integration of Hepatitis B virus DNA promotes major restructuring of human hepatocellular carcinoma genome architecture.

Most cancers are characterized by the somatic acquisition of genomic rearrangements during tumour evolution that eventually drive the oncogenesis. Here, using multiplatform sequencing technologies, we identify and characterize a remarkable mutational mechanism in human hepatocellular carcinoma caused by Hepatitis B virus, by which DNA molecules from the virus are inserted into the tumour genome causing dramatic changes in its configuration, including non-homologous chromosomal fusions, dicentric chromosomes and megabase-size telomeric deletions. This aberrant mutational mechanism, present in at least 8% of all HCC tumours, can provide the driver rearrangements that a cancer clone requires to survive and grow, including loss of relevant tumour suppressor genes. Most of these events are clonal and occur early during liver cancer evolution. Real-time timing estimation reveals some HBV-mediated rearrangements occur as early as two decades before cancer diagnosis. Overall, these data underscore the importance of characterising liver cancer genomes for patterns of HBV integration.

Cell Death in Hepatocellular Carcinoma: Pathogenesis and Therapeutic Opportunities.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and the third leading cause of cancer death worldwide. Closely associated with liver inflammation and fibrosis, hepatocyte cell death is a common trigger for acute and chronic liver disease arising from different etiologies, including viral hepatitis, alcohol abuse, and fatty liver. In this review, we discuss the contribution of different types of cell death, including apoptosis, necroptosis, pyroptosis, or autophagy, to the progression of liver disease and the development of HCC. Interestingly, inflammasomes have recently emerged as pivotal innate sensors with a highly pathogenic role in various liver diseases. In this regard, an increased inflammatory response would act as a key element promoting a pro-oncogenic microenvironment that may result not only in tumor growth, but also in the formation of a premetastatic niche. Importantly, nonparenchymal hepatic cells, such as liver sinusoidal endothelial cells, hepatic stellate cells, and hepatic macrophages, play an important role in establishing the tumor microenvironment, stimulating tumorigenesis by paracrine communication through cytokines and/or angiocrine factors. Finally, we update the potential therapeutic options to inhibit tumorigenesis, and we propose different mechanisms to consider in the tumor microenvironment field for HCC resolution.

Viral and immune factors associated with successful treatment withdrawal in HBeAg-negative chronic hepatitis B patients.

BACKGROUND & AIMS: Factors associated with a successful outcome upon nucleos(t)ide analogue (NA) treatment withdrawal in HBeAg-negative chronic hepatitis B (CHB) patients have yet to be clarified. The objective of this study was to analyse the HBV-specific T cell response, in parallel with peripheral and intrahepatic viral parameters, in patients undergoing NA discontinuation. METHODS: Twenty-seven patients without cirrhosis with HBeAg-negative CHB with complete viral suppression (>3 years) were studied prospectively. Intrahepatic HBV-DNA (iHBV-DNA), intrahepatic HBV-RNA (iHBV-RNA), and covalently closed circular DNA (cccDNA) were quantified at baseline. Additionally, serum markers (HBV-DNA, HBsAg, HBV core-related antigen [HBcrAg] and HBV-RNA) and HBV-specific T cell responses were analysed at baseline and longitudinally throughout follow-up. RESULTS: After a median follow-up of 34 months, 22/27 patients (82%) remained off-therapy, of whom 8 patients (30% of the total cohort) lost HBsAg. Baseline HBsAg significantly correlated with iHBV-DNA and iHBV-RNA, and these parameters were lower in patients who lost HBsAg. All patients had similar levels of detectable cccDNA regardless of their clinical outcome. Patients achieving functional cure had baseline HBsAg levels ≤1,000 IU/ml. Similarly, an increased frequency of functional HBV-specific CD8+ T cells at baseline was associated with sustained viral control off treatment. These HBV-specific T cell responses persisted, but did not increase, after treatment withdrawal. A similar, but not statistically significant trend, was observed for HBV-specific CD4+ T cell responses. CONCLUSIONS: Decreased cccDNA transcription and low HBsAg levels are associated with HBsAg loss upon NA discontinuation in patients with HBeAg-negative CHB. The presence of functional HBV-specific T cells at baseline are associated with a successful outcome after treatment withdrawal. LAY SUMMARY: Nucleos(t)ide analogue therapy can be discontinued in a high proportion of chronic hepatitis B patients without cirrhosis. The strength of HBV-specific immune T cell responses may contribute to successful viral control after antiviral treatment interruption. Our comprehensive study provides in-depth data on virological and immunological factors than can help guide individualised therapy in patients with chronic hepatitis B.

Chronic genotype 1 hepatitis C along with cirrhosis drives a persistent imprint in virus-specific CD8+ T cells after direct-acting antiviral therapies.

Chronic hepatitis C virus (HCV) infection impairs HCV CD8+ T-cell responses, while it could influence immune responses towards unrelated viruses/vaccines (e.g. cytomegalovirus, CMV, and influenza, Flu). The aim of our study was to delineate whether restoration of these virus-specific CD8+ T cells occurs after direct-acting antiviral (DAA) therapies and particularly in patients with cirrhosis. We performed longitudinal analysis (baseline, week 4, follow-up [FU] 12 and FU48) of virus-specific CD8+ T cells by multicolour flow cytometry in HCV-cirrhotic patients undergoing DAA therapy (n = 26) after in vitro expansion with immunodominant HCV, CMV and Flu epitopes restricted by HLA-A*02. HCV noncirrhotic patients (n = 9) and healthy individuals (n = 10) served as controls. We found that the proliferative capacity of HCV-specific CD8+ T cells increased from baseline up to FU48 in a significant proportion of cirrhotic and noncirrhotic patients. Nevertheless, these cells remained poor cytokine producers in both patient groups, regardless of the down-regulation of inhibitory co-regulatory receptors in HCV-cirrhotic patients at FU48. Likewise, high expression levels of these exhaustion markers were detected in CMV-/Flu-specific CD8+ T cells in HCV-cirrhotic patients at all time points, albeit without affecting their proliferative capacity or cytokine production. We conclude that DAA therapies induce restoration of the proliferative capacity of HCV-specific CD8+ T cells. However, these cells remain phenotypically and functionally impaired. Contrarily, the 'exhausted' phenotype in CMV-/Flu-specific CD8+ T cells in HCV-cirrhotic patients did not associate with their functions. Larger studier with longer follow-up may elucidate whether this complex interplay influences the outcome of cirrhotic patients.

Cirrhosis Hampers Early and Rapid Normalization of Natural Killer Cell Phenotype and Function in Hepatitis C Patients Undergoing Interferon-Free Therapy.

Background: Chronic hepatitis C virus (HCV) infection impairs natural killer (NK) cell phenotype and function. Whether restoration of NK cells occurs after successful interferon (IFN)-free therapies remains a controversial issue. Aim: To analyze how HCV-related liver cirrhosis impacts changes in NK cells prior and post-IFN-free therapies. Methods: NK cell analysis by multicolor flow cytometry was performed in HCV-infected patients with (n = 17) and without (n = 14) cirrhosis at baseline, week 4 during therapy, and weeks 12 and 48 after the end of therapy (FU12 and FU48, respectively). Non-HCV cirrhotic patients (n = 12) and healthy individuals (n = 12) served as controls. Results: At baseline, HCV cirrhotic patients presented an altered distribution of NK subsets (CD56dim and CD56bright) with higher expression of NKp46, HLA-DR, NKp30, KIR2DL2/L3, NKG2A, and CD85j receptors compared to healthy controls. All frequencies normalized by FU48, except for CD85j+ cells. Likewise, substantial alterations were detected in NK cell function assessed by (i) signal transducer and activator of transcription 1 (STAT1) and phosphorylated levels of STAT1 and STAT4, (ii) degranulation (CD107a), (iii) cytotoxicity [tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)], and (iv) cytokine production [IFN-γ and tumor necrosis factor-α (TNF-α)]. Of note, NK cell function at FU48 remained partially impaired. In contrast, non-cirrhotics showed normal baseline frequencies of HLA-DR-, NKG2A-, and CD85j-expressing NK cells. Importantly, altered baseline frequencies of NK cell subsets and NKp46+ CD56dim cells, as well as NK cell function, were rapidly and completely restored. Conclusions: NK cell phenotype alterations persist after HCV eradication in cirrhotic patients, while their function is only partially restored, compromising immune restoration and immunosurveillance.

The challenge of chronic hepatitis E in liver transplant recipients: Failure of sofosbuvir plus ribavirin therapy / Problema de la hepatitis E crónica en los receptores de trasplante de hígado: fracaso del tratamiento con sofosbuvir más ribavirina

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Deep-sequencing reveals broad subtype-specific HCV resistance mutations associated with treatment failure.

A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) α-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of "extra-target" RAS suggests the need for RAS screening in all three DAA target regions.

Low seroprevalence and zero incidence rate of hepatitis E in men who have sex with men during a hepatitis A outbreak.

Hepatitis E virus (HEV) and hepatitis A virus (HAV) are both secreted in feces. Despite HEV transmission in Europe is mainly zoonotic, person-to-person transmission has not been completely excluded. Men who have sex with men (MSM) constitute a high-risk group for HAV mostly due to oral sex. We investigated the potential transmission of HEV during an acute hepatitis A (AHA) outbreak mainly affecting MSM. One hundred and two patients were diagnosed with AHA. Sixty-nine (68%) self-reported to be MSM, 75% of whom had high-risk sexual behaviors and 46% had suffered previous sexually transmitted diseases. We collected serum from 85 (83%) patients during AHA. HEV-IgG seroprevalence was not different among MSM (7%) compared with non-MSM (8%) patients. Two patients had positive anti-HEV-IgM, but all samples tested negative for HEV-RNA. These results suggest that HEV does not spread by sexual contact or person-to-person in our area.

Barreras para la eliminación del virus de la hepatitis C en el colectivo de hombres que tienen relaciones sexuales con hombres infectados por el virus de la inmunodeficiencia humana: el elevado riesgo de reinfección. A propósito de un paciente con 6 episodios / Barriers to hepatitis C virus elimination in MSM living with HIV: The high risk of reinfection. Case study: A patient with 6 episodes

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Hepatitis C virus intrinsic molecular determinants may contribute to the development of cholestatic hepatitis after liver transplantation.

Cholestatic hepatitis C (CHC) is a severe form of hepatitis C virus (HCV) infection recurrence that leads to high graft loss rates early after liver transplantation (LT). To investigate the pathogenic mechanisms of CHC, we analysed HCV quasispecies in CHC patients compared to a control group (mild hepatitis C recurrence) by deep pyrosequencing. At the time of LT, NS5B quasispecies complexity was similar between the two groups but, after LT, it decreased more sharply in CHC patients than in the control group. Interestingly, the major variant before LT propagated efficiently and remained as the dominant sequence after LT in 62 % of CHC patients versus 11 % of controls (P=0.031). Sequence analysis of the complete non-structural region in a limited number of patients revealed a potential 12 aa signature specific to the CHC group. These data suggest that intrinsic molecular determinants in the circulating HCV quasispecies may provide a fitness advantage, contributing to the development of CHC.

Torque Teno Virus Is Associated With the State of Immune Suppression Early After Liver Transplantation.

The development of noninvasive biomarkers that reflect the state of immunosuppression (IS) remains an unmet need in liver transplantation (LT). Torque Teno virus (TTV) is a highly prevalent, nonpathogenic DNA virus whose plasma levels may be associated with the immune status of the host. The aim of this study was to assess the role of TTV as a biomarker of IS in LT recipients. TTV DNA in plasma was quantified by real-time polymerase chain reaction at different time points during the first year after transplant in a prospectively followed cohort of 63 de novo LT recipients, and any correlation between TTV DNA and biopsy-proven acute cellular rejection (ACR) and opportunistic infections was then evaluated. In addition, TTV DNA was studied in 10 longterm LT recipients in monotherapy with tacrolimus, 10 tolerant recipients, and 10 healthy controls. TTV was detected in the plasma of all patients. Among the 63 LT recipients, 20 episodes of ACR were diagnosed, and there were 28 opportunistic infections, 26 of them being cytomegalovirus (CMV) infections. TTV viremia was significantly lower during ACR (4.41 versus 5.95 log10 copies/mL; P = 0.002) and significantly higher during CMV infections (5.79 versus 6.59 log10 copies/mL; P = 0.009). The area under the receiver operating characteristic curve of TTV viral load for the diagnosis of moderate ACR was 0.869, with a sensitivity and negative predictive value of 100%, respectively, for a cutoff point of 4.75 log10 copies/mL. There were no statistically significant differences in TTV DNA in either longterm or tolerant patients and healthy controls. In conclusion, plasma TTV DNA levels are associated with immune-related events after LT and could constitute a potential biomarker of the state of IS during the first months after transplant.

Host phosphatidic acid phosphatase lipin1 is rate limiting for functional hepatitis C virus replicase complex formation.

Hepatitis C virus (HCV) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. HCV replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. We focused our attention on a phosphatidate phosphate (PAP) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. Lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. The best-characterized member of this family is lipin1, which cooperates with lipin2 to maintain glycerophospholipid homeostasis in the liver. Lipin1-deficient cell lines were generated by RNAi to study the role of this protein in different steps of HCV replication cycle. Using surrogate models that recapitulate different aspects of HCV infection, we concluded that lipin1 is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early HCV RNA replication. Infection studies in lipin1-deficient cells overexpressing wild type or phosphatase-defective lipin1 proteins suggest that lipin1 phosphatase activity is required to support HCV infection. Finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin1 may facilitate the generation of the membranous compartment that contains functional HCV replicase complexes.