N. Hansi

CXCR6 marks a novel subset of T-bet lo Eomes hi natural killer cells residing in human liver

Natural killer cells (NK) are highly enriched in the human liver, where they can regulate immunity and immunopathology. We probed them for a liver-resident subset, distinct from conventional bone-marrow-derived NK. CXCR6+ NK were strikingly enriched in healthy and diseased liver compared to blood (pu2009<u20090.0001). Human hepatic CXCR6+ NK had an immature phenotype (predominantly CD56brightCD16−CD57−), and expressed the tissue-residency marker CD69. CXCR6+ NK produced fewer cytotoxic mediators and pro-inflammatory cytokines than the non-liver-specific CXCR6− fraction. Instead CXCR6+ NK could upregulate TRAIL, a key death ligand in hepatitis pathogenesis. CXCR6 demarcated liver NK into two transcriptionally distinct populations: T-bethiEomeslo(CXCR6−) and T-betloEomeshi(CXCR6+); the latter was virtually absent in the periphery. The small circulating CXCR6+ subset was predominantly T-bethiEomeslo, suggesting its lineage was closer to CXCR6− peripheral than CXCR6+ liver NK. These data reveal a large subset of human liver-resident T-betloEomeshi NK, distinguished by their surface expression of CXCR6, adapted for hepatic tolerance and inducible anti-viral immunity.

IL-2high tissue-resident T cells in the human liver: Sentinels for hepatotropic infection

The liver provides a tolerogenic immune niche exploited by several highly prevalent pathogens as well as by primary and metastatic tumors. We have sampled healthy and hepatitis B virus (HBV)–infected human livers to probe for a subset of T cells specialized to overcome local constraints and mediate immunity. We characterize a population of T-betloEomesloBlimp-1hiHobitlo T cells found within the intrahepatic but not the circulating memory CD8 T cell pool expressing liver-homing/retention markers (CD69+CD103+ CXCR6+CXCR3+). These tissue-resident memory T cells (TRM) are preferentially expanded in patients with partial immune control of HBV infection and can remain in the liver after the resolution of infection, including compartmentalized responses against epitopes within all major HBV proteins. Sequential IL-15 or antigen exposure followed by TGF&bgr; induces liver-adapted TRM, including their signature high expression of exhaustion markers PD-1 and CD39. We suggest that these inhibitory molecules, together with paradoxically robust, rapid, cell-autonomous IL-2 and IFN&ggr; production, equip liver CD8 TRM to survive while exerting local noncytolytic hepatic immunosurveillance.

Distinct Metabolic Requirements of Exhausted and Functional Virus-Specific CD8 T Cells in the Same Host

Summary T cells undergo profound metabolic changes to meet the increased energy demands of maintaining an antiviral response. We postulated that differences in metabolic reprogramming would shape the efficacy of CD8 T cells mounted against persistent viral infections. We found that the poorly functional PD-1hi T cell response against hepatitis B virus (HBV) had upregulated the glucose transporter, Glut1, an effect recapitulated by oxygen deprivation to mimic the intrahepatic environment. Glut1hi HBV-specific T cells were dependent on glucose supplies, unlike the more functional cytomegalovirus (CMV)-specific T cells that could utilize oxidative phosphorylation in the absence of glucose. The inability of HBV-specific T cells to switch to oxidative phosphorylation was accompanied by increased mitochondrial size and lower mitochondrial potential, indicative of mitochondrial dysfunction. Interleukin (IL)-12, which recovers HBV-specific T cell effector function, increased their mitochondrial potential and reduced their dependence on glycolysis. Our findings suggest that mitochondrial defects limit the metabolic plasticity of exhausted HBV-specific T cells.

Hepatitis B virus–specific T cells associate with viral control upon nucleos(t)ide-analogue therapy discontinuation

BACKGROUND. The clinical management of chronic hepatitis B virus (HBV) patients is based exclusively on virological parameters that cannot independently determine in which patients nucleos(t)ide-analogue (NUC) therapy can be safely discontinued. NUCs efficiently suppress viral replication, but do not eliminate HBV. Thus, therapy discontinuation can be associated with virological and biochemical relapse and, consequently, therapy in the majority is life-long. METHODS. Since antiviral immunity is pivotal for HBV control, we investigated potential biomarkers for the safe discontinuation of NUCs within immune profiles of chronic HBV patients by utilizing traditional immunological assays (ELISPOT, flow cytometry) in conjunction with analyses of global non–antigen-specific immune populations (NanoString and CyTOF). Two distinct cohorts of 19 and 27 chronic HBV patients, respectively, were analyzed longitudinally prior to and after discontinuation of 2 different NUC therapy strategies. RESULTS. Absence of hepatic flares following discontinuation of NUC treatment correlated with the presence, during NUC viral suppression, of HBV core and polymerase-specific T cells that were contained within the ex vivo PD-1+ population. CONCLUSIONS. This study identifies the presence of functional HBV-specific T cells as a candidate immunological biomarker for safe therapy discontinuation in chronic HBV patients. Furthermore, the persistent and functional antiviral activity of PD-1+ HBV–specific T cells highlights the potential beneficial role of the expression of T cell exhaustion markers during human chronic viral infection. FUNDING. This work was funded by a Singapore Translational Research Investigator Award (NMRC/STaR/013/2012), the Eradication of HBV TCR Program (NMRC/TCR/014-NUHS/2015), the Singapore Immunology Network, the Wellcome Trust (107389/Z/15/Z), and a Barts and The London Charity (723/1795) grant.

Molecular Recalibration of PD-1+ Antigen-Specific T Cells from Blood and Liver

Checkpoint inhibitors and adoptive cell therapy provide promising options for treating solid cancers such as HBV-related HCC, but they have limitations. We tested the potential to combine advantages of each approach, genetically reprogramming T cells specific for viral tumor antigens to overcome exhaustion by down-modulating the co-inhibitory receptor PD-1. We developed a novel lentiviral transduction protocol to achieve preferential targeting of endogenous or TCR-redirected, antigen-specific CD8 T cells for shRNA knockdown of PD-1 and tested functional consequences for antitumor immunity. Antigen-specific and intrahepatic CD8 T cells transduced with lentiviral (LV)-shPD-1 consistently had a marked reduction in PD-1 compared to those transduced with a control lentiviral vector. PD-1 knockdown of human T cells rescued antitumor effector function and promoted killing of hepatoma cells in a 3D microdevice recapitulating the pro-inflammatory PD-L1hi liver microenvironment. However, upon repetitive stimulation, PD-1 knockdown drove T cell senescence and induction of other co-inhibitory pathways. We provide the proof of principle that T cells with endogenous or genetically engineered specificity for HBV-associated HCC viral antigens can be targeted for functional genetic editing. We show that PD-1 knockdown enhances immediate tumor killing but is limited by compensatory engagement of alternative co-inhibitory and senescence program upon repetitive stimulation.

Chronic Hepatitis B

A 45-year-old male Chinese university lecturer presented to his GP with malaise and dark urine. He was referred to the local infectious diseases clinic, and blood tests were as follows:

PTU-103 Characterisation of the immune profile in chronic hepatitis b patients following nucleos(t)ide discontinuation by cytof mass cytometry

Introduction Identifying chronic Hepatitis B virus (HBV) patients in whom nucleos (t)ide analogues (NUCs) can be safely discontinued with sustained immune control is a major unmet need of current treatment strategies. Predicting the onset of biochemical relapse or hepatic flares (HF) following NUC discontinuation is currently not possible. Through the utilisation of cytometry time of flight (CyTOF), a novel mass cytometry technology, in conjunction with traditional flow cytometry tools, the aim of this study was to establish an immune profile associated with the onset of HF upon withdrawal of NUC therapy. Method Cytokine and chemokine serum levels (IL-1β, IL-6, TNF-α, IL-10, CXCL-8, CXCL-10) were measured longitudinally using Luminex in chronic HBV patients who developed HF (n = 6) and in those who did not (n = 2) upon the discontinuation of NUCs. HBV-specific T cell responses were assessed after in vitro expansion of patient peripheral blood mononuclear cells with overlapping HBV peptide pools. An in-depth longitudinal analysis of the expression of 35 markers involved in the activation, differentiation, exhaustion and anti-viral effector function of T and NK cells was performed by CyTOF mass cytometry. Results The analysis of HBV-specific T cells using overlapping HBV peptides, shows that in patients with biochemical relapse after therapy withdrawal, there was no detection of functional antigen-specific T cells at all time points. The onset of HFs was, however, associated with a significant increase in the serum levels of CXCL-10 and IL-10. In contrast, a chronic HBV patient that controlled viral relapse without experiencing HF displayed functional circulating HBV-specific T cells. Preliminary analyses by CyTOF also revealed significant differences in terms of the frequency and functionality of the bulk of the T and NK cell populations both longitudinally (during and after discontinuation of therapy), as well as amongst patients characterised by relapse or immune control. Conclusion Consistent with previous studies, HFs are associated with an increase in the serum levels of CXCL10 and IL-10 while HBV-specific T cells remain undetectable in the periphery. The absence of circulating virus-specific CD8+T cells during HFs suggests that hepatic inflammation after NUC withdrawal in chronic HBV patients is induced independent of virus-specific CD8+T cells. CyTOF technology represents a major advance, allowing us to investigate multiple parameters, providing a unique insight into potential biomarkers associated with sustained immune control of HBV. Disclosure of interest None Declared.

PTU-107 Defining the low-risk inactive carrier in chronic hepatitis b with qhbsag: do the same rules apply in children and young adults?

Introduction A substantial proportion of e-Antigen negative chronic hepatitis B (CHB) under specialist follow-up are inactive carriers (IC) defined as those with low serum ALT and HBV DNA. Recent studies have described the potential utility of quantitative Hepatitis B surface antigen (qHBsAg) (<1,000 IU/ml) to represent a low-risk IC state, which is associated with reduced risk for disease progression and the development of HCC. Conversely higher qHBsAg levels (>1,000 IU/ml) are associated with an increase in risk of disease progression.1Accurate identification of low-risk IC’s would reduce the frequency of follow-up and the need for HCC screening in selected patients. We investigated whether a qHBsAg threshold (<1,000 IU/ml) representing a low-risk IC profile can be applied to children and young adults with CHB. Method Fifty-six consecutive treatment naïve eAg negative young patients (<30 years) considered IC’s (ALT <40, HBV DNA <2,000 IU/ml) over longitudinal follow-up were analysed; female = 38, median age = 26 years (range 12–30 years). A cohort of older patients (>30 years) based on the same parameters were included for comparison; female = 33%, median age = 42 years (range 31–61 years). HBsAg levels were quantified in all patients to determine any correlation between the qHBsAg threshold (<1,000 IU/ml) and age. Results In keeping with an IC disease profile, serum ALT and HBV DNA levels in both cohorts were similar (p = ns), however, qHBsAg levels were significantly higher in the <30s vs. >30s; mean qHBsAg 10,545 IU/ml vs. 5,278 IU/ml (p = 0.006). In accordance with this we detected a significant negative correlation with age and qHBsAg (Spearman Rho rs = -0.35, p = 0.0004). A significantly higher proportion of older patients had qHBsAg <1,000 IU/ml, (36% vs. 13%, p = <0.001), consistent with low-risk IC state. Conclusion The addition of qHBsAg to serum ALT and HBV DNA can enhance risk stratification. However, a threshold level (<1,000 IU/ml) cannot be safely utilised in young patients to define a low-risk IC state. The natural decline in qHBsAg with advancing age underlines the need for further studies to define an age limit above which this qHBsAg level (<1,000 IU/ml) can be used to identify low-risk ICs and streamline clinical monitoring. Disclosure of interest None Declared. Reference Tseng TC, Liu CJ, Yang HC, et al. High levels of hepatitis B surface antigen increase risk of hepatocellular carcinoma in patients with low HBV load. Gastroenterology 2012;142:1140–1149