Therapeutic potential of co-signaling receptor modulation in hepatitis B.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

Phenotypic CD8 T cell profiling in chronic hepatitis B to predict HBV-specific CD8 T cell susceptibility to functional restoration in vitro.

OBJECTIVE: Exhausted hepatitis B virus (HBV)-specific CD8 T cells in chronic HBV infection are broadly heterogeneous. Characterisation of their functional impairment may allow to distinguish patients with different capacity to control infection and reconstitute antiviral function. DESIGN: HBV dextramer+CD8 T cells were analysed ex vivo for coexpression of checkpoint/differentiation markers, transcription factors and cytokines in 35 patients with HLA-A2+chronic hepatitis B (CHB) and in 29 control HBsAg negative CHB patients who seroconverted after NUC treatment or spontaneously. Cytokine production was also evaluated in HBV peptide-stimulated T cell cultures, in the presence or absence of antioxidant, polyphenolic, PD-1/PD-L1 inhibitor and TLR-8 agonist compounds and the effect on HBV-specific responses was further validated on additional 24 HLA-A2 negative CHB patients. RESULTS: Severely exhausted HBV-specific CD8 T cell subsets with high expression of inhibitory receptors, such as PD-1, TOX and CD39, were detected only in a subgroup of chronic viraemic patients. Conversely, a large predominance of functionally more efficient HBV-specific CD8 T cell subsets with lower expression of coinhibitory molecules and better response to in vitro immune modulation, typically detected after resolution of infection, was also observed in a proportion of chronic viraemic HBV patients. Importantly, the same subset of patients who responded more efficiently to in vitro immune modulation identified by HBV-specific CD8 T cell analysis were also identified by staining total CD8 T cells with PD-1, TOX, CD127 and Bcl-2. CONCLUSIONS: The possibility to distinguish patient cohorts with different capacity to respond to immune modulatory compounds in vitro by a simple analysis of the phenotypic CD8 T cell exhaustion profile deserves evaluation of its clinical applicability.

Deregulated intracellular pathways define novel molecular targets for HBV-specific CD8 T cell reconstitution in chronic hepatitis B.

BACKGROUND & AIMS: In chronic HBV infection, elevated reactive oxygen species levels derived from dysfunctional mitochondria can cause increased protein oxidation and DNA damage in exhausted virus-specific CD8 T cells. The aim of this study was to understand how these defects are mechanistically interconnected to further elucidate T cell exhaustion pathogenesis and, doing so, to devise novel T cell-based therapies. METHODS: DNA damage and repair mechanisms, including parylation, CD38 expression, and telomere length were studied in HBV-specific CD8 T cells from chronic HBV patients. Correction of intracellular signalling alterations and improvement of antiviral T cell functions by the NAD precursor nicotinamide mononucleotide and by CD38 inhibition was assessed. RESULTS: Elevated DNA damage was associated with defective DNA repair processes, including NAD-dependent parylation, in HBV-specific CD8 cells of chronic HBV patients. NAD depletion was indicated by the overexpression of CD38, the major NAD consumer, and by the significant improvement of DNA repair mechanisms, and mitochondrial and proteostasis functions by NAD supplementation, which could also improve the HBV-specific antiviral CD8 T cell function. CONCLUSIONS: Our study delineates a model of CD8 T cell exhaustion whereby multiple interconnected intracellular defects, including telomere shortening, are causally related to NAD depletion suggesting similarities between T cell exhaustion and cell senescence. Correction of these deregulated intracellular functions by NAD supplementation can also restore antiviral CD8 T cell activity and thus represents a promising potential therapeutic strategy for chronic HBV infection. IMPACT AND IMPLICATIONS: Correction of HBV-specific CD8 T cell dysfunction is believed to represent a rational strategy to cure chronic HBV infection, which however requires a deep understanding of HBV immune pathogenesis to identify the most important targets for functional T cell reconstitution strategies. This study identifies a central role played by NAD depletion in the intracellular vicious circle that maintains CD8 T cell exhaustion, showing that its replenishment can correct impaired intracellular mechanisms and reconstitute efficient antiviral CD8 T cell function, with implications for the design of novel immune anti-HBV therapies. As these intracellular defects are likely shared with other chronic virus infections where CD8 exhaustion can affect virus clearance, these results can likely also be of pathogenetic relevance for other infection models.

Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B.

BACKGROUND & AIMS: In chronic HBV infection, mitochondrial functions and proteostasis are dysregulated in exhausted HBV-specific CD8 T cells. To better characterise the potential involvement of deregulated protein degradation mechanisms in T cell exhaustion, we analysed lysosome-mediated autophagy in HBV-specific CD8 T cells. Bioactive compounds able to simultaneously target both mitochondrial functions and proteostasis were tested to identify optimal combination strategies to reconstitute efficient antiviral CD8 T cell responses in patients with chronic HBV infection. METHODS: Lysosome-mediated degradation pathways were analysed by flow cytometry in virus-specific CD8 T cells from patients with chronic HBV infection. Mitochondrial function, intracellular proteostasis, and cytokine production were evaluated in HBV-peptide-stimulated T cell cultures, in the presence or absence of the polyphenols resveratrol (RSV) and oleuropein (OLE) and their metabolites, either alone or in combination with other bioactive compounds. RESULTS: HBV-specific CD8 T cells from patients with CHB showed impaired autophagic flux. RSV and OLE elicited a significant improvement in mitochondrial, proteostasis and antiviral functions in CD8 T cells. Cytokine production was also enhanced by synthetic metabolites, which correspond to those generated by RSV and OLE metabolism in vivo, suggesting that these polyphenols may also display an effect after transformation in vivo. Moreover, polyphenolic compounds improved the T cell revitalising effect of mitochondria-targeted antioxidants and of programmed cell death protein 1/programmed cell death ligand 1 blockade. CONCLUSIONS: Simultaneously targeting multiple altered intracellular pathways with the combination of mitochondria-targeted antioxidants and natural polyphenols may represent a promising immune reconstitution strategy for the treatment of chronic HBV infection. LAY SUMMARY: In chronic hepatitis B, antiviral T lymphocytes are deeply impaired, with many altered intracellular functions. In vitro exposure to polyphenols, such as resveratrol and oleuropein, can correct some of the deregulated intracellular pathways and improve antiviral T cell function. This effect can be further strengthened by the association of polyphenols with antioxidant compounds in a significant proportion of patients. Thus, the combination of antioxidants and natural polyphenols represents a promising strategy for chronic hepatitis B therapy.

The Good and the Bad of Natural Killer Cells in Virus Control: Perspective for Anti-HBV Therapy.

Immune modulatory therapies are widely believed to represent potential therapeutic strategies for chronic hepatitis B infection (CHB). Among the cellular targets for immune interventions, Natural Killer (NK) cells represent possible candidates because they have a key role in anti-viral control by producing cytokines and by exerting cytotoxic functions against virus-infected cells. However, in patients with chronic hepatitis B, NK cells have been described to be more pathogenic than protective with preserved cytolytic activity but with a poor capacity to produce anti-viral cytokines. In addition, NK cells can exert a regulatory activity and possibly suppress adaptive immune responses in the setting of persistent viral infections. Consequently, a potential drawback of NK-cell targeted modulatory interventions is that they can potentiate the suppressive NK cell effect on virus-specific T cells, which further causes impairment of exhausted anti-viral T cell functions. Thus, clinically useful NK-cell modulatory strategies should be not only suited to improve positive anti-viral NK cell functions but also to abrogate T cell suppression by NK cell-mediated T cell killing. This review outlines the main NK cell features with a particular focus on CHB infection. It describes different mechanisms involved in NK-T cell interplay as well as how NK cells can have positive anti-viral effector functions and negative suppressive effects on T cells activity. This review discusses how modulation of their balance can have potential therapeutic implications.

FluoroSpot assay to analyze SARS-CoV-2-specific T cell responses.

Monitoring antigen-specific T cell frequency and function is essential to assess the host immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we present a FluoroSpot assay for concurrently detecting ex vivo antiviral cytokine production by SARS-CoV-2-specific T cells following peptide stimulation. We then detail intracellular cytokine staining by flow cytometry to further validate the FluoroSpot assay results and define the specific T cell subpopulations. For complete details on the use and execution of this protocol, please refer to Tiezzi et al. (2023).1.

What Is the Current Status of Hepatitis B Virus Viro-Immunology?

The natural history of hepatitis B virus (HBV) infection is closely dependent on the dynamic interplay between the host immune response and viral replication. Spontaneous HBV clearance in acute self-limited infection is the result of an adequate and efficient antiviral immune response. Instead, it is widely recognized that in chronic HBV infection, immunologic dysfunction contributes to viral persistence. Long-lasting exposure to high viral antigens, upregulation of multiple co-inhibitory receptors, dysfunctional intracellular signaling pathways and metabolic alterations, and intrahepatic regulatory mechanisms have been described as features ultimately leading to a hierarchical loss of effector functions up to full T-cell exhaustion.

Natural heteroclitic-like peptides are generated by SARS-CoV-2 mutations.

Humoral immunity is sensitive to evasion by SARS-CoV-2 mutants, but CD8 T cells seem to be more resistant to mutational inactivation. By a systematic analysis of 30 spike variant peptides containing the most relevant VOC and VOI mutations that have accumulated overtime, we show that in vaccinated and convalescent subjects, mutated epitopes can have not only a neutral or inhibitory effect on CD8 T cell recognition but can also enhance or generate de novo CD8 T cell responses. The emergence of these mutated T cell function enhancing epitopes likely reflects an epiphenomenon of SARS-CoV-2 evolution driven by antibody evasion and increased virus transmissibility. In a subset of individuals with weak and narrowly focused CD8 T cell responses selection of these heteroclitic-like epitopes may bear clinical relevance by improving antiviral protection. The functional enhancing effect of these peptides is also worth of consideration for the future development of new generation, more potent COVID-19 vaccines.

Antigen Load and T Cell Function: A Challenging Interaction in HBV Infection.

Current treatment for chronic HBV infection is mainly based on nucleos(t)ide analogues, that in most cases need to be administered for a patient's lifetime. There is therefore a pressing need to develop new therapeutic strategies to shorten antiviral treatments. A severe dysfunction of virus-specific T cell responses contributes to virus persistence; hence, immune-modulation to reconstitute an efficient host antiviral response is considered a potential approach for HBV cure. In this perspective, a detailed understanding of the different causes of T cell exhaustion is essential for the design of successful functional T cell correction strategies. Among many different mechanisms which are widely believed to play a role in T cell dysfunction, persistent T cell exposure to high antigen burden, in particular HBsAg, is expected to influence T cell differentiation and function. Definitive evidence of the possibility to improve anti-viral T cell functions by antigen decline is, however, still lacking. This review aims at recapitulating what we have learned so far on the complex T cell-viral antigen interplay in chronic HBV infection.

Targeting Stress Sensor Kinases in Hepatocellular Carcinoma-Infiltrating Human NK Cells as a Novel Immunotherapeutic Strategy for Liver Cancer.

Natural killer (NK) cells may become functionally exhausted entering hepatocellular carcinoma (HCC), and this has been associated with tumor progression and poor clinical outcome. Hypoxia, low nutrients, immunosuppressive cells, and soluble mediators characterize the intratumor microenvironment responsible for the metabolic deregulation of infiltrating immune cells such as NK cells. HCC-infiltrating NK cells from patients undergoing liver resection for HCC were sorted, and genome-wide transcriptome profiling was performed. We have identified a marked general upregulation of gene expression profile along with metabolic impairment of glycolysis, OXPHOS, and autophagy as well as functional defects of NK cells. Targeting p38 kinase, a stress-responsive mitogen-activated protein kinase, we could positively modify the metabolic profile of NK cells with functional restoration in terms of TNF-α production and cytotoxicity. We found a metabolic and functional derangement of HCC-infiltrating NK cells that is part of the immune defects associated with tumor progression and recurrence. NK cell exhaustion due to the hostile tumor microenvironment may be restored with p38 inhibitors with a selective mechanism that is specific for tumor-infiltrating-not affecting liver-infiltrating-NK cells. These results may represent the basis for the development of a new immunotherapeutic strategy to integrate and improve the available treatments for HCC.

Unraveling the Multifaceted Nature of CD8 T Cell Exhaustion Provides the Molecular Basis for Therapeutic T Cell Reconstitution in Chronic Hepatitis B and C.

In chronic hepatitis B and C virus infections persistently elevated antigen levels drive CD8+ T cells toward a peculiar differentiation state known as T cell exhaustion, which poses crucial constraints to antiviral immunity. Available evidence indicates that T cell exhaustion is associated with a series of metabolic and signaling deregulations and with a very peculiar epigenetic status which all together lead to reduced effector functions. A clear mechanistic network explaining how intracellular metabolic derangements, transcriptional and signaling alterations so far described are interconnected in a comprehensive and unified view of the T cell exhaustion differentiation profile is still lacking. Addressing this issue is of key importance for the development of innovative strategies to boost host immunity in order to achieve viral clearance. This review will discuss the current knowledge in HBV and HCV infections, addressing how innate immunity, metabolic derangements, extensive stress responses and altered epigenetic programs may be targeted to restore functionality and responsiveness of virus-specific CD8 T cells in the context of chronic virus infections.

Pathogenetic Mechanisms of T Cell Dysfunction in Chronic HBV Infection and Related Therapeutic Approaches.

A great effort of research has been devoted in the last few years to developing new anti-HBV therapies of finite duration that also provide effective sustained control of virus replication and antigen production. Among the potential therapeutic strategies, immune-modulation represents a promising option to cure HBV infection and the adaptive immune response is a rational target for novel therapeutic interventions, in consideration of the key role played by T cells in the control of virus infections. HBV-specific T cells are severely dysfunctional in chronic HBV infection as a result of several inhibitory mechanisms which are simultaneously active within the chronically inflamed liver. Indeed, the liver is a tolerogenic organ harboring different non-parenchymal cell populations which can serve as antigen presenting cells (APC) but are poorly efficient in effector T cell priming, with propensity to induce T cell tolerance rather than T cell activation, because of a poor expression of co-stimulatory molecules, up-regulation of the co-inhibitory ligands PD-L1 and PD-L2 upon IFN stimulation, and production of immune regulatory cytokines, such as IL10 and TGF-ß. They include resident dendritic cells (DCs), comprising myeloid and plasmacytoid DCs, liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), hepatic stellate cells (HSCs) as well as the hepatocytes themselves. Additional regulatory mechanisms which contribute to T cell attrition in the chronically infected liver are the high levels of soluble mediators, such as arginase, indoleamine 2,3-dioxygenase (IDO) and suppressive cytokines, the up-regulation of inhibitory checkpoint receptor/ligand pairs, the expansion of regulatory cells, such as CD4+FOXp3+ Treg cells, myeloid-derived suppressor cells and NK cells. This review will deal with the interactions between immune cells and liver environment discussing the different mechanisms which contribute to T cell dysfunction in chronic hepatitis B, some of which are specifically activated in HBV infection and others which are instead common to chronic inflammatory liver diseases in general. Therapeutic interventions targeting dysregulated pathways and cellular functions will be also delineated.