BACKGROUND & AIMS:Virus-specific T-cell dysfunction is a hallmark of chronic hepatitis B (CHB), and its reversal may represent a potential strategy for achieving an HBV cure. A deeper understanding of the T-cell correlates of viral persistence and infection control is needed to clarify the role of CD8 T cells in CHB pathogenesis and to identify novel therapeutic targets for effective T-cell reconstitution. METHODS:Core- and polymerase-specific CD8 T cells from untreated, highly viremic, HBeAg-negative patients with CHB were analyzed by RNA sequencing and compared with those from patients who achieved a functional cure after nucleos(t)ide analogue treatment (rNUC) or acute hepatitis B (rACU). Histone acetylation levels, cytokine production, and cytotoxic activity were evaluated in T cells from patients with CHB following histone deacetylase inhibition. RESULTS:A 101-gene "resolution signature", shared by both resolution groups and distinguishing them from patients with CHB, was identified, along with 41 genes whose dysregulation was not reversed in rNUC patients. Although most dysregulated genes exhibited similar expression patterns in core- and polymerase-specific CD8 T cells from patients with CHB, approximately 30% showed discordant behavior. Targeting aberrant transcriptional regulation with entinostat significantly increased histone acetylation levels and improved T-cell function. CONCLUSIONS:Mechanisms underlying dysfunction in core- and polymerase-specific CD8 T cells are only partially shared between these two subsets, with no clear hierarchy in exhaustion severity between polymerase- and core-specific cells. A chronicity-associated, transcriptional scar-like signature persisted even after HBsAg loss. The resolution signature shared by polymerase- and core-specific CD8 T cells from rNUC and rACU patients identifies histone deacetylase inhibition as a promising immunotherapeutic strategy for CHB. IMPACT AND IMPLICATIONS:There is an urgent need to identify new therapeutic strategies that can achieve durable control of infection in people with chronic hepatitis B (CHB), as impairment of HBV-specific CD8 T cells is a major driver of viral persistence. In this study, transcriptional profiling of virus-specific T cells associated with functional cure identified epigenetic regulation of transcription as a potential target for restoring T-cell function in patients with CHB. In vitro treatment with entinostat significantly increased histone acetylation levels and improved T-cell function, supporting its potential use as an immunotherapeutic strategy to achieve HBV cure.

BackgroundHepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality, with limited curative options for advanced disease. Natural Killer (NK) cells are critical innate immune effectors, but their anti-tumor function is severely compromised by the immunosuppressive tumor immune microenvironment (TIME), particularly through transforming growth factor-beta (TGF-β). This study investigates the pivotal role of TGF-β signaling in modulating NK cell phenotypes and functions within the HCC TIME.Methodsto comprehensively assess TGF-β pathway activation and its impact on NK cells, tumor-infiltrating lymphocytes (TILs) and liver-infiltrating lymphocytes (LILs) were isolated from HCC patients undergoing curative resection. Phenotypic and functional analyses were performed, along with functional restoration experiments targeting TGF-β signaling.ResultsTumor-infiltrating NK cells (TINKs) exhibited significant activation of both canonical (SMAD-dependent) and non-canonical (TAK1/p38 MAPK) TGF-β signaling, with a predominance of the non-canonical pathway. This activation was associated with the emergence of an ILC1-like NK subset (CD103+/CD49a+), which was nearly absent in non-tumor liver tissue. These ILC1-like cells maintained strong cytokine production and expressed high levels of inhibitory receptors (PD-1, TIM-3, TIGIT), whereas conventional NK cells (cNKs; CD103−/CD49a−/CD9−) were functionally impaired. Notably, blocking TGF-β receptor binding and SMAD3 activation restored cNK functionality.Discussionour findings suggest that while non-canonical TGF-β signaling drives phenotypic reprogramming and contributes to NK cell dysfunction, canonical SMAD-dependent signaling remains a key therapeutic target for functional restoration. These results highlight the dual role of TGF-β in immune modulation and suggest that targeted pathway inhibition could enhance innate anti-tumor responses, opening new avenues for combination therapies in HCC.

Background & Aims:In chronic HBV infection, HBV-specific CD8 T cells are dysfunctional and comprise distinct subsets defined by phenotype and antigen specificity. We aimed to characterize the transcriptional and functional features of HBV-specific CD8 T-cell subsets in patients with HBeAg-negative chronic HBV infection who were either viremic (CHB) or had achieved spontaneous or nucleos(t)ide analogue (NUC)-induced HBsAg loss (RES), to better elucidate HBV-specific CD8 T-cell dysfunction and identify potential molecular targets for functional cure. Methods:Gene expression profiles of PD1hiCD127low/- and PD1+CD127+ memory-like (ML) core18-27-specific CD8 T-cell subsets were analyzed by Nanostring, adapted for low-input samples in 5 patients with HBeAg-negative CHB and 6 with RES. An expanded cohort of 23 patients with CHB and 22 with RES was evaluated for phenotypic and functional profiling. Selected deregulated genes were functionally validated in an additional cohort of 14 patients with HLA-A2-negative CHB. Results:Analysis of 84 genes concurrently expressed across all CD8 T-cell subsets identified an 11-gene signature describing a progressive transition from exhaustion-oriented PD1hiCD127low/- CD8 T cells in patients with CHB to memory-oriented ML CD8 cells in patients with RES, representing the two extremes of differentiation. Intermediate stages of memory differentiation were identified among ML CD8 T cells from patients with CHB, with high or low TOX expression (p <0.05 by Spearman's rank correlation). Higher frequencies of TOXlow ML CD8 cells in patients with CHB were associated with a greater serum HBsAg decline during NUC treatment compared to TOXhigh MLCHB (Δ slope p value = 0.003). Targeting selected deregulated genes with specific immune modulators significantly enhanced cytokine production by CD8 T cells, with response rates ranging from 30% to 86% of patients. Conclusions:Distinct exhaustion signatures characterize HBV-specific CD8 T-cell subsets and vary across disease phases. These findings support the development of individualized transcriptional and functional correction strategies and identify novel immune modulators with potential for immune-based anti-HBV therapies. Impact and implications:Exhausted HBV-specific CD8 T cells in chronic HBV infection are not a homogeneous population but comprise distinct subsets with differing capacities to control infection. This study identifies: (i) a transcriptional continuum of HBV-specific CD8 T-cell subsets spanning exhaustion to memory differentiation, reflecting disease progression and recovery in HBeAg-negative CHB; (ii) a core CD8 T-cell exhaustion gene signature characterized by progressively increased expression from memory-oriented to exhaustion-oriented subsets during active and resolution phases of disease; and (iii) the ability of targeted modulation of deregulated genes to restore antiviral CD8 T-cell function, with implications for the development of novel immune-based anti-HBV therapies. Overall, these findings advance our understanding of CD8 T-cell heterogeneity in chronic HBV infection and identify molecular targets for immunomodulatory strategies aimed at restoring CD8 T-cell functionality and achieving functional cure.

ObjectiveSelected populations of patients with chronic hepatitis B (CHB) may benefit from a combined use of pegylated interferon-alpha (pegIFN-α) and nucleos(t)ides (NUCs). The aim of our study was to assess the immunomodulatory effect of pegIFN-α on T and natural killer (NK) cell responses in NUC-suppressed patients to identify cellular and/or serological parameters to predict better T cell-restoring effect and better control of infection in response to pegIFN-α for a tailored application of IFN-α add-on.Design53 HBeAg-negative NUC-treated patients with CHB were randomised at a 1:1 ratio to receive pegIFN-α-2a for 48 weeks, or to continue NUC therapy and then followed up for at least 6 months maintaining NUCs. Serum hepatitis B surface antigen (HBsAg) and hepatitis B core‐related antigen (HBcrAg) levels as well as peripheral blood NK cell phenotype and function and HBV-specific T cell responses upon in vitro stimulation with overlapping HBV peptides were measured longitudinally before, during and after pegIFN-α therapy.ResultsTwo cohorts of pegIFN-α treated patients were identified according to HBsAg decline greater or less than 0.5 log at week 24 post-treatment. PegIFN-α add-on did not significantly improve HBV-specific T cell responses during therapy but elicited a significant multispecific and polyfunctional T cell improvement at week 24 post-pegIFN-α treatment compared with baseline. This improvement was maximal in patients who had a higher drop in serum HBsAg levels and a lower basal HBcrAg values.ConclusionsPegIFN-α treatment can induce greater functional T cell improvement and HBsAg decline in patients with lower baseline HBcrAg levels. Thus, HBcrAg may represent an easily and reliably applicable parameter to select patients who are more likely to achieve better response to pegIFN-α add-on to virally suppressed patients.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a threat to public health and the global economy, necessitating the development of various vaccination strategies. Mutations in the SPIKE protein gene, a crucial component of mRNA and adenovirus-based vaccines, raised concerns about vaccine efficacy, prompting the need for rapid vaccine updates. To address this, we leveraged PeptiCRAd, an oncolytic vaccine based on tumor antigen decorated oncolytic adenoviruses, creating a vaccine platform called PeptiVAX. First, we identified multiple CD8 T-cell epitopes from highly conserved regions across coronaviruses, expanding the range of T-cell responses to non-SPIKE proteins. We designed short segments containing the predicted epitopes presented by common HLA-Is in the global population. Testing the immunogenicity, we characterized T-cell responses to candidate peptides in peripheral blood mononuclear cells (PBMCs) from pre-pandemic healthy donors and ICU patients. As a proof of concept in mice, we selected a peptide with epitopes predicted to bind to murine MHC-I haplotypes. Our technology successfully elicited peptide-specific T-cell responses, unaffected by the use of unarmed adenoviral vectors or adeno-based vaccines encoding SPIKE. In conclusion, PeptiVAX represents a fast and adaptable SARS-CoV-2 vaccine delivery system that broadens T-cell responses beyond the SPIKE protein, offering potential benefits for vaccine effectiveness.

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 de-tecting 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

NK cells infiltrating Hepatocellular Carcinoma (HCC) may express residency markers such as Integrin Subunit Alpha 1 (CD49a) that have been associated with nurturing functions in the decidua, and characterized by the production of angiogenic factors as well as loss of cytotoxicity. CIBERSORT, a computational analysis method for quantifying cell fractions from bulk tissue gene expression profiles, was used to estimate the infiltrating immune cell composition of the tumor microenvironment from gene expression profiles of a large cohort of 225 HCCs in the public GEO database. Decidual-like CD49a+ NK cells, in addition to another 22 immune cell populations, were characterized and thoroughly investigated so that HCC cell heterogeneity in a large cohort of 225 HCCs from the public GEO database could be studied. An inverse correlation of the expression of CD49a+ NK-cells and CD8+ T-cells suggested a negative association with clinical outcomes. This result was confirmed in a further validation cohort of 100 HCC patients from The Cancer Genome Atlas, Liver Hepatocellular Carcinoma (TCGA-LIHC). Cox regression analysis did not identify CD49a+ cells as a variable independently associated with survival. However, a more abundant infiltrate of this subset was present in patients at a more advanced pathological and clinical HCC stage. In conclusion, we found that NK cells, with a decidual-like gene expression profile, are enriched in HCC, and their abundance increases not only in tumor size but also at advanced stages of the disease suggesting that these cells play a role in tumor growth. For this reason, these NK cells may represent a possible new target for immunotherapeutic approaches in HCC.

ObjectiveExhausted 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. DesignHBV 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. ResultsSeverely 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. ConclusionsThe 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.

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.

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.

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.