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OBJECTIVE: Selected 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. DESIGN: 53 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. RESULTS: Two 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. CONCLUSIONS: PegIFN-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.
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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.
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Hepatitis B Crónica , Hepatitis B , Humanos , Virus de la Hepatitis B , Antígenos de Superficie de la Hepatitis B , Replicación ViralRESUMEN
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.
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COVID-19 , Linfocitos T , Humanos , SARS-CoV-2 , CitocinasRESUMEN
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.
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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.
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Hepatitis B Crónica , Hepatitis B , Humanos , NAD/metabolismo , Linfocitos T CD8-positivos , Especies Reactivas de Oxígeno/metabolismo , Antivirales/uso terapéutico , Antivirales/metabolismo , Virus de la Hepatitis B , Hepatitis B/patologíaRESUMEN
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.
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Hepatitis B Crónica , Hepatitis B , Humanos , Hepatitis B Crónica/tratamiento farmacológico , Virus de la Hepatitis B , Antígeno HLA-A2/metabolismo , Antígeno HLA-A2/farmacología , Antígeno HLA-A2/uso terapéutico , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T CD8-positivosRESUMEN
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.
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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.
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Linfocitos T CD8-positivos/metabolismo , Hepatitis B Crónica/inmunología , Hepatitis C Crónica/inmunología , Linfocitos T/inmunología , HumanosRESUMEN
Chronically HBV infected subjects are more than 260 million worldwide; cirrhosis and liver cancer represent possible outcomes which affect around 700,000 patients per year. Both innate and adaptive immune responses are necessary for viral control and both have been shown to be defective in chronic patients. Metabolic remodeling is an essential process in T cell biology, particularly for T cell activation, differentiation and survival. Cellular metabolism relies on the conversion of nutrients into energy to support intracellular processes, and to generate fundamental intermediate components for cell proliferation and growth. Adaptive immune responses are the central mechanisms for the resolution of primary human infections leading to the activation of pathogen-specific B and T cell functions. In chronic HBV infection the anti-viral immune response fails to contain the virus and leads to persistent hepatic tissue damage which may finally result in liver cirrhosis and cancer. This T cell failure is associated with metabolic alterations suggesting that control of nutrient uptake and intracellular utilization as well as correct regulation of intracellular metabolic pathways are strategic for T cell differentiation during persistent chronic infections. This review will discuss some of the main features of the T cell metabolic processes which are relevant to the generation of an efficient antiviral response, with specific focus on their clinical relevance in chronic HBV infection in the perspective of possible strategies to correct deregulated metabolic pathways underlying T cell dysfunction of chronic HBV patients.
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Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Regulación de la Expresión Génica/inmunología , Virus de la Hepatitis B/inmunología , Inmunidad Adaptativa , Animales , Enfermedad Crónica , Regulación de la Expresión Génica/fisiología , Humanos , Hígado/inmunología , Hígado/virología , RatonesRESUMEN
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.
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Linfocitos T CD8-positivos/inmunología , Hepatitis B Crónica , Fitoquímicos/farmacología , Resveratrol/farmacología , Antioxidantes/farmacología , Células Cultivadas , Citocinas/biosíntesis , Virus de la Hepatitis B/patogenicidad , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/patología , Humanos , Factores Inmunológicos , Glucósidos Iridoides/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Mitocondrias Hepáticas/fisiología , Polifenoles/farmacología , Proteolisis/efectos de los fármacos , Deficiencias en la ProteostasisRESUMEN
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.
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Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia Adoptiva/métodos , Activación de Linfocitos , Tratamiento con ARN de Interferencia/métodos , Linfocitos T Reguladores/inmunología , Animales , Citocinas/metabolismo , Antígenos de Superficie de la Hepatitis B/sangre , Hepatitis B Crónica/sangre , Hepatitis B Crónica/virología , Humanos , Células Asesinas Naturales/inmunología , RatonesRESUMEN
Hepatitis C virus infection (HCV) represents a unique model to characterize, from early to late stages of infection, the T cell differentiation process leading to exhaustion of human CD8+ T cells. Here we show that in early HCV infection, exhaustion-committed virus-specific CD8+ T cells display a marked upregulation of transcription associated with impaired glycolytic and mitochondrial functions, that are linked to enhanced ataxia-telangiectasia mutated (ATM) and p53 signaling. After evolution to chronic infection, exhaustion of HCV-specific T cell responses is instead characterized by a broad gene downregulation associated with a wide metabolic and anti-viral function impairment, which can be rescued by histone methyltransferase inhibitors. These results have implications not only for treatment of HCV-positive patients not responding to last-generation antivirals, but also for other chronic pathologies associated with T cell dysfunction, including cancer.
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Linfocitos T CD8-positivos/inmunología , Hepatitis C/inmunología , Histona Metiltransferasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Enfermedad Aguda , Adolescente , Adulto , Anciano , Antivirales/farmacología , Antivirales/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Enfermedad Crónica , Epigénesis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Glucosa/metabolismo , Hepatitis C/sangre , Hepatitis C/genética , Hepatitis C/virología , Humanos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Análisis de Componente Principal , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Adulto JovenRESUMEN
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.
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Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/inmunología , Células Asesinas Naturales/inmunología , Antivirales/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/metabolismo , Virus de la Hepatitis B/inmunología , Humanos , Inmunoterapia , Células Asesinas Naturales/enzimología , Activación de Linfocitos/inmunologíaRESUMEN
Chronic hepatitis B virus (HBV) infection represents a worldwide public health concern with approximately 250 million people chronically infected and at risk of developing liver cirrhosis and hepatocellular carcinoma. Nucleos(t)ide analogues (NUC) are the most widely used therapies for HBV infection, but they often require long-lasting administration to avoid the risk of HBV reactivation at withdrawal. Therefore, there is an urgent need to develop novel treatments to shorten the duration of NUC therapy by accelerating virus control, and to complement the effect of available anti-viral therapies. In chronic HBV infection, virus-specific T cells are functionally defective, and this exhaustion state is a key determinant of virus persistence. Reconstitution of an efficient anti-viral T cell response may thus represent a rational strategy to treat chronic HBV patients. In this perspective, the enhancement of adaptive immune responses by a checkpoint inhibitor blockade, specific T cell vaccines, lymphocyte metabolism targeting, and autologous T cell engineering, including chimeric antigen receptor (CAR) and TCR-redirected T cells, constitutes a promising immune modulatory approach for a therapeutic restoration of protective immunity. The advances of the emerging immune-based therapies in the setting of the HBV research field will be outlined.
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Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Hepatitis B/terapia , Inmunoterapia , Ingeniería Genética , Hepatitis B/genética , Humanos , Linfocitos T/metabolismo , VacunaciónRESUMEN
BACKGROUND: Hepatitis C virus (HCV) persistence is associated with impaired CD8 functions. Whether functional restoration of CD8 T cells chronically exposed to antigen can be obtained once the antigen is removed remains to be clarified. OBJECTIVE: To determine whether clearance of HCV by antiviral treatment can fully restore the antiviral function of HCV-specific CD8 cells. DESIGN: Peripheral blood HCV-, Flu- and cytomegalovirus (CMV)-specific CD8 cells were quantified by tetramer staining in 28 patients whose HCV infection resolved after peginterferon or peginterferon/ribavirin treatment for either acute or chronic hepatitis and in eight subjects with acute HCV infection which resolved spontaneously for comparison. HCV-specific CD8 cells were evaluated for their phenotypic and functional characteristics by comparing different patient groups and CD8 cells with different viral specificities in the same patients. RESULTS: Sustained viral response (SVR) did not lead to full maturation of a functional memory CD8 cell response. In particular, SVR in chronic infection was associated with a greater level of T cell dysfunction than responders after acute infection, who showed HCV-specific CD8 responses comparable to those of spontaneous resolvers but weaker than those of Flu-specific CD8 cells. Higher programmed death (PD)-1 expression was detected on HCV than on Flu- and CMV-specific CD8 cells and the effect of PD-1/PD-L1 blockade was better in SVRs after chronic than after acute HCV infection. CONCLUSION: A better restoration of HCV-specific CD8 function was detectable after SVR in patients with acute hepatitis than in those with chronic disease. Thus, the difficulty in achieving a complete restoration of the antiviral T cell function should be considered in the design of immunomodulatory therapies.
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Antivirales/uso terapéutico , Proteínas Reguladoras de la Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/metabolismo , Ribavirina/uso terapéutico , Antivirales/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Femenino , Hepacivirus/inmunología , Hepatitis C/inmunología , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Humanos , Masculino , Ribavirina/farmacología , Carga Viral/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: The effect of IFN-α therapy on HBV-specific T-cell responses in HBeAg-negative, genotype D, chronic hepatitis B is largely undefined. Understanding to what extent IFN-α can modulate HBV-specific T-cells is important to define strategies to optimize IFN efficacy and to identify immunological parameters to predict response to therapy. METHODS: HBV-specific T-cell responses were analyzed longitudinally ex vivo and after expansion in vitro in 15 patients with genotype D, HBeAg-negative chronic hepatitis B treated with peginterferon-α-2a. HBV proteins and synthetic peptides were used to stimulate T-cell responses. Analysis of the CD4 and CD8 T-cell functions was performed by ELISPOT, intracellular cytokine and tetramer staining. The effect of anti-PD-L1 on T-cell functions was also analyzed. RESULTS: Ex vivo IFN-γ production by total HBV-specific T-cells was significantly greater before therapy in patients who showed HBV DNA <50 IU/ml at weeks 24 and/or 48 of therapy. No significant improvement of T-cell proliferation, Th1 cytokine production and cytotoxicity was observed during IFN therapy by both ex vivo and in vitro analysis. PD-1/PD-L1 blockade showed a modest improvement of cytokine production in a total of 15% of T-cell lines. CONCLUSIONS: IFN-α did not improve peripheral blood HBV-specific T-cell responses in the first 24 weeks of treatment, consistent either with a predominant antiviral/antiproliferative effect or with an immunomodulatory activity on other arms of the immune system which were not analyzed in our study. A better pre-treatment ex vivo IFN-γ production was associated with better chances to control HBV replication during therapy and represents a promising predictor of IFN efficacy.
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Antivirales/uso terapéutico , Antígenos e de la Hepatitis B/sangre , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Polietilenglicoles/uso terapéutico , Linfocitos T/inmunología , Adulto , Anciano , Citocinas/biosíntesis , Femenino , Antígenos de Superficie de la Hepatitis B/sangre , Hepatitis B Crónica/inmunología , Humanos , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/uso terapéuticoRESUMEN
BACKGROUND & AIMS: Hepatitis B virus (HBV) DNA detection in serum and/or in the liver of hepatitis B surface antigen (HBsAg)-negative patients with or without serologic markers of previous viral exposure is defined as occult HBV infection. Because the role of the adaptive response in keeping HBV replication under control in occult infection still is undefined, this study was performed to characterize the features of the HBV-specific T-cell response in this condition. METHODS: HBV-specific T-cell frequency and function were tested ex vivo and after in vitro expansion in 32 HBsAg-negative patients undergoing diagnostic liver biopsy for chronic hepatitis C: 18 with occult HBV infection (11 anti-HBc-negative and 7 anti-HBc-positive patients) defined by the detection of intrahepatic HBV DNA by polymerase chain reaction; 14 without detectable intrahepatic HBV DNA (5 anti-HBc-positive and 9 anti-HBc-negative patients). Six patients with chronic hepatitis B and 7 HBsAg-inactive carriers were studied for comparison. RESULTS: The presence or absence of serologic HBV markers defined 2 profiles of HBV-specific T-cell responses in occult infection. Anti-HBc-positive patients showed a T-cell response typical of protective memory, suggesting that this condition represents a resolved infection with immune-mediated virus control. In contrast, HBV-specific T cells in anti-HBc-negative patients did not readily expand and produce interferon-gamma in vitro, suggesting the possibility of a low-dose infection insufficient to allow maturation of protective memory. CONCLUSIONS: Our results suggest different mechanisms of control of viral replication in seropositive and seronegative occult infections. Additional studies aimed at understanding possible different clinical implications are needed.
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Linfocitos T CD8-positivos/inmunología , ADN Viral/análisis , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/inmunología , Inmunidad Celular/fisiología , Replicación Viral/genética , Adulto , Anciano , Biopsia , Linfocitos T CD8-positivos/metabolismo , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Genotipo , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Hepatitis B Crónica/patología , Hepatitis B Crónica/virología , Humanos , Hígado/patología , Hígado/virología , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , RecurrenciaRESUMEN
BACKGROUND & AIMS: The second slope of viral decline induced by interferon treatment has been suggested to be influenced mainly by the hepatitis C virus (HCV)-specific T-cell response; however, this hypothesis needs to be validated by results derived from experimental studies. METHODS: To address this issue, the HCV-specific T-cell response of 32 genotype-1-infected patients of the 270 patients enrolled in the dynamically individualized treatment of hepatitis C infection and correlates of viral/host dynamics phase III, open-label, randomized, multicenter trial was studied in relation to viral kinetics and treatment outcome. RESULTS: Greater proliferative responses by HCV-specific CD8 cells were found before treatment in patients with a fast viral decline and with a sustained viral response. However, no significant improvement of HCV-specific CD8 responses was observed in the first weeks of therapy in both rapid viral responder and non-rapid viral responder patients. A mild enhancement of proliferative T-cell responses and a partial restoration of the cytotoxic T-cell potential was expressed only late during treatment, likely favored by HCV clearance. CONCLUSIONS: Early restoration of an efficient T-cell response does not seem to be an essential requirement for a rapid viral decline in the first weeks of treatment. However, patients presenting a better HCV-specific CD8 cell proliferative potential at baseline are more likely to present a rapid and sustained viral response. Therefore, future treatment protocols should consider the development of strategies aimed at improving HCV-specific T-cell responses.
Asunto(s)
Antivirales/uso terapéutico , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Inmunidad Celular/efectos de los fármacos , Interferón-alfa/uso terapéutico , Polietilenglicoles/uso terapéutico , Ribavirina/uso terapéutico , Linfocitos T/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/virología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimioterapia Combinada , Ensayo de Inmunoadsorción Enzimática , Europa (Continente) , Hepacivirus/genética , Hepacivirus/crecimiento & desarrollo , Antígenos de la Hepatitis/genética , Antígenos de la Hepatitis/inmunología , Hepatitis C/diagnóstico , Hepatitis C/inmunología , Humanos , Interferón alfa-2 , Interferón-alfa/farmacología , Cinética , Activación de Linfocitos/efectos de los fármacos , Polietilenglicoles/farmacología , ARN Viral/sangre , Proteínas Recombinantes/inmunología , Ribavirina/farmacología , Linfocitos T/inmunología , Linfocitos T/virología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/virología , Resultado del TratamientoRESUMEN
UNLABELLED: The functional impairment of HCV-specific T cell responses is believed to be an important determinant of HCV persistence, but the functional T cell defects of patients with chronic hepatitis C (CH-C) are only partially defined. CD8 responses to HLA-A2-restricted epitopes of HCV and other unrelated viruses were studied in 23 HLA-A2-positive patients both ex vivo and after in vitro culture. Degranulation capacity, intracellular perforin, and granzyme-A content and cytokine production (IFN-gamma, TNF-alpha) by HCV- and non-HCV-specific CD8 cells were tested both ex vivo and in vitro, whereas cytolytic activity was studied after 10 days' expansion in vitro. Memory maturation and role of exhaustion were assessed ex vivo by HCV-specific CD8 staining for CD127 and PD-1, and in vitro after peripheral blood mononuclear cells (PBMC) culture in the presence of anti-PD-L1 monoclonal antibodies. IFN-gamma production and cytolytic activity were expressed less efficiently by HCV-specific than by non-HCV specific CD8 cells derived from the same CH-C patients. The amount of stored granzyme-A within single cells was always lower in HCV-specific CD8 cells, which were less efficient also in the release of lytic granules and in the production of TNF-alpha. The CD8 dysfunction was associated with high PD-1 expression by most HCV-specific CD8 cells, and PD-1/PD-L1 blockade by anti-PD-L1 antibodies in vitro was able to improve the HCV-specific CD8 function. CONCLUSION: Our study characterizes CD8 defects that may be important in maintaining HCV persistence; identification of strategies to correct these defects may help to define novel approaches to treat HCV infection.
Asunto(s)
Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/fisiopatología , Adulto , Anciano , Anticuerpos Monoclonales/farmacología , Antígenos CD/metabolismo , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/metabolismo , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Células Cultivadas , Femenino , Granzimas/metabolismo , Humanos , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Perforina , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptor de Muerte Celular Programada 1 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Radiofrequency thermal ablation (RFA) destroys tumoral tissue generating a local necrosis followed by marked inflammatory response with a dense T-cell infiltrate. In this study, we tested whether hepatocellular carcinoma thermal ablation can induce or enhance T-cell responses specific for hepatocellular carcinoma-associated antigens. Peripheral blood mononuclear cells derived from 20 patients with hepatocellular carcinoma were stimulated before and a month after RFA treatment with autologous hepatocellular carcinoma-derived protein lysates obtained before and immediately after RFA treatment. The effect of thermal ablation on memory T-cell responses to recall antigens [tetanus toxoid, protein purified derivative (PPD), Escherichia coli] was also assessed. T-cell reactivity was analyzed in an IFN-gamma enzyme-linked immunospot assay and by intracellular IFN-gamma staining. Treatment was followed by a significant increase of patients responsive either to tumor antigens derived from both the untreated hepatocellular carcinoma tissue (P < 0.05) and the necrotic tumor (P < 0.01) and by a higher frequency of circulating tumor-specific T cells. T-cell responses to recall antigens were also significantly augmented. Phenotypic analysis of circulating T and natural killer cells showed an increased expression of activation and cytotoxic surface markers. However, tumor-specific T-cell responses were not associated with protection from hepatocellular carcinoma relapse. Evidence of tumor immune escape was provided in one patient by the evidence that a new nodule of hepatocellular carcinoma recurrence was not recognized by T cells obtained at the time of RFA. In conclusion, RFA treatment generates the local conditions for activating the tumor-specific T-cell response. Although this effect is not sufficient for controlling hepatocellular carcinoma, it may represent the basis for the development of an adjuvant immunotherapy in patients undergoing RFA for primary and secondary liver tumors.