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Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.
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Linfocitos T CD8-positivos , Hepatitis B Crónica , Hígado , Animales , Humanos , Masculino , Ratones , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos/inmunología , Hepatocitos/virología , Hígado/inmunología , Hígado/virología , Fosforilación , Transducción de Señal , Activación de LinfocitosRESUMEN
Virus-specific T cells are critical to mediating viral control; however, Hepatitis B virus (HBV)-specific T cells among chronic Hepatitis B (CHB) patients are functionally exhausted. The inability to consistently measure the ex vivo functionality of HBV-specific T cells has prevented meaningful analysis during antiviral events such as HBeAg seroconversion, hepatic flares, and HBsAg loss. We optimized the traditional IFN-γ ELISpot assay to measure total ex vivo HBV-specific T cell frequencies using CHB PBMCs stimulated with HBV overlapping peptide (OLP) pools. This was then further adapted to assess individual antigen specificity (core, envelop, polymerase, X) and multifunctional HBV-specific T cells using a 3-analyte FluoroSpot assay. This protocol encompasses two major components: (1) PBMC handling/stimulation and (2) assay plate preparation and spot development. By performing this assay, ex vivo CHB patient T cell responses could be assessed longitudinally during immunotherapy or other important clinical events.
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Ensayo de Immunospot Ligado a Enzimas , Virus de la Hepatitis B , Hepatitis B Crónica , Linfocitos T , Humanos , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Virus de la Hepatitis B/inmunología , Ensayo de Immunospot Ligado a Enzimas/métodos , Linfocitos T/inmunología , Interferón gamma/metabolismo , Interferón gamma/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Leucocitos Mononucleares/metabolismoRESUMEN
BACKGROUND: Immunological studies on chronic hepatitis B virus (HBV) infection have convincingly shown immune dysfunction involving multiple cell types. The focus of the majority of studies has been on the role of T cells and showed an impaired functional T cell response to HBV. B cells have been evaluated more recently, but in contrast to T cells, more pronounced activation of circulating B cells has been reported. To gain more insight into the activation status of B cells, we investigated the activation gene profile of B cells in the blood and liver of chronic HBV patients. METHODS: RNA-seq and flow cytometric analysis was performed on peripheral blood B cells of immune active chronic HBV patients, comparing them with samples from healthy controls. In addition, gene expression profiles of B cells in the liver were analyzed by bulk and single-cell RNA-seq. RESULTS AND CONCLUSIONS: Our data show a distinctive B cell activation gene signature in the blood of immune active chronic HBV patients, characterized by a significant upregulation of immune-related genes, including IRF1, STAT1, STAT3, TAP1, and TAPBP. This peripheral activation profile was also observed in B cells from the liver by single cell RNA-seq showing upregulation of IRF1, CD83 and significantly higher CD69 expression, with naive and memory B cell subsets being the primary carriers of the signature. Our findings suggest that B cell gene profiles reflect responsiveness to HBV infection, these findings are relevant for clinical studies evaluating immunomodulatory treatment strategies for HBV.
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Studies have traditionally focused on the role of T cells in chronic hepatitis B (CHB), but recent evidence supports a role for B cells. The enrichment of so-called atypical memory (AtM) B cells, which show reduced signaling and impaired differentiation, is believed to be a characteristic feature of CHB, potentially contributing to the observed dysfunctional anti-HBsAg B-cell responses. Our study, involving 62 CHB patients across clinical phases, identified AtM B cells expressing IFNLR1 and interferon-stimulated genes. Contrary to previous reports, we found relatively low frequencies of AtM B cells in the liver, comparable to peripheral blood. However, liver plasma cell frequencies were significantly higher, particularly during phases with elevated viral loads and liver enzyme levels. Liver plasma cells exhibited signs of active proliferation, especially in the immune active phase. Our findings suggest a potential role for plasma cells, alongside potential implications and consequences of local proliferation, within the livers of CHB patients. While the significance of AtM B cells remains uncertain, further investigation is warranted to determine their responsiveness to interferons and their role in CHB.
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With the increasing momentum and success of monoclonal antibody therapy in conventional medical practices, there is a revived emphasis on the development of monoclonal antibodies targeting the hepatitis B surface antigen (anti-HBs) for the treatment of chronic hepatitis B (HBV) and hepatitis D (HDV). Combination therapies of anti-HBs monoclonal antibodies, and novel anti-HBV compounds and immunomodulatory drugs presenting a promising avenue to enhanced therapeutic outcomes in HBV/HDV cure regimens. In this review, we will cover the role of antibodies in the protection and clearance of HBV infection, the association of anti-HBV surface antigen antibodies (anti-HBs) in protection against HBV and how antibody effector functions, beyond neutralization, are likely necessary. Lastly, we will review clinical data from previous and ongoing clinical trials of passive antibody therapy to provide a state-of-the-are perspective on passive antibody therapies in combinations with additional novel agents.
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Hepatitis D , Inmunización Pasiva , Humanos , Hepatitis D/inmunología , Hepatitis D/tratamiento farmacológico , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/efectos de los fármacos , Anticuerpos contra la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/tratamiento farmacológico , Animales , Antígenos de Superficie de la Hepatitis B/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/inmunología , Hepatitis B/inmunología , Hepatitis B/prevención & control , Hepatitis B/tratamiento farmacológico , Antivirales/uso terapéutico , Virus de la Hepatitis Delta/inmunologíaRESUMEN
BACKGROUND AIMS: Pegylated interferon-α (PegIFNα) is of limited utility during immunotolerant or immune active phases of chronic hepatitis B infection but is being explored as part of new cure regimens. Low/absent levels of IFNα found in some patients receiving treatment are associated with limited/no virological responses. The study aimed to determine if sera from participants inhibit IFNα activity and/or contain therapy-induced anti-IFNα antibodies. APPROACH RESULTS: Pre-treatment, on-treatment, and post-treatment sera from 61 immunotolerant trial participants on PegIFNα/entecavir therapy and 88 immune active trial participants on PegIFNα/tenofovir therapy were screened for anti-IFNα antibodies by indirect ELISA. The neutralization capacity of antibodies was measured by preincubation of sera±recombinant human IFNα added to Huh7 cells with the measurement of interferon-stimulated gene (ISG)-induction by qPCR. Correlations between serum-induced ISG inhibition, presence, and titer of anti-IFNα antibodies and virological responses were evaluated. Preincubation of on-treatment serum from 26 immunotolerant (43%) and 13 immune active (15%) participants with recombinant-human IFNα markedly blunted ISG-induction in Huh7 cells. The degree of ISG inhibition correlated with IFNα antibody titer ( p < 0.0001; r = 0.87). On-treatment development of anti-IFNα neutralizing antibodies (nAbs) was associated with reduced quantitative HBsAg and qHBeAg declines ( p < 0.05) and inhibited IFNα bioactivity to 240 weeks after PegIFNα cessation. Children developed anti-IFNα nAbs more frequently than adults ( p = 0.004) but nAbs in children had less impact on virological responses. CONCLUSIONS: The development of anti-IFNα nAbs during PegIFNα treatment diminishes responses to antiviral therapy. Understanding how and why anti-IFNα antibodies develop may allow for the optimization of IFN-based therapy, which is critical given its renewed use in HBV-cure strategies.
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Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) and progresses faster in males than in females. We identify sex-based differences in kidney metabolism and in the blood metabolome of male and female individuals with diabetes. Primary human proximal tubular epithelial cells (PTECs) from healthy males displayed increased mitochondrial respiration, oxidative stress, apoptosis, and greater injury when exposed to high glucose compared with PTECs from healthy females. Male human PTECs showed increased glucose and glutamine fluxes to the TCA cycle, whereas female human PTECs showed increased pyruvate content. The male human PTEC phenotype was enhanced by dihydrotestosterone and mediated by the transcription factor HNF4A and histone demethylase KDM6A. In mice where sex chromosomes either matched or did not match gonadal sex, male gonadal sex contributed to the kidney metabolism differences between males and females. A blood metabolomics analysis in a cohort of adolescents with or without diabetes showed increased TCA cycle metabolites in males. In a second cohort of adults with diabetes, females without DKD had higher serum pyruvate concentrations than did males with or without DKD. Serum pyruvate concentrations positively correlated with the estimated glomerular filtration rate, a measure of kidney function, and negatively correlated with all-cause mortality in this cohort. In a third cohort of adults with CKD, male sex and diabetes were associated with increased plasma TCA cycle metabolites, which correlated with all-cause mortality. These findings suggest that differences in male and female kidney metabolism may contribute to sex-dependent outcomes in DKD.
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Diabetes Mellitus , Nefropatías Diabéticas , Insuficiencia Renal Crónica , Adolescente , Adulto , Humanos , Femenino , Masculino , Animales , Ratones , Caracteres Sexuales , Piruvatos , Glucosa , RiñónRESUMEN
Immune-mediated liver damage is the driver of disease progression in patients with chronic hepatitis B virus (HBV) infection. Liver damage is an Ag-independent process caused by bystander activation of CD8 T cells and NK cells. How bystander lymphocyte activation is initiated in chronic hepatitis B patients remains unclear. Periods of liver damage, called hepatic flares, occur unpredictably, making early events difficult to capture. To address this obstacle, we longitudinally sampled the liver of chronic hepatitis B patients stopping antiviral therapy and analyzed immune composition and activation using flow cytometry and single-cell RNA sequencing. At 4 wk after stopping therapy, HBV replication rebounded but no liver damage was detectable. There were no changes in cell frequencies at viral rebound. Single-cell RNA sequencing revealed upregulation of IFN-stimulated genes (ISGs) and proinflammatory cytokine migration inhibitory factor (MIF) at viral rebound in patients that go on to develop hepatic flares 6-18 wk after stopping therapy. The type I IFN signature was only detectable within the liver, and neither IFN-α/ß or ISG induction could be detected in the peripheral blood. In vitro experiments confirmed the type I IFN-dependent ISG profile whereas MIF was induced primarily by IL-12. MIF exposure further amplified inflammatory cytokine production by myeloid cells. Our data show that innate immune activation is detectable in the liver before clinically significant liver damage is evident. The combination of type I IFN and enhanced cytokine production upon MIF exposure represent the earliest immunological triggers of lymphocyte bystander activation observed in hepatic flares associated with chronic HBV infection.
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Hepatitis B Crónica , Hepatitis B , Humanos , Virus de la Hepatitis B , Hígado , Citocinas/metabolismo , Antivirales/uso terapéutico , Antivirales/metabolismoRESUMEN
BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which there is an unmet need to understand the cellular composition of the affected liver and how it underlies disease pathogenesis. We aimed to generate a comprehensive atlas of the PSC liver using multi-omic modalities and protein-based functional validation. METHODS: We employed single-cell and single-nucleus RNA sequencing (47,156 cells and 23,000 nuclei) and spatial transcriptomics (one sample by 10x Visium and five samples with Nanostring GeoMx DSP) to profile the cellular ecosystem in 10 PSC livers. Transcriptomic profiles were compared to 24 neurologically deceased donor livers (107,542 cells) and spatial transcriptomics controls, as well as 18,240 cells and 20,202 nuclei from three PBC livers. Flow cytometry was performed to validate PSC-specific differences in immune cell phenotype and function. RESULTS: PSC explants with parenchymal cirrhosis and prominent periductal fibrosis contained a population of cholangiocyte-like hepatocytes that were surrounded by diverse immune cell populations. PSC-associated biliary, mesenchymal, and endothelial populations expressed chemokine and cytokine transcripts involved in immune cell recruitment. Additionally, expanded CD4+ T cells and recruited myeloid populations in the PSC liver expressed the corresponding receptors to these chemokines and cytokines, suggesting potential recruitment. Tissue-resident macrophages, by contrast, were reduced in number and exhibited a dysfunctional and downregulated inflammatory response to lipopolysaccharide and interferon-γ stimulation. CONCLUSIONS: We present a comprehensive atlas of the PSC liver and demonstrate an exhaustion-like phenotype of myeloid cells and markers of chronic cytokine expression in late-stage PSC lesions. This atlas expands our understanding of the cellular complexity of PSC and has potential to guide the development of novel treatments. IMPACT AND IMPLICATIONS: Primary sclerosing cholangitis (PSC) is a rare liver disease characterized by chronic inflammation and irreparable damage to the bile ducts, which eventually results in liver failure. Due to a limited understanding of the underlying pathogenesis of disease, treatment options are limited. To address this, we sequenced healthy and diseased livers to compare the activity, interactions, and localization of immune and non-immune cells. This revealed that hepatocytes lining PSC scar regions co-express cholangiocyte markers, whereas immune cells infiltrate the scar lesions. Of these cells, macrophages, which typically contribute to tissue repair, were enriched in immunoregulatory genes and demonstrated a lack of responsiveness to stimulation. These cells may be involved in maintaining hepatic inflammation and could be a target for novel therapies.
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Colangitis Esclerosante , Humanos , Cicatriz/metabolismo , Cicatriz/patología , Ecosistema , Hígado/patología , Cirrosis Hepática/patología , Citocinas/metabolismo , Inflamación/metabolismo , Perfilación de la Expresión GénicaRESUMEN
BACKGROUND: There are no immunological biomarkers that predict control of chronic hepatitis B (CHB). The lack of immune biomarkers raises concerns for therapies targeting PD-1/PD-L1 because they have the potential for immune-related adverse events. Defining specific immune functions associated with control of HBV replication could identify patients likely to respond to anti-PD-1/PD-L1 therapies and achieve a durable functional cure. METHODS: We enrolled immunotolerant, HBeAg+ immune-active (IA+), HBeAg- immune-active (IA-), inactive carriers, and functionally cured patients to test ex vivo PD-1 blockade on HBV-specific T cell functionality. Peripheral blood mononuclear cells were stimulated with overlapping peptides covering HBV proteins +/-α-PD-1 blockade. Functional T cells were measured using a 2-color FluoroSpot assay for interferon-γ and IL-2. Ex vivo functional restoration was compared to the interferon response capacity assay, which predicts overall survival in cancer patients receiving checkpoint inhibitors. RESULTS: Ex vivo interferon-γ+ responses did not differ across clinical phases. IL-2+ responses were significantly higher in patients with better viral control and preferentially restored with PD-1 blockade. Inactive carrier patients displayed the greatest increase in IL-2 production, which was dominated by CD4 T cell and response to the HBcAg. The interferon response capacity assay significantly correlated with the degree of HBV-specific T cell restoration. CONCLUSIONS: IL-2 production was associated with better HBV control and superior to interferon-γ as a marker of T cell restoration following ex vivo PD-1 blockade. Our study suggests that responsiveness to ex vivo PD-1 blockade, or the interferon response capacity assay, may support stratification for α-PD-1 therapies.
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Hepatitis B Crónica , Humanos , Linfocitos T/metabolismo , Virus de la Hepatitis B , Interleucina-2 , Interferón gamma , Antígeno B7-H1 , Antígenos e de la Hepatitis B , Receptor de Muerte Celular Programada 1 , Leucocitos Mononucleares/metabolismo , BiomarcadoresRESUMEN
Background & Aims: Novel therapies for chronic hepatitis B (CHB), such as RNA interference, target all viral RNAs for degradation, whereas nucleoside analogues are thought to block reverse transcription with minimal impact on viral transcripts. However, limitations in technology and sampling frequency have been obstacles to measuring actual changes in HBV transcription in the liver of patients starting therapy. Methods: We used elective liver sampling with fine-needle aspirates (FNAs) to investigate the impact of treatment on viral replication in patients with CHB. Liver FNAs were collected from patients with CHB at baseline and 12 and 24 weeks after starting tenofovir alafenamide treatment. Liver FNAs were subjected to single-cell RNA sequencing and analysed using the Viral-Track method. Results: HBV was the only viral genome detected and was enriched within hepatocytes. The 5' sequencing technology identified protein-specific HBV transcripts and showed that tenofovir alafenamide therapy specifically reduced pre-genomic RNA transcripts with little impact on HBsAg or HBx transcripts. Infected hepatocytes displayed unique gene signatures associated with an immunological response to viral infection. Conclusions: Longitudinal liver sampling, combined with single-cell RNA sequencing, captured the dynamic impact of antiviral therapy on the replication status of HBV and revealed host-pathogen interactions at the transcriptional level in infected hepatocytes. This sequencing-based approach is applicable to early-stage clinical studies, enabling mechanistic studies of immunopathology and the effect of novel therapeutic interventions. Impact and Implications: Infection-dependent transcriptional changes and the impact of antiviral therapy on viral replication can be measured in longitudinal human liver biopsies using single-cell RNA sequencing data.
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A hepatitis C virus (HCV) vaccine is urgently needed. Vaccine development has been hindered by HCV's genetic diversity, particularly within the immunodominant hypervariable region 1 (HVR1). Here, we developed a strategy to elicit broadly neutralizing antibodies to HVR1, which had previously been considered infeasible. We first applied a unique information theory-based measure of genetic distance to evaluate phenotypic relatedness between HVR1 variants. These distances were used to model the structure of HVR1's sequence space, which was found to have five major clusters. Variants from each cluster were used to immunize mice individually, and as a pentavalent mixture. Sera obtained following immunization neutralized every variant in a diverse HCVpp panel (n = 10), including those resistant to monovalent immunization, and at higher mean titers (1/ID50 = 435) than a glycoprotein E2 (1/ID50 = 205) vaccine. This synergistic immune response offers a unique approach to overcoming antigenic variability and may be applicable to other highly mutable viruses.
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Hepacivirus , Hepatitis C , Animales , Ratones , Proteínas del Envoltorio Viral/genética , Inmunización , Inmunidad , Anticuerpos contra la Hepatitis C , Anticuerpos NeutralizantesRESUMEN
BACKGROUND AND AIMS: HBV infection is restricted to the liver, where it drives exhaustion of virus-specific T and B cells and pathogenesis through dysregulation of intrahepatic immunity. Our understanding of liver-specific events related to viral control and liver damage has relied almost solely on animal models, and we lack useable peripheral biomarkers to quantify intrahepatic immune activation beyond cytokine measurement. Our objective was to overcome the practical obstacles of liver sampling using fine-needle aspiration and develop an optimized workflow to comprehensively compare the blood and liver compartments within patients with chronic hepatitis B using single-cell RNA sequencing. APPROACH AND RESULTS: We developed a workflow that enabled multi-site international studies and centralized single-cell RNA sequencing. Blood and liver fine-needle aspirations were collected, and cellular and molecular captures were compared between the Seq-Well S 3 picowell-based and the 10× Chromium reverse-emulsion droplet-based single-cell RNA sequencing technologies. Both technologies captured the cellular diversity of the liver, but Seq-Well S 3 effectively captured neutrophils, which were absent in the 10× dataset. CD8 T cells and neutrophils displayed distinct transcriptional profiles between blood and liver. In addition, liver fine-needle aspirations captured a heterogeneous liver macrophage population. Comparison between untreated patients with chronic hepatitis B and patients treated with nucleoside analogs showed that myeloid cells were highly sensitive to environmental changes while lymphocytes displayed minimal differences. CONCLUSIONS: The ability to electively sample and intensively profile the immune landscape of the liver, and generate high-resolution data, will enable multi-site clinical studies to identify biomarkers for intrahepatic immune activity in HBV and beyond.
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Hepatitis B Crónica , Animales , Humanos , Hepatitis B Crónica/tratamiento farmacológico , Biopsia con Aguja Fina , Virus de la Hepatitis B/genética , Hígado/patología , Linfocitos T CD8-positivos , Biomarcadores , Análisis de Secuencia de ARNRESUMEN
Accumulation of activated immune cells results in nonspecific hepatocyte killing in chronic hepatitis B (CHB), leading to fibrosis and cirrhosis. This study aims to understand the underlying mechanisms in humans and to define whether these are driven by widespread activation or a subpopulation of immune cells. We enrolled CHB patients with active liver damage to receive antiviral therapy and performed longitudinal liver sampling using fine-needle aspiration to investigate mechanisms of CHB pathogenesis in the human liver. Single-cell sequencing of total liver cells revealed a distinct liver-resident, polyclonal CD8+ T cell population that was enriched at baseline and displayed a highly activated immune signature during liver damage. Cytokine combinations, identified by in silico prediction of ligand-receptor interaction, induced the activated phenotype in healthy liver CD8+ T cells, resulting in nonspecific Fas ligand-mediated killing of target cells. These results define a CD8+ T cell population in the human liver that can drive pathogenesis and a key pathway involved in their function in CHB patients.
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Hepatitis B Crónica , Humanos , Linfocitos T CD8-positivos , Cirrosis Hepática/patología , Antivirales/farmacología , Antivirales/uso terapéutico , Virus de la Hepatitis BRESUMEN
Individuals who spontaneously clear hepatitis C virus (HCV) infection have demonstrated evidence of partial protective immunity, whereas treatment-induced clearance provides little or no protection against reinfection. We aimed to investigate whether treatment of acute HCV infection with direct-acting antivirals (DAA) prevents establishment of, or reverses, T-cell exhaustion, leading to a virus-specific T-cell immune profile more similar to that seen in spontaneous clearance. The magnitude and breadth of HCV-specific T-cell responses before and after DAA or interferon-based therapy in acute or chronic HCV were compared to those of participants with spontaneous clearance of infection, using Enzyme-linked Immunospot (ELISPOT). PBMCs were available for 55 patients comprising 4 groups: spontaneous clearance (n = 17), acute interferon (n = 14), acute DAA (n = 13) and chronic DAA (n = 11). After controlling for sex, the magnitude of post-treatment HCV-specific responses after acute DAA treatment was greater than after chronic DAA or acute IFN treatment and similar to those found in spontaneous clearers. However, spontaneous clearers responded to more HCV peptide pools indicating greater breadth of response. In conclusion, early treatment with DAAs may prevent or reverse some degree of immune exhaustion and result in stronger HCV-specific responses post-treatment. However, individuals with spontaneous clearance had broader HCV-specific responses.
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Hepatitis C Crónica , Hepatitis C , Humanos , Hepacivirus , Antivirales/uso terapéutico , Antivirales/farmacología , Hepatitis C Crónica/tratamiento farmacológico , Interferones/uso terapéutico , InmunidadRESUMEN
A dysregulated adaptive immune system is a key feature of aging, and is associated with age-related chronic diseases and mortality. Most notably, aging is linked to a loss in the diversity of the T cell repertoire and expansion of activated inflammatory age-related T cell subsets, though the main drivers of these processes are largely unknown. Here, we find that T cell aging is directly influenced by B cells. Using multiple models of B cell manipulation and single-cell omics, we find B cells to be a major cell type that is largely responsible for the age-related reduction of naive T cells, their associated differentiation towards pathogenic immunosenescent T cell subsets, and for the clonal restriction of their T cell receptor (TCR). Accordingly, we find that these pathogenic shifts can be therapeutically targeted via CD20 monoclonal antibody treatment. Mechanistically, we uncover a new role for insulin receptor signaling in influencing age-related B cell pathogenicity that in turn induces T cell dysfunction and a decline in healthspan parameters. These results establish B cells as a pivotal force contributing to age-associated adaptive immune dysfunction and healthspan outcomes, and suggest new modalities to manage aging and related multi-morbidity.
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Interferons induced early after SARS-CoV-2 infection are crucial for shaping immunity and preventing severe COVID-19. We previously demonstrated that injection of pegylated interferon-lambda accelerated viral clearance in COVID-19 patients (NCT04354259). To determine if the viral decline is mediated by enhanced immunity, we assess in vivo responses to interferon-lambda by single cell RNA sequencing and measure SARS-CoV-2-specific T cell and antibody responses between placebo and interferon-lambda-treated patients. Here we show that interferon-lambda treatment induces interferon stimulated genes in peripheral immune cells expressing IFNLR1, including plasmacytoid dendritic cells and B cells. Interferon-lambda does not affect SARS-CoV-2-specific antibody levels or the magnitude of virus-specific T cells. However, we identify delayed T cell responses in older adults, suggesting that interferon-lambda can overcome delays in adaptive immunity to accelerate viral clearance in high-risk patients. Altogether, interferon-lambda offers an early COVID-19 treatment option for outpatients to boost innate antiviral defenses without dampening peripheral adaptive immunity.
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Tratamiento Farmacológico de COVID-19 , Interferones , Humanos , Anciano , SARS-CoV-2 , Anticuerpos Antivirales , Antivirales/farmacología , Antivirales/uso terapéutico , Linfocitos TRESUMEN
HepG2 cells reconstituted with Hepatitis B virus (HBV) entry receptor sodium taurocholate co-transporting polypeptide (NTCP) are widely used as a convenient in vitro cell culture infection model for HBV replication studies. As such, it is pertinent that HBV infectivity is maintained at steady-state levels for an accurate interpretation of in vitro data. However, variations in the HBV infection efficiency due to imbalanced NTCP expression levels in the HepG2 cell line may affect experimental results. In this study, we performed single cell-cloning of HepG2-NTCP-A3 parental cells via limiting dilution and obtained multiple subclones with increased permissiveness to HBV. Specifically, one subclone (HepG2-NTCP-A3/C2) yielded more than four-fold higher HBV infection compared to the HepG2-NTCP-A3 parental clone. In addition, though HBV infectivity was universally reduced in the absence of polyethylene glycol (PEG), subclone C2 maintained relatively greater permissiveness under PEG-free conditions, suggesting the functional heterogeneity within parental HepG2-NTCP-A3 may be exploitable in developing a PEG-free HBV infection model. The increased viral production correlated with increased intracellular viral antigen expression as evidenced through HBcAg immunofluorescence staining. Further, these subclones were found to express different levels of NTCP, albeit with no remarkable morphology or cell growth differences. In conclusion, we isolated the subclones of HepG2-NTCP-A3 which support efficient HBV production and thus provide an improved in vitro HBV infection model.