RESUMEN
Hepatocellular carcinoma (HCC) often develops following chronic hepatitis B virus (HBV) infection and responds poorly to immune checkpoint blockade. Here, we examined the antigen specificities of HCC-infiltrating T cells and their relevance to tumor control. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver, and tumor tissues from 46 HCC patients, we detected 91 different antigen-specific CD8+ T cell populations targeting HBV, neoantigen, tumor-associated, and disease-unrelated antigens. Parallel high-dimensional analysis delineated five distinct antigen-specific tissue-resident memory T (Trm) cell populations. Intratumoral and intrahepatic HBV-specific T cells were enriched for two Trm cell subsets that were PD-1loTOXlo, despite being clonally expanded. High frequencies of intratumoral terminally exhausted T cells were uncommon. Patients with tumor-infiltrating HBV-specific CD8+ Trm cells exhibited longer-term relapse-free survival. Thus, non-terminally exhausted HBV-specific CD8+ Trm cells show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/inmunología , Memoria Inmunológica/inmunología , Neoplasias Hepáticas/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Antígenos de Neoplasias/inmunología , Carcinoma Hepatocelular/patología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Neoplasias Hepáticas/patología , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/prevención & control , Receptor de Muerte Celular Programada 1/metabolismo , Células Tumorales CultivadasRESUMEN
The TRAIL pathway can mediate apoptosis of hepatic stellate cells to promote the resolution of liver fibrosis. However, TRAIL has the capacity to bind to regulatory receptors in addition to death-inducing receptors; their differential roles in liver fibrosis have not been investigated. Here we have dissected the contribution of regulatory TRAIL receptors to apoptosis resistance in primary human hepatic stellate cells (hHSC). hHSC isolated from healthy margins of liver resections from different donors expressed variable levels of TRAIL-R2/3/4 (but negligible TRAIL-R1) ex vivo and after activation. The apoptotic potential of TRAIL-R2 on hHSC was confirmed by lentiviral-mediated knockdown. A functional inhibitory role for TRAIL-R3/4 was revealed by shRNA knockdown and mAb blockade, showing that these regulatory receptors limit apoptosis of hHSC in response to both oligomerised TRAIL and NK cells. A close inverse ex vivo correlation between hHSC TRAIL-R4 expression and susceptibility to apoptosis underscored its central regulatory role. Our data provide the first demonstration of non-redundant functional roles for the regulatory TRAIL receptors (TRAIL-R3/4) in a physiological setting. The potential for these inhibitory TRAIL receptors to protect hHSC from apoptosis opens new avenues for prognostic and therapeutic approaches to the management of liver fibrosis.
Asunto(s)
Apoptosis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Anticuerpos Monoclonales/inmunología , Apoptosis/efectos de los fármacos , Células Cultivadas , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Humanos , Células Asesinas Naturales/inmunología , Hígado/citología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Miembro 10c de Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Miembro 10c de Receptores del Factor de Necrosis Tumoral/genética , Miembro 10c de Receptores del Factor de Necrosis Tumoral/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Receptores Señuelo del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores Señuelo del Factor de Necrosis Tumoral/genética , Receptores Señuelo del Factor de Necrosis Tumoral/metabolismoRESUMEN
NK cells are important antiviral effectors, highly enriched in the liver, with the potential to regulate immunopathogenesis in persistent viral infections. Here we examined whether changes in the NK pool are induced when patients with eAg-positive CHB are 'primed' with PegIFNα and importantly, whether these changes are sustained or further modulated long-term after switching to nucleos(t)ides (sequential NUC therapy), an approach currently tested in the clinic. Longitudinal sampling of a prospectively recruited cohort of patients with eAg+CHB showed that the cumulative expansion of CD56bright NK cells driven by 48-weeks of PegIFNα was maintained at higher than baseline levels throughout the subsequent 9 months of sequential NUCs. Unexpectedly, PegIFNα-expanded NK cells showed further augmentation in their expression of the activating NK cell receptors NKp30 and NKp46 during sequential NUCs. The expansion in proliferating, functional NK cells was more pronounced following sequential NUCs than in comparison cohorts of patients treated with de novo NUCs or PegIFNα only. Reduction in circulating HBsAg concentrations, a key goal in the path towards functional cure of CHB, was only achieved in those patients with enhancement of NK cell IFNγ and cytotoxicity but decrease in their expression of the death ligand TRAIL. In summary, we conclude that PegIFNα priming can expand a population of functional NK cells with an altered responsiveness to subsequent antiviral suppression by NUCs. Patients on sequential NUCs with a distinct NK cell profile show a decline in HBsAg, providing mechanistic insights for the further optimisation of treatment strategies to achieve sustained responses in CHB.
Asunto(s)
Virus de la Hepatitis B/inmunología , Hepatitis B/tratamiento farmacológico , Interferón-alfa/administración & dosificación , Células Asesinas Naturales/inmunología , Carga Viral/inmunología , Femenino , Hepatitis B/inmunología , Hepatitis B/patología , Antígenos de Superficie de la Hepatitis B/inmunología , Humanos , Células Asesinas Naturales/patología , Masculino , Receptor 1 Gatillante de la Citotoxidad Natural/inmunología , Receptor 3 Gatillante de la Citotoxidad Natural/inmunología , Estudios ProspectivosRESUMEN
Optimal immune activation of naïve CD8 T cells requires signal 1 mediated by the T cell receptor, signal 2 mediated by co-stimulation and signal 3 provided by pro-inflammatory cytokines. However, the potential for signal 3 cytokines to rescue anti-viral responses in functionally exhausted T cells has not been defined. We investigated the effect of using third signal cytokines IL-12 or IFN-α to rescue the exhausted CD8 T cell response characteristic of patients persistently infected with hepatitis B virus (HBV). We found that IL-12, but not IFN-α, potently augmented the capacity of HBV-specific CD8 T cells to produce effector cytokines upon stimulation by cognate antigen. Functional recovery mediated by IL-12 was accompanied by down-modulation of the hallmark inhibitory receptor PD-1 and an increase in the transcription factor T-bet. PD-1 down-regulation was observed in HBV but not CMV-specific T cells, in line with our finding that the highly functional CMV response was not further enhanced by IL-12. IL-12 enhanced a number of characteristics of HBV-specific T cells important for viral control: cytotoxicity, polyfunctionality and multispecificity. Furthermore, IL-12 significantly decreased the pro-apoptotic molecule Bim, which is capable of mediating premature attrition of HBV-specific CD8 T cells. Combining IL-12 with blockade of the PD-1 pathway further increased CD8 functionality in the majority of patients. These data provide new insights into the distinct signalling requirements of exhausted T cells and the potential to recover responses optimised to control persistent viral infections.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Interleucina-12/inmunología , Transducción de Señal/inmunología , Antivirales/inmunología , Antivirales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Linfocitos T CD8-positivos/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/virología , Regulación hacia Abajo , Regulación Viral de la Expresión Génica , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/virología , Humanos , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Interleucina-12/metabolismo , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Especificidad de la EspecieRESUMEN
Antiviral T cell responses in hepatotropic viral infections such as hepatitis B virus (HBV) are profoundly diminished and prone to apoptotic deletion. In this study, we investigate whether the large population of activated NK cells in the human liver contributes to this process. We show that in vitro removal of NK cells augments circulating CD8(+) T cell responses directed against HBV, but not against well-controlled viruses, in patients with chronic hepatitis B (CHB). We find that NK cells can rapidly eliminate HBV-specific T cells in a contact-dependent manner. CD8(+) T cells in the liver microcirculation are visualized making intimate contact with NK cells, which are the main intrahepatic lymphocytes expressing TNF-related apoptosis-inducing ligand (TRAIL) in CHB. High-level expression of the TRAIL death receptor TRAIL-R2 is found to be a hallmark of T cells exposed to the milieu of the HBV-infected liver in patients with active disease. Up-regulation of TRAIL-R2 renders T cells susceptible to caspase-8-mediated apoptosis, from which they can be partially rescued by blockade of this death receptor pathway. Our findings demonstrate that NK cells can negatively regulate antiviral immunity in chronic HBV infection and illustrate a novel mechanism of T cell tolerance in the human liver.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Hepatitis B Crónica/inmunología , Células Asesinas Naturales/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Adulto , Anciano , Apoptosis/fisiología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Estudios de Casos y Controles , Caspasa 8/metabolismo , Hepatitis B Crónica/metabolismo , Humanos , Tolerancia Inmunológica , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/virología , Hígado/citología , Hígado/metabolismo , Hígado/virología , Persona de Mediana Edad , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Regulación hacia Arriba , Adulto JovenRESUMEN
The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.