RESUMO
The pool of beta cell-specific CD8+ T cells in type 1 diabetes (T1D) sustains an autoreactive potential despite having access to a constant source of antigen. To investigate the long-lived nature of these cells, we established a DNA methylation-based T cell 'multipotency index' and found that beta cell-specific CD8+ T cells retained a stem-like epigenetic multipotency score. Single-cell assay for transposase-accessible chromatin using sequencing confirmed the coexistence of naive and effector-associated epigenetic programs in individual beta cell-specific CD8+ T cells. Assessment of beta cell-specific CD8+ T cell anatomical distribution and the establishment of stem-associated epigenetic programs revealed that self-reactive CD8+ T cells isolated from murine lymphoid tissue retained developmentally plastic phenotypic and epigenetic profiles relative to the same cells isolated from the pancreas. Collectively, these data provide new insight into the longevity of beta cell-specific CD8+ T cell responses and document the use of this methylation-based multipotency index for investigating human and mouse CD8+ T cell differentiation.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Células-Tronco Pluripotentes/fisiologia , Adolescente , Adulto , Animais , Autoantígenos/imunologia , Plasticidade Celular , Células Cultivadas , Metilação de DNA , Epigênese Genética , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica , Masculino , Camundongos , Análise de Célula Única , Adulto JovemRESUMO
AIMS/HYPOTHESIS: We hypothesised that islet beta cell antigen presentation in the gut along with a tolerising cytokine would lead to antigen-specific tolerance in type 1 diabetes. We evaluated this in a parallel open-label Phase 1b study using oral AG019, food-grade Lactococcus lactis bacteria genetically modified to express human proinsulin and human IL-10, as a monotherapy and in a parallel, randomised, double-blind Phase 2a study using AG019 in combination with teplizumab. METHODS: Adults (18-42 years) and adolescents (12-17 years) with type 1 diabetes diagnosed within 150 days were enrolled, with documented evidence of at least one autoantibody and a stimulated peak C-peptide level >0.2 nmol/l. Participants were allocated to interventions using interactive response technology. We treated 42 people aged 12-42 years with recent-onset type 1 diabetes, 24 with Phase 1b monotherapy (open-label) and 18 with Phase 2a combination therapy. In the Phase 2a study, after treatment of the first two open-label participants, all people involved were blinded to group assignment, except for the Data Safety Monitoring Board members and the unblinded statistician. The primary endpoint was safety and tolerability based on the incidence of treatment-emergent adverse events, collected up to 6 months post treatment initiation. The secondary endpoints were pharmacokinetics, based on AG019 detection in blood and faeces, and pharmacodynamic activity. Metabolic and immune endpoints included stimulated C-peptide levels during a mixed meal tolerance test, HbA1c levels, insulin use, and antigen-specific CD4+ and CD8+ T cell responses using an activation-induced marker assay and pooled tetramers, respectively. RESULTS: Data from 24 Phase 1b participants and 18 Phase 2a participants were analysed. No serious adverse events were reported and none of the participants discontinued AG019 due to treatment-emergent adverse events. No systemic exposure to AG019 bacteria, proinsulin or human IL-10 was demonstrated. In AG019 monotherapy-treated adults, metabolic variables were stabilised up to 6 months (C-peptide, insulin use) or 12 months (HbA1c) post treatment initiation. In participants treated with AG019/teplizumab combination therapy, all measured metabolic variables stabilised or improved up to 12 months and CD8+ T cells with a partially exhausted phenotype were significantly increased at 6 months. Circulating preproinsulin-specific CD4+ and CD8+ T cells were detected before and after treatment, with a reduction in the frequency of preproinsulin-specific CD8+ T cells after treatment with monotherapy or combination therapy. CONCLUSIONS/INTERPRETATION: Oral delivery of AG019 was well tolerated and safe as monotherapy and in combination with teplizumab. AG019 was not shown to interfere with the safety profile of teplizumab and may have additional biological effects, including changes in preproinsulin-specific T cells. These preliminary data support continuing studies with this agent alone and in combination with teplizumab or other systemic immunotherapies in type 1 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT03751007, EudraCT 2017-002871-24 FUNDING: This study was funded by Precigen ActoBio.
Assuntos
Diabetes Mellitus Tipo 1 , Adulto , Adolescente , Humanos , Interleucina-10 , Peptídeo C , Linfócitos T CD8-Positivos/metabolismo , Proinsulina , Método Duplo-CegoRESUMO
Soon after diagnosis with type 1 diabetes (T1D), many patients experience a period of partial remission. A longer partial remission is associated with a better response to treatment, but the mechanism is not known. The frequency of CD4+CD25+CD127hi (127-hi) cells, a cell subset with an anti-inflammatory Th2 bias, correlates positively with length of partial remission. The purpose of this study was to further characterize the nature of the Th2 bias in 127-hi cells. Single cell RNA sequencing paired with TCR sequencing of sorted 127-hi memory cells identifies clonally expanded Th2 clusters in 127-hi cells from T1D, but not from healthy donors. The Th2 clusters express GATA3, GATA3-AS1, PTGDR2, IL17RB, IL4R and IL9R. The existence of 127-hi Th2 cell clonal expansion in T1D suggests that disease factors may induce clonal expansion of 127-hi Th2 cells that prolong partial remission and delay disease progression.
Assuntos
Diabetes Mellitus Tipo 1 , Células Th2 , Humanos , Diabetes Mellitus Tipo 1/genéticaRESUMO
Hepatitis C virus (HCV) infection induces interferon (IFN)-stimulated genes (ISGs) and downstream innate immune responses. This study investigated whether baseline and on-treatment differences in these responses predict response versus virological breakthrough during therapy with direct-acting antivirals (DAAs). Thirteen HCV genotype 1b-infected patients who had previously failed a course of pegylated IFN/ribavirin were retreated with asunaprevir/daclatasvir for 24 weeks. After pretreatment biopsy, patients were randomized to undergo a second biopsy at week 2 or 4 on therapy. Microarray and NanoString analyses were performed on paired liver biopsies and analyzed using linear mixed models. As biomarkers for peripheral IFN responses, peripheral blood natural killer cells were assessed for phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and degranulation. Nine of 13 (69%) patients achieved sustained virological response at 12 weeks off therapy (SVR12), and 4 experienced virological breakthroughs between weeks 4 and 12. Patients who achieved SVR12 displayed higher ISG expression levels in baseline liver biopsies and a higher frequency of pSTAT1 and TRAIL-expressing, degranulating natural killer cells in baseline blood samples than those who experienced virological breakthrough. Comparing gene expression levels from baseline and on-therapy biopsies, 408 genes (±1.2-fold, P < 0.01) were differentially expressed. Genes down-regulated on treatment were predominantly ISGs. Down-regulation of ISGs was rapid and correlated with HCV RNA suppression. Conclusion: An enhanced IFN signature is observed at baseline in liver and blood of patients who achieve SVR12 compared to those who experience a virological breakthrough; the findings suggest that innate immunity may contribute to clearance of HCV during DAA therapy by preventing the emergence of resistance-associated substitutions that lead to viral breakthrough during DAA therapy.
Assuntos
Antivirais/uso terapêutico , Expressão Gênica , Hepatite C/tratamento farmacológico , Imidazóis/uso terapêutico , Imunidade Inata , Isoquinolinas/uso terapêutico , Sulfonamidas/uso terapêutico , Adulto , Idoso , Carbamatos , Estudos de Coortes , Quimioterapia Combinada , Feminino , Hepatite C/imunologia , Hepatite C/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Masculino , Pessoa de Meia-Idade , Pirrolidinas , RNA Mensageiro/metabolismo , Resultado do Tratamento , Valina/análogos & derivadosRESUMO
BACKGROUND & AIMS: Chronic hepatitis affects phenotypes of innate and adaptive immune cells. Mucosal-associated invariant T (MAIT) cells are enriched in the liver as compared with the blood, respond to intra-hepatic cytokines, and (via the semi-invariant T-cell receptor) to bacteria translocated from the gut. Little is known about the role of MAIT cells in livers of patients with chronic hepatitis C virus (HCV) infection and their fate after antiviral therapy. METHODS: We collected blood samples from 42 patients with chronic HCV infection who achieved a sustained virologic response after 12 weeks of treatment with sofosbuvir and velpatasvir. Mononuclear cells were isolated from blood before treatment, at weeks 4 and 12 during treatment, and 24 weeks after the end of treatment. Liver biopsies were collected from 37 of the patients prior to and at week 4 of treatment. Mononuclear cells from 56 blood donors and 10 livers that were not suitable for transplantation were used as controls. Liver samples were assessed histologically for inflammation and fibrosis. Mononuclear cells from liver and blood were studied by flow cytometry and analyzed for responses to cytokine and bacterial stimulation. RESULTS: The frequency of MAIT cells among T cells was significantly lower in blood and liver samples of patients with HCV infection than of controls (median, 1.31% vs 2.32% for blood samples, P = .0048; and median, 4.34% vs 13.40% for liver samples, P = .001). There was an inverse correlation between the frequency of MAIT cells in the liver and histologically determined levels of liver inflammation (r = -.5437, P = .0006) and fibrosis (r = -.5829, P = .0002). MAIT cells from the liver had higher levels of activation and cytotoxicity than MAIT cells from blood (P < .0001). Production of interferon gamma by MAIT cells was dependent on monocyte-derived interleukin 18, and was reduced in patients with HCV infection in response to T-cell receptor-mediated but not cytokine-mediated stimulation, as compared with controls. Anti-viral therapy rapidly decreased liver inflammation and MAIT cell activation and cytotoxicity, and increased the MAIT cell frequency among intra-hepatic but not blood T cells. The MAIT cell response to T-cell receptor-mediated stimulation did not change during the 12 weeks of antiviral therapy. CONCLUSIONS: In analyses of paired blood and liver samples from patients with chronic HCV infection before, during, and after antiviral therapy with sofosbuvir and velpatasvir, we found that intrahepatic MAIT cells are activated by monocyte-derived cytokines and depleted in HCV-induced liver inflammation.
Assuntos
Hepatite C Crônica/imunologia , Fígado/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Antivirais/uso terapêutico , Biópsia , Carbamatos/uso terapêutico , Estudos de Casos e Controles , Citocinas/imunologia , Combinação de Medicamentos , Hepatite C Crônica/sangue , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Contagem de Leucócitos , Fígado/efeitos dos fármacos , Fígado/virologia , Ativação Linfocitária , Monócitos/imunologia , Células T Invariantes Associadas à Mucosa/efeitos dos fármacos , Células T Invariantes Associadas à Mucosa/virologia , Comunicação Parácrina , Fenótipo , Sofosbuvir/uso terapêutico , Resposta Viral Sustentada , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.
Assuntos
Anemia Sideroblástica/genética , Anti-Inflamatórios/uso terapêutico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Síndromes de Imunodeficiência/genética , Mutação , Nucleotidiltransferases/genética , RNA de Transferência/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Anemia Sideroblástica/sangue , Criança , Pré-Escolar , Citocinas/sangue , Citocinas/genética , Deficiências do Desenvolvimento/genética , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Humanos , Imunofenotipagem , Masculino , Linhagem , Fenótipo , Fator de Necrose Tumoral alfa/análise , Sequenciamento do ExomaRESUMO
Hepatitis B virus (HBV) infects hepatocytes specifically and causes immune-mediated liver damage. How HBV interacts with the innate immunity at the early phase of infection, either with hepatocytes or other cells in the liver, remains controversial. To address this question, we utilized various human cell-culture models and humanized Alb-uPA/SCID mice. All these models were unable to mount an interferon (IFN) response despite robust HBV replication. To elucidate the mechanisms involved in the lack of IFN response, we examined whether HBV actively inhibits innate immune functions of hepatocytes. By treating HBV-infected cells with known inducers of the IFN signaling pathway, we observed no alteration of either sensing or downstream IFN response by HBV. We showed that the DNA innate sensing pathways are poorly active in hepatocytes, consistent with muted innate immune recognition of HBV. Upon exposure to high-level HBV, human macrophages could be activated with increased inflammatory cytokine expressions. CONCLUSION: HBV behaves like a "stealth" virus and is not sensed by, nor actively interferes with, the intrinsic innate immunity of infected hepatocytes. Macrophages are capable of sensing HBV, but require exposure to high HBV titers, potentially explaining the long "window period" during acute infection and HBV's propensity to chronic infection. (Hepatology 2017;66:1779-1793).
Assuntos
Vírus da Hepatite B/fisiologia , Hepatócitos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Macrófagos/fisiologia , Citocinas/metabolismo , Células Hep G2 , Hepatite B/imunologia , Humanos , Interferons/metabolismoRESUMO
OBJECTIVE: Chronic HCV infection is characterised by innate immune activation with increased interferon-stimulated genes (ISG) expression and by an altered phenotype of interferon-responsive natural killer (NK) cells. Here, we asked whether a rapid reduction in viremia by daclatasvir (DCV) and asunaprevir (ASV) improves the response to pegylated interferon (PegIFN) in patients who had previously failed a standard course of PegIFN/ribavirin (RBV) therapy. DESIGN: Twenty-two HCV-infected non-responders to previous PegIFN/RBV therapy were studied for IFN-responsiveness of NK cells during quadruple (QUAD) therapy with DCV, ASV, PegIFN and RBV. A direct comparison of early NK cell responses in PegIFN/RBV therapy and QUAD therapy was performed for seven patients using paired cryopreserved peripheral blood mononuclear cells (PBMC) from both treatment courses. As a validation cohort, nine DCV/ASV-treated patients were studied for their NK cell response to in vitro stimulation with IFNα. RESULTS: The 24â h virological response to QUAD therapy correlated with an increase in signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) expression in NK cells, and the STAT1/pSTAT1/TRAIL induction was greater during QUAD therapy than during previous PegIFN/RBV therapy. Successful QUAD therapy as well as successful IFN-free DCV/ASV regimen resulted in an improved functional NK cell response (degranulation and TRAIL expression) to in vitro stimulation with IFNα. CONCLUSIONS: IFN-responsiveness can be improved by inhibiting HCV replication and reducing the HCV-induced activation of the innate immune response. This may provide a rationale for clinical trials of a brief period of direct acting antiviral therapy followed by PegIFN/RBV therapy to reduce the overall treatment costs in low-income and middle-income countries. TRIAL REGISTRATION NUMBERS: NCT01888900 and NCT00718172.
Assuntos
Antivirais/uso terapêutico , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/imunologia , Imidazóis/uso terapêutico , Interferon-alfa/uso terapêutico , Isoquinolinas/uso terapêutico , Células Matadoras Naturais/imunologia , Sulfonamidas/uso terapêutico , Adulto , Idoso , Carbamatos , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Quimioterapia Combinada , Feminino , Hepacivirus/genética , Hepatite C Crônica/sangue , Humanos , Interferon-alfa/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação , Pirrolidinas , Retratamento , Ribavirina/uso terapêutico , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Resposta Viral Sustentada , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Valina/análogos & derivados , Carga ViralRESUMO
BACKGROUND & AIMS: Chronic hepatitis C virus infection activates an intrahepatic immune response, leading to increased expression of interferon (IFN)-stimulated genes and activation of natural killer (NK) cells-the most prevalent innate immune cell in the liver. We investigated whether the elimination of hepatitis C virus with direct-acting antiviral normalizes expression of IFN-stimulated genes and NK cell function. METHODS: We used multicolor flow cytometry to analyze NK cells from the liver and blood of 13 HCV-infected patients who did not respond to treatment with pegylated interferon and ribavirin. Samples were collected before and during IFN-free treatment with daclatasvir and asunaprevir and compared with samples from the blood of 13 healthy individuals (controls). Serum levels of chemokine C-X-C motif ligand (CXCL) 10 or CXCL11 were measured by enzyme-linked immunosorbent assay. RESULTS: Before treatment, all patients had increased levels of CXCL10 or CXCL11 and a different NK cell phenotype from controls, characterized by increased expression of HLA-DR, NKp46, NKG2A, CD85j, signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). NK cells from patients also had increased degranulation and decreased production of IFNγ and tumor necrosis factor α compared with NK cells from controls. Nine patients had an end-of-treatment response (undetectable virus) and 4 had virologic breakthrough between weeks 4 and 12 of therapy. A rapid decrease in viremia and level of inflammatory cytokines in all patients was associated with decreased activation of intrahepatic and blood NK cells; it was followed by restoration of a normal NK cell phenotype and function by week 8 in patients with undetectable viremia. This normalized NK cell phenotype was maintained until week 24 (end of treatment). CONCLUSIONS: Direct-acting antiviral-mediated clearance of HCV is associated with loss of intrahepatic immune activation by IFNα, which is indicated by decreased levels of CXCL10 and CXCL11 and normalization of NK cell phenotype and function.
Assuntos
Antivirais/farmacologia , Hepacivirus/imunologia , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Ribavirina/farmacologia , Idoso , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/imunologia , Hepatite C Crônica/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Masculino , Pessoa de Meia-Idade , RNA Viral/efeitos dos fármacos , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: Production of interferon (IFN)-γ by natural killer (NK) cells is attenuated during chronic infection with hepatitis C virus (HCV). We investigated whether this is due to intrinsic or extrinsic mechanisms of NK cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from patients with chronic HCV infection or uninfected blood donors (controls); NK cells and monocytes were isolated or eliminated. We cultured hepatoma cells that express luciferase-tagged subgenomic HCV replicons (Huh7/HCV replicon cells) or their HCV-negative counterparts (Huh7) with NK cells in the presence or absence of other populations of PBMCs. Antiviral activity, cytotoxicity, and cytokine production were assessed. RESULTS: NK cells produced greater amounts of IFN-γ when PBMC were cocultured with Huh7/HCV replicon cells than with Huh7 cells; NK cells and PBMCs from controls suppressed HCV replication to a greater extent than those from patients with chronic HCV infection. This antiviral effect was predominantly mediated by tumor necrosis factor (TNF)-α and IFN-γ. The antiviral activity of NK cells and their production of IFN-γ were reduced when they were used in coculture alone (rather than with PBMC), or after depletion of CD14(+) monocytes, after knockdown of the inflammasome in monocytes, or after neutralization of interleukin-18, which is regulated by the inflammasome. These findings indicate a role for monocytes in NK cell activation. Compared with control monocytes, monocytes from patients with chronic HCV infection had reduced TNF-α-mediated (direct) and reduced NK cell-mediated (indirect) antiviral effects. Control monocytes increased the antiviral effects of NK cells from patients with chronic HCV infection and their production of IFN-γ. CONCLUSIONS: Monocytes sense cells that contain replicating HCV and respond by producing interleukin-18 via the inflammasome and by activating NK cells. Patients with chronic HCV infection have reduced monocyte function, attenuating NK cell IFN-γ-mediated responses.
Assuntos
Hepacivirus/fisiologia , Inflamassomos/fisiologia , Interleucina-18/fisiologia , Células Matadoras Naturais/fisiologia , Monócitos/fisiologia , Replicação Viral/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Hepatite C Crônica/patologia , Hepatite C Crônica/fisiopatologia , Hepatócitos/patologia , Hepatócitos/fisiologia , Hepatócitos/virologia , Humanos , Interferon gama/fisiologia , Células Matadoras Naturais/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/fisiopatologia , Monócitos/patologiaRESUMO
UNLABELLED: Ribavirin (RBV) is an important component of interferon (IFN)-based and direct antiviral treatment regimens for hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host IFN response, has been proposed as RBV's mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN-α/ß receptor signaling, as NK cell cytotoxicity and IFN-γ production are regulated by signal transducer and activator of transcription (STAT)1- and STAT4-phosphorylation, respectively. Specifically, pSTAT1-dependent NK cell cytotoxicity increases and pSTAT4-dependent IFN-γ production decreases in response to endogenous, virus-induced IFN-α and during IFN-α-based therapy. To assess whether RBV has a direct effect on NK cells and/or improves the IFN-γ response of NK cells in the presence of IFN-α, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of RBV pretreatment, who all received subsequent pegylated (Peg)IFN/ribavirin combination therapy. During RBV pretreatment, both the frequency of CD56(dim) NK cells with cytotoxic effector functions and the frequency of CD56(bright) NK cells with the capacity to produce IFN-γ decreased (P = 0.049 and P = 0.001, respectively). In vitro or in vivo exposure of NK cells to RBV improved the pSTAT4 (P < 0.01) but not pSTAT1 response of NK cells to subsequent stimulation with IFN-α. This was associated with an increase in IFN-γ production but not cytotoxicity of NK cells during subsequent IFN-α-based therapy. The frequency of IFN-γ-producing NK cells was greater in fast second-phase virological responders than in slow responders. CONCLUSION: RBV enhances the pSTAT4 and IFN-γ response of NK cells to IFN-α-stimulation.
Assuntos
Antivirais/uso terapêutico , Hepacivirus , Hepatite C/tratamento farmacológico , Interferon-alfa/uso terapêutico , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Polietilenoglicóis/uso terapêutico , Ribavirina/uso terapêutico , Adulto , Antivirais/farmacologia , Quimioterapia Combinada , Feminino , Hepatite C/metabolismo , Hepatite C/patologia , Humanos , Técnicas In Vitro , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/patologia , Masculino , Pessoa de Meia-Idade , Polietilenoglicóis/farmacologia , Estudos Prospectivos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Ribavirina/farmacologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: HCV relies on host lipid metabolism to complete its life cycle and HCV core is crucial to this interaction. Liver secreted ANGPTL-3 is an LXR- and HNF-1α-regulated protein, which plays a key role in lipid metabolism by increasing plasma lipids via inhibition of lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3 by HCV core and identify the molecular mechanisms involved. METHODS: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein levels in HCV patients, the JFH-1 infectious system and liver cell lines. Transfections, chromatin immunoprecipitation and immunofluorescence delineated parts of the molecular mechanisms implicated in the core-mediated regulation of ANGPTL-3 gene expression. RESULTS: ANGPTL-3 gene expression was decreased in HCV-infected patients and the JFH-1 infectious system. mRNA and promoter activity levels were down-regulated by core. The response was lost when an HNF-1α element in ANGPTL-3 promoter was mutated, while loss of HNF-1α DNA binding to this site was recorded in the presence of HCV core. HNF-1α mRNA and protein levels were not altered by core. However, trafficking between nucleus and cytoplasm was observed and then blocked by an inhibitor of the HNF-1α-specific kinase Mirk/Dyrk1B. Transactivation of LXR/RXR signalling could not restore core-mediated down-regulation of ANGPTL-3 promoter activity. CONCLUSIONS: ANGPTL-3 is negatively regulated by HCV in vivo and in vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1α binding activity and blockage of LXR/RXR transactivation. The putative ensuing increase in serum lipid clearance and uptake by the liver may sustain HCV virus replication and persistence.
Assuntos
Angiopoietinas/genética , Hepacivirus/patogenicidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Adulto , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , DNA/metabolismo , Regulação para Baixo , Feminino , Humanos , Receptores X do Fígado , Masculino , Pessoa de Meia-Idade , Receptores Nucleares Órfãos/fisiologia , Regiões Promotoras Genéticas , Receptores X de Retinoides/fisiologiaRESUMO
Hepatitis C virus non-enveloped particles circulate in the serum of HCV-infected patients and are believed to be involved in viral persistence. It was previously demonstrated that recombinant HCVne particles can efficiently enter T cells. In this study we investigated the effect of this entry on the phenotype and function of PBMCs, focused on the CD4+ and CD8+ T-cells. We have generated recombinant HCVne in the absence of other viral proteins. PBMCs from healthy donors were sampled after incubation either with HCVne or the control at different time points. Levels of expression of CD107a, CD25, CTLA-4, and T regulatory cells were estimated and cytokine expression and secretion were also monitored. Peripheral T cells expressed elevated CD127. The intracellular expression of the inhibitory marker CTLA-4 (CD152) increased significantly on peripheral T cells at late hours post-treatment, compared to the respective non-treated group. Despite the fact that there was an initial immune response due to HCVne uptake, T cells were driven to a partial exhausted phenotype. A significant induction of CD4+CD25+(hi)CD127-regulatory T cells at late hours was observed. Consistently, Foxp3+CD4+ T cells were also increased. In parallel, a significant transcriptional activation and increased secretion of IL-2, IL-10, and IFN-γ, was recorded. Moreover, mRNA transcription of TGF-ß was considerably elevated. HCVne particles have the potential to shape the immune response by modifying specific phenotypic and functional markers mainly on CD4+ T cells and driving them to partial exhaustion as well as to Treg expansion.
Assuntos
Capsídeo/metabolismo , Regulação da Expressão Gênica , Leucócitos Mononucleares/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/virologia , Antígeno CTLA-4/metabolismo , Citocinas/metabolismo , Hepacivirus/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Leucócitos Mononucleares/virologia , Fenótipo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Human type 1 diabetes (T1D) is caused by autoimmune attack on the insulin-producing pancreatic beta cells by islet antigen-reactive T cells. How human islet antigen-reactive (IAR) CD4+ memory T cells from peripheral blood affect T1D progression in the pancreas is poorly understood. Here, we aim to determine if IAR T cells in blood could be detected in pancreas. We identify paired αß (TRA/TRB) T cell receptors (TCRs) in IAR T cells from the blood of healthy, at-risk, new-onset, and established T1D donors, and measured sequence overlap with TCRs in pancreata from healthy, at risk and T1D organ donors. We report extensive TRA junction sharing between IAR T cells and pancreas-infiltrating T cells (PIT), with perfect-match or single-mismatch TRA junction amino acid sequences comprising ~29% total unique IAR TRA junctions (942/3,264). PIT-matched TRA junctions were largely public and enriched for TRAV41 usage, showing significant nucleotide sequence convergence, increased use of germline-encoded versus non-templated residues in epitope engagement, and a potential for cross-reactivity. Our findings thus link T cells with distinctive germline-like TRA chains in the peripheral blood with T cells in the pancreas.
Assuntos
Diabetes Mellitus Tipo 1 , Pâncreas , Receptores de Antígenos de Linfócitos T alfa-beta , Humanos , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/sangue , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Pâncreas/imunologia , Masculino , Feminino , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T/imunologia , Células Germinativas/imunologia , Células Germinativas/metabolismo , Autoantígenos/imunologiaRESUMO
Distinct Natural Killer (NK)-like CD57+ and PD-1+ CD8+ exhausted-like T cell populations (Tex) have both been linked to beneficial immunotherapy response in autoimmune type 1 diabetes (T1D) patients. The origins and relationships between these cell types are poorly understood. Here we show that while PD-1+ and CD57+ Tex populations are epigenetically similar, CD57+ Tex cells display unique increased chromatin accessibility of inhibitory Killer Cell Immunoglobulin-like Receptor (iKIR) and other NK cell genes. PD-1+ and CD57+ Tex also show reciprocal expression of Inhibitory Receptors (IRs) and iKIRs accompanied by chromatin accessibility of Tcf1 and Tbet transcription factor target sites, respectively. CD57+ Tex show unappreciated gene expression heterogeneity and share clonal relationships with PD-1+ Tex, with these cells differentiating along four interconnected lineage trajectories: Tex-PD-1+, Tex-CD57+, Tex-Branching, and Tex-Fluid. Our findings demonstrate new relationships between Tex-like populations in human autoimmune disease and suggest that modulating common precursor populations may enhance response to autoimmune disease treatment.
Assuntos
Linfócitos T CD8-Positivos , Diabetes Mellitus Tipo 1 , Células Matadoras Naturais , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/genética , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/genética , Antígenos CD57/metabolismo , Linhagem da Célula/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Feminino , Masculino , AdultoRESUMO
UNLABELLED: Early, vigorous intrahepatic induction of interferon (IFN)-stimulated gene (ISG) induction is a feature of hepatitis C virus (HCV) infection, even though HCV inhibits the induction of type I IFNs in vitro. To identify the cytokines and cells that drive ISG induction and mediate antiviral activity during acute HCV infection, type I and III IFN responses were studied in (1) serial liver biopsies and plasma samples obtained from 6 chimpanzees throughout acute HCV infection and (2) primary human hepatocyte (PHH) cultures upon HCV infection. Type I IFNs were minimally induced at the messenger RNA (mRNA) level in the liver and were undetectable at the protein level in plasma during acute HCV infection of chimpanzees. In contrast, type III IFNs, in particular, interleukin (IL)-29 mRNA and protein, were strongly induced and these levels correlated with ISG expression and viremia. However, there was no association between intrahepatic or peripheral type III IFN levels and the outcome of acute HCV infection. Infection of PHH with HCV recapitulated strong type III and weak type I IFN responses. Supernatants from HCV-infected PHH cultures mediated antiviral activity upon transfer to HCV-replicon-containing cells. This effect was significantly reduced by neutralization of type III IFNs and less by neutralization of type I IFNs. Furthermore, IL-29 production by HCV-infected PHH occurred independently from type I IFN signaling and was not enhanced by the presence of plasmacytoid dendritic cells. CONCLUSION: Hepatocyte-derived type III IFNs contribute to ISG induction and antiviral activity, but are not the principal determinant of the outcome of HCV infection.
Assuntos
Doenças dos Símios Antropoides/imunologia , Regulação da Expressão Gênica/imunologia , Hepatite C/imunologia , Hepatite C/veterinária , Hepatócitos/imunologia , Interleucinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Doenças dos Símios Antropoides/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/genética , Quimiocina CXCL11/metabolismo , Técnicas de Cocultura , Células Dendríticas , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatócitos/metabolismo , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Interferons , Interleucinas/genética , Proteínas de Resistência a Myxovirus , Pan troglodytes , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Viremia/virologia , Replicação ViralRESUMO
UNLABELLED: Natural killer (NK) cells exhibit a polarized phenotype with increased cytotoxicity and decreased interferon gamma (IFN-γ) production in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is caused by type I interferon (IFN)-induced expression and phosphorylation levels of signal transducer and activator of transcription (STAT) molecules in NK cells and whether it affects the response and refractoriness of NK cells to IFN-α-based therapy of HCV. STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN-α-based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK cytotoxicity (tumor necrosis factor-apoptosis-inducing ligand [TRAIL] expression and degranulation) and decreased IFN-γ production. NK cells from patients with a greater than 2 log(10) first-phase HCV RNA decline to IFN-α-based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with a less than 2 log(10) first-phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (P = 0.024), but retained greater IFN-α responsiveness in vitro (P = 0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN-α during the second-phase virological response. CONCLUSION: These data show that IFN-α-induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells toward increased cytotoxicity and decreased IFN-γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN-α-based therapy.
Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral/imunologia , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/imunologia , Interferon-alfa/uso terapêutico , Células Matadoras Naturais , Polietilenoglicóis/uso terapêutico , Adulto , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/imunologia , Estudos de Coortes , Feminino , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Masculino , Pessoa de Meia-Idade , Fosforilação/imunologia , Estudos Prospectivos , Proteínas Recombinantes/uso terapêutico , Ribavirina/uso terapêutico , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT4/imunologia , Fator de Transcrição STAT4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Human islet antigen reactive CD4 + memory T cells (IAR T cells) from peripheral blood have been studied extensively for their role in the pathogenesis of autoimmune type 1 diabetes (T1D). However, IAR T cells are rare, and it remains poorly understood how they affect T1D progression in the pancreas. Using single cell RNA-sequencing coupled with a multiplexed activation induced marker (AIM) enrichment assay, we identified paired TCR alpha/beta (TRA/TRB) T cell receptors (TCRs) in IAR T cells from the blood of healthy, at-risk, new onset, and established T1D donors. Using TCR sequences as barcodes, we measured infiltration of IAR T cells from blood into pancreas of organ donors with and without T1D. We detected extensive TCR sharing between IAR T cells from peripheral blood and pancreatic infiltrating T cells (PIT), with perfectly matched or single mismatched TRA junctions and J gene regions, comprising ~ 34% of unique IAR TCRs. PIT-matching IAR T cells had public TRA chains that showed increased use of germline-encoded residues in epitope engagement and a propensity for cross-reactivity. The link with T cells in the pancreas implicates autoreactive IAR T cells with shared TRA junctions and increased levels in blood with the prediabetic and new onset phases of T1D progression.
RESUMO
Teplizumab has been approved for the delay of the onset of type 1 diabetes and may modulate new onset disease. We found that patients who were EBV positive at baseline had a more robust response to drug in two clinical trials and therefore postulated that latent virus has general effects in modifying immune responses. We compared the phenotypes, transcriptomes, and development of peripheral blood cells before and after teplizumab treatment. Higher number of Tregs and partially exhausted CD8 + T cells were found in EBV seropositive individuals at the baseline in the TN10 trial and AbATE trial. Single cell transcriptomics and functional assays identified downregulation of the T cell receptor and other signaling pathways before treatment. Impairments in function of adaptive immune cells were enhanced by teplizumab treatment in EBV seropositive individuals. Our data indicate that EBV can impair signaling pathways generally in immune cells, that broadly redirect cell differentiation.
RESUMO
Variation in the preservation of ß cell function in clinical trials in type 1 diabetes (T1D) has emphasized the need to define biomarkers to predict treatment response. The T1DAL trial targeted T cells with alefacept (LFA-3-Ig) and demonstrated C-peptide preservation in approximately 30% of new-onset T1D individuals. We analyzed islet antigen-reactive (IAR) CD4+ T cells in PBMC samples collected prior to treatment from alefacept- and placebo-treated individuals using flow cytometry and single-cell RNA sequencing. IAR CD4+ T cells at baseline had heterogeneous phenotypes. Transcript profiles formed phenotypic clusters of cells along a trajectory based on increasing maturation and activation, and T cell receptor (TCR) chains showed clonal expansion. Notably, the frequency of IAR CD4+ T cells with a memory phenotype and a unique transcript profile (cluster 3) were inversely correlated with C-peptide preservation in alefacept-treated, but not placebo-treated, individuals. Cluster 3 cells had a proinflammatory phenotype characterized by expression of the transcription factor BHLHE40 and the cytokines GM-CSF and TNF-α, and shared TCR chains with effector memory-like clusters. Our results suggest IAR CD4+ T cells as a potential baseline biomarker of response to therapies targeting the CD2 pathway and warrant investigation for other T cell-related therapies.