RESUMEN
BACKGROUND & AIMS: Dysfunctional CD8(+) T cells are believed to contribute to the ability of hepatitis C virus (HCV) to evade the immune response. Most studies have focused on the effector functions of HCV-specific CD8(+) T cells or their surface expression of inhibitory receptors. There is currently no information available about the ex vivo ability of HCV-specific CD8(+) T cells to inhibit viral replication (antiviral efficacy). METHODS: To analyze the antiviral efficacy of virus-specific CD8(+) T cells ex vivo, we used an immunologic model based on a cell line that expresses HLA-A*02 and contains a stably replicating HCV reporter replicon. We isolated HCV-specific CD8(+) T cells from 18 HLA-A*02-positive patients with chronic HCV infection and 15 subjects with resolved HCV infection (7 spontaneous, 8 after therapy). Replicon cells were labeled with virus-specific peptides; inhibition of HCV replication was determined by measuring luciferase activity after 72 hours of coculture with virus-specific CD8(+) T cells. RESULTS: HCV-specific CD8(+) T cells from patients with chronic HCV infection had a significantly lower antiviral efficacy than influenza-, Epstein-Barr virus-, and cytomegalovirus-specific CD8(+) T cells. Antiviral efficacy was associated with the ability of virus-specific CD8(+) T cells to secrete interferon gamma. The antiviral efficacy of HCV-specific CD8(+) T cells was linked to surface expression of CD127 and PD-1. The cytokines interleukin-2, interleukin-7, and interleukin-15 increased the antiviral efficacy of CD127-positive but not of CD127-negative, HCV-specific CD8(+) T cells. Spontaneous, but not antiviral therapy-induced, viral clearance was associated with increased antiviral efficacy. CONCLUSIONS: The ability of CD8(+) T cells to inhibit HCV replication ex vivo is associated with their ability to secrete interferon gamma and their surface expression of CD127 and PD-1.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Inmunidad Celular/inmunología , Replicación Viral/inmunología , Adulto , Anciano , Antivirales/uso terapéutico , Células Cultivadas , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Hepacivirus/genética , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-7/biosíntesis , Subunidad alfa del Receptor de Interleucina-7/inmunología , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/biosíntesis , Carga Viral/inmunologíaRESUMEN
Dipeptidylpeptidase IV (CD26) is a multifunctional ectoenzyme involved in T cell activation that has been implicated in autoimmune pathophysiology. Because IL-17-producing CD4(+) T cells (Th17 cells) are important mediators of autoimmune disease, we analyzed the expression of CD26 and its enzymatic function on human Th17 cells. Analysis of CD26 expression on different CD4(+) T helper subsets showed that CD26 expression is highest on CD4(+) T cells producing type 17 cytokines (e.g., IL-22, IL-17, GM-CSF, or TNF) compared with Th1, Th2, and regulatory T cells. Phenotypic analysis revealed that CD26(++)CD4(+) T cells express the type 17 differentiation molecules CD161, CCR6, lL-23R, and retinoic acid-related orphan receptor-γt. Furthermore, sorted CD26(++)CD4(+) T cells contain >90-98% of Th17 cells, indicating that CD26(++) T cells harbor the Th17 lineage. A comparison with CD161 and CCR6 indicated that analysis of CD26 coexpression may improve the phenotypic characterization of Th17 cells. Of note, CD26(++) Th17 cells are enriched in the inflamed tissue of patients with hepatitis and inflammatory bowel disease. Functional analysis in migration assays revealed that CD26 expressed on Th17 cells is enzymatically active. Indeed, CD26 negatively regulates the chemotactic CD4(+) T cell response to the inflammatory chemokines CXCL9-12 that can be restored by pharmacological blockade of the enzymatic center of CD26. In summary, these results strongly suggest that CD26 may contribute to the orchestration of the immune response by Th17 cells in human inflammatory diseases. They also suggest that the phenotypic analysis of Th17 cells may be facilitated by determination of CD26 expression.
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Dipeptidil Peptidasa 4/biosíntesis , Células Th17/enzimología , Células Th17/inmunología , Regulación hacia Arriba/inmunología , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Dipeptidil Peptidasa 4/sangre , Dipeptidil Peptidasa 4/metabolismo , Hepatitis Autoinmune/enzimología , Hepatitis Autoinmune/inmunología , Hepatitis Autoinmune/patología , Hepatitis Viral Humana/enzimología , Hepatitis Viral Humana/inmunología , Hepatitis Viral Humana/patología , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/enzimología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Interleucina-17/biosíntesis , Linfocitos T Reguladores/enzimología , Linfocitos T Reguladores/inmunología , Células TH1/enzimología , Células TH1/inmunología , Células Th2/enzimología , Células Th2/inmunologíaRESUMEN
CD8(+) T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8(+) T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8(+)CD161(+) T cells in healthy donors and those with hepatitis C virus and defined a population of CD8(+) T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor gamma-t, P = 6 x 10(-9); RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 x 10(-7); IL-18 receptor, P = 4 x 10(-6)), and chemokine receptors (e.g., CCR6, P = 3 x 10(-8); CXCR6, P = 3 x 10(-7); CCR2, P = 4 x 10(-7)). CD161(+)CD8(+) T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-gamma and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8(+) T cells in normal humans and a substantial fraction of tissue-infiltrating CD8(+) T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.
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Linfocitos T CD8-positivos/metabolismo , Hepacivirus/inmunología , Hepatitis C/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Subgrupos de Linfocitos T/inmunología , Alanina Transaminasa/metabolismo , Linfocitos T CD8-positivos/clasificación , Línea Celular , Citocinas/metabolismo , Sangre Fetal/citología , Sangre Fetal/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunofenotipificación , Luciferasas , Receptores de Citocinas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND & AIMS: Perforin plays a central role in the immunopathogenesis of different viral infections. However, its role in hepatitis C virus (HCV) infection has not been fully understood. Here, we analyzed two closely related questions: first, is CD8+ T cell-mediated killing of HCV-replicating human hepatoma cells mediated by perforin? Second, if so, do HCV-specific CD8+ T cells obtained from chronically HCV infected patients express and upregulate perforin? METHODS: Susceptibility of HCV-replicating human hepatoma cells to the cytotoxic pathway was tested in vitro by addition of perforin substitute streptolysin O and granzyme B and by co-culture experiments with a perforin-expressing HCV-specific CD8+ T cell clone in the presence of perforin or caspase inhibitors. HCV-specific CD8+ T cells were obtained and analyzed for perforin expression and differentiation markers ex vivo from 12 chronically infected patients and 12 patients with resolved HCV infection. RESULTS: HCV-replicating human hepatoma cells were susceptible to cytotoxic killing in vitro and a dominant role of perforin in HCV-specific CD8+ T cell-mediated cytolysis was observed. However, HCV-specific CD8+ T cells obtained ex vivo from chronically HCV infected patients expressed only low levels of perforin and showed an impaired ability to upregulate perforin. This was tightly linked to the distinct differentiation stage of HCV-specific CD8+ T cell differentiation ex vivo since early and intermediate differentiated HCV-specific CD8+ T cells only showed weak perforin expression in contrast to late differentiated CD8+ T cells that displayed strong perforin expression. CONCLUSIONS: Our results suggest that perforin plays a dominant role in CD8+ T cell-mediated lysis of HCV-replicating human hepatoma cells but that lysis may be limited in human chronic viral infection by the low perforin expression of early/intermediate differentiated HCV-specific CD8+ T cells.
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Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/virología , Hepacivirus/crecimiento & desarrollo , Hepatitis C Crónica/inmunología , Hepatocitos/citología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Adulto , Anciano , Linfocitos T CD8-positivos/metabolismo , Carcinoma Hepatocelular , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Células Cultivadas , Femenino , Hepatocitos/virología , Humanos , Neoplasias Hepáticas , Masculino , Persona de Mediana Edad , Perforina , Proteínas Citotóxicas Formadoras de Poros/metabolismoRESUMEN
Exhausted CD8+ T cell responses during chronic viral infections are defined by a complex expression pattern of inhibitory receptors. However, very little information is currently available about the coexpression patterns of these receptors on human virus-specific CD8+ T cells and their correlation with antiviral functions, T cell differentiation and antigen recognition. We addressed these important aspects in a cohort of 38 chronically HCV infected patients and found a coexpression of inhibitory receptors such as 2B4, CD160 and KLRG1 in association with PD-1 in about half of the HCV-specific CD8+ T cell responses. Importantly, this exhaustive phenotype was associated with low and intermediate levels of CD127 expression, an impaired proliferative capacity, an intermediate T cell differentiation stage and absence of sequence variations within the corresponding epitopes, indicating ongoing antigen triggering. In contrast, a low expression of inhibitory receptors by the remaining HCV-specific CD8+ T cells occurred in concert with a CD127hi phenotype, an early T cell differentiation stage and presence of viral sequence variations within the corresponding epitopes. In sum, these results suggest that T cell exhaustion contributes to the failure of about half of HCV-specific CD8+ T cell responses and that it is determined by a complex interplay of immunological (e.g. T cell differentiation) and virological (e.g. ongoing antigen triggering) factors.
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Antígenos CD/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Hepatitis C/inmunología , Lectinas Tipo C/metabolismo , Receptores Inmunológicos/metabolismo , Transactivadores/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Citometría de Flujo , Proteínas Ligadas a GPI , Hepacivirus/aislamiento & purificación , Hepatitis C/virología , Humanos , Activación de Linfocitos , Receptor de Muerte Celular Programada 1 , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Carga ViralRESUMEN
Hepatitis C virus infection is a major cause of chronic liver disease. CD4(+) T cells play a key role in disease outcome. However, the critical functions and associated phenotypes of intrahepatic CD4(+) T cells are not well defined. We have previously shown that CD8(+) T cells expressing the C type lectin CD161 are highly enriched in the human liver, especially during chronic hepatitis. These cells are associated with a type 17 differentiation pattern and express cytokines including IL-17A, IL-22, and IFN-γ. We therefore analyzed expression of CD161 on CD4(+) T cells in blood and liver and addressed the relevant phenotype and functional capacity of these populations. We observed marked enrichment of CD161(+)CD4(+) T cells in the liver during chronic hepatitis such that they are the dominant subtype (mean 55% of CD4(+) T cells). IL-22 and IL-17 secreting CD4(+) T cells were readily found in the livers of HCV(+) and NASH donors, although not enriched compared to blood. There was, however, specific enrichment of a novel subset of IL-22/IFN-γ dual secretors (p = 0.02) compared to blood, a result reconfirmed with direct ex vivo analyses. These data indicate the dominance of CD161(+) expressing lymphocyte populations within the hepatic infiltrate, associated with a distinct cytokine profile. Given their documented roles as antiviral and hepatoprotective cytokines respectively, the impact of co-secretion of IFN-γ and IL-22 in the liver may be particularly significant.
RESUMEN
It has been shown previously that suppressive virus-specific FoxP3+ regulatory CD8+ T cells can be expanded from human peripheral blood mononuclear cells after in vitro antigen-specific stimulation. This study extended this finding by analysing the mechanisms of virus-specific FoxP3+ regulatory CD8+ T-cell generation during peptide-specific expansion in vitro. It was shown that hepatitis C virus (HCV)-, influenza virus (FLU)-, Epstein-Barr virus (EBV)- and cytomegalovirus (HCMV)-specific FoxP3+ regulatory CD8+ T cells could be expanded differentially from the blood of chronically HCV-infected patients following in vitro peptide-specific stimulation. The different ability of virus-specific CD8+ T-cell populations to express FoxP3 after continuous antigen stimulation in vitro correlated significantly with the ex vivo differentiation status. Indeed, CD27+ CD28+ CD57- HCV-, FLU- and EBV-specific CD8+ T cells displayed a significantly higher ability to give rise to FoxP3+ regulatory CD8+ T cells compared with CD27- CD28- CD57+ HCMV-specific CD8+ T cells. Similar T-cell receptor expression patterns of FoxP3+ versus FoxP3- CD8+ T cells of the same antigen specificity indicated that both cell populations were probably expanded from the same virus-specific CD8+ T-cell precursor. In addition, no specific antigen-presenting cell populations were required for the generation of FoxP3+ CD8+ T cells, as CD8+-selected virus-specific FoxP3+ CD8+ T cells could be expanded by peptide presentation in the absence of antigen-presenting cells. Taken together, these results suggest that the ability to expand FoxP3+ regulatory CD8+ T cells from virus-specific CD8+ T cells differs among distinct virus-specific CD8+ T-cell populations depending on the differentiation status.