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Immunotherapies targeting the "don't eat me" myeloid checkpoint constituted by CD47 SIRPα interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti-CD47-SIRPα therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies and the strong ADCC (antibody-dependent cellular cytotoxicity) effector function of polymorphonuclear cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb-dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRPα checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRPα blockade, and ROS induction. Although previous studies had concluded that disruption of SIRPα signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRPα also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRPα immune checkpoint and to design agents able to enhance myeloid cell function while limiting adverse effects on healthy cells able to participate in the anti-tumor immune response.
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Antígenos de Diferenciación , Antígeno CD47 , NADPH Oxidasas , Neoplasias , Receptores Inmunológicos , Linfocitos T , Trogocitosis , Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos de Diferenciación/inmunología , Antígeno CD47/inmunología , Activación Enzimática , Humanos , Recuento de Linfocitos , NADPH Oxidasas/inmunología , NADPH Oxidasas/metabolismo , Neoplasias/inmunología , Neoplasias/terapia , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Trogocitosis/inmunologíaRESUMEN
PMN-MDSCs support tumor progression and resistance to ICI therapy through their suppressive functions but their heterogeneity limits their use as biomarkers in cancer. Our aim was to investigate the phenotypic and functional subsets of PMN-MDSCs to identify biomarkers of response to ICI therapy. We isolated low-density CD15+ PMNs from patients with metastatic melanoma and assessed their immune-suppressive capacities. Expression of CD10 and CD16 was used to identify mature and immature subsets and correlate them to inhibition of T cell proliferation or direct cytotoxicity. Frequencies of the PMN-MDSCs subsets were next correlated to the radiological response of 36 patients receiving ICI therapy. Mature activated cells constituted the major population of PMN-MDSCs. They were found in a higher proportion in the pre-treatment blood of patients non responders to ICI. A subset of immature cells characterized by intermediate levels of CD10 and CD16, the absence of expression of SIRPα and a strong direct cytotoxicity to T cells was increased in patients responding to ICI. The paradoxical expansion of such cells during ICI therapy suggests a role of PMNs in the inflammatory events associated to efficient ICI therapy and the usefulness of their monitoring in patients care.
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BACKGROUND: Knee osteoarthritis (OA) is a painful condition affecting >250 million people worldwide and is a leading cause of disability. Intra-articular (IA) corticosteroids and/or oral opioids are often recommended for the management of knee OA pain. There are, however, concerns regarding their safety and tolerability. STUDY QUESTION: Do patients diagnosed with knee OA show a decrease in opioids or IA corticosteroid injections prescribed/administered in hospitals following hylan G-F 20 treatment? STUDY DESIGN: This case-crossover, retrospective study using Health Facts®, a de-identified electronic health records database, enrolled patients ≥18 years with knee OA treated with hylan G-F 20 between January 1, 2000 and March 31, 2016, with data within 6 months before/after treatment. MEASURES AND OUTCOMES: Primary endpoints compared days on opioids, amounts of opioids, and number of IA corticosteroid injections before/after hylan G-F 20 treatment via paired t-tests. RESULTS: A total of 513 patients were qualified for analysis. In the opioid cohort, the average total number of days on opioids (N = 50; 5.0 vs 13.5 days; P = 0.007) and average total amount of opioids (N = 44; 165.4 morphine mg equivalents [MME] vs 493.7 MME; P = 0.013) were lower 6 months after hylan G-F 20 treatment than 6 months before treatment. In the IA corticosteroid cohort, the average number of IA corticosteroid injections decreased after hylan G-F 20 treatment (N = 36; 0.56 in the 6-month follow-up vs 1.39 before treatment; P < 0.0001). Additional time frames of 1-5 months before and after treatment were examined; similar conclusions were drawn for patients with >2 months of data. CONCLUSION: Patients with knee OA previously treated with opioids or IA corticosteroid injections who received hylan G-F 20 demonstrated statistically significant decreases in these medications >2 months following hylan G-F 20 treatment versus >2 months before treatment.
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A better characterization of T-cell subsets in the microenvironment of classical Hodgkin lymphoma (cHL) would help to develop immunotherapies. Using multicolor flow cytometry, we identified in 6 of 43 cHL tissue samples a previously unrecognized subset of CD8 T cells coexpressing CXCR5 and inducible T-cell costimulator (ICOS) molecules (CD8CXCR5+ICOS+). These cells shared phenotypic features with follicular helper T (TFH) cells including low CCR7 expression together with high expression of B-cell lymphoma-6, programmed cell death 1, B and T lymphocyte attenuator, CD200, and OX40. They had deficient cytotoxicity, low interferon-γ secretion, and common functional properties with intratumoral CD4+ TFH cells, such as production of interleukin-4 (IL-4), IL-21, CXCL13, and capacity to sustain B cells. Gene profiling analysis showed a significant similarity between the signatures of CD8CXCR5+ICOS+ T cells and CD4+ TFH cells. Benign lymphadenitis tissues (n = 8) were devoid of CD8CXCR5+ICOS+ cells. Among the 35 B-cell lymphoma tissues analyzed, including follicular lymphomas (n = 13), diffuse large cell lymphomas (n = 12), marginal zone lymphomas (MZLs; n = 3), mantle cell lymphomas (n = 3), and chronic lymphocytic leukemias (n = 4), only 1 MZL sample contained CD8CXCR5+ICOS+ cells. Lymphoma tumors with CD8CXCR5+ICOS+ cells shared common histopathological features including residual germinal centers, and contained high amounts of activated CD8CXCR5-ICOS+ cells. These data demonstrate a CD8 T-cell differentiation pathway leading to the acquisition of some TFH similarities. They suggest a particular immunoediting process with global CD8 activation acting mainly, but not exclusively, in HL tumors.
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Linfocitos T CD8-positivos/metabolismo , Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/metabolismo , Proteína Coestimuladora de Linfocitos T Inducibles/biosíntesis , Proteínas de Neoplasias/biosíntesis , Receptores CXCR5/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Diferenciación Celular , Citocinas/metabolismo , Femenino , Enfermedad de Hodgkin/patología , Humanos , MasculinoRESUMEN
Natural-killer (NK) cells are important immune effectors during a viral infection. Latent CMV infection is widely spread and was demonstrated to shape the NK cell repertoire through the NKG2C receptor. An expansion of NKG2C+ NK cells has been reported during primary HIV infection (PHI), but their role is not known. We previously found a correlation between the maturation state of the NK cell compartment and a lower viral load by studying patients from the ANRS 147 Optiprim trial. We investigated here extensively the NKG2C+ NK cells at the time of PHI and its evolution after 3 months of early antiretroviral therapy (combination antiretroviral therapy (cART)). Multiparametric cytometry combined with bioinformatics was used to determine subsets. NKbright NKG2C+ progenitor, NKdim NKG2C+ effector and NKdim NKG2C+CD57+ memory-like populations were identified. Two groups of patients were unraveled according to the distribution of the NKG2C+ subsets skewed toward either progenitor/effector or memory-like phenotype. Patients with high NKG2C+CD57+ NK cell frequencies showed lower HIV-RNA, lower immune activation, higher pDC counts and reached more rapidly undetectable levels of HIV-RNA at M1 under cART. NKG2C+CD57+ NK cell frequency was the only factor strongly correlated to low viral load among other clinical features. While the patients were cytomegalovirus (CMV) infected, there was no sign of reactivation of CMV during PHI suggesting that memory-like NK cells were already present at the time of HIV infection and constituted a preexisting immune response able to contribute to natural control of HIV. This parameter appears to be a good candidate in the search of predictive markers to monitor HIV remission.
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Natural killer (NK) cells are major effectors of the innate immune response. Despite an overall defect in their function associated with chronic human immunodeficiency virus (HIV) infection, their role in primary HIV infection is poorly understood. We investigated the modifications of the NK cell compartment in patients from the ANRS-147-Optiprim trial, a study designed to examine the benefits of intensive combination antiretroviral therapy (cART) in patients with acute or early primary HIV infection. Multiparametric flow cytometry combined with bioinformatics analyses identified the NK phenotypes in blood samples from 30 primary HIV-infected patients collected at inclusion and after 3 months of cART. NK phenotypes were revealed by co-expression of CD56/CD16/NKG2A/NKG2C and CD57, five markers known to delineate stages of NK maturation. Three groups of patients were formed according to their distributions of the 12 NK cell phenotypes identified. Their virological and immunological characteristics were compared along with the early outcome of cART. At inclusion, HIV-infected individuals could be grouped into those with predominantly immature/early differentiated NK cells and those with predominantly mature NK cells. Several virological and immunological markers were improved in patients with mature NK profiles, including lower HIV viral loads, lower immune activation markers on NK and dendritic cell (DC), lower levels of plasma IL-6 and IP-10, and a trend to normal DC counts. Whereas all patients showed a decrease of viremia higher than 3 log10 copies/ml after 3 months of treatment, patients with a mature NK profile at inclusion reached this threshold more rapidly than patients with an immature NK profile (70 vs. 38%). In conclusion, a better early response to cART is observed in patients whose NK profile is skewed to maturation at inclusion. Whether the mature NK cells contributed directly or indirectly to HIV control through a better immune environment under cART is unknown. The NK maturation status of primary infected patients should be considered as a relevant marker of an immune process contributing to the early outcome of cART that could help in the management of HIV-infected patients.
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The prognosis of follicular lymphoma (FL) patients is suspected to be influenced by tumor-infiltrating regulatory T cells (Treg). The mechanism of Treg enrichment in FL and their impact on malignant FL B cells remains to be elucidated. We analyzed 46 fresh lymph node biopsy samples, including FL (n = 20), diffuse large B-cell lymphoma (n = 10), classical Hodgkin lymphoma (n = 9), and reactive lymphadenitis (n = 7). Using multicolor flow cytometry and cell sorting, we observed an accumulation of CD25(high)CD127(low/neg) Tregs in FL tissues. These Tregs comprised activated ICOS(+) Tregs that were able to suppress not only conventional T cells, but also FL B cells. These FL B cells were able to express ICOSL in vitro and to generate CD25(high)FoxP3(high) Tregs expressing ICOS. Treg generation was associated with ICOS/ICOSL engagement and was abrogated by antagonist anti-ICOS and anti-ICOSL antibodies. Interactions between Tregs and FL B cells resulted in ICOSL downregulation on FL B cells. Our results highlight a key role for Tregs in FL pathogenesis and suggest that targeting the ICOS/ICOSL pathway may be a promising immunotherapy for FL treatment. Cancer Res; 76(16); 4648-60. ©2016 AACR.
Asunto(s)
Ligando Coestimulador de Linfocitos T Inducibles/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfoma de Células B/inmunología , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Separación Celular , Citometría de Flujo , Humanos , Inmunohistoquímica , Ligando Coestimulador de Linfocitos T Inducibles/antagonistas & inhibidores , Proteína Coestimuladora de Linfocitos T Inducibles/antagonistas & inhibidores , Ratones , Reacción en Cadena de la Polimerasa , Transducción de Señal/inmunología , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
Understanding immune alterations in cancer patients is a major challenge and requires precise phenotypic study of immune subsets. Improvement of knowledge regarding the biology of natural killer (NK) cells and technical advances leads to the generation of high dimensional dataset. High dimensional flow cytometry requires tools adapted to complex dataset analyses. This study presents an example of NK cell maturation analysis in Healthy Volunteers (HV) and patients with Acute Myeloid Leukemia (AML) with an automated procedure using the FLOCK algorithm. This procedure enabled to automatically identify NK cell subsets according to maturation profiles, with 2D mapping of a four-dimensional dataset. Differences were highlighted in AML patients compared to HV, with an overall increase of NK maturation. Among patients, a strong heterogeneity in NK cell maturation defined three distinct profiles. Overall, automatic gating with FLOCK algorithm is a recent procedure, which enables fast and reliable identification of cell populations from high-dimensional cytometry data. Such tools are necessary for immune subset characterization and standardization of data analyses. This tool is adapted to new immune cell subsets discovery, and may lead to a better knowledge of NK cell defects in cancer patients. Overall, 2D mapping of NK maturation profiles enabled fast and reliable identification of NK cell subsets.
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Q fever endocarditis is characterized by a defective cell-mediated immune response, which may be associated with the dysregulation of circulating subsets of immune cells. In this study, we found that naïve CD8(+) T lymphocytes and CD56dim natural killer cells were decreased patients whereas central memory CD8(+) T lymphocytes were increased. It is likely that these different subsets of immune cells play a role in the immunosuppression accompanying Q fever endocarditis.
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Sangre/inmunología , Linfocitos T CD8-positivos/inmunología , Endocarditis Bacteriana/inmunología , Células Asesinas Naturales/inmunología , Subgrupos Linfocitarios/inmunología , Fiebre Q/inmunología , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Recuento de Linfocitos , Masculino , Persona de Mediana EdadRESUMEN
Q fever endocarditis, a severe complication of Q fever, is associated with a defective immune response, the mechanisms of which are poorly understood. We hypothesized that Q fever immune deficiency is related to altered distribution and activation of circulating monocyte subsets. Monocyte subsets were analyzed by flow cytometry in peripheral blood mononuclear cells from patients with Q fever endocarditis and controls. The proportion of classical monocytes (CD14(+)CD16(-) monocytes) was similar in patients and controls. In contrast, the patients with Q fever endocarditis exhibited a decrease in the non-classical and intermediate subsets of monocytes (CD16(+) monocytes). The altered distribution of monocyte subsets in Q fever endocarditis was associated with changes in their activation profile. Indeed, the expression of HLA-DR, a canonical activation molecule, and PD-1, a co-inhibitory molecule, was increased in intermediate monocytes. This profile was not restricted to CD16(+) monocytes because CD4(+) T cells also overexpressed PD-1. The mechanism leading to the overexpression of PD-1 did not require the LPS from C. burnetii but involved interleukin-10, an immunosuppressive cytokine. Indeed, the incubation of control monocytes with interleukin-10 led to a higher expression of PD-1 and neutralizing interleukin-10 prevented C. burnetii-stimulated PD-1 expression. Taken together, these results show that the immune suppression of Q fever endocarditis involves a cross-talk between monocytes and CD4(+) T cells expressing PD-1. The expression of PD-1 may be useful to assess chronic immune alterations in Q fever endocarditis.
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Endocarditis Bacteriana/inmunología , Interleucina-10/sangre , Monocitos/inmunología , Receptor de Muerte Celular Programada 1/sangre , Fiebre Q/inmunología , Adulto , Anciano , Coxiella burnetii/inmunología , Endocarditis Bacteriana/microbiología , Femenino , Expresión Génica , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Fiebre Q/microbiología , Linfocitos T Reguladores/inmunologíaRESUMEN
The elimination of hepatitis C virus (HCV) in > 50% of chronically infected patients by treatment with IFN-α suggests that plasmacytoid dendritic cells (pDCs), major producers of IFN-α, play an important role in the control of HCV infection. However, despite large amounts of Toll-like receptor 7-mediated IFN-α, produced by pDCs exposed to HCV-infected hepatocytes, HCV still replicates in infected liver. Here we show that HCV envelope glycoprotein E2 is a novel ligand of pDC C-type lectin immunoreceptors (CLRs), blood DC antigen 2 (BDCA-2) and DC-immunoreceptor (DCIR). HCV particles inhibit, via binding of E2 glycoprotein to CLRs, production of IFN-α and IFN-λ in pDCs exposed to HCV-infected hepatocytes, and induce in pDCs a rapid phosphorylation of Akt and Erk1/2, in a manner similar to the crosslinking of BDCA-2 or DCIR. Blocking of BDCA-2 and DCIR with Fab fragments of monoclonal antibodies preserves the capacity of pDCs to produce type I and III IFNs in the presence of HCV particles. Thus, negative interference of CLR signaling triggered by cell-free HCV particles with Toll-like receptor signaling triggered by cell-associated HCV results in the inhibition of the principal pDC function, production of IFN.
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Células Dendríticas/inmunología , Interferones/inmunología , Lectinas Tipo C/inmunología , Glicoproteínas de Membrana/inmunología , Receptores Inmunológicos/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Células COS , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Células Dendríticas/metabolismo , Células Dendríticas/virología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Citometría de Flujo , Hepacivirus/inmunología , Hepacivirus/metabolismo , Hepacivirus/fisiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Interferones/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligandos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 7/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The development of polychromatic cytometry has contributed to significant progress in the field of human immunology. Although numerous functional studies of rare cell populations have been performed using this technology, here we used polychromatic cytometry to explore the dynamics of complex cellular systems implicated in innate immunity. We used PBMC stimulated with live influenza virus as an experimental model. We studied the time course of activation of PBMC, which contain DC, monocytes, and NK cells, all of which are, in addition to their innate immune properties, susceptible to Flu infection. We developed 12 color panels to investigate intracellular expression of IFN-α, TNF-α, IL-12, IL-6, IFN-γ, CD107, and influenza virus nucleoprotein simultaneously in these cell populations. These panels allowed reproducible determination of activation markers induced in DC after their direct exposure to various stimulations or in NK cells by indirect DC-mediated activation within the complex cellular environment. The ability to use a low number of cells and reduced quantities of reagents permitted us to perform kinetic experiments. The power of polychromatic cytometry associated with bioinformatic tools allowed us to analyze the multiple functional data generated as dynamic clustering maps. These maps present a readily understandable view of activation events induced in different populations of PBMC. In addition, it reveals new information on the coordination of the complex pathways induced and on the cellular interactions that sustained indirect DC-mediated NK cell activation. Our work shows that polychromatic cytometry is a tool for discoveries in unexplored complex cell systems, at the crossroads of immunology and virology. © 2012 International Society for Advancement of Cytometry.
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Citometría de Flujo/métodos , Animales , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Perros , Humanos , Inmunidad Innata , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismoRESUMEN
Plasmacytoid dendritic cells (pDCs) respond to viral infection by production of alpha interferon (IFN-α), proinflammatory cytokines, and cell differentiation. The elimination of hepatitis C virus (HCV) in more than 50% of chronically infected patients by treatment with IFN-α suggests that pDCs can play an important role in the control of HCV infection. pDCs exposed to HCV-infected hepatoma cells, in contrast to cell-free HCV virions, produce large amounts of IFN-α. To further investigate the molecular mechanism of HCV sensing, we studied whether exposure of pDCs to HCV-infected hepatoma cells activates, in parallel to interferon regulatory factor 7 (IRF7)-mediated production of IFN-α, nuclear factor kappa B (NF-κB)-dependent pDC responses, such as expression of the differentiation markers CD40, CCR7, CD86, and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and secretion of the proinflammatory cytokines TNF-α and interleukin 6 (IL-6). We demonstrate that exposure of pDCs to HCV-infected hepatoma cells surprisingly did not induce phosphorylation of NF-κB or cell surface expression of CD40, CCR7, CD86, or TRAIL or secretion of TNF-α and IL-6. In contrast, CpG-A and CpG-B induced production of TNF-α and IL-6 in pDCs exposed to the HCV-infected hepatoma cells, showing that cell-associated virus did not actively inhibit Toll-like receptor (TLR)-mediated NF-κB phosphorylation. Our results suggest that cell-associated HCV signals in pDCs via an endocytosis-dependent mechanism and IRF7 but not via the NF-κB pathway. In spite of IFN-α induction, cell-associated HCV does not induce a full functional response of pDCs. These findings contribute to the understanding of evasion of immune responses by HCV.
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Células Dendríticas/inmunología , Células Dendríticas/virología , Hepacivirus/fisiología , Hepatitis C/inmunología , FN-kappa B/inmunología , Transducción de Señal , Línea Celular Tumoral , Células Cultivadas , Hepacivirus/genética , Hepatitis C/genética , Hepatitis C/virología , Humanos , Interferón-alfa/genética , Interferón-alfa/inmunología , FN-kappa B/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
B and T lymphocyte attenuator (BTLA), like its relative programmed cell death-1 (PD-1), is a receptor that negatively regulates murine T cell activation. However, its expression and function on human T cells is currently unknown. We report in this study on the expression of BTLA in human T cell subsets as well as its regulation on virus-specific T cells during primary human CMV infection. BTLA is expressed on human CD4(+) T cells during different stages of differentiation, whereas on CD8(+) T cells, it is found on naive T cells and is progressively downregulated in memory and differentiated effector-type cells. During primary CMV infection, BTLA was highly induced on CMV-specific CD8(+) T cells immediately following their differentiation from naive cells. After control of CMV infection, BTLA expression went down on memory CD8(+) cells. Engagement of BTLA by mAbs blocked CD3/CD28-mediated T cell proliferation and Th1 and Th2 cytokine secretion. Finally, in vitro blockade of the BTLA pathway augmented, as efficient as anti-PD-1 mAbs, allogeneic as well as CMV-specific CD8(+) T cell proliferation. Thus, our results suggest that, like PD-1, BTLA provides a potential target for enhancing the functional capacity of CTLs in viral infections.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Citomegalovirus/inmunología , Epítopos de Linfocito T/inmunología , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Animales , Antígenos CD/biosíntesis , Antígenos CD/fisiología , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/fisiología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/prevención & control , Citotoxicidad Inmunológica/genética , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Femenino , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Receptor de Muerte Celular Programada 1 , Receptores Inmunológicos/antagonistas & inhibidores , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Linfocitos T Citotóxicos/virología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
B- and T-lymphocyte attenuator (BTLA) is a lymphoid receptor that inhibits lymphocyte activation on interaction with its ligand, herpesvirus entry mediator (HVEM). We developed monoclonal antibodies against BTLA and HVEM to study their expression using immunohistochemical and flow cytometric analyses in human tissues. In reactive lymph nodes, they were both expressed in interfollicular T cells and in B cells from mantle and marginal zones. Within germinal centers, B cells were negative, whereas T follicular helper (TFH) cells were BTLA+ and follicular dendritic cells were HVEM+. BTLA was strongly expressed in chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL, 19 of 19 positive) when compared with other small B-cell lymphomas, including follicular lymphoma (0 of 24 positive), mantle cell lymphoma (0 of 10 positive), and marginal zone lymphoma (0 of 5 positive). Our results suggest that down-regulation of the BTLA-HVEM pathway may be involved in germinal center B-cell activation. The specific high expression of BTLA in B-CLL/SLL represents a new potential diagnostic tool. The BTLA positivity of TFH cells may be a basis for designing future immunotherapies.
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Leucemia Linfocítica Crónica de Células B/inmunología , Receptores Inmunológicos/biosíntesis , Miembro 14 de Receptores del Factor de Necrosis Tumoral/biosíntesis , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos B/inmunología , Regulación hacia Abajo , Humanos , Inmunohistoquímica , Ratones , Receptores Inmunológicos/genética , Miembro 14 de Receptores del Factor de Necrosis Tumoral/genética , Miembro 14 de Receptores del Factor de Necrosis Tumoral/inmunologíaRESUMEN
DNA methylation of retroviral promoters and enhancers localized in the provirus 5' long terminal repeat (LTR) is considered to be a mechanism of transcriptional suppression that allows retroviruses to evade host immune responses and antiretroviral drugs. However, the role of DNA methylation in the control of HIV-1 latency has never been unambiguously demonstrated, in contrast to the apparent importance of transcriptional interference and chromatin structure, and has never been studied in HIV-1-infected patients. Here, we show in an in vitro model of reactivable latency and in a latent reservoir of HIV-1-infected patients that CpG methylation of the HIV-1 5' LTR is an additional epigenetic restriction mechanism, which controls resistance of latent HIV-1 to reactivation signals and thus determines the stability of the HIV-1 latency. CpG methylation acts as a late event during establishment of HIV-1 latency and is not required for the initial provirus silencing. Indeed, the latent reservoir of some aviremic patients contained high proportions of the non-methylated 5' LTR. The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable. In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5' LTR in viremic patients. However, even dense methylation of the HIV-1 5'LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-alpha, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid. Tight but incomplete control of HIV-1 latency by CpG methylation might have important implications for strategies aimed at eradicating HIV-1 infection.
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Islas de CpG , Metilación de ADN , VIH-1/fisiología , Latencia del Virus/fisiología , Adulto , Anciano , Linfocitos T CD4-Positivos/metabolismo , Cromatina/fisiología , Células Clonales , Clonación Molecular , Femenino , Infecciones por VIH/metabolismo , Duplicado del Terminal Largo de VIH , Seronegatividad para VIH , VIH-1/genética , Humanos , Células Jurkat , Masculino , Persona de Mediana Edad , Regiones Promotoras Genéticas , Provirus/genética , Provirus/metabolismo , Viremia/genética , Viremia/metabolismoRESUMEN
Plasmacytoid dendritic cells (pDCs) are responsible for the production of type I IFN during viral infection. Viral elimination by IFN-alpha-based therapy in more than 50% of patients chronically infected with hepatitis C virus (HCV) suggests a possible impairment of production of endogenous IFN-alpha by pDCs in infected individuals. In this study, we investigated the impact of HCV on pDC function. We show that exposure of pDCs to patient serum- and cell culture-derived HCV resulted in production of IFN-alpha by pDCs isolated from some donors, although this production was significantly lower than that induced by influenza and human herpesvirus type 1 (HHV-1). Using specific inhibitors we demonstrate that endocytosis and endosomal acidification were required for IFN-alpha production by pDCs in response to cell culture-derived HCV. HCV and noninfectious HCV-like particles inhibited pDC-associated production of IFN-alpha stimulated with Toll-like receptor 9 (TLR9) agonists (CpG-A or HHV-1) but not that of IFN-alpha stimulated with TLR7 agonists (resiquimod or influenza virus). The blockade of TLR9-mediated production of IFN-alpha, effective only when pDCs were exposed to virus prior to or shortly after CpG-A stimulation, was already detectable at the IFN-alpha transcription level 2 h after stimulation with CpG-A and correlated with down-regulation of the transcription factor IRF7 expression and of TLR9 expression. In conclusion, rapidly and early occurring particle-host cell protein interaction during particle internalization and endocytosis followed by blockade of TLR9 function could result in less efficient sensing of HCV RNA by TLR7, with impaired production of IFN-alpha. This finding is important for our understanding of HCV-DC interaction and immunopathogenesis of HCV infection.
Asunto(s)
Células Dendríticas/virología , Hepacivirus/metabolismo , Herpesvirus Humano 1/metabolismo , Virus de la Influenza A/metabolismo , Interferón-alfa/metabolismo , Células Dendríticas/metabolismo , Endocitosis , Hepacivirus/genética , Herpesvirus Humano 1/genética , Humanos , Virus de la Influenza A/genética , Leucocitos Mononucleares/metabolismo , Receptor Toll-Like 9/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: The homeless are at very high risk for both respiratory diseases and poor access to health care delivery systems. The aim of this study was to assess the contribution of a shelter-based survey to identify respiratory diseases in the homeless population in Marseilles, in order to further develop preventive interventions. METHODS: A prevalence survey of respiratory diseases was conducted in two homeless shelters in Marseilles, in February 2005. A multidisciplinary team including infectious diseases specialists, lung specialists, residents, nurses, physiotherapists and X-ray technicians visited the two shelters. Interview, physical examination, sputum sampling for Mycobacterium tuberculosis detection, nasal swabs for virus detection and chest X-rays were performed in shelters. Chest X-rays were subsequently analysed by a pneumonologist and subjects found to have radiological abnormalities were hospitalized for further investigations. RESULTS: Of the 221 homeless persons enrolled, 110/221 (50%) had at least one respiratory manifestations. Chest X-rays were abnormal in 14 persons. Active tuberculosis was diagnosed in two, influenza in two, respiratory syncytial virus infection in two, acute pneumonia in one, asthma in two, lung cancer in one, acute bronchitis in 23, chronic bronchitis in 42, and exacerbation of chronic obstructive pulmonary disease in 22 persons, respectively. CONCLUSION: Our shelter-based survey, including clinical evaluation and chest radiographic screening, shows that this approach can be useful to control and prevent respiratory diseases among the homeless. However, further studies are necessary to determine the magnitude of influenza in the homeless, the impact of influenza immunization and optimal frequency of interventions in shelters.
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Personas con Mala Vivienda , Tamizaje Masivo , Infecciones del Sistema Respiratorio/epidemiología , Adulto , Femenino , Francia/epidemiología , Encuestas Epidemiológicas , Humanos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Infecciones del Sistema Respiratorio/clasificación , Infecciones del Sistema Respiratorio/diagnóstico , Adulto JovenRESUMEN
The persistence of human immunodeficiency virus type 1 (HIV-1) in memory CD4+ T cells is a major obstacle to the eradication of the virus with current antiretroviral therapy. Here, we investigated the effect of the activation status of CD4+ T cells on the predominance of R5 and X4 HIV-1 variants in different subsets of CD4+ T cells in ex vivo-infected human lymphoid tissues and peripheral blood mononuclear cells (PBMCs). In these cell systems, we examined the sensitivity of HIV replication to reverse transcriptase inhibitors. We demonstrate that R5 HIV-1 variants preferentially produced productive infection in HLA-DR- CD62L- CD4+ T cells. These cells were mostly in the G1b phase of the cell cycle, divided slowly, and expressed high levels of CCR5. In contrast, X4 HIV-1 variants preferentially produced productive infection in activated HLA-DR+ CD62L+ CD4+ T cells, which expressed high levels of CXCR4. The abilities of the nucleoside reverse transcriptase inhibitors (NRTI) zidovudine and lamivudine to stop HIV-1 replication were 20 times greater in activated T cells than in slowly dividing HLA-DR- CD62L- CD4+ T cells. This result, demonstrated both in a highly physiologically relevant ex vivo lymphoid tissue model and in PBMCs, correlated with higher levels of thymidine kinase mRNA in activated than in slowly dividing HLA-DR- CD62L- CD4+ T cells. The non-NRTI nevirapine was equally efficient in both cell subsets. The lymphoid tissue and PBMC-derived cell systems represent well-defined models which could be used as new tools for the study of the mechanism of resistance to HIV-1 inhibitors in HLA-DR- CD62L- CD4+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Inhibidores de la Transcriptasa Inversa/farmacología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Farmacorresistencia Viral Múltiple , Infecciones por VIH/virología , VIH-1/metabolismo , Antígenos HLA-DR , Humanos , Selectina L/genética , Leucocitos Mononucleares , Activación de Linfocitos , Tonsila Palatina , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Especificidad de la Especie , Replicación Viral/efectos de los fármacosRESUMEN
OBJECTIVES: To determine whether increased glucose metabolism is the potential cause of the decreased plasma glucose curve determined after oral glucose tolerance testing in horses with lower motor neuron degeneration. ANIMALS: 3 horses with signs suggestive of lower motor neuron degeneration, 1 horse with malignant melanoma with multiple metastases, and an obese but otherwise healthy horse. Procedures-Glucose metabolism was assessed by use of the hyperglycemic clamp and euglycemic hyperinsulinemic clamp techniques. RESULTS: Mean rate of glucose metabolism of horses with lower motor neuron degeneration was significantly greater (mean, 3.7 times greater than control horses; range, 2.1 to 4.8 times greater) than that reported in 5 healthy control horses (41 +/- 13 micromol/kg/min vs 11 +/- 4.5 micromol/kg/min, respectively). In addition, one of the affected horses, an 8-year-old warmblood gelding, had a 5.6-times increased sensitivity to exogenously administered insulin, compared with that reported in 5 healthy control horses. Pancreatic insulin secretion was not insufficient in horses with lower motor neuron degeneration. Findings in the 2 diseased control horses were unremarkable. CONCLUSIONS AND CLINICAL RELEVANCE: Increased glucose metabolism in horses with lower motor neuron degeneration may be the cause of the decreased plasma glucose curve detected after oral glucose tolerance testing. This finding could aid in developing supportive treatments with respect to adequate glucose and vitamin E supplementation.