RESUMO
Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.
Assuntos
Linfoma de Burkitt , Infecções por Vírus Epstein-Barr , Animais , Camundongos , Herpesvirus Humano 4/genética , Infecções por Vírus Epstein-Barr/complicações , Aflatoxina B1/toxicidade , Ligantes , Linfoma de Burkitt/metabolismo , Quimiocinas , CarcinogêneseRESUMO
BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.
Assuntos
COVID-19 , Coinfecção , Hepatite B , Hepatite D , Humanos , Camundongos , Animais , Vírus Delta da Hepatite/fisiologia , Vírus da Hepatite B/fisiologia , Interferons , Antígenos da Hepatite delta/metabolismo , Hepatite D/complicações , Hepatite B/complicações , Replicação Viral/fisiologia , COVID-19/complicações , SARS-CoV-2/genética , RNA Viral/genéticaRESUMO
Here, we report a novel experimental setup to perform adoptive transfer of gene-edited B cells using humanized immune system mice by infusing autologous HIS mouse-derived human B cells "educated" in a murine context and thus rendered tolerant to the host. The present approach presents two advantages over the conventional humanized PBMC mouse models: (i) it circumvents the risk of xenogeneic graft-versus-host reaction and (ii) it mimics more closely human immune responses, thus favoring clinical translation. We show that the frequencies and numbers of transduced B cells in recipient's spleens one week post-transfer are within the range of the size of the pre-immune B cell population specific for a given protein antigen in the mouse. They are also compatible with the B cell numbers required to elicit a sizeable immune response upon immunization. Altogether, our findings pave the way for future studies aiming at assessing therapeutic interventions involving B cell reprogramming for instance by an antibody transgene in a "humanized" hematopoietic setting.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucócitos Mononucleares , Transferência Adotiva , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos SCIDRESUMO
Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.
Assuntos
Carcinoma Hepatocelular/virologia , Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Neoplasias Hepáticas/virologia , Proteínas Repressoras/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas de Ciclo Celular/genética , Vírus da Hepatite B/genética , Hepatócitos/virologia , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteômica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Fatores de Processamento de Serina-Arginina/genética , Proteínas do Core Viral/genética , Replicação ViralRESUMO
Cell therapy has emerged as an attractive therapeutic option in organ transplantation. During the last decade, the therapeutic potency of Treg immunotherapy has been shown in various preclinical animal models and safety was demonstrated in first clinical trials. However, there are still critical open questions regarding specificity, survival, and migration to the target tissue so the best Treg population for infusion into patients is still under debate. Recent advances in CAR technology hold the promise for Treg-functional superiority. Another exciting strategy is the generation of B-cell antibody receptor (BAR) Treg/cytotoxic T cells to specifically regulate or deplete alloreactive memory B cells. Finally, B cells are also capable of immune regulation, making them promising candidates for immunomodulatory therapeutic strategies. This article summarizes available literature on cell-based innovative therapeutic approaches aiming at modulating alloimmune response for transplantation. Crucial areas of investigation that need a joined effort of the transplant community for moving the field toward successful achievement of tolerance are highlighted.
Assuntos
Motivação , Transplante de Órgãos , Animais , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Tolerância Imunológica , Imunoterapia Adotiva , Linfócitos T ReguladoresRESUMO
Nowadays, cancers still represent a significant health burden, accounting for around 10 million deaths per year, due to ageing populations and inefficient treatments for some refractory cancers. Immunotherapy strategies that modulate the patient's immune system have emerged as good treatment options. Among them, the adoptive transfer of B cells selected ex vivo showed promising results, with a reduction in tumor growth in several cancer mouse models, often associated with antitumoral immune responses. Aside from the benefits of their intrinsic properties, including antigen presentation, antibody secretion, homing and long-term persistence, B cells can be modified prior to reinfusion to increase their therapeutic role. For instance, B cells have been modified mainly to boost their immuno-stimulatory activation potential by forcing the expression of costimulatory ligands using defined culture conditions or gene insertion. Moreover, tumor-specific antigen presentation by infused B cells has been increased by ex vivo antigen loading (peptides, RNA, DNA, virus) or by the sorting/ engineering of B cells with a B cell receptor specific to tumor antigens. Editing of the BCR also rewires B cell specificity toward tumor antigens, and may trigger, upon antigen recognition, the secretion of antitumor antibodies by differentiated plasma cells that can then be recognized by other immune components or cells involved in tumor clearance by antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity for example. With the expansion of gene editing methodologies, new strategies to reprogram immune cells with whole synthetic circuits are being explored: modified B cells can sense disease-specific biomarkers and, in response, trigger the expression of therapeutic molecules, such as molecules that counteract the tumoral immunosuppressive microenvironment. Such strategies remain in their infancy for implementation in B cells, but are likely to expand in the coming years.
Assuntos
Linfócitos B/metabolismo , Edição de Genes/métodos , Animais , Anticorpos/metabolismo , Apresentação de Antígeno/genética , Apresentação de Antígeno/fisiologia , Humanos , Imunoterapia , Imunoterapia Adotiva/métodosRESUMO
Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.
Assuntos
Evolução Molecular , Hepacivirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Hepatite C/metabolismo , Hepatite C/patologia , Hepatite C/virologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Células Tumorais Cultivadas , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Replicação ViralRESUMO
The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver.
Assuntos
Técnicas de Cultura de Células , Meios de Cultura/química , Hepatócitos/fisiologia , Fígado/citologia , Células-Tronco , Animais , Estudos de Casos e Controles , Masculino , Camundongos Endogâmicos NOD , Cultura Primária de CélulasRESUMO
Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.
Assuntos
Regulação da Expressão Gênica , Vírus da Hepatite B/patogenicidade , Hepatite B/virologia , Provírus/patogenicidade , Receptores Citoplasmáticos e Nucleares/metabolismo , Replicação Viral , Animais , DNA Viral/genética , Feminino , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/patologia , Vírus da Hepatite B/genética , Humanos , Técnicas In Vitro , Ligantes , Camundongos , Camundongos Endogâmicos C3H , Provírus/genéticaRESUMO
Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) and current treatments for chronic hepatitis B and HCC are suboptimal. Herein, we identified cellular serine/threonine Polo-like-kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study was to demonstrate the proviral role of PLK1 in HBV biosynthesis and validate PLK1 inhibition a potential antiviral strategy. To this end, we employed physiologically relevant HBV infection models of primary human hepatocytes (PHHs) and differentiated HepaRG cells in conjunction with pharmacologic PLK1 inhibitors, small interfering RNA (siRNA)-mediated knockdown, and overexpression of constitutively active PLK1 (PLK1CA ). In addition, a humanized liver Fah-/- /Rag2-/- /Il2rg-/- (FRG) mouse model was used to determine the antiviral effect of PLK1 inhibitor BI-2536 on HBV infection in vivo. Finally, in vitro PLK1 kinase assays and site-directed mutagenesis were employed to demonstrate that HBV core protein (HBc) is a PLK1 substrate. We demonstrated that HBV infection activated cellular PLK1 in PHHs and differentiated HepaRG cells. PLK1 inhibition by BI-2536 or siRNA-mediated knockdown suppressed HBV DNA biosynthesis, whereas overexpression of PLK1CA increased it, suggesting that the PLK1 effects on viral biosynthesis are specific and that PLK1 is a proviral cellular factor. Significantly, BI-2536 administration to HBV-infected humanized liver FRG mice strongly inhibited HBV infection, validating PLK1 as an antiviral target in vivo. The proviral action of PLK1 is associated with the biogenesis of the nucleocapsid, as BI-2536 leads to its decreased intracellular formation/accumulation. In this respect, our studies identified HBc as a PLK1 substrate in vitro, and mapped PLK1 phosphorylation sites on this protein. CONCLUSION: PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and antitumoral strategies against HBV infection and HBV-mediated carcinogenesis. (Hepatology 2017;66:1750-1765).
Assuntos
Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/uso terapêutico , Proteínas do Core Viral/metabolismo , Replicação Viral , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Hepatócitos/enzimologia , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Fosforilação , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/farmacologia , Quinase 1 Polo-LikeRESUMO
Lipoprotein components are crucial factors for hepatitis C virus (HCV) assembly and entry. As hepatoma cells producing cell culture-derived HCV (HCVcc) particles are impaired in some aspects of lipoprotein metabolism, it is of upmost interest to biochemically and functionally characterize the in vivo produced viral particles, particularly regarding how lipoprotein components modulate HCV entry by lipid transfer receptors such as scavenger receptor BI (SR-BI). Sera from HCVcc-infected liver humanized FRG mice were separated by density gradients. Viral subpopulations, termed HCVfrg particles, were characterized for their physical properties, apolipoprotein association, and infectivity. We demonstrate that, in contrast to the widely spread distribution of apolipoproteins across the different HCVcc subpopulations, the most infectious HCVfrg particles are highly enriched in apoE, suggesting that such apolipoprotein enrichment plays a role for entry of in vivo derived infectious particles likely via usage of apolipoprotein receptors. Consistent with this salient feature, we further reveal previously undefined functionalities of SR-BI in promoting entry of in vivo produced HCV. First, unlike HCVcc, SR-BI is a particularly limiting factor for entry of HCVfrg subpopulations of very low density. Second, HCVfrg entry involves SR-BI lipid transfer activity but not its capacity to bind to the viral glycoprotein E2. In conclusion, we demonstrate that composition and biophysical properties of the different subpopulations of in vivo produced HCVfrg particles modulate their levels of infectivity and receptor usage, hereby featuring divergences with in vitro produced HCVcc particles and highlighting the powerfulness of this in vivo model for the functional study of the interplay between HCV and liver components.
Assuntos
Hepacivirus/metabolismo , Hepatite C/metabolismo , Fígado/virologia , Internalização do Vírus , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Hepacivirus/genética , Hepatite C/genética , Hepatite C/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Hematopoietic stem cell (HSC)-based gene therapy holds promise for the cure of many diseases. The field is now moving toward the use of lentiviral vectors (LVs) as evidenced by 4 successful clinical trials. These trials used vesicular-stomatitis-virus-G protein (VSV-G)-LVs at high doses combined with strong cytokine-cocktail stimulation to obtain therapeutically relevant transduction levels; however, they might compromise the HSC character. Summarizing all these disadvantages, alternatives to VSV-G-LVs are urgently needed. We generated here high-titer LVs pseudotyped with a baboon retroviral envelope glycoprotein (BaEV-LVs), resistant to human complement. Under mild cytokine prestimulation to preserve the HSC characteristics, a single BaEV-LV application at a low dose, resulted in up to 90% of hCD34(+) cell transduction. Even more striking was that these new BaEV-LVs allowed, at low doses, efficient transduction of up to 30% of quiescent hCD34(+) cells, whereas high-dose VSV-G-LVs were insufficient. Importantly, reconstitution of NOD/Lt-SCID/γc(-/-) (NSG) mice with BaEV-LV-transduced hCD34(+) cells maintained these high transduction levels in all myeloid and lymphoid lineages, including early progenitors. This transduction pattern was confirmed or even increased in secondary NSG recipient mice. This suggests that BaEV-LVs efficiently transduce true HSCs and could improve HSC-based gene therapy, for which high-level HSC correction is needed for life-long cure.
Assuntos
Betaretrovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Células-Tronco Hematopoéticas , Lentivirus/genética , Transdução Genética , Proteínas do Envelope Viral/genética , Animais , Antígenos CD34 , Linhagem Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Macaca , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCIDRESUMO
The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.
Assuntos
Transfusão de Sangue , Terapia Genética , Proteína HMGA2/metabolismo , Globinas beta/genética , Globinas beta/metabolismo , Talassemia beta/genética , Talassemia beta/terapia , Adolescente , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Pré-Escolar , Células Clonais/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Proteína HMGA2/genética , Homeostase , Humanos , Lentivirus/genética , Masculino , MicroRNAs/genética , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Tempo , Ativação Transcricional , Adulto Jovem , Talassemia beta/metabolismoRESUMO
The development of lentiviral vectors (LVs) for expression of a specific antibody can be achieved through the transduction of mature B-cells. This approach would provide a versatile tool for active immunotherapy strategies for infectious diseases or cancer, as well as for protein engineering. Here, we created a lentiviral expression system mimicking the natural production of these two distinct immunoglobulin isoforms. We designed a LV (FAM2-LV) expressing an anti-HCV-E2 surface glycoprotein antibody (AR3A) as a membrane-anchored Ig form or a soluble Ig form, depending on the B-cell maturation status. FAM2-LV induced high-level and functional membrane expression of the transgenic antibody in a nonsecretory B-cell line. In contrast, a plasma cell (PC) line transduced with FAM2-LV preferentially produced the secreted transgenic antibody. Similar results were obtained with primary B-cells transduced ex vivo. Most importantly, FAM2-LV transduced primary B-cells efficiently differentiated into PCs, which secreted the neutralizing anti-HCV E2 antibody upon adoptive transfer into immunodeficient NSG (NOD/SCIDγc(-/-)) recipient mice. Altogether, these results demonstrate that the conditional FAM2-LV allows preferential expression of the membrane-anchored form of an antiviral neutralizing antibody in B-cells and permits secretion of a soluble antibody following B-cell maturation into PCs in vivo.
Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Vetores Genéticos , Imunoglobulina G/imunologia , Ativação Linfocitária , Animais , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Hepacivirus/imunologia , Humanos , Lentivirus , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos NOD , Transporte Proteico , Receptores de IgG/metabolismo , Transdução Genética , Proteínas do Envelope Viral/imunologiaRESUMO
Although Epstein-Barr virus (EBV) infection is widely distributed, certain EBV-driven malignancies are geographically restricted. EBV-associated Burkitt's lymphoma (eBL) is endemic in children living in sub-Saharan Africa. This population is heavily exposed to food contaminated with the mycotoxin aflatoxin B1 (AFB1). Here, we show that exposure to AFB1 in in vitro and in vivo models induces activation of the EBV lytic cycle and increases EBV load, two events that are associated with an increased risk of eBL in vivo. AFB1 treatment leads to the alteration of cellular gene expression, with consequent activations of signaling pathways, e.g. PI3K, that in turn mediate reactivation of the EBV life cycle. Finally, we show that AFB1 triggers EBV-driven cellular transformation both in primary human B cells and in a humanized animal model. In summary, our data provide evidence for a role of AFB1 as a cofactor in EBV-mediated carcinogenesis.
Assuntos
Aflatoxina B1/toxicidade , Linfócitos B/virologia , Linfoma de Burkitt/virologia , Exposição Ambiental , Infecções por Vírus Epstein-Barr/patologia , Herpesvirus Humano 4/efeitos dos fármacos , Animais , Linfócitos B/patologia , Linfoma de Burkitt/induzido quimicamente , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Feminino , Herpesvirus Humano 4/fisiologia , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Transdução de Sinais , Ativação Viral , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: The role of innate immunity in general and of type I interferon (IFN-I) in particular in HTLV-1 pathogenesis is still a matter of debate. ADAR1-p150 is an Interferon Stimulated Gene (ISG) induced by IFN-I that can edit viral RNAs. We therefore investigated whether it could play the role of an anti-HTLV factor. RESULTS: We demonstrate here that ADAR1 is also expressed in the absence of IFN stimulation in activated primary T-lymphocytes that are the natural target of this virus and in HTLV-1 or HTLV-2 chronically infected T-cells. ADAR1 expression is also increased in primary lymphocytes obtained from HTLV-1 infected individuals. We show that ADAR1 enhances HTLV-1 and HTLV-2 infection in T-lymphocytes and that this proviral effect is independent from its editing activity. ADAR1 expression suppresses IFN-α inhibitory effect on HTLV-1 and HTLV-2 and acts through the repression of PKR phosphorylation. DISCUSSION: This study demonstrates that two interferon stimulated genes, i.e. PKR and ADAR1 have opposite effects on HTLV replication in vivo. The balanced expression of those proteins could determine the fate of the viral cycle in the course of infection.
Assuntos
Adenosina Desaminase/metabolismo , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Vírus Linfotrópico T Tipo 2 Humano/fisiologia , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , eIF-2 Quinase/antagonistas & inibidores , Células Cultivadas , Humanos , Inibição Psicológica , Dados de Sequência Molecular , Análise de Sequência de DNA , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
Cancer remains a leading cause of death worldwide, but immunotherapies hold promises to cure it by awaking the patient's immune system to provide long-term protection. Cell therapies, involving the infusion of immune cells, either directly or genetically modified, are being developed to recognize and destroy cancer cells. Here, we explored the potential of a new synthetic circuit to reprogram B cells to cure cancers. This circuit consists in a sensor (a membrane-anchored IgG1), a transducer (a fragment of the NR4A1 promoter) and an effector molecule. Upon recognition of its target, this sensor triggers signaling pathways leading to the activation of the transducer and to effector expression (here, a reporter molecule). We showed that this circuit could discriminate tumors expressing the target antigen from those that did not, in a dose dependent manner in vitro. Going further, we replaced the original membrane-anchored sensor by an immunoglobulin expression cassette that can not only be membrane-anchored but also be secreted depending on B-cell maturation status. This allowed concomitant activation of the circuit and secretion of transgenic antibodies directed against the targeted antigen. Of note, these antibodies could correctly bind their target and were recognized by FcR expressed at the surface of immune cells, which should synergically amplify the action of the effector. The potential of reprogrammed B cells remains to be assessed in vivo by implementing a therapeutic effector. In the future, B-cell reprogramming platforms should allow personalized cancer treatment by adapting both the sensor and the therapeutic effectors to patients.
Assuntos
Linfócitos B , Humanos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Animais , Camundongos , Neoplasias/terapia , Neoplasias/imunologia , Neoplasias/metabolismo , Linhagem Celular Tumoral , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Imunoglobulina G/imunologia , Antígenos de Neoplasias/imunologia , Reprogramação Celular/genética , Imunoterapia/métodos , Transdução de SinaisRESUMO
A challenge for gene therapy of genetic diseases is to maintain corrected cell populations in subjects undergoing transplantation in cases in which the corrected cells do not have intrinsic selective advantage over nontransduced cells. For inherited hematopoietic disorders, limitations include inefficient transduction of stem cell pools, the requirement for toxic myelosuppression, and a lack of optimal methods for cell selection after transduction. Here, we have designed a lentiviral vector that encodes human ß-globin and a truncated erythropoietin receptor, both under erythroid-specific transcriptional control. This truncated receptor confers enhanced sensitivity to erythropoietin and a benign course in human carriers. Transplantation of marrow transduced with the vector into syngenic thalassemic mice, which have elevated plasma erythropoietin levels, resulted in long-term correction of the disease even at low ratios of transduced/untransduced cells. Amplification of the red over the white blood cell lineages was self-controlled and averaged â¼ 100-fold instead of â¼ 5-fold for ß-globin expression alone. There was no detectable amplification of white blood cells or alteration of hematopoietic homeostasis. Notwithstanding legitimate safety concerns in the context of randomly integrating vectors, this approach may prove especially valuable in combination with targeted integration or in situ homologous recombination/repair and may lower the required level of pretransplantation myelosuppression.
Assuntos
Terapia Genética/métodos , Talassemia beta/terapia , Animais , Sequência de Bases , Primers do DNA/genética , Modelos Animais de Doenças , Eritropoese/genética , Expressão Gênica , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Homeostase , Humanos , Lentivirus/genética , Camundongos , Receptores da Eritropoetina/genética , Proteínas Recombinantes/genética , Transplante Isogênico , Globinas beta/genética , Talassemia beta/sangue , Talassemia beta/genéticaRESUMO
The expansion of genetic engineering has brought a new dimension for synthetic immunology. Immune cells are perfect candidates because of their ability to patrol the body, interact with many cell types, proliferate upon activation, and differentiate in memory cells. This study aimed at implementing a new synthetic circuit in B cells, allowing the expression of therapeutic molecules in a temporally and spatially restricted manner that is induced by the presence of specific antigens. This should enhance endogenous B cell functions in terms of recognition and effector properties. We developed a synthetic circuit encoding a sensor (a membrane-anchored B cell receptor targeting a model antigen), a transducer (a minimal promoter induced by the activated sensor), and effector molecules. We isolated a 734-bp-long fragment of the NR4A1 promoter, specifically activated by the sensor signaling cascade in a fully reversible manner. We demonstrate full antigen-specific circuit activation as its recognition by the sensor induced the activation of the NR4A1 promoter and the expression of the effector. Overall, such novel synthetic circuits offer huge possibilities for the treatment of many pathologies, as they are completely programmable; thus, the signal-specific sensors and effector molecules can be adapted to each disease.
RESUMO
A lentiviral vector encoding ß-globin flanked by insulator elements has been used to treat ß-thalassemia (ß-Thal) successfully in one human subject. However, a clonal expansion was observed after integration in the HMGA2 locus, raising the question of how commonly lentiviral integration would be associated with possible insertional activation. Here, we report correcting ß-Thal in a murine model using the same vector and a busulfan-conditioning regimen, allowing us to investigate efficacy and clonal evolution at 9.2 months after transplantation of bone marrow cells. The five gene-corrected recipient mice showed near normal levels of hemoglobin, reduced accumulation of reticulocytes, and normalization of spleen weights. Mapping of integration sites pretransplantation showed the expected favored integration in transcription units. The numbers of gene-corrected long-term repopulating cells deduced from the numbers of unique integrants indicated oligoclonal reconstitution. Clonal abundance was quantified using a Mu transposon-mediated method, indicating that clones with integration sites near growth-control genes were not enriched during growth. No integration sites involving HMGA2 were detected. Cells containing integration sites in genes became less common after prolonged growth, suggesting negative selection. Thus, ß-Thal gene correction in mice can be achieved without expansion of cells harboring vectors integrated near genes involved in growth control.