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The genes mapping at the HLA region show high density, strong linkage disequilibrium and high polymorphism, which affect the association of HLA class I and class II genes with autoimmunity. We focused on the HLA haplotypes, genomic structures consisting of an array of specific alleles showing some degrees of genetic association with different autoimmune disorders. GWASs in many pathologies have identified variants in either the coding loci or the flanking regulatory regions, both in linkage disequilibrium in haplotypes, that are frequently associated with increased risk and may influence gene expression. We discuss the relevance of the HLA gene expression because the level of surface heterodimers determines the number of complexes presenting self-antigen and, thus, the strength of pathogenic autoreactive T cells immune response.
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Enfermedades Autoinmunes , Autoinmunidad , Antígenos HLA , Haplotipos , Humanos , Autoinmunidad/genética , Autoinmunidad/inmunología , Antígenos HLA/genética , Antígenos HLA/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Predisposición Genética a la Enfermedad , Desequilibrio de Ligamiento , Regulación de la Expresión GénicaRESUMEN
Background: Celiac disease (CD) is a chronic immuno-mediated enteropathy caused by dietary gluten in genetically susceptible individuals carrying HLA (Human Leukocytes Antigen) genes that encode for DQ2.5 and DQ8 molecules. TRAFD1 (TRAF-type zinc finger domain 1) is a gene recently found associated with CD and defined as a master regulator of IFNγ signalling and of MHC class I antigen processing/presentation. There is no specific drug therapy and the only effective treatment is the gluten-free diet (GFD). The great majority of celiac patients when compliant with GFD have a complete remission of symptoms and recovery of gut mucosa architecture and function. Until now, very few studies have investigated molecular differences occurring in CD patients upon the GFD therapy. Methods: We looked at the expression of both HLA DQ2.5 and TRAFD1 risk genes in adult patients with acute CD at the time of and in treated patients on GFD. Specifically, we measured by qPCR the HLA-DQ2.5 and TRAFD1 mRNAs on peripheral blood mononuclear cells (PBMC) from the two groups of patients. Results: When we compared the HLA-DQ mRNA expression, we didn't find significant variation between the two groups of patients, thus indicating that GFD patients have the same capability to present gliadin antigens to cognate T cells as patients with active disease. Conversely, TRAFD1 was more expressed in PBMC from treated CD subjects. Notably, TRAFD1 transcripts significantly increased in the patients analyzed longitudinally during the GFD, indicating a role in the downregulation of gluten-induced inflammatory pathways. Conclusion: Our study demonstrated that HLA-DQ2.5 and TRAFD1 molecules are two important mediators of anti-gluten immune response and inflammatory process.
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Macrophages have both a protective and pathological role in many autoimmune and inflammatory diseases. Macrophages phenotype is regulated by the environment that affects their polarization toward a pro- or anti-inflammatory phenotype. We describe a protocol for in vitro differentiation of macrophages from blood peripheral monocytes, that may be adopted to study different pathologies. Here, we are interested to study the phenotype of macrophages differentiated from patients affected by acute celiac disease (CD) or subjects following a gluten free diet (GFD), after in vitro gliadin challenge. We assess the pro-inflammatory phenotype of these macrophages by cytokines quantization on the cell supernatant. Moreover, our proposed protocol allows the preparation of total RNA to analyze the expression profile of many genes.
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Células Sanguíneas , Macrófagos , Diferenciación Celular , FenotipoRESUMEN
Kidney transplanted recipients (KTR) are at high risk of severe SARS-CoV-2 infection due to immunosuppressive therapy. Although several studies reported antibody production in KTR after vaccination, data related to immunity to the Omicron (B.1.1.529) variant are sparse. Herein, we analyzed anti-SARS-CoV-2 immune response in seven KTR and eight healthy controls after the second and third dose of the mRNA vaccine (BNT162b2). A significant increase in neutralizing antibody (nAb) titers were detected against pseudoviruses expressing the Wuhan-Hu-1 spike (S) protein after the third dose in both groups, although nAbs in KTR were lower than controls. nAbs against pseudoviruses expressing the Omicron S protein were low in both groups, with no increase after the 3rd dose in KTR. Reactivity of CD4+ T cells after boosting was observed when cells were challenged with Wuhan-Hu-1 S peptides, while Omicron S peptides were less effective in both groups. IFN-γ production was detected in KTR in response to ancestral S peptides, confirming antigen-specific T cell activation. Our study demonstrates that the 3rd mRNA dose induces T cell response against Wuhan-Hu-1 spike peptides in KTR, and an increment in the humoral immunity. Instead, humoral and cellular immunity to Omicron variant immunogenic peptides were low in both KTR and healthy vaccinated subjects.
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Vacunas contra la COVID-19 , COVID-19 , Humanos , Vacuna BNT162 , COVID-19/prevención & control , SARS-CoV-2/genética , Anticuerpos Neutralizantes , Riñón , Anticuerpos Antivirales , Vacunas de ARNmRESUMEN
In human cells BRAF oncogene is invariably expressed as a mix of two coding transcripts: BRAF-ref and BRAF-X1. These two mRNA isoforms, remarkably different in the sequence and length of their 3'UTRs, are potentially involved in distinct post-transcriptional regulatory circuits. Herein, we identify PARP1 among the mRNA Binding Proteins that specifically target the X1 3'UTR in melanoma cells. Mechanistically, PARP1 Zinc Finger domain down-regulates BRAF expression at the translational level. As a consequence, it exerts a negative impact on MAPK pathway, and sensitizes melanoma cells to BRAF and MEK inhibitors, both in vitro and in vivo. In summary, our study unveils PARP1 as a negative regulator of the highly oncogenic MAPK pathway in melanoma, through the modulation of BRAF-X1 expression.
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Melanoma , Proteínas Proto-Oncogénicas B-raf , Humanos , Vemurafenib , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Indoles/farmacología , Sulfonamidas/farmacología , Melanoma/genética , Melanoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Sistema de Señalización de MAP Quinasas , Poli(ADP-Ribosa) Polimerasa-1/genéticaRESUMEN
Macrophages play an important role in the pathogenesis of celiac disease (CD) because they are involved in both inflammatory reaction and antigen presentation. We analyzed the expression of CD-associated HLA-DQ2.5 risk alleles on macrophages isolated by two cohorts of adult patients, from the U.S. and Italy, at different stages of disease and with different genotypes. After isolating and differentiating macrophages from PBMC, we assessed the HLA genotype and quantified the HLA-DQ2.5 mRNAs by qPCR, before and after gliadin stimulation. The results confirmed the differences in expression between DQA1*05:01 and DQB1*02:01 predisposing alleles and the non-CD associated alleles, as previously shown on other types of APCs. The gliadin challenge confirmed the differentiation of macrophages toward a proinflammatory phenotype, but above all, it triggered an increase of DQA1*05:01 mRNA, as well as a decrease of the DQB1*02:01 transcript. Furthermore, we observed a decrease in the DRB1 genes expression and a downregulation of the CIITA transactivator. In conclusion, our findings provide new evidences on the non-coordinated regulation of celiac disease DQ2.5 risk genes and support the hypothesis that gliadin could interfere in the three-dimensional arrangement of chromatin at the HLA locus.
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Celiac disease (CeD) is a chronic immuno-mediated enteropathy caused by dietary gluten with marked autoimmunity traits. The human leukocyte antigen (HLA) class II heterodimers represent the main predisposing factor, although environmental agents, as viral infection, gut microbiota, and dietary regimen, also contribute to CeD risk. These molecules are involved in autoimmunity as they present self-antigens to autoreactive T cells that have escaped the thymic negative selection. In CeD, the HLA class II risk alleles, DQA1*05-DQB1*02 and DQA1*03-DQB1*03, encode for DQ2.5 and DQ8 heterodimers, and, furthermore, disease susceptibility was found strictly dependent on the dose of these genes. This finding questioned how the expression of HLA-DQ risk genes, and of relative surface protein on antigen-presenting cells, might be relevant for the magnitude of anti-gluten inflammatory response in CeD patients, and impact the natural history of disease, its pathomechanisms, and compliance to dietary treatment. In this scenario, new personalized medical approaches will be desirable to support an early, accurate, and non-invasive diagnosis, and to define genotype-guided preventive and therapeutic strategies for CeD. To reach this goal, a stratification of genetic risk, disease outcome, and therapy compliance based on HLA genotypes, DQ2.5/DQ8 expression measurement and magnitude of T cell response to gluten is mandatory. IMPACT: This article revises the current knowledge on how different HLA haplotypes, carrying the DQ2.5/DQ8 risk alleles, impact the onset of CeD. This review discusses how the expression of susceptibility HLA-DQ genes can determine the risk assessment, outcome, and prevention of CeD. The recent insights on the environmental factors contributing to CeD in childhood are reviewed. This review discusses the use of HLA risk gene expression as a tool to support medical precision approaches for an early and non-invasive diagnosis of CeD, and to define genotype-guided preventive and therapeutic strategies.
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Enfermedad Celíaca/diagnóstico , Genes MHC Clase II , Pruebas Genéticas , Antígenos HLA-DQ/genética , Medicina de Precisión , Enfermedad Celíaca/dietoterapia , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Toma de Decisiones Clínicas , Dieta Sin Gluten , Diagnóstico Precoz , Predisposición Genética a la Enfermedad , Glútenes/inmunología , Antígenos HLA-DQ/inmunología , Humanos , Fenotipo , Valor Predictivo de las Pruebas , Pronóstico , Medición de Riesgo , Factores de Riesgo , Linfocitos T/inmunologíaRESUMEN
The DR5-DQ7/DR7-DQ2 genotype is very frequent among patients affected by celiac disease (CD), in Europe. This genotype, associated to high risk of CD, carries the HLA-DQA1*05 and HLA-DQB1*02 predisposing alleles, in trans configuration. The alleles encode the DQ2.5 heterodimer responsible of gluten peptide presentation on the surface of antigen-presenting cells (APCs), and consequent pathogenic CD4+ T cell activation. We demonstrated that DR5/DR7 APCs induce an anti-gluten CD4+ T cell response, of comparable intensity to that observed with APCs carrying DR1/DR3 genotype, which risk alleles are in cis configuration. In addition, we showed that DR5/DR7 APCs from celiac patients stimulated an effector CD4+ T cell response higher with respect to that induced by DR5/DR7 APCs from healthy subjects. To explain these findings, we assessed the DQ2.5 RNA and protein quantity. We showed that the expression of DQA1*05 and DQB1*02 risk alleles is much higher than the expression of non-CD-associated alleles, in agreement with the previous results obtained with DR1/DR3 genotype. The differential expression of transcripts influences the quantity of DQα1*05 and DQß1*02 chains and, as consequence, the cell surface density of DQ2.5 heterodimers. Moreover, both RNA and proteins, are more abundant in APCs from celiac patients than controls. Finally, to unravel the mechanism regulating the expression of predisposing DQA1*05 and DQB1*02 alleles, we quantified the new synthetized RNA and found that the differential expression is explained by their transcription rate. Our results confirmed that the strength of antigen-specific CD4+ T cell response is mainly determined by the amount of gluten in the diet and provided a new possible approach for a personalized diagnosis and for risk stratification.
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Alelos , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Expresión Génica , Predisposición Genética a la Enfermedad/genética , Glútenes/inmunología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DR/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Células Presentadoras de Antígenos/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , HumanosRESUMEN
BACKGROUND: Many pseudogenes possess biological activities and play important roles in the pathogenesis of various types of cancer including bladder cancer (BlCa), which still lacks suitable molecular biomarkers. Recently, pseudogenes were found to be significantly enriched in a pan-cancer classification based on the Cancer Genome Atlas gene expression data. Among them, the top-ranking pseudogene was the proliferation-associated 2G4 pseudogene 4 (PA2G4P4). METHODS: Genomic and transcript features of PA2G4P4 were determined by GeneBank database analysis followed by 5' RACE experiments. Therefore, we conducted a retrospective molecular study on a cohort of 45 patients of BlCa. PA2G4P4 expression was measured by RT-qPCR, whereas PA2G4P4 transcript distribution was analyzed by in situ hybridization on both normal and cancerous histological sections and compared to the immunolocalization of its parental PA2G4/EBP1 protein. Finally, we tested the effects of PA2G4P4 depletion on proliferation, migration, and death of BlCa cells. RESULTS: We showed for the first time PA2G4P4 overexpression in BlCa tissues and in cell lines. PA2G4P4 distribution strictly overlaps PA2G4/EBP1 protein localization. Moreover, we showed that PA2G4P4 knockdown affects both proliferation and migration of BlCa cells, highlighting its potential oncogenic role. CONCLUSIONS: PA2G4P4 may play a functional role as an oncogene in BlCa development, suggesting it as a good candidate for future investigation and new clinical applications.
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HLA class II genes encode highly polymorphic heterodimeric proteins functioning to present antigens to T cells and stimulate a specific immune response. Many HLA genes are strongly associated with autoimmune diseases as they stimulate self-antigen specific CD4+ T cells driving pathogenic responses against host tissues or organs. High expression of HLA class II risk genes is associated with autoimmune diseases, influencing the strength of the CD4+ T-mediated autoimmune response. The expression of HLA class II genes is regulated at both transcriptional and post-transcriptional levels. Protein components of the RNP complex binding the 3'UTR and affecting mRNA processing have previously been identified. Following on from this, the regulation of HLA-DQ2.5 risk genes, the main susceptibility genetic factor for celiac disease (CD), was investigated. The DQ2.5 molecule, encoded by HLA-DQA1*05 and HLA-DQB1*02 alleles, presents the antigenic gluten peptides to CD4+ T lymphocytes, activating the autoimmune response. The zinc-finger protein Tristetraprolin (TTP) or ZFP36 was identified to be a component of the RNP complex and has been described as a factor modulating mRNA stability. The 3'UTR of CD-associated HLA-DQA1*05 and HLA-DQB1*02 mRNAs do not contain canonical TTP binding consensus sequences, therefore an in silico approach focusing on mRNA secondary structure accessibility and stability was undertaken. Key structural differences specific to the CD-associated mRNAs were uncovered, allowing them to strongly interact with TTP through their 3'UTR, conferring a rapid turnover, in contrast to lower affinity binding to HLA non-CD associated mRNA.
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Enfermedad Celíaca/genética , Cadenas alfa de HLA-DQ/genética , Cadenas beta de HLA-DQ/genética , Estabilidad del ARN , Tristetraprolina/metabolismo , Regiones no Traducidas 3' , Enfermedad Celíaca/metabolismo , Línea Celular Tumoral , Cadenas alfa de HLA-DQ/metabolismo , Cadenas beta de HLA-DQ/metabolismo , Humanos , ARN Mitocondrial/química , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Tristetraprolina/genéticaRESUMEN
HLA gene expression has an important role in the autoimmune disease predisposition. We investigated the mRNA expression profile of the risk alleles HLA-DRB1*15 and HLA-DRB1*13 in a cohort of subjects both multiple sclerosis (MS) patients and healthy controls. Moreover, we explored the expression of the allele HLA-DRB1*11 that is very frequent in our cohort from southern Italy. We found that the expression of MS-associated alleles in heterozygous MS patients was always higher than the nonassociated alleles. The differential risk allele expression occurred also in nonaffected subjects, though with a lower increment compared to MS patients.
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Cadenas HLA-DRB1/sangre , Cadenas HLA-DRB1/genética , Esclerosis Múltiple/genética , Adolescente , Adulto , Anciano , Alelos , Estudios de Cohortes , Femenino , Frecuencia de los Genes , Heterocigoto , Humanos , Italia , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Adulto JovenRESUMEN
To date, the study of the impact of major hystocompatibility complex on autoimmunity has been prevalently focused on structural diversity of MHC molecules in binding and presentation of (auto)antigens to cognate T cells. Recently, a number of experimental evidences suggested new points of view to investigate the complex relationships between MHC gene expression and the individual predisposition to autoimmune diseases. Irrespective of the nature of the antigen, a threshold of MHC-peptide complexes needs to be reached, as well as a threshold of T cell receptors engaged is required, for the activation and proliferation of autoantigen-reactive T cells. Moreover, it is well known that increased expression of MHC class II molecules may alter the T cell receptor repertoire during thymic development, and affect the survival and expansion of mature T cells. Many evidences confirmed that the level of both transcriptional and post-transcriptional regulation are involved in the modulation of the expression of MHC class II genes and that both contribute to the predisposition to autoimmune diseases. Here, we aim to focus some of these regulative aspects to better clarify the role of MHC class II genes in predisposition and development of autoimmunity.
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Enfermedades Autoinmunes/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Linfocitos T/inmunología , Animales , Presentación de Antígeno , Enfermedades Autoinmunes/genética , Autoinmunidad/genética , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunomodulación , Polimorfismo Genético , Procesamiento Postranscripcional del ARN , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
In the recent years, the incidence of inflammatory bowel disease (IBD) has dramatically increased in young subjects, however, the pathogenesis of paediatric IBD is poorly investigated. In this study we aimed to evaluate the cytokine pattern and the phenotype of cytokine producing cells in the intestinal mucosa of paediatric patients affected by Crohn's disease (CD) or ulcerative colitis (UC) and of non-IBD healthy controls (HC). Cytokine (IL-15, TNF-α, INF-γ) production was analyzed at basal condition and after mitogen stimulation either intracellularly by flow cytometry or in intestinal cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). A higher frequency of enterocytes (EpCam+ cells) was observed in UC patients compared to CD or HC. An expansion of enterocytes producing IL-15 and TNF-α were found in IBD patients compared to HC. A marked expression of IL-15 in the intestinal epithelium of IBD patients was further confirmed by immunohistochemistry. Myeloid dendritic (CD11c+) cells producing TNF-α and INF-γ were increased in IBD biopsies. Unexpectedly, only after a strong mitogen stimulus, as phytohaemagglutinin, the frequency of CD3+ cells producing IFN-γ was increased in IBD compared to control intestinal mucosa. Interestingly, functional studies performed on organ cultures of intestinal biopsies with neutralizing anti-IL-15 monoclonal antibody showed a marked reduction of mononuclear cell activation, proliferation of crypt enterocytes, as well as a reduction of TNF-α release in organ culture supernatants. In conclusion, we found that in the gut mucosa of IBD children both enterocytes and dendritic cells produce proinflammatory cytokines. The over-expression of IL-15 by enterocytes in IBD intestine and the reduced IBD inflammation by IL-15 blockage suggests that this cytokine could be a therapeutic target in IBD.
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Citocinas/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Adolescente , Biomarcadores/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Niño , Células Dendríticas/metabolismo , Enterocitos/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Linfocitos T/metabolismoRESUMEN
HLA genes represent the main risk factor in autoimmune disorders. In celiac disease (CD), the great majority of patients carry the HLA DQA1*05 and DQB1*02 alleles, both of which encode the DQ2.5 molecule. The formation of complexes between DQ2.5 and gluten peptides on antigen-presenting cells (APCs) is necessary to activate pathogenic CD4(+) T lymphocytes. It is widely accepted that the DQ2.5 genes establish the different intensities of anti-gluten immunity, depending whether they are in a homozygous or a heterozygous configuration. Here, we demonstrated that HLA DQA1*05 and DQB1*02 gene expression is much higher than expression of non-CD-associated genes. This influences the protein levels and causes a comparable cell surface exposure of DQ2.5 heterodimers between DQ2.5 homozygous and heterozygous celiac patients. As a consequence, the magnitude of the anti-gluten CD4(+) T cell response is strictly dependent on the antigen dose and not on the DQ2.5 gene configuration of APCs. Furthermore, our findings support the concept that the expression of DQ2.5 genes is an important risk factor in celiac disease. The preferential expression of DQ2.5 alleles provides a new functional explanation of why these genes are so frequently associated with celiac disease and with other autoimmune disorders.
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Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Expresión Génica , Predisposición Genética a la Enfermedad , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Alelos , Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Epítopos de Linfocito T/inmunología , Genotipo , Glútenes/inmunología , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Humanos , Virus de la Influenza A/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Riesgo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Many solid tumours including melanoma, glioblastoma, and breast carcinomas express MHC class II molecules (MHC II). The surface expression of these molecules confers to non-hematopoietic tumour cells the role of non-professional antigen presenting cells and the ability to potentially stimulate tumour-specific CD4+ T cell response. We studied EBP1, an ErbB3 binding protein, and the effects of p48 and p42 isoforms on the MHC II expression in U87 glioblastoma, M14 melanoma and MCF7 mammary carcinoma cell lines. We found that overexpression of p48 increases MHC II transcription in U87 and M14, through upregulation of CIITA transactivator and STAT1 phosphorylation. In addition, p48 protein influences MHC II expression by increasing mRNA stability. In melanoma and glioblastoma cell lines, p48 isoform functions as oncogene promoting tumour growth, while p42 isoform, that does not affect MHC II expression, acts as a tumour suppressor by blocking cell growth and inducing apoptosis. In contrast, p48 seems to act as tumour suppressor in breast carcinoma inhibiting proliferation, favouring apoptosis, and inducing a slight increase of MHC II expression similar to p42. Our data highlight the tissue specificity function of EBP1 isoforms and demonstrate that only the oncogene p48 activates MHC II expression in human solid tumours, via STAT1 phosphorylation, in order to affect tumour progression by triggering specific immune response.
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Proteínas Adaptadoras Transductoras de Señales/genética , Genes MHC Clase II , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Unión al ARN/genética , Factor de Transcripción STAT1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Especificidad de Órganos , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Major histocompatibility complex class II (MHCII) molecules are heterodimeric surface proteins involved in the presentation of exogenous antigens during the adaptive immune response. We demonstrate the existence of a fine level of regulation, coupling the transcription and processing of mRNAs encoding α and ß chains of MHCII molecules, mediated through binding of their Untraslated Regions (UTRs) to the same ribonucleoproteic complex (RNP). We propose a dynamic model, in the context of the 'MHCII RNA operon' in which the increasing levels of DRA and DRB mRNAs are docked by the RNP acting as a bridge between 5'- and 3'-UTR of the same messenger, building a loop structure and, at the same time, joining the two chains, thanks to shared common predicted secondary structure motifs. According to cell needs, as during immune surveillance, this RNP machinery guarantees a balanced synthesis of DRA and DRB mRNAs and a consequent balanced surface expression of the heterodimer.
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Regulación de la Expresión Génica , Cadenas alfa de HLA-DR/genética , Cadenas beta de HLA-DR/química , Regiones no Traducidas 5' , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Línea Celular Tumoral , ADN Complementario/metabolismo , Antígenos HLA-DR/análisis , Cadenas alfa de HLA-DR/química , Cadenas alfa de HLA-DR/metabolismo , Cadenas beta de HLA-DR/genética , Cadenas beta de HLA-DR/metabolismo , Humanos , Modelos Genéticos , Proteínas del Factor Nuclear 90/antagonistas & inhibidores , Motivos de Nucleótidos , Multimerización de Proteína , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Ribonucleoproteínas/metabolismo , Transcripción GenéticaRESUMEN
Major histocompatibility complex class II mRNAs encode heterodimeric proteins involved in the presentation of exogenous antigens during an immune response. Their 3'UTRs bind a protein complex in which we identified two factors: EBP1, an ErbB3 receptor-binding protein and DRBP76, a double-stranded RNA binding nuclear protein, also known as nuclear factor 90 (NF90). Both are well-characterized regulatory factors of several mRNA molecules processing. Using either EBP1 or DRBP76/NF90-specific knockdown experiments, we established that the two proteins play a role in regulating the expression of HLA-DRA, HLA-DRB1 and HLA-DQA1 mRNAs levels. Our study represents the first indication of the existence of a functional unit that includes different transcripts involved in the adaptive immune response. We propose that the concept of 'RNA operon' may be suitable for our system in which MHCII mRNAs are modulated via interaction of their 3'UTR with same proteins.
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Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Proteínas del Factor Nuclear 90/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/fisiología , Células Presentadoras de Antígenos/inmunología , Línea Celular Tumoral , Citoplasma/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas del Factor Nuclear 90/antagonistas & inhibidores , Proteínas del Factor Nuclear 90/fisiología , Operón , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/fisiologíaRESUMEN
The efficacy of a new vaccine-delivery vector, based on the filamentous bacteriophage fd displaying a single-chain antibody fragment known to bind the mouse DC surface molecule DEC-205, is reported. We demonstrate both in vitro and in vivo an enhanced receptor-mediated uptake of phage particles expressing the anti-DEC-205 fragment by DCs. We also report that DCs targeted by fd virions in the absence of other stimuli produce IFN-α and IL-6, and acquire a mature phenotype. Moreover, DC-targeting with fd particles double-displaying the anti-DEC-205 fragment on the pIII protein and the OVA(257-264) antigenic determinant on the pVIII protein induced potent inhibition of the growth of the B16-OVA tumor in vivo. This protection was much stronger than other immunization strategies and similar to that induced by adoptively transferred DCs. Since targeting DEC-205 in the absence of DC activation/maturation agents has previously been described to result in tolerance, the ability of fd bacteriophages to induce a strong tumor-specific immune response by targeting DCs through DEC-205 is unexpected, and further validates the potential employment of this safe, versatile and inexpensive delivery system for vaccine formulation.
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Vacunas contra el Cáncer , Células Dendríticas/metabolismo , Inovirus/inmunología , Anticuerpos de Cadena Única/metabolismo , Virión/metabolismo , Animales , Antígenos CD/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Diferenciación Celular , Células Dendríticas/inmunología , Células Dendríticas/patología , Células Dendríticas/virología , Enterobacteriaceae/virología , Inovirus/patogenicidad , Interferón gamma/metabolismo , Interleucina-6/metabolismo , Lectinas Tipo C/inmunología , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , Terapia Molecular Dirigida , Ovalbúmina/genética , Ovalbúmina/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores de Superficie Celular/inmunología , Anticuerpos de Cadena Única/genética , Transgenes/genética , Carga Tumoral , Vacunación , Virión/inmunología , Virión/patogenicidadRESUMEN
The ability of fd bacteriophage particles to trigger different arms of the immune system has been previously shown by us with particular emphasis on the ability of phages to raise CTL responses in vitro and in vivo. Here we show that fd virions in the absence of adjuvants are able to evoke a DTH reaction mediated by antigen specific CD8+ T cells. In addition, we analyzed the induction of CTL responses in mice depleted of CD4+ T cells, and we observed that short-term secondary CTL responses were induced in the absence of CD4+ T cells while induction of long-term memory CTLs required the presence of CD4+ T lymphocytes. These results examine the cellular mechanism at the basis of fd efficiency and provide new elements to further validate the use of fd particles for eliciting and monitoring antigen-specific CTLs.