RESUMEN
Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis.
Asunto(s)
Transformación Celular Viral , Infecciones por Virus de Epstein-Barr/patología , Herpesvirus Humano 4/patogenicidad , Linfoma de Células B/patología , Células Plasmáticas/patología , Animales , Diferenciación Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Fibroblastos , Herpesvirus Humano 4/metabolismo , Humanos , Linfoma de Células B/virología , Ratones , Ratones Noqueados , Células Plasmáticas/virología , Cultivo Primario de Células , Transactivadores/genética , Transactivadores/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/metabolismoRESUMEN
The germinal center reaction is essential for efficient humoral immunity, but it can also give rise to B cell lymphomas. Cre/loxP-mediated conditional gene knock-out or knock-in can be used for the genetic manipulation of germinal center B cells in vivo. Here we present a novel allele, Cγ1-CreERT2, that allows for timed activation of Cre recombinase in a small fraction of germinal center B cells. This allele will be useful to study normal and malignant germinal center B cell development in vivo.
Asunto(s)
Linfocitos B/fisiología , Técnicas de Sustitución del Gen/métodos , Técnicas de Inactivación de Genes/métodos , Centro Germinal/inmunología , Integrasas/genética , Alelos , Animales , Diferenciación Celular , Humanos , RatonesRESUMEN
Epstein-Barr Virus (EBV) infects human B cells and drives them into continuous proliferation. Two key viral factors in this process are the latent membrane proteins LMP1 and LMP2A, which mimic constitutively activated CD40 receptor and B-cell receptor signaling, respectively. EBV-infected B cells elicit a powerful T-cell response that clears the infected B cells and leads to life-long immunity. Insufficient immune surveillance of EBV-infected B cells causes life-threatening lymphoproliferative disorders, including mostly germinal center (GC)-derived B-cell lymphomas. We have modeled acute EBV infection of naive and GC B cells in mice through timed expression of LMP1 and LMP2A. Although lethal when induced in all B cells, induction of LMP1 and LMP2A in just a small fraction of naive B cells initiated a phase of rapid B-cell expansion followed by a proliferative T-cell response, clearing the LMP-expressing B cells. Interfering with T-cell activity prevented clearance of LMP-expressing B cells. This was also true for perforin deficiency, which in the human causes a life-threatening EBV-related immunoproliferative syndrome. LMP expression in GC B cells impeded the GC reaction but, upon loss of T-cell surveillance, led to fatal B-cell expansion. Thus, timed expression of LMP1 together with LMP2A in subsets of mouse B cells allows one to study major clinically relevant features of human EBV infection in vivo, opening the way to new therapeutic approaches.
Asunto(s)
Linfocitos B/virología , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Proteínas de la Matriz Viral/genética , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Antígenos CD40/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica , Centro Germinal/inmunología , Centro Germinal/metabolismo , Herpesvirus Humano 4/patogenicidad , Humanos , Ratones , Perforina/deficiencia , Perforina/genética , Linfocitos T/inmunología , Linfocitos T/patología , Linfocitos T/virología , Proteínas de la Matriz Viral/biosíntesisRESUMEN
Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells.
Asunto(s)
Linfocitos B/metabolismo , Sistemas CRISPR-Cas , Edición Génica/métodos , Macrófagos/metabolismo , Mutagénesis , Linfocitos T/metabolismo , Animales , Diferenciación Celular/genética , Células Cultivadas , Activación de Linfocitos/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Plasmáticas/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity. Here, we used an adeno-associated virus-based CRISPR-Cas9 system with an inhibitor of nonhomologous end joining to repair such mutations in potentially long-lived T cells ex vivo. Repaired CD8 memory T cells efficiently cured lethal hyperinflammation in a mouse model of Epstein-Barr virus-triggered FHL2, a subtype caused by perforin-1 (Prf1) deficiency. Furthermore, repair of PRF1 and Munc13-4 (UNC13D)-whose deficiency causes the FHL subtype FHL3-in mutant memory T cells from two critically ill patients with FHL restored T cell cytotoxicity. These results provide a starting point for the treatment of genetic T cell immune dysregulation syndromes with repaired autologous T cells.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Linfohistiocitosis Hemofagocítica , Animales , Ratones , Humanos , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/terapia , Sistemas CRISPR-Cas , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/terapia , Células T de Memoria , Herpesvirus Humano 4 , Proteínas de la Membrana/genéticaRESUMEN
Most people infected by EBV acquire specific immunity, which then controls latent infection throughout their life. Immune surveillance of EBV-infected cells by cytotoxic CD4+ T cells has been recognized; however, the molecular mechanism of generating cytotoxic effector T cells of the CD4+ subset remains poorly understood. Here we compared phenotypic features and the transcriptome of EBV-specific effector-memory CD4+ T cells and CD8+ T cells in mice and found that both T cell types show cytotoxicity and, to our surprise, widely similar gene expression patterns relating to cytotoxicity. Similar to cytotoxic CD8+ T cells, EBV-specific cytotoxic CD4+ T cells from human peripheral blood expressed T-bet, Granzyme B, and Perforin and upregulated the degranulation marker, CD107a, immediately after restimulation. Furthermore, T-bet expression in cytotoxic CD4+ T cells was highly correlated with Granzyme B and Perforin expression at the protein level. Thus, differentiation of EBV-specific cytotoxic CD4+ T cells is possibly controlled by mechanisms shared by cytotoxic CD8+ T cells. T-bet-mediated transcriptional regulation may explain the similarity of cytotoxic effector differentiation between CD4+ T cells and CD8+ T cells, implicating that this differentiation pathway may be directed by environmental input rather than T cell subset.
RESUMEN
Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes.
Asunto(s)
Desoxirribonucleasas/genética , Desoxirribonucleasas/fisiología , Embrión de Mamíferos/citología , Marcación de Gen/métodos , Ingeniería Genética/métodos , Recombinación Genética/genética , Activación Transcripcional/genética , Activación Transcripcional/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , ADN/genética , Embrión de Mamíferos/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Integrasas/fisiología , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/fisiología , Ratones , Modelos Animales , Datos de Secuencia Molecular , Células 3T3 NIH , Oocitos/citología , Oocitos/fisiologíaRESUMEN
Epstein-Barr virus (EBV) is a γ herpes virus endemic in humans and transforming human B lymphocytes. It causes a variety of human pathologies ranging from infectious mononucleosis upon acute infection to EBV-driven B-cell lymphomas. In humans, EBV-infected cells are under powerful immune surveillance by T and NK cells. If this immune surveillance is compromised as in immunosuppressed (AIDS- or posttransplantation) patients, the virus can spread from rare, EBV-containing cells and cause life-threatening pathologies. We have found that EBV immune surveillance and lymphomagenesis can be modeled in mice by targeted expression of key EBV proteins in the B-cell lineage. As EBV does not infect mouse B cells and mice have thus not coevolved with the virus, EBV exploits basic modes of the host immune response to optimize its coexistence with the host.