RESUMO
Hematopoiesis is a continuous process of blood cell production driven by hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Proliferation and differentiation of HSPCs are regulated by complex transcriptional networks. In order to identify transcription factors with key roles in HSPC-mediated hematopoietic reconstitution, we developed an efficient and robust CRISPR/Cas9-based in vivo genetic screen. Using this experimental system, we identified the TFDP1 transcription factor to be essential for HSPC proliferation and post-transplant hematopoiesis. We further discovered that E2F4, an E2F transcription factor, serves as a binding partner of TFDP1 and is required for HSPC proliferation. Deletion of TFDP1 caused downregulation of genes associated with the cell cycle, with around 50% of these genes being identified as direct targets of TFDP1 and E2F4. Thus, our study expands the transcriptional network governing hematopoietic development through an in vivo CRISPR/Cas9-based genetic screen and identifies TFDP1/E2F4 as positive regulators of cell cycle genes in HSPCs.
RESUMO
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.
Assuntos
Infecções por Vírus Epstein-Barr , Linfo-Histiocitose Hemofagocítica , Animais , Camundongos , Humanos , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/terapia , Sistemas CRISPR-Cas , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/terapia , Células T de Memória , Herpesvirus Humano 4 , Proteínas de Membrana/genéticaRESUMO
The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38-68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200-350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy. Graphical overview.
RESUMO
While CRISPR-Cas9 is key for the development of gene therapy, its potential off-target mutations are still a major concern. Here, we establish a "spacer-nick" gene correction approach that combines the Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200 to 350 bp. In combination with adeno-associated virus (AAV) serotype 6 template delivery, our approach led to efficient HDR in human hematopoietic stem and progenitor cells (HSPCs including long-term HSCs) and T cells, with minimal NHEJ-mediated on-target mutations. Using spacer-nick, we developed an approach to repair disease-causing mutations occurring in the HBB, ELANE, IL7R, and PRF1 genes. We achieved gene correction efficiencies of 20 to 50% with minimal NHEJ-mediated on-target mutations. On the basis of in-depth off-target assessment, frequent unintended genetic alterations induced by classical CRISPR-Cas9 were significantly reduced or absent in the HSPCs treated with spacer-nick. Thus, the spacer-nick gene correction approach provides improved safety and suitability for gene therapy.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Hematopoéticas , Dependovirus , Edição de Genes , Terapia Genética , Humanos , MutaçãoRESUMO
Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANEL172P mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, â¼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.
Assuntos
Sistemas CRISPR-Cas/genética , Síndrome Congênita de Insuficiência da Medula Óssea/terapia , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Elastase de Leucócito/genética , Mutação , Neutropenia/congênito , Alelos , Animais , Diferenciação Celular/genética , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Éxons , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células HEK293 , Humanos , Interleucina-3/genética , Interleucina-3/metabolismo , Camundongos , Camundongos Transgênicos , Neutropenia/genética , Neutropenia/patologia , Neutropenia/terapia , Neutrófilos/metabolismo , RNA Guia de Cinetoplastídeos/genética , Transfecção , Resultado do TratamentoRESUMO
Mutations that accumulate in self-renewing hematopoietic stem and progenitor cells (HSPCs) can cause severe blood disorders. To model such disorders in mice, we developed a CRISPR/Cas9/adeno-associated virus (AAV)-based system to knock in and repair genes by homologous recombination in mouse HSPCs. Here, we provide a step-by-step protocol to achieve high efficiency of gene knockin in mouse HSPCs, while maintaining engraftment capacity. This approach enables the functional study of hematopoietic disease mutations in vivo, without requiring germline mutagenesis. For complete details on the use and execution of this protocol, please refer to Tran et al. (2019).
Assuntos
Sistemas CRISPR-Cas , Técnicas de Introdução de Genes/métodos , Células-Tronco Hematopoéticas/citologia , Recombinação Homóloga , Células-Tronco/citologia , Animais , CamundongosRESUMO
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.
Assuntos
Transformação Celular Viral , Infecções por Vírus Epstein-Barr/patologia , Herpesvirus Humano 4/patogenicidade , Linfoma de Células B/patologia , Plasmócitos/patologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Fibroblastos , Herpesvirus Humano 4/metabolismo , Humanos , Linfoma de Células B/virologia , Camundongos , Camundongos Knockout , Plasmócitos/virologia , Cultura Primária de Células , Transativadores/genética , Transativadores/metabolismo , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/metabolismoRESUMO
Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis.
Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Introdução de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular , CamundongosRESUMO
B2 cells engage in classical antibody responses, whereas B1 cells are considered carriers of innate immunity, biased toward recognizing epitopes present on the surfaces of common pathogens and self antigens. To explore the role of B cell antigen receptor (BCR) specificity in driving B1 cell differentiation, we developed a transgenic system allowing us to change BCR specificity in B cells in an inducible and programmed manner. Mature B2 cells differentiated into bona fide B1 cells upon acquisition of a B1 cell-typical self-reactive BCR through a phase of proliferative expansion. Thus, B2 cells have B1 cell differentiation potential in addition to their classical capacity to differentiate into memory and plasma cells, and B1 differentiation can be instructed by BCR-mediated self-reactivity, in the absence of B1-lineage precommitment.
Assuntos
Subpopulações de Linfócitos B/citologia , Diferenciação Celular/imunologia , Plasticidade Celular/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Plasticidade Celular/genética , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , TranscriptomaRESUMO
Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3' end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes , Motivos de Nucleotídeos/genética , RNA Guia de Cinetoplastídeos/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
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.
Assuntos
Linfócitos B/fisiologia , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Centro Germinativo/imunologia , Integrases/genética , Alelos , Animais , Diferenciação Celular , Humanos , CamundongosRESUMO
Forkhead box class O1 (FOXO1) acts as a tumor suppressor in solid tumors. The oncogenic phosphoinositide-3-kinase (PI3K) pathway suppresses FOXO1 transcriptional activity by enforcing its nuclear exclusion upon AKT-mediated phosphorylation. We show here abundant nuclear expression of FOXO1 in Burkitt lymphoma (BL), a germinal center (GC) B-cell-derived lymphoma whose pathogenesis is linked to PI3K activation. Recurrent FOXO1 mutations, which prevent AKT targeting and lock the transcription factor in the nucleus, are used by BL to circumvent mutual exclusivity between PI3K and FOXO1 activation. Using genome editing in human and mouse lymphomas in which MYC and PI3K cooperate synergistically in tumor development, we demonstrate proproliferative and antiapoptotic activity of FOXO1 in BL and identify its nuclear localization as an oncogenic event in GC B-cell-derived lymphomagenesis.
Assuntos
Linfócitos B , Linfoma de Burkitt , Núcleo Celular , Transformação Celular Neoplásica , Proteína Forkhead Box O1 , Centro Germinativo , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Edição de Genes , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Camundongos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
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.
Assuntos
Linfócitos B/metabolismo , Sistemas CRISPR-Cas , Edição de Genes/métodos , Macrófagos/metabolismo , Mutagênese , Linfócitos T/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Ativação Linfocitária/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmócitos/metabolismo , Reprodutibilidade dos TestesRESUMO
Phosphatidylinositol 3' OH kinase (PI3K) signaling and FOXO transcription factors play opposing roles at several B cell developmental stages. We show here abundant nuclear FOXO1 expression in the proliferative compartment of the germinal center (GC), its dark zone (DZ), and PI3K activity, downregulating FOXO1, in the light zone (LZ), where cells are selected for further differentiation. In the LZ, however, FOXO1 was expressed in a fraction of cells destined for DZ reentry. Upon FOXO1 ablation or induction of PI3K activity, GCs lost their DZ, owing at least partly to downregulation of the chemokine receptor CXCR4. Although this prevented proper cyclic selection of cells in GCs, somatic hypermutation and proliferation were maintained. Class switch recombination was partly lost due to a failure of switch region targeting by activation-induced deaminase (AID).
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Fatores de Transcrição Forkhead/imunologia , Centro Germinativo/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Animais , Linfócitos B/citologia , Separação Celular , Cromatografia Líquida , Citidina Desaminase/imunologia , Citometria de Fluxo , Imunofluorescência , Proteína Forkhead Box O1 , Regulação da Expressão Gênica/imunologia , Centro Germinativo/citologia , Switching de Imunoglobulina/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Mutantes , Reação em Cadeia da Polimerase , Hipermutação Somática de Imunoglobulina/imunologia , Espectrometria de Massas em TandemRESUMO
The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.
Assuntos
Sistemas CRISPR-Cas/genética , Reparo do DNA por Junção de Extremidades/genética , Engenharia Genética/métodos , Adenoviridae/genética , Proteínas E4 de Adenovirus/biossíntese , Proteínas E4 de Adenovirus/genética , Animais , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP , DNA Ligases/genética , Regulação da Expressão Gênica , Genoma Humano , Recombinação Homóloga/genética , Humanos , Camundongos , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
Multiple myeloma is a bone marrow plasma cell tumor which is supported by the external growth factors APRIL and IL-6, among others. Recently, we identified eosinophils and megakaryocytes to be functional components of the micro-environmental niches of benign bone marrow plasma cells and to be important local sources of these cytokines. Here, we investigated whether eosinophils and megakaryocytes also support the growth of tumor plasma cells in the MOPC315.BM model for multiple myeloma. As it was shown for benign plasma cells and multiple myeloma cells, IL-6 and APRIL also supported MOPC315.BM cell growth in vitro, IL-5 had no effect. Depletion of eosinophils in vivo by IL-5 blockade led to a reduction of the early myeloma load. Consistent with this, myeloma growth in early stages was retarded in eosinophil-deficient ΔdblGATA-1 mice. Late myeloma stages were unaffected, possibly due to megakaryocytes compensating for the loss of eosinophils, since megakaryocytes were found to be in contact with myeloma cells in vivo and supported myeloma growth in vitro. We conclude that eosinophils and megakaryocytes in the niches for benign bone marrow plasma cells support the growth of malignant plasma cells. Further investigations are required to test whether perturbation of these niches represents a potential strategy for the treatment of multiple myeloma.
Assuntos
Medula Óssea/patologia , Proliferação de Células , Eosinófilos/citologia , Megacariócitos/citologia , Mieloma Múltiplo/patologia , Animais , Técnicas de Cocultura , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Plasma cells are of crucial importance for long-term immune protection. It is thought that long-lived plasma cells survive in specialized niches in the bone marrow. Here we demonstrate that bone marrow eosinophils localized together with plasma cells and were the key providers of plasma cell survival factors. In vitro, eosinophils supported the survival of plasma cells by secreting the proliferation-inducing ligand APRIL and interleukin-6 (IL-6). In eosinophil-deficient mice, plasma cell numbers were much lower in the bone marrow both at steady state and after immunization. Reconstitution experiments showed that eosinophils were crucial for the retention of plasma cells in the bone marrow. Moreover, depletion of eosinophils induced apoptosis in long-lived bone marrow plasma cells. Our findings demonstrate that the long-term maintenance of plasma cells in the bone marrow requires eosinophils.
Assuntos
Medula Óssea/patologia , Eosinófilos/metabolismo , Interleucina-6/metabolismo , Plasmócitos/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Transferência Adotiva , Animais , Antígenos de Diferenciação/biossíntese , Apoptose/imunologia , Remoção de Componentes Sanguíneos , Medula Óssea/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , Eosinófilos/imunologia , Eosinófilos/patologia , Memória Imunológica , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmócitos/citologia , Plasmócitos/imunologia , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologiaRESUMO
OBJECTIVE: Elevated levels of BAFF and APRIL are characteristic of patients with systemic lupus erythematosus (SLE). The reasons for enhanced cytokine production are not well understood. This study was undertaken to identify the cells responsible for the overproduction of these cytokines. METHODS: BAFF expression was analyzed on peripheral blood mononuclear cells by multiparameter flow cytometry and in tissue samples by immunofluorescence staining. The levels of BAFF and APRIL mRNA were quantified in sorted B cells. In vitro cultures were used to analyze whether B cell survival and differentiation was supported by autocrine BAFF and/or APRIL. RESULTS: Aberrant activation of B cells in patients with SLE was associated with a significant up-regulation of BAFF expression in naive, memory, and plasma cells. Furthermore, strong expression of BAFF and APRIL was found in plasma cells from the lymph node, bone marrow, and kidney. The levels of BAFF and APRIL mRNA in CD19+ B cells correlated both with the titer of anti-double stranded DNA antibodies and with the SLE Disease Activity Index. In vitro experiments demonstrated that B cells released functional BAFF/APRIL upon activation. CONCLUSION: Our data show that B cells contribute to the enhanced levels of circulating BAFF and APRIL. The aberrant up-regulation of these cytokines may initiate a vicious circle in which enhanced levels of BAFF and APRIL act in an autocrine manner to reinforce the systemic activation of the humoral immune system.
Assuntos
Fator Ativador de Células B/metabolismo , Linfócitos B/metabolismo , Sistema Imunitário/fisiopatologia , Lúpus Eritematoso Sistêmico/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Anticorpos Antinucleares/metabolismo , Linfócitos B/patologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Estudos de Casos e Controles , Feminino , Humanos , Rim/metabolismo , Rim/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Lúpus Eritematoso Sistêmico/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Masculino , RNA Mensageiro/metabolismo , Índice de Gravidade de DoençaRESUMO
B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in peripheral B cell survival, maturation, and differentiation. BAFF and APRIL are produced by a variety of cell types such as macrophages/monocytes and dendritic cells. Our analysis shows that BAFF mRNA is also expressed in all B cell subsets isolated from bone marrow, spleen, and peritoneal cavity of BALB/c mice. APRIL expression is restricted to early stages of B cell development in the bone marrow and the peritoneal B1 subset. Stimulation of B2 and B1 cells with LPS or CpG-oligodeoxynucleotides induced MyD88-dependent plasma cell differentiation and intracellular expression of BAFF and APRIL. Furthermore, activation of B cells up-regulated membrane expression of BAFF. The finding that in vitro activation of B cells is inhibited by the antagonist transmembrane activator and calcium modulator ligand interactor Ig, indicates that BAFF and/or APRIL are released into the culture supernatants. It shows that B cell survival, proliferation, and differentiation are supported by an autocrine pathway. In vivo activation of B cells with a T-dependent Ag- induced BAFF expression in germinal center B cells. In (NZB x NZW)F(1) mice with established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of BAFF. In (NZB x NZW)F(1) mice, the continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to the development of autoimmune disease.