RESUMO
Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through nontemplated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had nonphysiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted the generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for the generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Vacinas , Camundongos , Humanos , Animais , Anticorpos Amplamente Neutralizantes , Anticorpos Neutralizantes , HIV-1/genética , Anticorpos Anti-HIV , DNA Nucleotidilexotransferase , Regiões Determinantes de Complementaridade/genética , Infecções por HIV/prevenção & controleRESUMO
Antibodies against fetal red blood cell (RBC) antigens can cause hemolytic disease of the fetus and newborn (HDFN). Reductions in HDFN due to anti-RhD antibodies have been achieved through use of Rh immune globulin (RhIg), a polyclonal antibody preparation that causes antibody-mediated immunosuppression (AMIS), thereby preventing maternal immune responses against fetal RBCs. Despite the success of RhIg, it is only effective against 1 alloantigen. The lack of similar interventions that mitigate immune responses toward other RBC alloantigens reflects an incomplete understanding of AMIS mechanisms. AMIS has been previously attributed to rapid antibody-mediated RBC removal, resulting in B-cell ignorance of the RBC alloantigen. However, our data demonstrate that antibody-mediated RBC removal can enhance de novo alloimmunization. In contrast, inclusion of antibodies that possess the ability to rapidly remove the target antigen in the absence of detectable RBC clearance can convert an augmented antibody response to AMIS. These results suggest that the ability of antibodies to remove target antigens from the RBC surface can trigger AMIS in situations in which enhanced immunity may otherwise occur. In doing so, these results hold promise in identifying key antibody characteristics that can drive AMIS, thereby facilitating the design of AMIS approaches toward other RBC antigens to eliminate all forms of HDFN.
Assuntos
Eritroblastose Fetal , Eritrócitos , Feminino , Recém-Nascido , Humanos , Eritrócitos/metabolismo , Anticorpos , Tolerância Imunológica , Terapia de Imunossupressão , Imunoglobulina rho(D) , Isoantígenos , IsoanticorposRESUMO
BACKGROUND: Sickle cell disease is characterized by hemolytic anemia, pain, and progressive organ damage. A high level of erythrocyte fetal hemoglobin (HbF) comprising α- and γ-globins may ameliorate these manifestations by mitigating sickle hemoglobin polymerization and erythrocyte sickling. BCL11A is a repressor of γ-globin expression and HbF production in adult erythrocytes. Its down-regulation is a promising therapeutic strategy for induction of HbF. METHODS: We enrolled patients with sickle cell disease in a single-center, open-label pilot study. The investigational therapy involved infusion of autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes a short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA (shmiR), allowing erythroid lineage-specific knockdown. Patients were assessed for primary end points of engraftment and safety and for hematologic and clinical responses to treatment. RESULTS: As of October 2020, six patients had been followed for at least 6 months after receiving BCH-BB694 gene therapy; median follow-up was 18 months (range, 7 to 29). All patients had engraftment, and adverse events were consistent with effects of the preparative chemotherapy. All the patients who could be fully evaluated achieved robust and stable HbF induction (percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.3%), with HbF broadly distributed in red cells (F-cells 58.9 to 93.6% of untransfused red cells) and HbF per F-cell of 9.0 to 18.6 pg per cell. Clinical manifestations of sickle cell disease were reduced or absent during the follow-up period. CONCLUSIONS: This study validates BCL11A inhibition as an effective target for HbF induction and provides preliminary evidence that shmiR-based gene knockdown offers a favorable risk-benefit profile in sickle cell disease. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656).
Assuntos
Anemia Falciforme/terapia , Hemoglobina Fetal/biossíntese , Terapia Genética , Interferência de RNA , Proteínas Repressoras/genética , gama-Globinas/metabolismo , Adolescente , Adulto , Anemia Falciforme/genética , Criança , Regulação para Baixo , Feminino , Hemoglobina Fetal/genética , Técnicas de Silenciamento de Genes , Vetores Genéticos , Humanos , Masculino , Projetos Piloto , RNA Interferente Pequeno , Proteínas Repressoras/metabolismo , Transplante Autólogo , Adulto Jovem , gama-Globinas/genéticaRESUMO
The adaptors DOCK8 and MyD88 have been linked to serological memory. Here we report that DOCK8-deficient patients had impaired antibody responses and considerably fewer CD27(+) memory B cells. B cell proliferation and immunoglobulin production driven by Toll-like receptor 9 (TLR9) were considerably lower in DOCK8-deficient B cells, but those driven by the costimulatory molecule CD40 were not. In contrast, TLR9-driven expression of AICDA (which encodes the cytidine deaminase AID), the immunoglobulin receptor CD23 and the costimulatory molecule CD86 and activation of the transcription factor NF-κB, the kinase p38 and the GTPase Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.
Assuntos
Linfócitos B/imunologia , Fatores de Troca do Nucleotídeo Guanina/imunologia , Memória Imunológica/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Receptor Toll-Like 9/imunologia , Adolescente , Animais , Diferenciação Celular/imunologia , Criança , Pré-Escolar , Citometria de Fluxo , Quinase 2 de Adesão Focal/imunologia , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Fosforilação , Fator de Transcrição STAT3/imunologia , Quinases da Família src/imunologiaRESUMO
A promising treatment for ß-hemoglobinopathies is the de-repression of γ-globin expression leading to increased fetal hemoglobin (HbF) by targeting BCL11A. Here, we aim to improve a lentivirus vector (LV) containing a single BCL11A shmiR (SS) to further increase γ-globin induction. We engineered a novel LV to express two shmiRs simultaneously targeting BCL11A and the γ-globin repressor ZNF410. Erythroid cells derived from human HSCs transduced with the double shmiR (DS) showed up to a 70% reduction of both BCL11A and ZNF410 proteins. There was a consistent and significant additional 10% increase in HbF compared to targeting BCL11A alone in erythroid cells. Erythrocytes differentiated from SCD HSCs transduced with the DS demonstrated significantly reduced in vitro sickling phenotype compared to the SS. Erythrocytes differentiated from transduced HSCs from ß-thalassemia major patients demonstrated improved globin chain balance by increased γ-globin with reduced microcytosis. Reconstitution of DS-transduced cells from Berkeley SCD mice was associated with a statistically larger reduction in peripheral blood hemolysis markers compared with the SS vector. Overall, these results indicate that the DS LV targeting BCL11A and ZNF410 can enhance HbF induction for treating ß-hemoglobinopathies and could be used as a model to simultaneously and efficiently target multiple gene products.
Assuntos
Hemoglobina Fetal , Hemoglobinopatias , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Hemoglobinopatias/genética , Hemoglobinopatias/terapia , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , gama-Globinas/genéticaRESUMO
During B-cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cµ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200 kilobases downstream in the same transcriptional orientation as V(D)J and Cµ exons. Long repetitive switch (S) regions precede Cµ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cµ with a downstream CH (ref. 2). Activation-induced cytidine deaminase (AID) initiates CSR by promoting deamination lesions within Sµ and a downstream acceptor S region; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sµ DSB to the downstream end of an acceptor S-region DSB. However, the relative frequency of deletional to inversional CSR junctions has not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved is unknown. To address this question, we adapt high-throughput genome-wide translocation sequencing into a highly sensitive DSB end-joining assay and apply it to endogenous AID-initiated S-region DSBs in mouse B cells. We show that CSR is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis Igh organizational features in combination with frequent S-region DSBs initiated by AID. We further implicate ATM-dependent DSB-response factors in enforcing this mechanism and provide an explanation of why CSR is so reliant on the 53BP1 DSB-response factor.
Assuntos
Linfócitos B/metabolismo , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Switching de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos B/enzimologia , Linfócitos B/imunologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desaminação , Camundongos , Deleção de Sequência/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Éxons VDJ/genéticaRESUMO
Emerging cellular therapies require the collection of peripheral blood hematopoietic stem cells (HSC) by apheresis for in vitro manipulation to accomplish gene addition or gene editing. These therapies require relatively large numbers of HSCs within a short time frame to generate an efficacious therapeutic product. This review focuses on the principal factors that affect collection outcomes, especially relevant to gene therapy for sickle cell disease.
Assuntos
Anemia Falciforme/terapia , Edição de Genes/métodos , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Condicionamento Pré-Transplante/métodos , Anemia Falciforme/patologia , HumanosRESUMO
A proportion of homologous recombination (HR) events in mammalian cells resolve by "long tract" gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Neoplasias Ovarianas/genética , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Cromátides/genética , Reparo do DNA por Junção de Extremidades/genética , Feminino , Conversão Gênica/genética , Instabilidade Genômica/genética , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , Neoplasias Ovarianas/patologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1006410.].
RESUMO
Loss-of-function mutations in the signal transducer and activator of transcription 3 gene (stat3) result in autosomal dominant hyper-IgE syndrome (AD-HIES), a condition in which patients have recurrent debilitating infections, including frequent pneumococcal and staphylococcal pneumonias. stat3 mutations cause defective adaptive TH17 cellular responses, an immune mechanism believed to be critical for clearance of pneumococcal colonization and diminished antibody responses. Here we wished to evaluate the role of stat3 in the clearance of pneumococcal carriage and immunity using mice with a stat3 mutation recapitulating AD-HIES. We show here that naive AD-HIES mice have prolonged nasal carriage of pneumococcus compared to WT mice. Mutant and wild-type mice were then immunized with a pneumococcal whole-cell vaccine (WCV) that provides TH17-mediated protection against pneumococcal colonization and antibody-mediated protection against pneumonia and sepsis. WCV-immunized AD-HIES mice made significantly less pneumococcus-specific interleukin-17A (IL-17A) and antibody than WT mice. The WCV-elicited protection against colonization was abrogated in AD-HIES mice, but immunization with WCV still protected AD-HIES mice against aspiration pneumonia/sepsis. Taken together, our results suggest that impaired clearance of nasopharyngeal carriage due to poor adaptive IL-17A responses may contribute to the increased rates of pneumococcal respiratory infection in AD-HIES patients.
Assuntos
Síndrome de Job/genética , Síndrome de Job/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Células Th17/imunologia , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Streptococcus pneumoniae/imunologiaRESUMO
In this issue of Blood, Arthur et al uncover that HLA alloantibodies cannot solely account for the immune mechanism in platelet refractoriness.
Assuntos
Plaquetas/imunologia , Linfócitos T CD8-Positivos/imunologia , Transfusão de Plaquetas , Animais , HumanosRESUMO
During an immune response, B cells undergo rapid proliferation and activation-induced cytidine deaminase (AID)-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here, we report that exogenous and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patient samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress.
Assuntos
Linfócitos B/citologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/imunologia , Citidina Desaminase/genética , Dano ao DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos da radiação , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/efeitos da radiação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica/imunologia , Centro Germinativo/citologia , Humanos , Switching de Imunoglobulina/fisiologia , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Plasmócitos/citologia , Plasmócitos/imunologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: The B-cell receptor transmembrane activator and calcium modulator ligand interactor (TACI) is important for T-independent antibody responses. One in 200 blood donors are heterozygous for the TACI A181E mutation. OBJECTIVE: We sought to investigate the effect on B-cell function of TACI A181E heterozygosity in reportedly healthy subjects and of the corresponding TACI A144E mutation in mice. METHODS: Nuclear factor κB (NF-κB) activation was measured by using the luciferase assay in 293T cells cotransfected with wild-type and mutant TACI. TACI-driven proliferation, isotype switching, and antibody responses were measured in B cells from heterozygous TACI A144E knock-in mice. Mouse mortality was monitored after intranasal pneumococcal challenge. RESULTS: Levels of natural antibodies to the pneumococcal polysaccharide component phosphocholine were significantly lower in A181E-heterozygous than TACI-sufficient Swedish blood donors never immunized with pneumococcal antigens. Although overexpressed hTACI A181E and mTACI A144E acted as dominant-negative mutations in transfectants, homozygosity for A144E in mice resulted in absent TACI expression in B cells, indicating that the mutant protein is unstable when naturally expressed. A144E heterozygous mice, such as TACI+/- mice, expressed half the normal level of TACI on their B cells and exhibited similar defects in a proliferation-inducing ligand-driven B-cell activation, antibody responses to TNP-Ficoll, production of natural antibodies to phosphocholine, and survival after intranasal pneumococcal challenge. CONCLUSION: These results suggest that TACI A181E heterozygosity results in TACI haploinsufficiency with increased susceptibility to pneumococcal infection. This has important implications for asymptomatic TACI A181E carriers.
Assuntos
Pneumonia Pneumocócica/genética , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Técnicas de Introdução de Genes , Células HEK293 , Haploinsuficiência , Heterozigoto , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia Pneumocócica/imunologia , Reação em Cadeia da Polimerase , Proteína Transmembrana Ativadora e Interagente do CAML/imunologiaRESUMO
Although the BBAP E3 ligase and its binding partner BAL are overexpressed in chemotherapy-resistant lymphomas, the role of these proteins in DNA damage responses remains undefined. Because BAL proteins modulate promoter-coupled transcription and contain structural motifs associated with chromatin remodeling and DNA repair, we reasoned that the BBAP E3 ligase might target nucleosomal proteins. Herein, we demonstrate that BBAP selectively monoubiquitylates histone H4 lysine 91 and protects cells exposed to DNA-damaging agents. Disruption of BBAP-mediated monoubiquitylation of histone H4K91 is associated with the loss of chromatin-associated H4K20 methylase, mono- and dimethyl H4K20, and a delay in the kinetics of 53BP1 foci formation at sites of DNA damage. Because 53BP1 localizes to DNA damage sites, in part, via an interaction with dimethyl H4K20, these data directly implicate BBAP in the monoubiquitylation and additional posttranslational modification of histone H4 and an associated DNA damage response.
Assuntos
Dano ao DNA/fisiologia , Histonas/metabolismo , Lisina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Células HeLa , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metilação/efeitos dos fármacos , Nucleossomos/metabolismo , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/fisiologiaRESUMO
Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation, microcephaly, and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4), Artemis, and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders, we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity, but not Artemis deficiency, were associated with markedly reduced reprogramming efficiency, which could be partially rescued by genetic complementation. Moreover, we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest, particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-, but not Artemis- or DNA-PK-mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming, with a major role for LIG4 and DNA-PKcs and a minor, if any, for Artemis.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Células-Tronco Pluripotentes Induzidas/citologia , Catálise , Ciclo Celular , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA , Endonucleases , Fibroblastos/metabolismo , Fibroblastos/patologia , Células-Tronco Hematopoéticas/citologia , Humanos , Mutação , Proteínas Nucleares/metabolismo , FenótipoRESUMO
The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-ß (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cε germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cε GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-κB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iε promoter. Addition of the NF-κB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-κB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cε locus and class switching to IgE.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina E/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Citidina Desaminase/metabolismo , Immunoblotting , Switching de Imunoglobulina/efeitos dos fármacos , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Cadeias épsilon de Imunoglobulina/genética , Cadeias épsilon de Imunoglobulina/imunologia , Cadeias épsilon de Imunoglobulina/metabolismo , Cadeias gama de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/metabolismo , Interleucina-4/imunologia , Interleucina-4/farmacologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Fenilenodiaminas/imunologia , Fenilenodiaminas/farmacologia , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismoRESUMO
BACKGROUND: The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID). OBJECTIVE: We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency. METHODS: We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of γH2AX accumulation was studied after ionizing radiation. RESULTS: Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect. CONCLUSIONS: Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.
Assuntos
Linfócitos B/fisiologia , Mutação/genética , Proteínas Nucleares/genética , Imunodeficiência Combinada Severa/genética , Adolescente , Adulto , Alelos , Linfócitos B/efeitos da radiação , Linhagem Celular Transformada , Criança , Pré-Escolar , Análise Mutacional de DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA , Endonucleases , Heterozigoto , Histonas/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas Oncogênicas v-abl/genética , Proteínas Oncogênicas v-abl/metabolismo , Fenótipo , Tolerância a Radiação/genética , Radiação Ionizante , Recombinação V(D)J/genética , Adulto JovemRESUMO
Progenitor B cells reside in complex bone marrow (BM) microenvironments where they receive signals for growth and maturation. We reported previously that the CXCL12-focal adhesion kinase (FAK)-VLA4 pathway plays an important role in progenitor B cell adhesion and migration. In this study, we have conditionally targeted in B cells FAK, and found that the numbers of progenitor pro-B, pre-B, and immature B cells are reduced by 30-40% in B cell-specific FAK knockout mice. When cultured in methylcellulose with IL-7 ± CXCL12, Fak-deleted pro-B cells yield significantly fewer cells and colonies. Using in situ quantitative imaging cytometry, we establish that in longitudinal femoral BM sections, pro-B cells are preferentially localized in close proximity to the endosteum of the metaphyses and the diaphysis. Fak deletion disrupts the nonrandom distribution of pro-B cells and induces the mobilization of pro-B cells to the periphery in vivo. These effects of Fak deletion on pro-B cell mobilization and localization in BM are amplified under inflammatory stress, that is, after immunization with nitrophenol-conjugated chicken γ-globulin in alum. Collectively, these studies suggest the importance of FAK in regulating pro-B cell homeostasis and maintenance of their spatial distribution in BM niches.