RESUMO
The single-nucleotide polymorphism rs1990760 in the gene encoding the cytosolic viral sensor IFIH1 results in an amino-acid change (A946T; IFIH1T946) that is associated with multiple autoimmune diseases. The effect of this polymorphism on both viral sensing and autoimmune pathogenesis remains poorly understood. Here we found that human peripheral blood mononuclear cells (PBMCs) and cell lines expressing the risk variant IFIH1T946 exhibited heightened basal and ligand-triggered production of type I interferons. Consistent with those findings, mice with a knock-in mutation encoding IFIH1T946 displayed enhanced basal expression of type I interferons, survived a lethal viral challenge and exhibited increased penetrance in autoimmune models, including a combinatorial effect with other risk variants. Furthermore, IFIH1T946 mice manifested an embryonic survival defect consistent with enhanced responsiveness to RNA self ligands. Together our data support a model wherein the production of type I interferons driven by an autoimmune risk variant and triggered by ligand functions to protect against viral challenge, which probably accounts for its selection within human populations but provides this advantage at the cost of modestly promoting the risk of autoimmunity.
Assuntos
Autoimunidade/genética , Infecções por Cardiovirus/genética , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Adolescente , Adulto , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Southern Blotting , Infecções por Cardiovirus/imunologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Vírus da Encefalomiocardite/imunologia , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Immunoblotting , Helicase IFIH1 Induzida por Interferon/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Viroses/genética , Viroses/imunologia , Adulto JovemRESUMO
Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.
Assuntos
Anticorpos Biespecíficos , Antígenos CD19 , Plasmócitos , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Anticorpos Biespecíficos/farmacologia , Animais , Camundongos , Antígenos CD19/imunologia , Antígenos CD19/genética , Antígenos CD19/metabolismo , Plasmócitos/metabolismo , Plasmócitos/imunologia , Linhagem Celular Tumoral , Linfócitos T/imunologia , Linfócitos T/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Complexo CD3/genética , Ativação Linfocitária/imunologia , Citotoxicidade ImunológicaRESUMO
Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Variação Genética , Guanilato Ciclase/genética , Síndromes de Imunodeficiência/genética , Adenina/análogos & derivados , Adenina/farmacologia , Proteína 10 de Linfoma CCL de Células B/genética , Linfócitos B/citologia , Linhagem Celular , Diploide , Éxons , Genes Dominantes , Humanos , Células Jurkat , Linfoma/genética , Subunidade p50 de NF-kappa B/genética , Piperidinas/farmacologia , Polimorfismo de Nucleotídeo Único , Doenças da Imunodeficiência Primária/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Mast cells are involved in many distinct pathologic conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. OBJECTIVE: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. METHODS: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by real-time quantitative PCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. RESULTS: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins were small GTPases, receptors, and transporters. One such cell surface protein was CD98 heavy chain (CD98hc), encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. CONCLUSIONS: Glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, CD98hc plays an important role in mast cell function.
Assuntos
Cadeia Pesada da Proteína-1 Reguladora de Fusão/análise , Mastócitos/química , Proteínas de Membrana/análise , Proteoma , Animais , Células Cultivadas , Cadeia Pesada da Proteína-1 Reguladora de Fusão/fisiologia , Mastócitos/fisiologia , CamundongosRESUMO
Folliculin interacting protein 1 (Fnip1) is a cytoplasmic protein originally discovered through its interaction with the master metabolic sensor 5' AMP-activated protein kinase (AMPK) and Folliculin, a protein mutated in individuals with Birt-Hogg-Dubé Syndrome. In response to low energy, AMPK stimulates catabolic pathways such as autophagy to enhance energy production while inhibiting anabolic pathways regulated by the mechanistic target of rapamycin complex 1 (mTORC1). We previously found that constitutive disruption of Fnip1 in mice resulted in a lack of peripheral B cells because of a block in B cell development at the pre-B cell stage. Both AMPK and mTORC1 were activated in Fnip1-deficient B cell progenitors. In this study, we found inappropriate mTOR localization at the lysosome under nutrient-depleted conditions. Ex vivo lysine or arginine depletion resulted in increased apoptosis. Genetic inhibition of AMPK, inhibition of mTORC1, or restoration of cell viability with a Bcl-xL transgene failed to rescue B cell development in Fnip1-deficient mice. Fnip1-deficient B cell progenitors exhibited increased nuclear localization of transcription factor binding to IgHM enhancer 3 (TFE3) in developing B cells, which correlated with an increased expression of TFE3-target genes, increased lysosome numbers and function, and increased autophagic flux. These results indicate that Fnip1 modulates autophagy and energy response pathways in part through the regulation of AMPK, mTORC1, and TFE3 in B cell progenitors.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Linfócitos B/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Transporte/metabolismo , Homeostase , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation remains largely unknown. We have engineered the naturally occurring MBT domain repeats of L3MBTL1 to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or dimethylated lysine. The domain is broadly specific for methylated lysine ("pan-specific") and can be applied to any biological system. We have used our approach to demonstrate that SIRT1 is a substrate of the methyltransferase G9a both in vitro and in cells, to perform proteome-wide detection and enrichment of methylated proteins, and to identify candidate in-cell substrates of G9a and the related methyltransferase GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.
Assuntos
Lisina/genética , Lisina/metabolismo , Proteoma/genética , Proteoma/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Insetos , Metilação , Estrutura Terciária de Proteína , Proteômica/métodos , Células Sf9 , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Edição de Genes , Engenharia Genética , Plasmócitos/imunologia , Plasmócitos/metabolismo , Reparo de DNA por Recombinação , Animais , Biomarcadores , Proteína 9 Associada à CRISPR , Citocinas/metabolismo , Dependovirus/genética , Loci Gênicos , Vetores Genéticos/genética , Humanos , Imunoterapia , Camundongos , Fenótipo , Polimorfismo de Nucleotídeo Único , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Receptores CCR5/genética , Transdução GenéticaRESUMO
BACKGROUND: Respiratory viral infection in early childhood, including that from respiratory syncytial virus (RSV), has been previously associated with the development of asthma. OBJECTIVE: We aimed to determine whether ex vivo RSV infection of bronchial epithelial cells (BECs) from children with asthma would induce specific gene expression patterns and whether such patterns were associated with lung function among BEC donors. METHODS: Primary BECs from carefully characterized children with asthma (n = 18) and matched healthy children without asthma (n = 8) were differentiated at an air-liquid interface for 21 days. Air-liquid interface cultures were infected with RSV for 96 hours and RNA was subsequently isolated from BECs. In each case, we analyzed gene expression using RNA sequencing and assessed differences between conditions by linear modeling of the data. BEC donors completed spirometry to measure lung function. RESULTS: RSV infection of BECs from subjects with asthma, compared with uninfected BECs from subjects with asthma, led to a significant increase in expression of 6199 genes. There was significantly greater expression of 195 genes in BECs from children with asthma and airway obstruction (FEV1/forced vital capacity < 0.85 and FEV1 < 100% predicted) than in BECs from children with asthma without obstruction, or in BECs from healthy children. These specific genes were found to be highly enriched for viral response genes induced in parallel with types I and III interferons. CONCLUSIONS: BECs from children with asthma and with obstructive physiology exhibit greater expression of types I and III interferons and interferon-stimulated genes than do cells from children with normal lung function, and expression of interferon-associated genes correlates with the degree of airway obstruction. These findings suggest that an exaggerated interferon response to viral infection by airway epithelial cells may be a mechanism leading to lung function decline in a subset of children with asthma.
Assuntos
Asma/imunologia , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Pulmão/fisiologia , Mucosa Respiratória/fisiologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Adolescente , Asma/complicações , Células Cultivadas , Criança , Feminino , Humanos , Imunidade Inata , Interferon Tipo I/genética , Interferon gama/genética , Masculino , Infecções por Vírus Respiratório Sincicial/complicações , Análise de Sequência de RNA , Espirometria , TranscriptomaRESUMO
Bronchial epithelial cells (BECs) from healthy children inhibit human lung fibroblast (HLF) expression of collagen and fibroblast-to-myofibroblast transition (FMT), whereas asthmatic BECs do so less effectively, suggesting that diminished epithelial-derived regulatory factors contribute to airway remodeling. Preliminary data demonstrated that secretion of the activin A inhibitor follistatin-like 3 (FSTL3) by healthy BECs was greater than that by asthmatic BECs. We sought to determine the relative secretion of FSTL3 and activin A by asthmatic and healthy BECs, and whether FSTL3 inhibits FMT. To quantify the abundance of the total proteome FSTL3 and activin A in supernatants of differentiated BEC cultures from healthy children and children with asthma, we performed mass spectrometry and ELISA. HLFs were cocultured with primary BECs and then HLF expression of collagen I and α-smooth muscle actin (α-SMA) was quantified by qPCR, and FMT was quantified by flow cytometry. Loss-of-function studies were conducted using lentivirus-delivered shRNA. Using mass spectrometry and ELISA results from larger cohorts, we found that FSTL3 concentrations were greater in media conditioned by healthy BECs compared with asthmatic BECs (4,012 vs. 2,553 pg/ml; P = 0.002), and in media conditioned by asthmatic BECs from children with normal lung function relative to those with airflow obstruction (FEV1/FVC ratio < 0.8; n = 9; 3,026 vs. 1,922 pg/ml; P = 0.04). shRNA depletion of FSTL3 in BECs (n = 8) increased HLF collagen I expression by 92% (P = 0.001) and α-SMA expression by 88% (P = 0.02), and increased FMT by flow cytometry in cocultured HLFs, whereas shRNA depletion of activin A (n = 6) resulted in decreased α-SMA (22%; P = 0.01) expression and decreased FMT. Together, these results indicate that deficient FSTL3 expression by asthmatic BECs impairs epithelial regulation of HLFs and FMT.
Assuntos
Asma/patologia , Epitélio/metabolismo , Epitélio/patologia , Proteínas Relacionadas à Folistatina/deficiência , Pulmão/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Actinas/metabolismo , Ativinas/metabolismo , Adolescente , Sequência de Aminoácidos , Criança , Colágeno Tipo I/metabolismo , Células Epiteliais/metabolismo , Feminino , Proteínas Relacionadas à Folistatina/química , Proteínas Relacionadas à Folistatina/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Masculino , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: Mast cells are significantly involved in IgE-mediated allergic reactions; however, their roles in health and disease are incompletely understood. OBJECTIVE: We aimed to define the proteome contained in mast cell releasates on activation to better understand the factors secreted by mast cells that are relevant to the contribution of mast cells in diseases. METHODS: Bone marrow-derived cultured mast cells (BMCMCs) and peritoneal cell-derived mast cells were used as "surrogates" for mucosal and connective tissue mast cells, respectively, and their releasate proteomes were analyzed by mass spectrometry. RESULTS: Our studies showed that BMCMCs and peritoneal cell-derived mast cells produced substantially different releasates following IgE-mediated activation. Moreover, we observed that the transglutaminase coagulation factor XIIIA (FXIIIA) was one of the most abundant proteins contained in the BMCMC releasates. Mast cell-deficient mice exhibited increased FXIIIA plasma and activity levels as well as reduced bleeding times, indicating that mast cells are more efficient in their ability to downregulate FXIIIA than in contributing to its amounts and functions in homeostatic conditions. We found that human chymase and mouse mast cell protease-4 (the mouse homologue of human chymase) had the ability to reduce FXIIIA levels and function via proteolytic degradation. Moreover, we found that chymase deficiency led to increased FXIIIA amounts and activity, as well as reduced bleeding times in homeostatic conditions and during sepsis. CONCLUSIONS: Our study indicates that the mast cell protease content can shape its releasate proteome. Moreover, we found that chymase plays an important role in the regulation of FXIIIA via proteolytic degradation.
Assuntos
Quimases/metabolismo , Fator XIII/metabolismo , Mastócitos/metabolismo , Animais , Medula Óssea , Células Cultivadas , Homeostase/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peritônio , Proteólise , Proteoma , Sepse/imunologiaRESUMO
Previous work has shown conflicting roles for Tec family kinases in regulation of TLR-dependent signaling in myeloid cells. In the present study, we performed a detailed investigation of the role of the Tec kinases Btk and Tec kinases in regulating TLR signaling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less proinflammatory cytokines in response to TLR stimulation than do wild-type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more proinflammatory cytokines than do wild-type cells. We then compared the phosphoproteome regulated by Tec kinases and LPS in primary peritoneal and bone marrow-derived macrophages. From this analysis we determined that Tec kinases regulate different signaling programs in these cell types. In additional studies using bone marrow-derived macrophages, we found that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signaling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signaling in many types of myeloid cells. However, our data also support a cell type-specific TLR inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signaling via PI3K.
Assuntos
Macrófagos/imunologia , Fosfoproteínas/imunologia , Proteínas Tirosina Quinases/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Cavidade Peritoneal/citologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfoproteínas/genética , Fosforilação , Cultura Primária de Células , Proteínas Tirosina Quinases/genética , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
The intermediate mesoderm (IM) is the embryonic source of all kidney tissue in vertebrates. The factors that regulate the formation of the IM are not yet well understood. Through investigations in the chick embryo, the current study identifies and characterizes Vg1/Nodal signaling (henceforth referred to as 'Nodal-like signaling') as a novel regulator of IM formation. Excess Nodal-like signaling at gastrulation stages resulted in expansion of the IM at the expense of the adjacent paraxial mesoderm, whereas inhibition of Nodal-like signaling caused repression of IM gene expression. IM formation was sensitive to levels of the Nodal-like pathway co-receptor Cripto and was inhibited by a truncated form of the secreted molecule cerberus, which specifically blocks Nodal, indicating that the observed effects are specific to the Nodal-like branch of the TGFß signaling pathway. The IM-promoting effects of Nodal-like signaling were distinct from the known effects of this pathway on mesoderm formation and left-right patterning, a finding that can be attributed to specific time windows for the activities of these Nodal-like functions. Finally, a link was observed between Nodal-like and BMP signaling in the induction of IM. Activation of IM genes by Nodal-like signaling required an active BMP signaling pathway, and Nodal-like signals induced phosphorylation of Smad1/5/8, which is normally associated with activation of BMP signaling pathways. We postulate that Nodal-like signaling regulates IM formation by modulating the IM-inducing effects of BMP signaling.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Rim/embriologia , Mesoderma/embriologia , Ligantes da Sinalização Nodal/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Embrião de Galinha , Clonagem Molecular , Eletroporação , Imunofluorescência , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Rim/citologia , Fosforilação , Proteínas Smad/metabolismoRESUMO
Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of ß-catenin-responsive transcription (ß-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of ß-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/ß-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.
Assuntos
Melanoma/metabolismo , Proteína Quinase C/genética , Via de Sinalização Wnt/genética , Proteína Wnt3A/metabolismo , Apoptose , Linhagem Celular Tumoral , Receptores Frizzled/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/patologia , Fosforilação , Proteína Quinase C/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Proteína Wnt3A/antagonistas & inibidores , Proteína Wnt3A/genética , beta Catenina/metabolismoRESUMO
Posttransplant lymphoproliferative disease (PTLD) is a major therapeutic challenge that has been difficult to study using human cells because of a lack of suitable models for mechanistic characterization. Here, we show that ex vivo-differentiated B cells isolated from a subset of healthy donors can elicit pathologies similar to PTLD when transferred into immunodeficient mice. The primary driver of PTLD-like pathologies were IgM-producing plasmablasts with Epstein-Barr virus (EBV) genomes that expressed genes commonly associated with EBV latency. We show that a small subset of EBV+ peripheral blood-derived B cells expressing self-reactive, nonmutated B cell receptors (BCRs) expand rapidly in culture in the absence of BCR stimulation. Furthermore, we found that in vitro and in vivo expansion of EBV+ plasmablasts required BCR signaling. Last, treatment of immunodeficient mice with the BCR pathway inhibitor, ibrutinib, delays onset of PTLD-like pathologies in vivo. These data have implications for the diagnosis and care of transplant recipients who are at risk of developing PTLD.
Assuntos
Infecções por Vírus Epstein-Barr , Transtornos Linfoproliferativos , Humanos , Animais , Camundongos , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/terapia , Herpesvirus Humano 4 , Transtornos Linfoproliferativos/terapia , Transdução de Sinais , Linfócitos BRESUMO
WTX is a tumor suppressor protein that is lost or mutated in up to 30% of cases of Wilms tumor. Among its known functions, WTX interacts with the ß-transducin repeat containing family of ubiquitin ligase adaptors and promotes the ubiquitination and degradation of the transcription factor ß-catenin, a key control point in the WNT/ß-catenin signaling pathway. Here, we report that WTX interacts with a second ubiquitin ligase adaptor, KEAP1, which functions to regulate the ubiquitination of the transcription factor NRF2, a key control point in the antioxidant response. Surprisingly, we find that unlike its ability to promote the ubiquitination of ß-catenin, WTX inhibits the ubiquitination of NRF2. WTX and NRF2 compete for binding to KEAP1, and thus loss of WTX leads to rapid ubiquitination and degradation of NRF2 and a reduced response to cytotoxic insult. These results expand our understanding of the molecular mechanisms of WTX and reveal a novel regulatory mechanism governing the antioxidant response.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antioxidantes/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Tumor de Wilms/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Ligação Competitiva/fisiologia , Cromossomos Humanos X/genética , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Fosforilação/fisiologia , RNA Interferente Pequeno/genética , Serina/metabolismo , Ativação Transcricional/fisiologia , Proteínas Supressoras de Tumor/genética , Ubiquitinação/fisiologia , Tumor de Wilms/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
Bispecific antibodies are an important tool for the management and treatment of acute leukemias. Advances in genome-engineering have enabled the generation of human plasma cells that secrete therapeutic proteins and are capable of long-term in vivo engraftment in humanized mouse models. As a next step towards clinical translation of engineered plasma cells (ePCs) towards cancer therapy, here we describe approaches for the expression and secretion of bispecific antibodies by human plasma cells. We show that human ePCs expressing either fragment crystallizable domain deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of specific primary human cell subsets and B-acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19 bispecific secretion by ePCs and prevents self-targeting. Further, anti-CD19 bispecific-ePCs elicited tumor eradication in vivo following local delivery in flank-implanted Raji lymphoma cells. Finally, immunodeficient mice engrafted with anti-CD19 bispecific-ePCs and autologous T cells potently prevented in vivo growth of CD19+ acute lymphoblastic leukemia in patient-derived xenografts. Collectively, these findings support further development of ePCs for use as a durable, local delivery system for the treatment of acute leukemias, and potentially other cancers.
RESUMO
The secreted products of cells drive many functions in vivo; however, methods to link this functional information to surface markers and transcriptomes have been lacking. By accumulating secretions close to secreting cells held within cavity-containing hydrogel nanovials, we demonstrate workflows to analyze the amount of IgG secreted from single human B cells and link this information to surface markers and transcriptomes from the same cells. Measurements using flow cytometry and imaging flow cytometry corroborate the association between IgG secretion and CD38/CD138. By using oligonucleotide-labeled antibodies we find that upregulation of pathways for protein localization to the endoplasmic reticulum and mitochondrial oxidative phosphorylation are most associated with high IgG secretion, and uncover surrogate plasma cell surface markers (e.g., CD59) defined by the ability to secrete IgG. Altogether, this method links quantity of secretion with single-cell sequencing (SEC-seq) and enables researchers to fully explore the links between genome and function, laying the foundation for discoveries in immunology, stem cell biology, and beyond.
Assuntos
Linfócitos B , Plasmócitos , Humanos , Membrana Celular , Biomarcadores/metabolismo , Imunoglobulina G/metabolismoRESUMO
Macroautophagy/autophagy proteins have been linked with the development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to the complex roles of these proteins in multiple immune cell types. We have previously shown that a form of noncanonical autophagy induced by ITGAV/alpha(v) integrins regulates B cell activation by viral and self-antigens, in mice. Here, we investigate the involvement of this pathway in B cells from human tissues. Our data reveal that autophagy is specifically induced in the germinal center and memory B cell subpopulations of human tonsils and spleens. Transcriptomic analysis show that the induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of ITGAV/alpha(v) integrin-dependent autophagy in human B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells, we found that ITGAV/alpha(v)-dependent autophagy limits activation of specific pathways related to B cell responses, while promoting others. These data provide new mechanistic links for autophagy and B-cell-mediated immune dysregulation in diseases such as lupus.
Assuntos
Autofagia , Integrina alfaV , Humanos , Animais , Camundongos , Integrina alfaV/genética , Integrina alfaV/metabolismo , Transcriptoma , Linfócitos B/metabolismo , Mitocôndrias/metabolismoRESUMO
Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells. The EµB29 enhancer/promoter in the LV limited gene expression in non-B cell lineages. By engineering a knob-in-hole-reversed (KiHR) modification in the CH3-Fc eCD4-Ig domain, we reduced interactions between eCD4-Ig and endogenous B cell immunoglobulin G proteins, which improved HIV-1 neutralization potency. Unlike previous approaches in non-lymphoid cells, eCD4-Ig-KiHR produced in B cells promoted HIV-1 neutralizing protection without requiring exogenous TPST2, a tyrosine sulfation enzyme required for eCD4-Ig-KiHR function. This finding indicated that B cell machinery is well suited to produce therapeutic proteins. Lastly, to overcome the inefficient transduction efficiency associated with VSV-G LV delivery to primary B cells, an optimized measles pseudotyped LV packaging methodology achieved up to 75% transduction efficiency. Overall, our findings support the utility of B cell gene therapy platforms for therapeutic protein delivery.
RESUMO
In the avian embryo, endothelial cells originate from several sources, including the lateral plate and somite mesoderm. In this study, we show that Gata transcription factors are expressed in the lateral plate and in vasculogenic regions of the avian somite and are able to promote a vascular endothelial fate when ectopically expressed in somite precursors. A fusion of GATA4 to the transcriptional activator VP16 promoted endothelium formation, indicating that GATA transcription factors promote vasculogenesis via activation of downstream targets, while a fusion of GATA4 to the transcriptional repressor engrailed repressed expression of Vascular Endothelial Growth Factor Receptor 2, a marker of endothelial precursors. These findings indicate a role for GATA transcription factors in the differentiation of the endothelium.