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1.
Cell ; 178(4): 949-963.e18, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31353221

RESUMO

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.


Assuntos
Neoplasias da Mama/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Cinamatos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Antagonistas do Receptor de Estrogênio/uso terapêutico , Feminino , Fulvestranto/uso terapêutico , Células HEK293 , Xenoenxertos , Humanos , Indazóis/farmacologia , Ligantes , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Polimorfismo de Nucleotídeo Único , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
2.
Nat Immunol ; 22(5): 571-585, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33903764

RESUMO

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.


Assuntos
Células Dendríticas Foliculares/imunologia , Fibroblastos/imunologia , Linfonodos/imunologia , Células Estromais/imunologia , Idoso , Animais , Apoptose/genética , Apoptose/imunologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Dendríticas Foliculares/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Humanos , Imunidade Celular/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfonodos/citologia , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Análise de Célula Única , Células Estromais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
3.
Nature ; 618(7967): 1072-1077, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37196676

RESUMO

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.


Assuntos
Anticorpos Monoclonais , Membrana Celular , Inflamação , Fígado , Fatores de Crescimento Neural , Traumatismo por Reperfusão , Animais , Camundongos , Alanina Transaminase , Alarminas , Anticorpos Monoclonais/imunologia , Aspartato Aminotransferases , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/imunologia , Moléculas de Adesão Celular Neuronais/ultraestrutura , Morte Celular , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Concanavalina A , Galactosamina , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Inflamação/patologia , Lactato Desidrogenases , Fígado/patologia , Microscopia Eletrônica , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/ultraestrutura , Infiltração de Neutrófilos , Traumatismo por Reperfusão/patologia
4.
Nature ; 611(7934): 148-154, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36171287

RESUMO

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.


Assuntos
Fibroblastos Associados a Câncer , Proteínas de Membrana , Miofibroblastos , Neoplasias Pancreáticas , Células Estromais , Animais , Humanos , Camundongos , Fibroblastos Associados a Câncer/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteínas de Membrana/metabolismo , Miofibroblastos/metabolismo , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Receptores de Fatores de Crescimento Transformadores beta , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Antígeno B7-H1
5.
Nat Immunol ; 15(2): 161-7, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24362890

RESUMO

CD11b(+) dendritic cells (DCs) seem to be specialized for presenting antigens via major histocompatibility (MHC) class II complexes to stimulate helper T cells, but the genetic and regulatory basis for this is not established. Conditional deletion of Irf4 resulted in loss of CD11b(+) DCs, impaired formation of peptide-MHC class II complexes and defective priming of helper T cells but not of cytotoxic T lymphocyte (CTL) responses. Gene expression and chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analyses delineated an IRF4-dependent regulatory module that programs enhanced MHC class II antigen presentation. Expression of the transcription factor IRF4 but not of IRF8 restored the ability of IRF4-deficient DCs to efficiently process and present antigen to MHC class II-restricted T cells and promote helper T cell responses. We propose that the evolutionary divergence of IRF4 and IRF8 facilitated the specialization of DC subsets for distinct modes of antigen presentation and priming of helper T cell versus CTL responses.


Assuntos
Apresentação de Antígeno/genética , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fatores Reguladores de Interferon/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Fatores Reguladores de Interferon/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/genética , Transgenes/genética
6.
Nature ; 554(7693): 544-548, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29443960

RESUMO

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor ß (TGFß) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFß-blocking and anti-PD-L1 antibodies reduced TGFß signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFß shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Neoplasias Urológicas/tratamento farmacológico , Neoplasias Urológicas/imunologia , Urotélio/patologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Anticorpos Monoclonais Humanizados , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Estudos de Coortes , Colágeno/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Imunoterapia , Camundongos , Mutação , Metástase Neoplásica , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Resultado do Tratamento , Microambiente Tumoral/imunologia , Neoplasias Urológicas/genética , Neoplasias Urológicas/patologia , Urotélio/efeitos dos fármacos , Urotélio/imunologia
7.
Nature ; 527(7578): 323-8, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26536114

RESUMO

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.


Assuntos
Antibacterianos/farmacologia , Bacteriemia , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Espaço Intracelular/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologia , Animais , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Portador Sadio/tratamento farmacológico , Portador Sadio/microbiologia , Desenho de Fármacos , Feminino , Imunoconjugados/química , Espaço Intracelular/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Testes de Sensibilidade Microbiana , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fagossomos/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade , Vancomicina/uso terapêutico
8.
Artigo em Inglês | MEDLINE | ID: mdl-30104274

RESUMO

There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill Escherichia coli through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Antibacterianos/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Escherichia coli/efeitos dos fármacos
9.
Bioconjug Chem ; 29(7): 2468-2477, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-29856915

RESUMO

Despite the recent success of antibody-drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody-FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.


Assuntos
Transporte Biológico , Transferência Ressonante de Energia de Fluorescência , Imunoconjugados/farmacocinética , Permeabilidade da Membrana Celular , Reagentes de Ligações Cruzadas/química , Humanos , Imunoconjugados/metabolismo , Peptídeos
12.
Blood ; 122(22): 3678-90, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-23886837

RESUMO

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.


Assuntos
Proteínas de Transporte/fisiologia , Endotélio Vascular/embriologia , Endotélio Vascular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Junções Intercelulares/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neovascularização Fisiológica , Gravidez , Interferência de RNA , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia
13.
Mol Pharm ; 12(6): 1717-29, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25853436

RESUMO

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.


Assuntos
Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Oligopeptídeos/uso terapêutico , Animais , Antineoplásicos/química , Western Blotting , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Imunoconjugados/química , Imuno-Histoquímica , Camundongos , Camundongos SCID , Oligopeptídeos/química , Ratos , Ratos Sprague-Dawley , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
14.
PLoS Pathog ; 8(3): e1002572, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22412374

RESUMO

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was -300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/virologia , Vírus da Influenza A/imunologia , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Classe II , Humanos , Memória Imunológica/imunologia , Internalização do Vírus
15.
Blood ; 120(10): 2011-20, 2012 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-22791285

RESUMO

Dendritic cells (DCs) can capture extracellular antigens and load resultant peptides on to MHC class I molecules, a process termed cross presentation. The mechanisms of cross presentation remain incompletely understood, particularly in primary human DCs. One unknown is the extent to which antigen delivery to distinct endocytic compartments determines cross presentation efficiency, possibly by influencing antigen egress to the cytosol. We addressed the problem directly and quantitatively by comparing the cross presentation of identical antigens conjugated with antibodies against different DC receptors that are targeted to early or late endosomes at distinct efficiencies. In human BDCA1+ and monocyte-derived DCs, CD40 and mannose receptor targeted antibody conjugates to early endosomes, whereas DEC205 targeted antigen primarily to late compartments. Surprisingly, the receptor least efficient at internalization, CD40, was the most efficient at cross presentation. This did not reflect DC activation by CD40, but rather its relatively poor uptake or intra-endosomal degradation compared with mannose receptor or DEC205. Thus, although both early and late endosomes appear to support cross presentation in human DCs, internalization efficiency, especially to late compartments, may be a negative predictor of activity when selecting receptors for vaccine development.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Endocitose/imunologia , Endossomos/imunologia , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/metabolismo , Antígenos CD/imunologia , Antígenos CD/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Endossomos/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunidade Inata , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Peptídeos/imunologia , Peptídeos/metabolismo , Cultura Primária de Células , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo
16.
Nat Cell Biol ; 9(5): 523-30, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17417625

RESUMO

Zebrafish somitogenesis is governed by a segmentation clock that generates oscillations in expression of several Notch pathway genes, including her1, her7 and deltaC. Using a combination of pharmacological inhibition and Mendelian genetics, we show that DeltaD and DeltaC, two Notch ligands, represent functionally distinct signals within the segmentation clock. Using high-resolution fluorescent in situ hybridization, the oscillations were divided into phases based on eight distinct subcellular patterns of mRNA localization for 140,000 cells. her1, her7 and deltaC expression was examined in wild-type, deltaD(-/-) and deltaC(-/-) embryos. We identified areas within the tailbud where the clock is set up in the progenitor cells (priming), where the clock starts running (initiation), and where the clocks of neighbouring cells are entrained (synchronization). We find that the clocks of motile cells are primed by deltaD in a progenitor zone in the posterior tailbud and that deltaD is required for cells to initiate oscillations on exiting this zone. Oscillations of adjacent cells are synchronized and amplified by deltaC in the posterior presomitic mesoderm as cell movement subsides and cells maintain stable neighbour relationships.


Assuntos
Relógios Biológicos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Somitos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Relógios Biológicos/efeitos dos fármacos , Movimento Celular , Dipeptídeos/farmacologia , Células-Tronco Embrionárias/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Microinjeções , Mutação , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Somitos/efeitos dos fármacos , Fatores de Tempo , Técnicas de Cultura de Tecidos , Fatores de Transcrição/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
17.
Proc Natl Acad Sci U S A ; 108(7): 2759-64, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21273506

RESUMO

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.


Assuntos
Síndrome de Bardet-Biedl/fisiopatologia , Proteínas de Transporte/metabolismo , Centrossomo/metabolismo , Cílios/fisiologia , Transdução de Sinais/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Análise de Variância , Animais , Síndrome de Bardet-Biedl/metabolismo , Imunofluorescência , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Espectrometria de Massas , Membranas/crescimento & desenvolvimento , Imagem com Lapso de Tempo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Peixe-Zebra
18.
J Biol Chem ; 287(29): 24082-91, 2012 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-22613716

RESUMO

Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.


Assuntos
Anticorpos/uso terapêutico , Melanócitos/metabolismo , Melanoma/tratamento farmacológico , Melanossomas/metabolismo , Antígeno gp100 de Melanoma/metabolismo , Animais , Anticorpos/química , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia de Fluorescência , Oligopeptídeos/química , Oligopeptídeos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Antígeno gp100 de Melanoma/genética
19.
Proc Natl Acad Sci U S A ; 107(9): 4287-92, 2010 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-20142498

RESUMO

In response to inflammatory stimuli, dendritic cells (DCs) trigger the process of maturation, a terminal differentiation program required to initiate T-lymphocyte responses. A hallmark of maturation is down-regulation of endocytosis, which is widely assumed to restrict the ability of mature DCs to capture and present antigens encountered after the initial stimulus. We found that mature DCs continue to accumulate antigens, especially by receptor-mediated endocytosis and phagocytosis. Internalized antigens are transported normally to late endosomes and lysosomes, loaded onto MHC class II molecules (MHCII), and then presented efficiently to T cells. This occurs despite the fact that maturation results in the general depletion of MHCII from late endocytic compartments, with MHCII enrichment being typically thought to be a required feature of antigen processing and peptide loading compartments. Internalized antigens can also be cross-presented on MHC class I molecules, without any reduction in efficiency relative to immature DCs. Thus, although mature DCs markedly down-regulate their capacity for macropinocytosis, they continue to capture, process, and present antigens internalized via endocytic receptors, suggesting that they may continuously initiate responses to newly encountered antigens during the course of an infection.


Assuntos
Células Dendríticas/imunologia , Endocitose , Receptores Imunológicos/imunologia , Animais , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Fagocitose
20.
J Immunol ; 182(6): 3573-82, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19265136

RESUMO

Chitin is a ubiquitous polysaccharide in fungi, insects, and parasites. We hypothesized that chitin is a size-dependent regulator of innate immunity. To test this hypothesis, we characterized the effects of chitins of different sizes on murine bronchoalveolar or peritoneal macrophages. In these studies, large chitin fragments were inert, while both intermediate-sized chitin (40-70 microm) and small chitin (SC; <40 microm, largely 2-10 microm) stimulated TNF elaboration. In contrast, only SC induced IL-10 elaboration. The effects of intermediate-sized chitin were mediated by pathways that involve TLR2, dectin-1, and NF-kappaB. In contrast, the effects of SC were mediated by TLR2-dependent and -independent, dectin-1-dependent pathways that involved the mannose receptor and spleen tyrosine kinase. Chitin contains size-dependent pathogen-associated molecular patterns that stimulate TLR2, dectin-1, and the mannose receptor, differentially activate NF-kappaB and spleen tyrosine kinase, and stimulate the production of pro- and anti-inflammatory cytokines.


Assuntos
Quitina/química , Quitina/fisiologia , Regulação da Expressão Gênica/imunologia , Interleucina-10/biossíntese , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Células Cultivadas , Citocinas/biossíntese , Citocinas/fisiologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/química , Mediadores da Inflamação/fisiologia , Interleucina-10/genética , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peso Molecular , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/genética
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