Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.052
Filtrar
1.
Biophys Chem ; 265: 106441, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32745829

RESUMO

The possibility of immobilizing a protein with antigenic properties on a solid support offers significant possibilities in the development of immunosensors and vaccine formulations. For both applications, the orientation of the antigen should ensure ready accessibility of the antibodies to the epitope. However, an experimental assessment of the orientational preferences necessarily proceeds through the preparation/isolation of the antigen, the immobilization on different surfaces and one or more biophysical characterization steps. To predict a priori whether favorable orientations can be achieved or not would allow one to select the most promising experimental routes, partly mitigating the time cost towards the final product. In this manuscript, we apply a simple computational model, based on united-residue modelling, to the prediction of the orientation of the receptor binding domain of the SARS-CoV-2 spike protein on surfaces commonly used in lateral-flow devices. These calculations can account for the experimental observation that direct immobilization on gold gives sufficient exposure of the epitope to obtain a response in immunochemical assays.


Assuntos
Betacoronavirus/metabolismo , Epitopos/química , Modelos Moleculares , Glicoproteína da Espícula de Coronavírus/metabolismo , Antígenos/química , Antígenos/imunologia , Antígenos/metabolismo , Epitopos/imunologia , Simulação de Acoplamento Molecular , Domínios Proteicos , Dióxido de Silício/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Propriedades de Superfície
2.
Nat Commun ; 11(1): 3734, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709894

RESUMO

Medullary thymic epithelial cells (mTEC) contribute to the development of T cell tolerance by expressing and presenting tissue-restricted antigens (TRA), so that developing T cells can assess the self-reactivity of their antigen receptors prior to leaving the thymus. mTEC are a heterogeneous population of cells that differentially express TRA. Whether mTEC subsets induce distinct autoreactive T cell fates remains unclear. Here, we establish bacterial artificial chromosome (BAC)-transgenic mouse lines with biased mTEClo or mTEChi expression of model antigens. The transgenic lines support negative selection of antigen-specific thymocytes depending on antigen dose. However, model antigen expression predominantly by mTEClo supports TCRαß+ CD8αα intraepithelial lymphocyte development; meanwhile, mTEChi-restricted expression preferentially induces Treg differentiation of antigen-specific cells in these models to impact control of infectious agents and tumor growth. In summary, our data suggest that mTEC subsets may have a function in directing distinct mechanisms of T cell tolerance.


Assuntos
Antígenos/imunologia , Diferenciação Celular/imunologia , Células Epiteliais/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos/metabolismo , Infecções Bacterianas , Medula Óssea , Linhagem Celular Tumoral , Feminino , Tolerância Imunológica , Linfonodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Linfócitos T/metabolismo , Timócitos/imunologia , Fatores de Transcrição/genética
3.
Int J Nanomedicine ; 15: 2685-2697, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32368049

RESUMO

Background: Nanocarriers could deliver significantly higher amounts of antigen to antigen-presenting cells (APCs), which have great potential to stimulate humoral and cellular response in cancer immunotherapy. Thereafter, silica solid nanosphere (SiO2) was prepared, and a model antigen (ovalbumin, OVA) was covalently conjugated on the surface of SiO2 to form nanovaccine (OVA@SiO2). And the application of OVA@SiO2 for cancer immunotherapy was evaluated. Materials and Methods: SiO2 solid nanosphere was prepared by the Stöber method, then successively aminated by aminopropyltriethoxysilane and activated with glutaraldehyde. OVA was covalently conjugated on the surface of activated SiO2 to obtain nanovaccine (OVA@SiO2). Dynamic light scattering, scanning electron microscope, and transmission electron microscope were conducted to identify the size distribution, zeta potential and morphology of OVA@SiO2. The OVA loading capacity was investigated by varying glutaraldehyde concentration. The biocompatibility of OVA@SiO2 to DC2.4 and RAW246.7 cells was evaluated by a Cell Counting Kit-8 assay. The uptake of OVA@SiO2 by DC2.4 and its internalization pathway were evaluated in the absence or presence of different inhibitors. The activation and maturation of bone marrow-derived DC cells by OVA@SiO2 were also investigated. Finally, the in vivo transport of OVA@SiO2 and its toxicity to organs were appraised. Results: All results indicated the successful covalent conjugation of OVA on the surface of SiO2. The as-prepared OVA@SiO2 possessed high antigen loading capacity, which had good biocompatibility to APCs and major organs. Besides, OVA@SiO2 facilitated antigen uptake by DC2.4 cells and its cytosolic release. Noteworthily, OVA@SiO2 significantly promoted the maturation of dendritic cells and up-regulation of cytokine secretion by co-administration of adjuvant CpG-ODN. Conclusion: The as-prepared SiO2 shows promising potential for use as an antigen delivery carrier.


Assuntos
Antígenos/metabolismo , Vacinas Anticâncer/farmacologia , Imunoterapia/métodos , Nanosferas/química , Ovalbumina/química , Adjuvantes Imunológicos/administração & dosagem , Animais , Apresentação do Antígeno , Antígenos/administração & dosagem , Antígenos/química , Antígenos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/química , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Nanosferas/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Ovalbumina/imunologia , Ovalbumina/farmacocinética , Células RAW 264.7 , Dióxido de Silício/química
4.
Nat Commun ; 11(1): 1817, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286311

RESUMO

Dendritic cells (DCs) constitute a specialized population of immune cells that present exogenous antigen (Ag) on major histocompatibility complex (MHC) class I molecules to initiate CD8 + T cell responses against pathogens and tumours. Although cross-presentation depends critically on the trafficking of Ag-containing intracellular vesicular compartments, the molecular machinery that regulates vesicular transport is incompletely understood. Here, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in their DCs exhibit a major impairment in cross-presentation and thus a poor in vivo anti-tumour response. We find that kinesin-1 critically regulates antigen cross-presentation in DCs, by controlling Ag degradation, the endosomal pH, and MHC-I recycling. Mechanistically, kinesin-1 appears to regulate early endosome maturation by allowing the scission of endosomal tubulations. Our results highlight kinesin-1's role as a molecular checkpoint that modulates the balance between antigen degradation and cross-presentation.


Assuntos
Apresentação do Antígeno/imunologia , Células Dendríticas/metabolismo , Endossomos/metabolismo , Cinesina/metabolismo , Ácidos/metabolismo , Animais , Antígenos/metabolismo , Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Proliferação de Células , Endocitose , Antígenos de Histocompatibilidade Classe I/metabolismo , Cinesina/deficiência , Camundongos Knockout , Camundongos Transgênicos , Microtúbulos/metabolismo , Neoplasias/patologia , Ovalbumina/imunologia , Solubilidade
5.
PLoS One ; 15(4): e0230943, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240230

RESUMO

Pericellular and extracellular proteoglycans play an important role in modulating morphogen gradients and signal transductions. Chondroitin sulfate proteoglycan 4 (Cspg4) is a membrane spanning proteoglycan expressed in immature progenitor cells and cancer cells. Cspg4 participates in cellular events such as proliferation, migration and signal transduction, and these events are generally important for embryo development. In this study, we characterized Cspg4 for its roles in zebrafish embryonic development. Our results demonstrated that cspg4 was maternally expressed from 0 to 3 hours post fertilization (hpf) and expressed in the anterior and posterior embryo end after 9 hpf. Knocking-down cspg4 resulted in a shorter anterior-posterior axis than control embryo, which could be rescued by co-injecting wnt11 mRNA suggesting that Cspg4 regulates body axis organization through modulating the Wnt/planar cell polarity signaling pathway. In addition, overexpressing cspg4 caused cyclopia. The Cspg4 transmembrane domain mutant embryo phenocopied the global over-expression of cspg4 mRNA and led to cyclopia with a very low penetrance. Our results demonstrated that the quantitatively and spatially accurate distribution of Cspg4 is critical for body axis and midline development during gastrulation.


Assuntos
Antígenos/metabolismo , Polaridade Celular/fisiologia , Proteoglicanas/metabolismo , Via de Sinalização Wnt/fisiologia , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/fisiologia , RNA Mensageiro/metabolismo
6.
Immunity ; 52(3): 487-498.e6, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32155411

RESUMO

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.


Assuntos
Antígenos/imunologia , Butirofilinas/imunologia , Células Germinativas/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Antígenos/metabolismo , Antígenos CD/química , Antígenos CD/imunologia , Antígenos CD/metabolismo , Butirofilinas/química , Butirofilinas/metabolismo , Células CHO , Cricetinae , Cricetulus , Células Germinativas/metabolismo , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/metabolismo
7.
PLoS Comput Biol ; 16(3): e1007714, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163410

RESUMO

Antigen recognition by T-cells is guided by the T-cell receptor (TCR) heterodimer formed by α and ß chains. A huge diversity of TCR sequences should be maintained by the immune system in order to be able to mount an effective response towards foreign pathogens, so, due to cooperative binding of α and ß chains to the pathogen, any constraints on chain pairing can have a profound effect on immune repertoire structure, diversity and antigen specificity. By integrating available structural data and paired chain sequencing results we were able to show that there are almost no constraints on pairing in TCRαß complexes, allowing naive T-cell repertoire to reach the highest possible diversity. Additional analysis reveals that the specific choice of contacting amino acids can still have a profound effect on complex conformation. Moreover, antigen-driven selection can distort the uniform landscape of chain pairing, while small, yet significant, differences in the pairing can be attributed to various specialized T-cell subsets such as MAIT and iNKT T-cells, as well as other TCR sets specific to certain antigens.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta , Aminoácidos , Animais , Antígenos/química , Antígenos/metabolismo , Biologia Computacional , Bases de Dados de Proteínas , Humanos , Camundongos , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia
8.
Nat Med ; 26(3): 430-440, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066977

RESUMO

Adjuvants are central to the efficacy of subunit vaccines. Aluminum hydroxide (alum) is the most commonly used vaccine adjuvant, yet its adjuvanticity is often weak and mechanisms of triggering antibody responses remain poorly understood. We demonstrate that site-specific modification of immunogens with short peptides composed of repeating phosphoserine (pSer) residues enhances binding to alum and prolongs immunogen bioavailability. The pSer-modified immunogens formulated in alum elicited greatly increased germinal center, antibody, neutralizing antibody, memory and long-lived plasma cell responses compared to conventional alum-adsorbed immunogens. Mechanistically, pSer-immunogen:alum complexes form nanoparticles that traffic to lymph nodes and trigger B cell activation through multivalent and oriented antigen display. Direct uptake of antigen-decorated alum particles by B cells upregulated antigen processing and presentation pathways, further enhancing B cell activation. These data provide insights into mechanisms of action of alum and introduce a readily translatable approach to significantly improve humoral immunity to subunit vaccines using a clinical adjuvant.


Assuntos
Adjuvantes Imunológicos/farmacologia , Hidróxido de Alumínio/farmacologia , Imunidade Humoral/efeitos dos fármacos , Peptídeos/imunologia , Engenharia de Proteínas , Animais , Apresentação do Antígeno/efeitos dos fármacos , Antígenos/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Endocitose/efeitos dos fármacos , Epitopos/imunologia , Imunização , Memória Imunológica/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/química , Peptídeos/química , Fosfosserina/metabolismo
9.
Transfusion ; 60(4): 688-693, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31930726

RESUMO

BACKGROUND: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT). STUDY DESIGN AND METHODS: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages. RESULTS: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients. CONCLUSION: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn.


Assuntos
Imunoglobulina G/imunologia , Testes Imunológicos/normas , Sistema do Grupo Sanguíneo de Kell/imunologia , Fagocitose/imunologia , Antígenos/imunologia , Antígenos/metabolismo , Eritrócitos/imunologia , Hemólise/imunologia , Humanos , Alótipos de Imunoglobulina/imunologia , Isoanticorpos/imunologia
10.
Elife ; 82019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31815664

RESUMO

Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in synapses of primary mouse B cells and human B cell lines forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction.


Assuntos
Citoesqueleto de Actina/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Formação de Anticorpos/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos/metabolismo , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Sinapses Imunológicas/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/metabolismo
11.
Cell Rep ; 29(13): 4223-4235.e5, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875534

RESUMO

Immunoglobulin D (IgD) is an ancient antibody with dual membrane-bound and fluid-phase antigen receptor functions. The biology of secreted IgD remains elusive. Here, we demonstrate that teleost IgD+IgM- plasmablasts constitute a major lymphocyte population in some mucosal surfaces, including the gut mucosa. Remarkably, secreted IgD binds to gut commensal bacteria, which in turn stimulate IgD gene transcription in gut B cells. Accordingly, secreted IgD from gut as well as gill mucosae, but not the spleen, show a V(D)J gene configuration consistent with microbiota-driven clonal expansion and diversification, including mild somatic hypermutation. By showing that secreted IgD establishes a mutualistic relationship with commensals, our findings suggest that secreted IgD may play an evolutionary conserved role in mucosal homeostasis.


Assuntos
Linfócitos B/imunologia , Imunoglobulina D/genética , Imunoglobulina M/metabolismo , Intestinos/imunologia , Mutação/genética , Oncorhynchus mykiss/imunologia , Sequência de Aminoácidos , Animais , Antígenos/metabolismo , Células Clonais , Regiões Determinantes de Complementaridade/imunologia , Microbioma Gastrointestinal , Brânquias/imunologia , Imunoglobulina D/química , Intestinos/microbiologia , Subpopulações de Linfócitos/imunologia , Oncorhynchus mykiss/microbiologia , Hipermutação Somática de Imunoglobulina/genética , Baço/metabolismo , Transcrição Genética , Recombinação V(D)J/genética
12.
Cell Rep ; 29(13): 4435-4446.e9, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875551

RESUMO

Uptake of apoptotic cells (ACs) by dendritic cells (DCs) and induction of a tolerogenic DC phenotype is an important mechanism for establishing peripheral tolerance to self-antigens. The receptors involved and underlying signaling pathways are not fully understood. Here, we identify Dectin-1 as a crucial tolerogenic receptor binding with nanomolar affinity to the core domain of several annexins (annexin A1, A5, and A13) exposed on ACs. Annexins bind to Dectin-1 on a site distinct from the interaction site of pathogen-derived ß-glucans. Subsequent tolerogenic signaling induces selective phosphorylation of spleen tyrosine kinase (SYK), causing activation of NADPH oxidase-2 and moderate production of reactive oxygen species. Thus, mice deficient for Dectin-1 develop autoimmune pathologies (autoantibodies and splenomegaly) and generate stronger immune responses (cytotoxic T cells) against ACs. Our data describe an important immunological checkpoint system and provide a link between immunosuppressive signals of ACs and maintenance of peripheral immune tolerance.


Assuntos
Anexinas/metabolismo , Apoptose , Lectinas Tipo C/metabolismo , NADPH Oxidase 2/metabolismo , Tolerância Periférica , Envelhecimento/metabolismo , Animais , Anexinas/química , Antígenos/metabolismo , Autoimunidade , Sítios de Ligação , Sequência Conservada/genética , Drosophila , Feminino , Humanos , Imunossupressão , Células Jurkat , Masculino , Camundongos Knockout , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Quinase Syk/metabolismo , beta-Glucanas/metabolismo
13.
J Immunol Methods ; 474: 112669, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614128

RESUMO

Detection of anti-drug antibodies is a critical step in the development of large molecule biopharmaceuticals. In the case of multicomponent/multifunctional molecules, such as fusion proteins and protein conjugates such as covalent polyethylene glycol (PEG)~protein conjugates, it is useful to further characterize anti-drug antibody (ADA) binding to key domains of the drug. The detection of anti-PEG antibodies poses special challenges that if overlooked can result in underreporting antibody responses. Here we describe the development and characterization of a novel ELISA to detect anti-PEG antibodies that provides a more complete interpretation of anti-PEG than other published methods. Being specific to the PEG moiety alone, this method is intended to detect anti-PEG antibodies independent of the protein to which PEG is conjugated. Based upon early indications that our assay could detect anti-PEG antibodies at a surprisingly high frequency in the general population, our emphasis throughout method development and validation was to ensure that non-specific signals and unintended interactions were not falsely contributing to detection of anti-PEG antibodies. Techniques, including orthogonal methods used to ensure that this ELISA detected antibodies specific to PEG included competition, immunodepletion, immunoprecipitation/western blot and an Octet kinetic binding analysis. The validated ELISA can detect 100 ng/mL of an anti-PEG IgG positive control and 800 ng/mL of an anti-PEG IgM positive control in the presence of 7.5 µg/mL of the PEGylated therapeutic (MW 64 kDa). The intra-assay percent co-efficient of variation (CV) and inter-assay CV of the low positive control samples in the screening method were 4.1 to 7.2% and 16.7 to 17.7%, respectively. Additional assay performance parameters that were validated are also described. When the validated assay was applied to a population of 200 healthy blood donors with no known exposure to biopharmaceutical PEG conjugates it indicated a pre-existing anti-PEG antibody prevalence of 97.5%. We suggest this surprising result is a consequence of exposure to PEG additives in everyday products, such as cosmetics, processed foods and over-the-counter (OTC) pharmaceuticals.


Assuntos
Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Polietilenoglicóis , Antígenos/metabolismo , Sítios de Ligação de Anticorpos , Humanos , Cinética , Polietilenoglicóis/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Tensoativos/química
14.
Biomed Pharmacother ; 119: 109430, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31518874

RESUMO

B-cell activating factor (BAFF) plays a key role in the normal regulation of B cell development and immune response. Its abnormal expression level is accompanied by the occurrence of various autoimmune diseases. Therefore, BAFF is an effective target for the treatment of such diseases. Here, we report a new anti-BAFF monoclonal antibody. Based on improved in vitro immunization method, we used a recombinant BAFF containing unnatural amino acid p-nitro-phenylalanine (pNO2Phe) as an antigen to trigger immune response in vitro. The plasma cells were sorted by flow cytometry (FACS), and the antibody library was constructed based on the sorted plasma cells. The high affinity antigen-binding fragments were panned by phage display technology, and finally the anti-BAFF human IgG was obtained. The antibody demonstrated its ability to neutralize BAFF effectively both in vitro and in vivo. We propose that this novel full-length human anti-BAFF monoclonal antibody is a promising therapeutic candidate for the treatment of autoimmune diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Fator Ativador de Células B/imunologia , Imunização , Animais , Anticorpos Neutralizantes/imunologia , Antígenos/metabolismo , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Células Dendríticas/metabolismo , Feminino , Humanos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plasmócitos/metabolismo , Proteínas Recombinantes/metabolismo
15.
Immunol Cell Biol ; 97(10): 931-940, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31420892

RESUMO

Individual CD4+ T cells can become one of a number of helper (Th) lineages with distinct effector functions. However, whether biases in Th potential exist prior to antigen encounter is unknown. Studies have identified cell-intrinsic functional heterogeneity among naïve T cells that can be parsed based on the strength of T-cell receptor (TCR) interactions with self-peptide. Here, using CD5 levels as a surrogate for the strength of these basal TCR signals, we sought to identify pre-existing effector biases in the CD4+ T-cell lineage. We show that ex vivo-activated CD5lo CD4+ T cells produce greater amounts of the Th1 cytokine interferon-gamma (IFNγ) than their CD5hi counterparts. In addition, a greater percentage of CD5lo effector CD4+ T cells produce IFNγ in both polyclonal and monoclonal CD4+ T-cell populations after antigen challenge in vivo. These results suggest that differential IFNγ production potential exists among CD4+ T cells prior to activation and independent of TCR affinity for foreign antigen.


Assuntos
Antígenos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Interferon gama/biossíntese , Animais , Antígenos CD/metabolismo , Linhagem Celular , Ativação Linfocitária/imunologia , Masculino , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/metabolismo
16.
Immunohorizons ; 3(8): 389-401, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427364

RESUMO

The germinal center (GC) is the anatomical site where humoral immunity evolves. B cells undergo cycles of proliferation and selection to produce high-affinity Abs against Ag. Direct linkage of a TLR9 agonist (CpG) to a T-dependent Ag increases the number of GC B cells. We used a T-dependent Ag complexed with CpG and a genetic model for ablating the TLR9 signaling adaptor molecule MyD88 specifically in B cells (B-MyD88- mice) together with transcriptomics to determine how this innate pathway positively regulates the GC. GC B cells from complex Ag-immunized B-MyD88- mice were defective in inducing gene expression signatures downstream of c-Myc and mTORC1. In agreement with the latter gene signature, ribosomal protein S6 phosphorylation was increased in GC B cells from wild-type mice compared with B-MyD88- mice. However, GC B cell expression of a c-Myc protein reporter was enhanced by CpG attached to Ag in both wild-type and B-MyD88- mice, indicating a B cell-extrinsic effect on c-Myc protein expression combined with a B cell-intrinsic enhancement of gene expression downstream of c-Myc. Both mTORC1 activity and c-Myc are directly induced by T cell help, indicating that TLR9 signaling in GC B cells either enhances their access to T cell help or directly influences these pathways to further enhance the effect of T cell help. Taken together, these findings indicate that TLR9 signaling in the GC could provide a surrogate prosurvival stimulus, "TLR help," thus lowering the threshold for selection and increasing the magnitude of the GC response.


Assuntos
Antígenos/química , Linfócitos B/imunologia , Centro Germinativo/metabolismo , Ligantes , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor Toll-Like 9/agonistas , Animais , Antígenos/metabolismo , Ativação Linfocitária/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Transcriptoma , gama-Globulinas/imunologia
17.
mBio ; 10(4)2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455651

RESUMO

Using multiple viral systems, and performing silencing approaches, overexpression approaches, and experiments in knockout cells, we report, for the first time, that interferon (IFN)-induced protein 44 (IFI44) positively affects virus production and negatively modulates innate immune responses induced after viral infections. Moreover, IFI44 is able to rescue poly(I·C)- and IFN-mediated inhibition of virus growth. Furthermore, we report a novel interaction of IFI44 with the cellular factor FK506-binding protein 5 (FKBP5), which binds to cellular kinases such as the inhibitor of nuclear factor kappa B (IκB) kinases (IKKα, IKKß, and IKKε). Importantly, in the presence of FKBP5, IFI44 decreases the ability of IKKß to phosphorylate IκBα and the ability of IKKε to phosphorylate interferon regulatory factor 3 (IRF-3), providing a novel mechanism for the function of IFI44 in negatively modulating IFN responses. Remarkably, these new IFI44 functions may have implications for diseases associated with excessive immune signaling and for controlling virus infections mediated by IFN responses.IMPORTANCE Innate immune responses mediated by IFN and inflammatory cytokines are critical for controlling virus replication. Nevertheless, exacerbated innate immune responses could be detrimental for the host and feedback mechanisms are needed to maintain the cellular homeostasis. In this work, we describe a completely novel function for IFI44 in negatively modulating the innate immune responses induced after viral infections. We show that decreasing IFI44 expression by using small interfering RNAs (siRNAs) or by generating knockout (KO) cells impairs virus production and increases the levels of IFN responses. Moreover, we report a novel interaction of IFI44 with the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the inhibitor of nuclear factor kappa B (IκB) kinases IKKα, IKKß, and IKKε. Our data indicate that binding of IFI44 to FKBP5 decreased the phosphorylation of IRF-3 and IκBα mediated by IKKε and IKKß, respectively, providing a likely explanation for the function of IFI44 in negatively modulating IFN responses. These results provide new insights into the induction of innate immune responses and suggest that IFI44 is a new potential antiviral target for reducing virus replication.


Assuntos
Antígenos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Interações Hospedeiro-Patógeno , Imunidade Inata , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , Proteínas de Ligação a Tacrolimo/metabolismo , Antígenos/genética , Citocinas/imunologia , Proteínas do Citoesqueleto/genética , Humanos , Interferons/imunologia , Fosforilação , Infecções por Vírus de RNA/virologia , Vírus de RNA/fisiologia , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/genética , Replicação Viral
18.
Commun Biol ; 2: 304, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428692

RESUMO

Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem. Here we use high-throughput single-cell B-cell receptor sequencing (scBCR-seq) to obtain accurately paired full-length variable regions in a massively parallel fashion. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion. In addition, we immunized rats with chicken ovalbumin and profiled antigen-reactive B cells from lymph nodes of immunized animals. The scBCR-seq data recovered 81% (n = 56/69) of B-cell lineages identified from hybridomas generated from the same set of B cells subjected to scBCR-seq. Importantly, scBCR-seq identified an additional 710 candidate lineages not recovered as hybridomas. We synthesized, expressed and tested 93 clones from the identified lineages and found that 99% (n = 92/93) of the clones were antigen-reactive. Our results establish scBCR-seq as a powerful tool for antibody discovery.


Assuntos
Anticorpos/metabolismo , Antígenos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Receptores de Antígenos de Linfócitos B/genética , Análise de Célula Única , Animais , Células Germinativas/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Ratos , Reprodutibilidade dos Testes
19.
PLoS Biol ; 17(8): e3000420, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31433805

RESUMO

Dendritic cells (DCs) play pivotal roles in T-cell homeostasis and activation, and metabolic programing has been recently linked to DC development and function. However, the metabolic underpinnings corresponding to distinct DC functions remain largely unresolved. Here, we demonstrate a special metabolic-epigenetic coupling mechanism orchestrated by tuberous sclerosis complex subunit 1 (TSC1)-mechanistic target of rapamycin (mTOR) for homeostatic DC function. Specific ablation of Tsc1 in the DC compartment (Tsc1DC-KO) largely preserved DC development but led to pronounced reduction in naïve and memory-phenotype cluster of differentiation (CD)8+ T cells, a defect fully rescued by concomitant ablation of mTor or regulatory associated protein of MTOR, complex 1 (Rptor) in DCs. Moreover, Tsc1DC-KO mice were unable to launch efficient antigen-specific CD8+ T effector responses required for containing Listeria monocytogenes and B16 melanomas. Mechanistically, our data suggest that the steady-state DCs tend to tune down de novo fatty acid synthesis and divert acetyl-coenzyme A (acetyl-CoA) for histone acetylation, a process critically controlled by TSC1-mTOR. Correspondingly, TSC1 deficiency elevated acetyl-CoA carboxylase 1 (ACC1) expression and fatty acid synthesis, leading to impaired epigenetic imprinting on selective genes such as major histocompatibility complex (MHC)-I and interleukin (IL)-7. Remarkably, tempering ACC1 activity was able to divert cytosolic acetyl-CoA for histone acetylation and restore the gene expression program compromised by TSC1 deficiency. Taken together, our results uncover a crucial role for TSC1-mTOR in metabolic programing of the homeostatic DCs for T-cell homeostasis and implicate metabolic-coupled epigenetic imprinting as a paradigm for DC specification.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Animais , Antígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/fisiologia , Células Dendríticas/imunologia , Epigênese Genética , Homeostase , Listeria monocytogenes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/imunologia , Proteínas Supressoras de Tumor/genética
20.
Elife ; 82019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31290744

RESUMO

B lymphocytes use B cell receptors (BCRs) to recognize antigens. It is still not clear how BCR transduces antigen-specific physical signals upon binding across cell membrane for the conversion to chemical signals, triggering downstream signaling cascades. It is hypothesized that through a series of conformational changes within BCR, antigen engagement in the extracellular domain of BCR is transduced to its intracellular domain. By combining site-specific labeling methodology and FRET-based assay, we monitored conformational changes in the extracellular domains within BCR upon antigen engagement. Conformational changes within heavy chain of membrane-bound immunoglobulin (mIg), as well as conformational changes in the spatial relationship between mIg and Igß were observed. These conformational changes were correlated with the strength of BCR activation and were distinct in IgM- and IgG-BCR. These findings provide molecular mechanisms to explain the fundamental aspects of BCR activation and a framework to investigate ligand-induced molecular events in immune receptors.


Assuntos
Antígenos/metabolismo , Linfócitos B/fisiologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligação Proteica , Conformação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA