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1.
Mol Immunol ; 114: 553-560, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31521019

RESUMO

Cell surface display is a useful platform to examine the interactions between two proteins of interest, such as immune receptors and ligands. This technique is also useful for studies on the immune receptors of lower vertebrates and invertebrates. However, in many cases, the commonly used cell culture temperature is relatively high for proteins from such organisms. Since insect cells can be cultured at lower temperatures than many other cells, and since they are equipped with "quality control" system, which is advantageous for the presentation of properly folded proteins, we anticipated that the insect cell surface display system could be more suitable for that type of research. In the present study, multiple cloning site of the commercially available expression vector pIB/V5-His was modified, and whether this vector could be useful to present fish immune-related membrane proteins was investigated. Using this plasmid, fugu's CD8α and CC chemokine receptor 7 could be presented on the cell surface. The clones of the lamprey variable lymphocyte receptors obtained previously by the yeast surface display (YSD) system as hen's egg lysozyme (HEL) binders also could be presented on the cell surface and bound to HEL. These results suggest that functional immune-related membrane proteins can be presented on the insect cell surface, indicating that this system is useful for immunological studies on exothermal animals.


Assuntos
Membrana Celular/imunologia , Proteínas de Peixes/imunologia , Peixes/imunologia , Insetos/imunologia , Proteínas de Membrana/imunologia , Animais , Células Cultivadas , Galinhas/imunologia , Citometria de Fluxo/métodos , Lampreias/imunologia , Ligantes , Muramidase/imunologia , Receptores Imunológicos/imunologia
2.
EBioMedicine ; 47: 329-340, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31474552

RESUMO

BACKGROUND: The objective of the current study was to study the molecular mechanism(s) underlying cardiac troponin I autoantibody (cTnIAAb) binding to cardiomyocyte and resultant myocardial damage/dysfunction. METHODS: cTnIAAb was purified from serum of 10 acute myocardial infarction (AMI) patients with left ventricular remodeling. Recombinant human cTnI was used to generate three mouse-derived monoclonal anti-cTnI antibodies (cTnImAb1, cTnImAb2, and cTnImAb3). The target proteins in cardiac myocyte membrane bound to cTnImAb and effect of cTnIAAb and cTnImAb on apoptosis and myocardial function were determined. FINDINGS: We found that cTnIAAb/cTnImAb1 directly bound to the cardiomyocyte membraneα-Enolase (ENO1) and triggered cell apoptosis via increased expression of ENO1 and Bax, decreased expression of Bcl2, subsequently activating Caspase8, Caspase 3, phosphatase and tensin homolog (PTEN) while inhibiting Akt activity. This cTnIAAb-ENO1-PTEN-Akt signaling axis contributed to increased myocardial apoptosis, myocardial collagen deposition, and impaired systolic dysfunction. INTERPRETATION: Results obtained in this study indicate that cTnIAAb is involved in the process of ventricular remodeling after myocardial injury. FUND: The National Natural Science Foundation of China (Grant#: 81260026).


Assuntos
Autoanticorpos/imunologia , Miocardite/etiologia , Miocardite/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Troponina I/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Autoanticorpos/efeitos adversos , Biomarcadores , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Testes de Função Cardíaca , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Miocardite/diagnóstico , Miocardite/fisiopatologia , Miocárdio/imunologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo
3.
Int J Mol Sci ; 20(15)2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31387209

RESUMO

Galectin-1 (Gal-1) is a 14 kDa protein that has been well characterized for promoting cancer metastasis and tumor immune evasion. By localizing to the cancer cell surface, Gal-1 induces T cell apoptosis through binding T cell surface receptors. The transmembrane protein, Sushi Domain Containing 2 (SUSD2), has been previously shown to be required for Gal-1 surface presentation in breast cancer cells. Western immunoblot analysis revealed that SUSD2 is cleaved into two fragments. However, the significance of this cleavage for Gal-1 surface localization has not been investigated. To define the location of cleavage, a mutagenesis analysis of SUSD2 was performed. Our studies demonstrated that SUSD2 is cleaved at its glycine-aspartic acid-proline-histidine (GDPH) amino acid sequence. Generation of a noncleavable SUSD2 mutant (GDPH∆-SUSD2) showed that SUSD2 cleavage was required for SUSD2 and Gal-1 plasma membrane localization. Noncleavable cysteine mutants were also unable to present Gal-1 at the cell surface, further demonstrating that SUSD2 cleavage is required for Gal-1 surface presentation. Treatment with the serine protease inhibitor, Pefabloc SC, inhibited SUSD2 cleavage in a dose dependent manner, suggesting that SUSD2 is cleaved by a serine protease. Therefore, identification and inhibition of this protease may provide a new therapeutic tool for inhibiting SUSD2 and Gal-1's combined tumorigenic function in breast cancer.


Assuntos
Motivos de Aminoácidos , Apresentação do Antígeno/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Galectina 1/imunologia , Glicoproteínas de Membrana/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Neoplasias da Mama/genética , Membrana Celular/imunologia , Membrana Celular/metabolismo , Dissulfetos , Retículo Endoplasmático/metabolismo , Feminino , Galectina 1/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteólise
4.
Nat Commun ; 10(1): 3452, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31388002

RESUMO

Bacteria have been extensively utilized for bioimaging, diagnosis and therapy given their unique characteristics including genetic manipulation, rapid proliferation and disease site targeting specificity. However, clinical translation of bacteria for these applications has been largely restricted by their unavoidable side effects and low treatment efficacies. Engineered bacteria for biomedical applications ideally need to generate only a low inflammatory response, show slow elimination by macrophages, low accumulation in normal organs, and almost unchanged inherent bioactivities. Here we describe a set of stealth bacteria, cell membrane coated bacteria (CMCB), meeting these requirement. Our findings are supported by evaluation in multiple mice models and ultimately demonstrate the potential of CMCB to serve as efficient tumor imaging agents. Stealth bacteria wrapped up with cell membranes have the potential for a myriad of bacterial-mediated biomedical applications.


Assuntos
Bactérias/genética , Engenharia Celular/métodos , Diagnóstico por Imagem/métodos , Sistemas de Liberação de Medicamentos/métodos , Neoplasias/diagnóstico por imagem , Animais , Bactérias/imunologia , Linhagem Celular Tumoral/transplante , Membrana Celular/imunologia , Modelos Animais de Doenças , Eritrócitos/ultraestrutura , Feminino , Humanos , Proteínas Luminescentes/administração & dosagem , Proteínas Luminescentes/farmacocinética , Macrófagos , Masculino , Camundongos , Cultura Primária de Células , Probióticos , Estudo de Prova de Conceito , Distribuição Tecidual
5.
Folia Histochem Cytobiol ; 57(3): 116-126, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31388982

RESUMO

INTRODUCTION: Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a selective suppressor of the immune response that has been linked to the evasion of immune surveillance by cancer cells. However, the exact prognostic impact of RCAS1 on epithelial ovarian cancer (EOC) has not been fully elucidated. The main aim of our study was to evaluate the influence of RCAS1 immunoreactivity (RCAS1-Ir) in EOC cells and in tumor stroma cells on patient overall survival. We also focused on RCAS1-Ir and the structure of the tumor stroma. MATERIAL AND METHODS: RCAS1-Ir was evaluated by means of immunohistochemistry in 67 patients with EOC. We distinguished cytoplasmic and membranous immunoreactivity patterns. RESULTS: We found that high cytoplasmic RCAS1-Ir in cancer cells was associated with more than a two-time shortened period of overall survival. Membranous RCAS1-Ir in cancer cells, as well as in tumor stroma macrophages and fibroblasts, did not correlate with patient survival. RCAS1-Ir in the cytoplasm of cancer cells was positively correlated with the degree of tumor stroma infiltration by fibroblasts and macrophages, but not with RCAS1-Ir in these cells. On the other hand, membranous RCAS1-Ir in cancer cells was positively correlated with RCAS1-Ir in fibroblasts and macrophages, but not with their quantity. CONCLUSIONS: Due to their different impacts on patient prognosis and tumor stroma structure, it seems that cytoplasmic and membranous RCAS1-Ir in EOC cells may have different biological functions.


Assuntos
Antígenos de Neoplasias/imunologia , Carcinoma Epitelial do Ovário/diagnóstico , Neoplasias Ovarianas/diagnóstico , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Epitelial do Ovário/imunologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Citoplasma/imunologia , Feminino , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Pessoa de Meia-Idade , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Prognóstico
6.
Immunol Rev ; 291(1): 44-56, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31402497

RESUMO

T cells are central players of our immune system, as their functions range from killing tumorous and virus-infected cells to orchestrating the entire immune response. In order for T cells to divide and execute their functions, they must be activated by antigen-presenting cells (APCs) through a cell-cell junction. Extracellular interactions between receptors on T cells and their ligands on APCs trigger signaling cascades comprised of protein-protein interactions, enzymatic reactions, and spatial reorganization events, to either stimulate or repress T cell activation. Plasma membrane is the major platform for T cell signaling. Recruitment of cytosolic proteins to membrane-bound receptors is a common critical step in many signaling pathways. Membranes decrease the dimensionality of protein-protein interactions to enable weak yet biologically important interactions. Membrane resident proteins can phase separate into micro-islands that promote signaling by enriching or excluding signal regulators. Moreover, some membrane lipids can either mediate or regulate cell signaling by interacting with signaling proteins. While it is critical to investigate T cell signaling in a cellular environment, the large number of signaling pathways involved and potential crosstalk have made it difficult to obtain precise, quantitative information on T cell signaling. Reconstitution of purified proteins to model membranes provides a complementary avenue for T cell signaling research. Here, I review recent progress in studying T cell signaling using membrane reconstitution approaches.


Assuntos
Membrana Celular/metabolismo , Ativação Linfocitária , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Comunicação Celular , Membrana Celular/química , Membrana Celular/imunologia , Espaço Extracelular , Humanos , Ligantes , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Superfície Celular/metabolismo
7.
Immunol Rev ; 291(1): 75-90, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31402506

RESUMO

To efficiently initiate activation responses against rare ligands in the microenvironment, lymphocytes employ sophisticated mechanisms involving signaling amplification. Recently, a signaling amplification mechanism initiated from phosphatidylinositol (PI) 4, 5-biphosphate [PI(4,5)P2] hydrolysis and synthesis for sustained B cell activation has been reported. Antigen and B cell receptor (BCR) recognition triggered the prompt reduction of PI(4,5)P2 density within the BCR microclusters, which led to the positive feedback for the synthesis of PI(4,5)P2 outside of the BCR microclusters. At single molecule level, the diffusion of PI(4,5)P2 was slow, allowing for the maintenance of a PI(4,5)P2 density gradient between the inside and outside of the BCR microclusters and the persistent supply of PI(4,5)P2 from outside to inside of the BCR microclusters. Here, we review studies that have contributed to uncovering the molecular mechanisms of PI(4,5)P2-derived signaling amplification model. Based on these studies, we proposed a "gasoline engine model" in which the activation of B cell signaling inside the microclusters is similar to the working principle of burning gasoline within the engine chamber of a gasoline engine. We also discuss the evidences showing the potential universality of this model and future prospects.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Ativação Linfocitária/imunologia , Fosfatidilinositóis/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Membrana Celular/imunologia , Humanos , Transdução de Sinais
8.
Nat Commun ; 10(1): 3199, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324770

RESUMO

Most cancer vaccines are unsuccessful in eliciting clinically relevant effects. Without using exogenous antigens and adoptive cells, we show a concept of utilizing biologically reprogrammed cytomembranes of the fused cells (FCs) derived from dendritic cells (DCs) and cancer cells as tumor vaccines. The fusion of immunologically interrelated two types of cells results in strong expression of the whole tumor antigen complexes and the immunological co-stimulatory molecules on cytomembranes (FMs), allowing the nanoparticle-supported FM (NP@FM) to function like antigen presenting cells (APCs) for T cell immunoactivation. Moreover, tumor-antigen bearing NP@FM can be bio-recognized by DCs to induce DC-mediated T cell immunoactivation. The combination of these two immunoactivation pathways offers powerful antitumor immunoresponse. Through mimicking both APCs and cancer cells, this cytomembrane vaccine strategy can develop various vaccines toward multiple tumor types and provide chances for accommodating diverse functions originating from the supporters.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Membrana Celular/imunologia , Nanopartículas/uso terapêutico , Animais , Fusão Celular , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Feminino , Imunoterapia , Ativação Linfocitária , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Transcriptoma , Transplante Heterólogo
9.
Hematology ; 24(1): 544-551, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31315540

RESUMO

Objective: Immunoglobulin D (IgD) levels are often elevated in patients with autoimmune diseases. However, the oncogenic activities of IgD and IgD receptor (IgDR) in diffuse large B-cell lymphoma (DLBCL) have not been reported in detail. Therefore, we aimed to investigate the expression of IgD and IgDR in patients with DLBCL. Methods: Membrane IgD (mIgD) and IgDR expression in tissue samples was analyzed using IHC, mIgD and IgDR expression on peripheral blood mononuclear cells (PBMCs) was analyzed by FCM, and secreted IgD (sIgD) level was analyzed by ELISA. Fisher's exact test and Spearman correlation analysis were used to evaluate the relationship between IgD, IgDR, and clinical parameters. Results: The pathological lymph nodes of 34 patients with DLBCL were studied, and mIgD and IgDR expression was found in 16 and 19 patients. mIgD and IgDR expression was upregulated in patients with DLBCL and mIgD expression was significantly associated with IgDR expression. Further correlation analysis showed that mIgD expression was correlated with serum ß2-MG level and Hans algorithm as germinal center B (GCB), whereas IgDR expression correlated with serum LDH level, IPI score and GCB. ELISA showed that sIgD level was significantly increased in DLBCL patients and it correlated with serum ß2-MG and LDH levels. FCM showed that mIgD and IgDR expression in PBMCs of patients with DLBCL was significantly higher than that in healthy controls. Conclusion: Our findings suggest that overexpression of IgD and IgDR is an abnormal activation state in DLBCL.


Assuntos
Regulação Neoplásica da Expressão Gênica , Imunoglobulina D/biossíntese , Leucócitos Mononucleares/química , Receptores Fc/biossíntese , Estudos de Casos e Controles , Linhagem Celular Tumoral , Membrana Celular/imunologia , Feminino , Humanos , Imunoglobulina D/análise , Imunoglobulina D/genética , L-Lactato Desidrogenase/sangue , Linfonodos/química , Linfonodos/patologia , Linfoma Difuso de Grandes Células B/sangue , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pseudolinfoma/sangue , Pseudolinfoma/patologia , Receptores Fc/análise , Receptores Fc/genética , Regulação para Cima , Microglobulina beta-2/análise
10.
Adv Mater ; 31(32): e1902542, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31183900

RESUMO

Current cancer immunotherapies including chimeric antigen receptor (CAR)-based therapies and checkpoint immune inhibitors have demonstrated significant clinical success, but always suffer from immunotoxicity and autoimmune disease. Recently, nanomaterial-based immunotherapies are developed to precisely control in vivo immune activation in tumor tissues for reducing immune-related adverse events. However, little consideration has been put on the spatial modulation of interactions between immune cells and cancer cells to optimize the efficacy of cancer immunotherapies. Herein, a rational design of immunomodulating nanoparticles is demonstrated that can in situ modify the tumor cell surface with natural killer cell (NK cell)-activating signals to achieve in situ activation of tumor-infiltrating NK cells, as well as direction of their antitumor immunity toward tumor cells. Using these immunomodulating nanoparticles, the remarkable inhibition of tumor growth is observed in mice without noticeable side effects. This study provides an accurate immunomodulation strategy that achieves safe and effective antitumor immunity through in situ NK cell activation in tumors. Further development by constructing interactions with various immune cells can potentially make this nanotechnology become a general platform for the design of advanced immunotherapies for cancer treatments.


Assuntos
Membrana Celular/imunologia , Fatores Imunológicos/química , Células Matadoras Naturais/metabolismo , Nanopartículas/química , Resinas Acrílicas/química , Animais , Ácidos Borônicos/química , Linhagem Celular Tumoral , Reagentes para Ligações Cruzadas/química , Portadores de Fármacos/química , Imunoglobulina G/química , Imunoterapia , Células Matadoras Naturais/imunologia , Camundongos , Transplante de Neoplasias , Polímeros/química , Soroalbumina Bovina/química , Propriedades de Superfície
11.
Int J Immunogenet ; 46(5): 307-320, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31183978

RESUMO

The detection and semiquantitative measurement of circulating human leucocyte antigen (HLA)-specific antibodies is essential for the management of patients before and after transplantation. In addition, the pretransplant cross-match to assess the reactivity of recipient HLA antibody against donor lymphocytes has long been the gold standard to prevent hyperacute rejection. Whilst both of these tests assume that recipient HLA-specific antibody is the only variable in the assessment of transplant risk, this is not the case. Transplant immunologists recognize that some HLA antigens are expressed at levels a magnitude lower than others (e.g., HLA-C, HLA-DQ), but within loci, and between different cell types there are many factors that influence HLA expression in both resting and activated cells. HLA is not usually expressed without the specific promoter proteins NLRC5, for HLA class I, and CIITA, for class II. The quantity of HLA protein production is then affected by factors including promoter region polymorphisms, alternative exon splice sites, methylation and microRNA-directed degradation. Different loci are influenced by multiple combinations of these control mechanisms making prediction of HLA regulation difficult, but an ability to measure the cellular expression of each HLA antigen, in conjunction with knowledge of circulating HLA-specific antibody, would lead to a more informed algorithm to assess transplant risk.


Assuntos
Regulação da Expressão Gênica , Antígenos HLA/genética , Antígenos HLA/imunologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Epigênese Genética , Variação Genética , Humanos , Isoanticorpos/imunologia , Polimorfismo Genético , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , Imunologia de Transplantes
12.
Sensors (Basel) ; 19(8)2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31010258

RESUMO

A new electrochemical immunosensor for cancer cell detection based on a specific interaction between the metastasis-related antigen of epithelial cell adhesion molecule (EpCAM) on the cell membrane and its monoclonal antibody (Anti-EpCAM) immobilized on a gold electrode has been developed. The amino-terminated polyamidoamine dendrimer (G6 PAMAM) was first covalently attached to the 3-mercaptopropionic acid (MPA)-functionalized gold electrode to obtain a thin film, and then completely carboxylated by succinic anhydride (SA). Next, the Anti-EpCAM was covalently bound with the G6 PAMAM to obtain a stable recognition layer. In the presence of the EpCAM expressing hepatocellular carcinomas cell line of HepG2, the specific immune recognition (Anti-EpCAM/EpCAM) led to an obvious change of the electron transfer ability. The properties of the layer-by-layer assembly process was examined by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The final determination of HepG2 cells was performed in the presence of the reversible [Fe(CN)6]3-/4- redox couple using impedance technique. Based on the advantages of PAMAM nanomaterial and immune reaction, a linear response to HepG2 cells ranging from 1 × 104 to 1 × 106 cells mL-1 with a calculated detection limit of 2.1 × 103 cells mL-1 was obtained. We expect this method can provide a potential tool for cancer cell monitoring and protein expression analysis.


Assuntos
Técnicas Biossensoriais , Molécula de Adesão da Célula Epitelial/genética , Imunoensaio , Neoplasias Hepáticas/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Membrana Celular/química , Membrana Celular/imunologia , Dendrímeros/química , Espectroscopia Dielétrica , Molécula de Adesão da Célula Epitelial/química , Molécula de Adesão da Célula Epitelial/imunologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/imunologia , Nanoestruturas/química , Poliaminas/química
13.
ACS Appl Mater Interfaces ; 11(10): 9850-9859, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30788951

RESUMO

Chemo-immunotherapy is an important tool to overcome tumor immune suppression in cancer immunotherapy. Herein, we report a surface-layer (S-layer) protein-enhanced immunotherapy strategy based on cell membrane-coated S-CM-HPAD nanoparticles for the effective malignant tumor therapy and metastasis inhibition. The S-CM-HPAD NPs could effectively deliver the tumor antigen, DOX, and immunoadjuvant to the homotypic tumor by the homotypic targeting ability of the coated cell membrane. In addition to its ability to induce tumor cell death, the loaded DOX could enhance the immunotherapy response by inhibition of myeloid-derived suppressor cells (MDSCs). Because of the intrinsic adjuvant property and capability to surface display epitopes and proteins, the S-layers localized on the surface of S-CM-HPAD NPs potentiated the immune response to the antigen. The results confirmed that the protective immunity against tumor occurrence was promoted effectively by prompting proliferation of lymphocytes and secretion of cytokine caused by the tumor-associated antigen and adjuvant. The excellent combinational therapeutic effects on the inhibition of tumor growth and metastasis in the melanoma tumor models demonstrated that the S-layer-enhanced immunotherapeutic method is a promising strategy for tumor immunotherapy of malignant tumor growth and metastasis.


Assuntos
Antígenos de Neoplasias/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Imunoconjugados/administração & dosagem , Melanoma Experimental/tratamento farmacológico , Nanopartículas/administração & dosagem , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/química , Membrana Celular/química , Membrana Celular/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunoconjugados/química , Imunoconjugados/imunologia , Melanoma Experimental/patologia , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Nanopartículas/química , Metástase Neoplásica , Propriedades de Superfície
14.
J Cell Sci ; 132(4)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30745330

RESUMO

The essential function of the T cell receptor (TCR) is to translate the engagement of peptides on the major histocompatibility complex (pMHC) into appropriate intracellular signals through the associated cluster of differentiation 3 (CD3) complex. The spatial organization of the TCR-CD3 complex in the membrane is thought to be a key regulatory element of signal transduction, raising the question of how receptor clustering impacts on TCR triggering. How signal transduction at the TCR-CD3 complex encodes the quality and quantity of pMHC molecules is not fully understood. This question can be approached by reconstituting T cell signaling in model and cell membranes and addressed by single-molecule imaging of endogenous proteins in T cells. We highlight such methods and further discuss how TCR clustering could affect pMHC rebinding rates, the local balance between kinase and phosphatase activity and/or the lipid environment to regulate the signal efficiency of the TCR-CD3 complex. We also examine whether clustering could affect the conformation of cytoplasmic CD3 tails through a biophysical mechanism. Taken together, we highlight how the spatial organization of the TCR-CD3 complex - addressed by reconstitution approaches - has emerged as a key regulatory element in signal transduction of this archetypal immune receptor.


Assuntos
Complexo CD3/imunologia , Complexo Principal de Histocompatibilidade , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Complexo CD3/química , Complexo CD3/metabolismo , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Humanos , Antígenos Comuns de Leucócito/química , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Modelos Biológicos , Ligação Proteica , Transporte Proteico , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Imagem Individual de Molécula/métodos , Linfócitos T/metabolismo , Linfócitos T/ultraestrutura
15.
J Immunol ; 202(5): 1417-1427, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683703

RESUMO

The random gene segment rearrangement during B cell development ensures Ab repertoire diversity. Because this process might generate autoreactive specificities, it has been proposed that stringent selection mechanisms prevent the development of autoreactive B cells. However, conventional assays to identify autoreactive B cells usually employ in vitro-generated Abs, which differ from membrane-bound BCRs. In this study, we used a cell-based assay to investigate the autoreactivity of membrane-bound BCRs derived from different B cell developmental stages of human peripheral blood. Contrasted to soluble Ab counterparts, only a few of the tested BCRs were autoreactive, although the cell-based assay sensitively detects feeble Ag recognition of a germline-reverted murine BCR that was selected after OVA immunization of mice, whereas conventional assays failed to do so. Together, these data suggest that proper identification of autoreactive B cells requires the membrane-bound BCR, as the soluble Ab may largely differ from its BCR counterpart in Ag binding.


Assuntos
Imunoglobulina M/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Membrana Celular/imunologia , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
16.
J Virol ; 93(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30674630

RESUMO

Lymphocyte antigen 6E (LY6E) is a GPI-anchored, interferon-inducible protein that has been shown to modulate viral infection in a cell type-dependent manner. Our recent work showed that LY6E promotes HIV-1 infection in some high-CD4-expressing cells, including human peripheral blood mononuclear cells (PBMCs) and the SupT1 cell line. In this work, we provide evidence that LY6E inhibits HIV-1 entry and spread in low-CD4-expressing Jurkat cells and human monocyte-derived macrophages (MDMs) through downregulation of the viral receptor CD4. We found that knockdown of LY6E in Jurkat cells and MDMs increases HIV-1 infection, yet overexpression of LY6E in Jurkat cells inhibits HIV-1 entry and replication. LY6E was found to be colocalized with CD4 on the plasma membrane of Jurkat cells and MDMs and enhances CD4 internalization. We artificially manipulated the CD4 level in Jurkat and SupT1 cells and found that overexpression of CD4 in Jurkat cells overcomes the inhibitory effect of LY6E; conversely, blocking the function of CD4 in SupT1 with a neutralizing antibody eliminates the enhancement of LY6E on HIV-1 entry. The CD4-dependent inhibitory phenotype of LY6E in low-CD4-expressing human MDMs can be recapitulated for a panel of transmitted founder viruses and laboratory-adapted HIV-1 strains. Given that HIV-1 can target low-CD4-expressing cells during acute infection yet replicates efficiently in high-CD4-expressing T cells at the late stage of disease, our observation that LY6E differentially modulates HIV-1 replication in a CD4-dependent manner has implications for understanding the complex roles of interferon (IFN)-induced proteins in AIDS pathogenesis.IMPORTANCE The role of IFN-induced genes (ISGs) in viral infection remains incompletely understood. While most ISGs are antiviral, some ISGs have been shown to promote viral infection, including HIV-1 infection. We previously showed that IFN-inducible LY6E protein promotes HIV-1 infection in human PMBCs and high-CD4-expressing SupT1 cells. Here we found that LY6E inhibits HIV-1 entry and replication in low-CD4-expressing MDMs and Jurkat cells. Mechanistically, we demonstrated that LY6E downregulates the cell surface receptor CD4, thus impairing the virus binding to target cells. This is in contrast to the situation of high-CD4-expressing cells, where LY6E predominantly promotes viral membrane fusion. The opposing role of IFN-inducible LY6E in modulating HIV-1 infection highlights the complex roles of ISGs in viral infection and viral pathogenesis.


Assuntos
Antígenos de Superfície/imunologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/virologia , Proteínas Ligadas por GPI/imunologia , Células HEK293 , Infecções por HIV/virologia , Células HeLa , Humanos , Interferons/imunologia , Células Jurkat , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Macrófagos/imunologia , Macrófagos/virologia , Internalização do Vírus , Replicação Viral/imunologia
17.
Nat Commun ; 10(1): 56, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30610190

RESUMO

CD1 proteins are expressed on dendritic cells, where they display lipid antigens to T-cell receptors (TCRs). Here we describe T-cell autoreactivity towards ubiquitous human membrane phospholipids presented by CD1b. These T-cells discriminate between two major types of lipids, sphingolipids and phospholipids, but were broadly cross-reactive towards diverse phospholipids including phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. The crystal structure of a representative TCR bound to CD1b-phosphatidylcholine provides a molecular mechanism for this promiscuous recognition. We observe a lateral escape channel in the TCR, which shunted phospholipid head groups sideways along the CD1b-TCR interface, without contacting the TCR. Instead the TCR recognition site involved the neck region phosphate that is common to all major self-phospholipids but absent in sphingolipids. Whereas prior studies have focused on foreign lipids or rare self-lipids, we define a new molecular mechanism of promiscuous recognition of common self-phospholipids including those that are known targets in human autoimmune disease.


Assuntos
Antígenos CD1/química , Fosfolipídeos/química , Receptores de Antígenos de Linfócitos T/química , Linfócitos T/fisiologia , Apresentação do Antígeno , Ligação Competitiva , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Cristalografia por Raios X , Humanos , Modelos Imunológicos , Simulação de Acoplamento Molecular
18.
Methods Mol Biol ; 1834: 75-83, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30324437

RESUMO

The complement system is a central part of the innate immune system. It defends the human body against infections and helps with the clearance of apoptotic particles and cellular debris. The importance of the complement system in physiology is reflected by autoreactive diseases that occur due to loss of functions of complement regulators as identified in age-related macular degeneration or gain of functions in complement convertases like C3 glomerulopathy. The chapter aims to provide methods to study complement regulation on a molecular level. Here we describe a set of in vitro assays, the combined techniques of ELISA and immunoblotting, to determine complement activation and regulation on surfaces. The methods allow to follow part of the complement activation cascade and to determine the activity of complement regulators like factor H.


Assuntos
Western Blotting , Membrana Celular/imunologia , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Ensaio de Imunoadsorção Enzimática , Membrana Celular/metabolismo , Proteínas do Sistema Complemento/metabolismo , Humanos
19.
Immunology ; 156(3): 270-276, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30460991

RESUMO

CD5 and CD6 are related surface receptors that limit and promote T-cell responses. Co-stimulatory effects of CD6 depend on binding a cell surface ligand, CD166, and recruitment of the intracellular adaptor proteins GADS and SLP-76 by C-terminal phosphotyrosines. We have continued to identify interactions of CD5 and CD6 to understand their roles in T-cell activation. In a screen to identify binding partners for peptides containing a cytoplasmic sequence, SDSDY conserved between CD5 and CD6, we identified ezrin radixin moesin (ERM) proteins, which link plasma membrane proteins to actin. Purified radixin FERM domain bound directly to CD5 and CD6 SDSDY peptides in a phosphorylation-dependent manner (KD = 0·5-2 µm) at 37°. In human T-cell blasts, mutation of the CD6 SDSDY sequence enhanced CD69 expression in response to CD3 monoclonal antibody. In this proximal readout, interactions of the SDSDY sequence were dominant compared with the C-terminal tyrosines of CD6. In contrast, in a more downstream readout, interleukin-2 expression, in response to immobilized CD3 and CD6 monoclonal antibodies, the C-terminal tyrosines were dominant. The data suggest that varying functional effects of CD6 and potentially CD5 depend on interactions of different cytoplasmic regions with the cytoskeleton and alter depending on the stimuli.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD5/imunologia , Proteínas de Ligação a DNA/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Actinas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Membrana Celular/imunologia , Citoplasma/imunologia , Citoesqueleto/imunologia , Humanos , Fosforilação/imunologia , Ratos , Tirosina/imunologia
20.
Immunol Lett ; 205: 16-24, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30439478

RESUMO

Extracellular nucleotides, mainly ATP, but also ADP, UTP, UDP and UDP-sugars, adenosine, and adenine base participate in the "purinergic signalling" pathway, an ubiquitous system of cell-to-cell communication. Fundamental pathophysiological processes such as tissue homeostasis, wound healing, neurodegeneration, immunity, inflammation and cancer are modulated by purinergic signalling. Nucleotides can be released from cells via unspecific or specific mechanisms. A non-regulated nucleotide release can occur from damaged or dying cells, whereas exocytotic granules, plasma membrane-derived microvesicles, membrane channels (connexins, pannexins, calcium homeostasis modulator (CALHM) channels and P2X7 receptor) or specific ATP binding cassette (ABC) transporters are involved in the controlled release. Four families of specific receptors, i.e. nucleotide P2X and P2Y receptors, adenosine P1 receptors, and the adenine-selective P0 receptor, and several ecto- nucleotidases are essential components of the "purinergic signalling" pathway. Thanks to the activity of ecto-nucleotidases, ATP (and possibly other nucleotides) are degraded into additional messenger molecules with specific action. The final biological effects depend on the type and amount of released nucleotides, their modification by ecto-nucleotidases, and their possible cellular re-uptake. Overall, these processes confer a remarkable level of selectivity and plasticity to purinergic signalling that makes this network one of the most relevant extracellular messenger systems in higher organisms.


Assuntos
Nucleosídeos/metabolismo , Nucleotídeos/metabolismo , Receptores Purinérgicos/metabolismo , Transdução de Sinais , Adenosina Trifosfatases/metabolismo , Animais , Membrana Celular/enzimologia , Membrana Celular/imunologia , Humanos , Transdução de Sinais/imunologia
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