Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
1.
Front Immunol ; 13: 799919, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35432331

RESUMO

Tγδ17 cells have emerged as a key population in the development of inflammatory and autoimmune conditions such as psoriasis. Thus, the therapeutic intervention of Tγδ17 cells can exert protective effects in this type of pathologies. Tγδ cells commit to IL-17 production during thymus development, and upon immune challenge, additional extrathymic signals induce the differentiation of uncommitted Tγδ cells into Tγδ17 effector cells. Despite the interest in Tγδ17 cells during the past 20 years, the role of TCR signaling in the generation and function of Tγδ17 cells has not been completely elucidated. While some studies point to the notion that Tγδ17 differentiation requires weak or no TCR signaling, other works suggest that Tγδ17 require the participation of specific kinases and adaptor molecules downstream of the TCR. Here we have examined the differentiation and pathogenic function of Tγδ17 cells in "knockin" mice bearing conservative mutations in the CD3ε polyproline rich sequence (KI-PRS) with attenuated TCR signaling due to lack of binding of the essential adaptor Nck. KI-PRS mice presented decreased frequency and numbers of Tγδ17 cells in adult thymus and lymph nodes. In the Imiquimod model of skin inflammation, KI-PRS presented attenuated skin inflammation parameters compared to wild-type littermates. Moreover, the generation, expansion and effector function Tγδ17 cells were impaired in KI-PRS mice upon Imiquimod challenge. Thus, we conclude that an intact CD3ε-PRS sequence is required for optimal differentiation and pathogenic function of Tγδ17 cells. These data open new opportunities for therapeutic targeting of specific TCR downstream effectors for treatment of Tγδ17-mediated diseases.


Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T , Animais , Imiquimode , Inflamação/metabolismo , Camundongos , Mutação , Peptídeos
2.
Br J Pharmacol ; 179(9): 1839-1856, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-33817782

RESUMO

Metabolism is dynamically regulated to accompany immune cell function, and altered immunometabolism can result in impaired immune responses. Concomitantly, the pharmacological manipulation of metabolic processes offers an opportunity for therapeutic intervention in inflammatory disorders. The nicotinamide adenine dinucleotide (NAD+ ) is a critical metabolic intermediate that serves as enzyme cofactor in redox reactions, and is also used as a co-substrate by many enzymes such as sirtuins, adenosine diphosphate ribose transferases and synthases. Through these activities, NAD+ metabolism regulates a broad spectrum of cellular functions such as energy metabolism, DNA repair, regulation of the epigenetic landscape and inflammation. Thus, the manipulation of NAD+ availability using pharmacological compounds such as NAD+ precursors can have immune-modulatory properties in inflammation. Here, we discuss how the NAD+ metabolism contributes to the immune response and inflammatory conditions, with a special focus on multiple sclerosis, inflammatory bowel diseases and inflammageing. LINKED ARTICLES: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.


Assuntos
NAD , Sirtuínas , Autoimunidade , Metabolismo Energético , Imunidade , NAD/metabolismo , Sirtuínas/metabolismo
3.
J Invest Dermatol ; 141(6): 1522-1532.e3, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33181141

RESUMO

Allergic contact dermatitis, also known as contact hypersensitivity, is a frequent T-cell‒mediated inflammatory skin disease characterized by red, itchy, swollen, and cracked skin. It is caused by the direct contact with an allergen and/or irritant hapten. Galectin-1 (Gal-1) is a ß-galactoside‒binding lectin, which is highly expressed in several types of immune cells. The role of endogenous Gal-1 in contact hypersensitivity is not known. We found that Gal-1‒deficient mice display more sustained and prolonged skin inflammation than wild-type mice after oxazolone treatment. Gal-1‒deficient mice have increased CD8+ T cells and neutrophilic infiltration in the skin. After the sensitization phase, Gal-1‒depleted mice showed an increased frequency of central memory CD8+ T cells and IFN-γ secretion by CD8+ T cells. The absence of Gal-1 does not affect the migration of transferred CD4+ and CD8+ T cells from the blood to the lymph nodes or to the skin. The depletion of CD4+ T lymphocytes as well as adoptive transfer experiments demonstrated that endogenous expression of Gal-1 on CD8+ T lymphocytes exerts a major role in the control of contact hypersensitivity model. These data underscore the protective role of endogenous Gal-1 in CD8+ but not CD4+ T cells in the development of allergic contact dermatitis.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Dermatite Alérgica de Contato/imunologia , Galectina 1/deficiência , Pele/patologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/metabolismo , Dermatite Alérgica de Contato/patologia , Modelos Animais de Doenças , Feminino , Galectina 1/genética , Humanos , Masculino , Camundongos , Oxazolona/administração & dosagem , Oxazolona/imunologia , Pele/imunologia
4.
Cells ; 9(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32906785

RESUMO

The interleukin 23 (IL-23) is a key pro-inflammatory cytokine in the development of chronic inflammatory diseases, such as psoriasis, inflammatory bowel diseases, multiple sclerosis, or rheumatoid arthritis. The pathological consequences of excessive IL-23 signaling have been linked to its ability to promote the production of inflammatory mediators, such as IL-17, IL-22, granulocyte-macrophage colony-stimulating (GM-CSF), or the tumor necrosis factor (TNFα) by target populations, mainly Th17 and IL-17-secreting TCRγδ cells (Tγδ17). Due to their pivotal role in inflammatory diseases, IL-23 and its downstream effector molecules have emerged as attractive therapeutic targets, leading to the development of neutralizing antibodies against IL-23 and IL-17 that have shown efficacy in different inflammatory diseases. Despite the success of monoclonal antibodies, there are patients that show no response or partial response to these treatments. Thus, effective therapies for inflammatory diseases may require the combination of multiple immune-modulatory drugs to prevent disease progression and to improve quality of life. Alternative strategies aimed at inhibiting intracellular signaling cascades using small molecule inhibitors or interfering peptides have not been fully exploited in the context of IL-23-mediated diseases. In this review, we discuss the current knowledge about proximal signaling events triggered by IL-23 upon binding to its membrane receptor to bring to the spotlight new opportunities for therapeutic intervention in IL-23-mediated pathologies.


Assuntos
Interleucina-23/metabolismo , Humanos , Transdução de Sinais
5.
Science ; 368(6497): 1371-1376, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32439659

RESUMO

The effect of immunometabolism on age-associated diseases remains uncertain. In this work, we show that T cells with dysfunctional mitochondria owing to mitochondrial transcription factor A (TFAM) deficiency act as accelerators of senescence. In mice, these cells instigate multiple aging-related features, including metabolic, cognitive, physical, and cardiovascular alterations, which together result in premature death. T cell metabolic failure induces the accumulation of circulating cytokines, which resembles the chronic inflammation that is characteristic of aging ("inflammaging"). This cytokine storm itself acts as a systemic inducer of senescence. Blocking tumor necrosis factor-α signaling or preventing senescence with nicotinamide adenine dinucleotide precursors partially rescues premature aging in mice with Tfam-deficient T cells. Thus, T cells can regulate organismal fitness and life span, which highlights the importance of tight immunometabolic control in both aging and the onset of age-associated diseases.


Assuntos
Senilidade Prematura/imunologia , Proteínas de Ligação a DNA/deficiência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/deficiência , Multimorbidade , Linfócitos T/metabolismo , Fatores de Transcrição/deficiência , Senilidade Prematura/genética , Senilidade Prematura/prevenção & controle , Animais , Síndrome da Liberação de Citocina/imunologia , Proteínas de Ligação a DNA/genética , Feminino , Deleção de Genes , Inflamação/genética , Inflamação/imunologia , Longevidade , Masculino , Camundongos , Camundongos Mutantes , Proteínas Mitocondriais/genética , NAD/administração & dosagem , NAD/farmacologia , Aptidão Física , Linfócitos T/ultraestrutura , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores
6.
PLoS Biol ; 18(3): e3000646, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203518

RESUMO

Interleukin 23 (IL-23) triggers pathogenic features in pro-inflammatory, IL-17-secreting T cells (Th17 and Tγδ17) that play a key role in the development of inflammatory diseases. However, the IL-23 signaling cascade remains largely undefined. Here, we used quantitative phosphoproteomics to characterize IL-23 signaling in primary murine Th17 cells. We quantified 6,888 phosphorylation sites in Th17 cells and found 168 phosphorylations regulated upon IL-23 stimulation. IL-23 increased the phosphorylation of the myosin regulatory light chain (RLC), an actomyosin contractibility marker, in Th17 and Tγδ17 cells. IL-23-induced RLC phosphorylation required Janus kinase 2 (JAK2) and Rho-associated protein kinase (ROCK) catalytic activity, and further study of the IL-23/ROCK connection revealed an unexpected role of IL-23 in the migration of Tγδ17 and Th17 cells through ROCK activation. In addition, pharmacological inhibition of ROCK reduced Tγδ17 recruitment to inflamed skin upon challenge with inflammatory agent Imiquimod. This work (i) provides new insights into phosphorylation networks that control Th17 cells, (ii) widely expands the current knowledge on IL-23 signaling, and (iii) contributes to the increasing list of immune cells subsets characterized by global phosphoproteomic approaches.


Assuntos
Inflamação/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Células Th17/metabolismo , Animais , Movimento Celular , Imiquimode/farmacologia , Inflamação/patologia , Subunidade p19 da Interleucina-23/genética , Janus Quinase 2 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Proteômica/métodos , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Serina/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo
7.
Front Immunol ; 8: 938, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28848546

RESUMO

Phosphorylation is the most abundant post-translational modification, regulating several aspects of protein and cell function. Quantitative phosphoproteomics approaches have expanded the scope of phosphorylation analysis enabling the quantification of changes in thousands of phosphorylation sites simultaneously in two or more conditions. These approaches offer a global view of the impact of cellular perturbations such as extracellular stimuli or gene ablation in intracellular signaling networks. Such great potential also brings on a new challenge: to identify, among the thousands of phosphorylations found in global phosphoproteomics studies, the small subset of site-specific phosphorylations expected to be functionally relevant. This review focus on updating and integrating findings on T lymphocyte signaling generated using global phosphoproteomics approaches, drawing attention on the biological relevance of the obtained data.

8.
Nat Commun ; 8: 15620, 2017 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-28555668

RESUMO

Glucose and glycolysis are important for the proinflammatory functions of many immune cells, and depletion of glucose in pathological microenvironments is associated with defective immune responses. Here we show a contrasting function for glucose in dendritic cells (DCs), as glucose represses the proinflammatory output of LPS-stimulated DCs and inhibits DC-induced T-cell responses. A glucose-sensitive signal transduction circuit involving the mTOR complex 1 (mTORC1), HIF1α and inducible nitric oxide synthase (iNOS) coordinates DC metabolism and function to limit DC-stimulated T-cell responses. When multiple T cells interact with a DC, they compete for nutrients, which can limit glucose availability to the DCs. In such DCs, glucose-dependent signalling is inhibited, altering DC outputs and enhancing T-cell responses. These data reveal a mechanism by which T cells regulate the DC microenvironment to control DC-induced T-cell responses and indicate that glucose is an important signal for shaping immune responses.


Assuntos
Células Dendríticas/imunologia , Glucose/metabolismo , Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/imunologia , Técnicas de Cocultura , Células Dendríticas/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação , Interferon gama/metabolismo , Lipopolissacarídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais , Linfócitos T/citologia
9.
EMBO J ; 34(15): 2008-24, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26136212

RESUMO

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses.


Assuntos
Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Interleucina-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Análise de Variância , Animais , Western Blotting , Clonagem Molecular , Citometria de Fluxo , Leupeptinas , Camundongos , Camundongos Transgênicos , Mutagênese , Proteínas Proto-Oncogênicas c-myc/imunologia , Piridinas , Pirimidinas , Reação em Cadeia da Polimerase em Tempo Real
10.
Sci Signal ; 7(348): ra99, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25336615

RESUMO

Protein kinase D2 (PKD2) is a serine and threonine kinase that is activated in T cells by diacylglycerol and protein kinase C in response to stimulation of the T cell receptor (TCR) by antigen. We quantified the activation of PKD2 at the single-cell level and found that this kinase acts as a sensitive digital amplifier of TCR engagement, enabling CD8(+) T cells to match the production of inflammatory cytokines to the quality and quantity of TCR ligands. There was a digital response pattern of PKD2 activation in response to TCR engagement, such that increasing the concentration and potency of TCR ligands increased the number of cells that exhibited activated PKD2. However, for each cell that responded to TCR stimulation, the entire cellular pool of PKD2 (~400,000 molecules) was activated. Moreover, PKD2 acted as an amplification checkpoint for antigen-stimulated digital cytokine responses and translated the differential strength of TCR signaling to determine the number of naïve CD8(+) T cells that became effector cells. Together, these results provide insights into PKD family kinases and how they act digitally to amplify signaling networks controlled by the TCR.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas Quinases/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Camundongos , Camundongos Knockout , Proteína Quinase D2 , Proteínas Quinases/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética
11.
Mol Cell Proteomics ; 13(12): 3544-57, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25266776

RESUMO

The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages.


Assuntos
Redes Reguladoras de Genes , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Linfócitos T Citotóxicos/metabolismo , Canais de Cátion TRPP/genética , Animais , Anticorpos/farmacologia , Complexo CD3/genética , Complexo CD3/metabolismo , Isótopos de Carbono , Regulação da Expressão Gênica , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Marcação por Isótopo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Isótopos de Nitrogênio , Especificidade de Órgãos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/genética , Fosforilação , Proteoma/genética , Transdução de Sinais , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Canais de Cátion TRPP/deficiência
12.
Nat Immunol ; 15(9): 808-14, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25137455

RESUMO

T lymphocyte proliferation and differentiation are controlled by signaling pathways initiated by the T cell antigen receptor. Here we explore how key serine-threonine kinases and their substrates mediate T cell signaling and coordinate T cell metabolism to meet the metabolic demands of participating in an immune response.


Assuntos
Diferenciação Celular/imunologia , Proliferação de Células/fisiologia , Proteínas Serina-Treonina Quinases/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Humanos , Ativação Linfocitária/imunologia
13.
Biochem J ; 442(3): 649-59, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22233340

RESUMO

PKD (protein kinase D) 2 is a serine/threonine kinase activated by diacylglycerol in response to engagement of antigen receptors in lymphocytes. To explore PKD2 regulation and function in TCR (T-cell antigen receptor) signal transduction we expressed TCR complexes with fixed affinity for self antigens in the T-cells of PKD2-null mice or mice deficient in PKD2 catalytic activity. We also developed a single cell assay to quantify PKD2 activation as T-cells respond to developmental stimuli or engagement of α/ß TCR complexes in vivo. Strikingly, PKD2 loss caused increases in thymic output, lymphadenopathy and splenomegaly in TCR transgenic mice. The precise magnitude and timing of PKD2 activation during T-cell development is thus critical to regulate thymic homoeostasis. PKD2-null T-cells that exit the thymus have a normal transcriptome, but show a limited and abnormal transcriptional response to antigen. Transcriptional profiling reveals the full consequences of PKD2 loss and maps in detail the selective, but critical, function for PKD2 in signalling by α/ß mature TCR complexes in peripheral T-cells.


Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Canais de Cátion TRPP/genética , Animais , Diferenciação Celular , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Linfócitos T/imunologia , Canais de Cátion TRPP/metabolismo
14.
Blood ; 118(2): 416-24, 2011 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-21527521

RESUMO

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.


Assuntos
Plaquetas/metabolismo , Ativação Plaquetária/genética , Proteína Quinase C/fisiologia , Proteínas Quinases/fisiologia , Trombose/genética , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Camundongos , Camundongos Knockout , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Agregação Plaquetária/fisiologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase D2 , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombose/metabolismo
15.
Nat Immunol ; 12(4): 352-61, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21399638

RESUMO

Here we report an unbiased analysis of the cytotoxic T lymphocyte (CTL) serine-threonine phosphoproteome by high-resolution mass spectrometry. We identified approximately 2,000 phosphorylations in CTLs, of which approximately 450 were controlled by T cell antigen receptor (TCR) signaling. A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators. A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus. Dephosphorylation of HDAC7 resulted in its accumulation in the nucleus and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.


Assuntos
Histona Desacetilases/metabolismo , Fosfoproteínas/metabolismo , Proteômica/métodos , Transdução de Sinais , Linfócitos T Citotóxicos/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Cromatografia Líquida , Citosol/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histona Desacetilases/genética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , Fosforilação , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/citologia
16.
Biochem J ; 432(1): 153-63, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20819079

RESUMO

Mammalian PKD (protein kinase D) isoforms have been implicated in the regulation of diverse biological processes in response to diacylglycerol and PKC (protein kinase C) signalling. To compare the functions of PKD1 and PKD2 in vivo, we generated mice deficient in either PKD1 or PKD2 enzymatic activity, via homozygous expression of PKD1(S744A/S748A) or PKD2(S707A/S711A) 'knockin' alleles. We also examined PKD2-deficient mice generated using 'gene-trap' technology. We demonstrate that, unlike PKD1, PKD2 catalytic activity is dispensable for normal embryogenesis. We also show that PKD2 is the major PKD isoform expressed in lymphoid tissues, but that PKD2 catalytic activity is not essential for the development of mature peripheral T- and B-lymphocytes. PKD2 catalytic activity is, however, required for efficient antigen receptor-induced cytokine production in T-lymphocytes and for optimal T-cell-dependent antibody responses in vivo. Our results reveal a key in vivo role for PKD2 in regulating the function of mature peripheral lymphocytes during adaptive immune responses. They also confirm the functional importance of PKC-mediated serine phosphorylation of the PKD catalytic domain for PKD activation and downstream signalling and reveal that different PKD family members have unique and non-redundant roles in vivo.


Assuntos
Linfócitos/metabolismo , Tecido Linfoide/metabolismo , Proteína Quinase C/metabolismo , Proteínas Quinases/metabolismo , Animais , Western Blotting , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Linfócitos/citologia , Linfócitos/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteína Quinase C/genética , Proteína Quinase D2 , Proteínas Quinases/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo
17.
Blood ; 110(13): 4331-40, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17823309

RESUMO

The T-cell receptor beta (TCRbeta)/pre-TCRalpha (pTalpha) pre-TCR complex (pre-TCR) signals the expansion and differentiation of de-veloping thymocytes. Functional pro-perties of the pre-TCR rely on its unique pTalpha chain, which suggests the participation of specific intracellular adaptors. However, pTalpha-interacting molecules remain unknown. Here, we identified a polyproline-arginine sequence in the human pTalpha cytoplasmic tail that interacted in vitro with SH3 domains of the CIN85/CMS family of adaptors, and mediated the recruitment of multiprotein complexes involving all (CMS, CIN85, and CD2BP3) members. Supporting the physiologic relevance of this interaction, we found that 1 such adaptor, CMS, interacted in vivo with human pTalpha, and its expression was selectively up-regulated during human thymopoiesis in pre-TCR-activated thymocytes. Upon activation, pre-TCR clustering was induced, and CMS and polymerized actin were simultaneously recruited to the pre-TCR activation site. CMS also associated via its C-terminal region to the actin cytoskeleton in the endocytic compartment, where it colocalized with internalized pTalpha in traffic to lysosomal degradation. Notably, deletion of the pTalpha CIN85/CMS-binding motif impaired pre-TCR-mediated Ca(2+) mobilization and NFAT transcriptional activity, and precluded activation induced by overexpression of a CMS-SH3 N-terminal mutant. These results provide the first molecular evidence for a pTalpha intracellular adaptor involved in pre-TCR function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação , Sinalização do Cálcio , Linhagem Celular , Proteínas do Citoesqueleto/genética , Citosol , Humanos , Complexos Multiproteicos , Fatores de Transcrição NFATC/metabolismo , Timo/citologia , Transfecção
18.
J Biol Chem ; 279(23): 24485-92, 2004 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-15060077

RESUMO

We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.


Assuntos
Complexo CD3/química , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/química , Western Blotting , Diferenciação Celular , Linhagem Celular Transformada , Membrana Celular/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Glicosilação , Humanos , Microscopia Confocal , Fenótipo , Testes de Precipitina , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais
19.
J Biol Chem ; 278(16): 14507-13, 2003 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-12473666

RESUMO

Engagement of the alpha beta T cell receptor (TCR) by its ligand results in the down-modulation of TCR cell surface expression, which is thought to be a central event in T cell activation. On the other hand, pre-TCR signaling is a key process in alpha beta T cell development, which appears to proceed in a constitutive and ligand-independent manner. Here, comparative analyses on the dynamics of pre-TCR and TCR cell surface expression show that unligated pre-TCR complexes expressed on human pre-T cells behave as engaged TCR complexes, i.e. they are rapidly internalized and degraded in lysosomes and proteasomes but do not recycle back to the cell surface. Thus, pre-TCR down-regulation takes place constitutively without the need for extracellular ligation. By using TCR alpha/p Tau alpha chain chimeras, we demonstrate that prevention of recycling and induction of degradation are unique pre-TCR properties conferred by the cytoplasmic domain of the pT alpha chain. Finally, we show that pre-TCR internalization is a protein kinase C-independent process that involves the combination of src kinase-dependent and -independent pathways. These data suggest that constitutive pre-TCR down-modulation regulates pre-TCR surface expression levels and hence the extent of ligand-independent signaling through the pre-TCR.


Assuntos
Citoplasma/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Biotinilação , Linhagem Celular , Membrana Celular/metabolismo , Dimerização , Regulação para Baixo , Citometria de Fluxo , Proteínas de Fluorescência Verde , Humanos , Ligantes , Proteínas Luminescentes/metabolismo , Lisossomos/metabolismo , Microscopia Confocal , Testes de Precipitina , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de Tempo , Transfecção
20.
Semin Immunol ; 14(5): 325-34, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12220933

RESUMO

Considerable progress has recently been made in defining the role that pre-antigen receptor complexes, namely the pre-T and pre-B cell receptors, play in lymphocyte development. It is now established that these receptors direct, in a similar way, the survival, expansion, clonality and further differentiation of pre-T and pre-B lymphocytes, respectively. However, less is known about the mechanisms which ensure that only minute amounts of pre-TCR and pre-BCR reach the plasma membrane of developing lymphocytes. In this review, we discuss the implications of recent experimental approaches which address the developmental regulation of human pre-TCR expression and the molecular mechanisms that control surface pre-TCR expression levels.


Assuntos
Diferenciação Celular/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Animais , Membrana Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Humanos , Glicoproteínas de Membrana/genética , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T/imunologia , Transcrição Gênica/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA