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1.
Sci Rep ; 9(1): 16382, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690814

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

2.
Front Immunol ; 10: 2209, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31572401

RESUMO

Complement split products (CSPs), such as the fragments C4d and C3d, which are generated as a consequence of complement regulatory processes, are established markers for disease activity in autoimmunity or antibody-mediated graft rejection. Since immunoglobulin-like transcript 4 (ILT4) was previously shown to interact with soluble CSPs, but not with CSPs covalently-bound to target surfaces following classical complement activation, the present study aimed to identify novel cellular receptors interacting with covalently-deposited CSPs. By applying an unbiased screening approach using a cDNA mammalian expression library generated from human monocyte-derived dendritic cells and probed with recombinant human C4d, we identified neuropilin-1 (NRP1) as a novel receptor for C4d, C3d, and iC3b. NRP1, a highly conserved type 1 transmembrane protein, plays important roles in the development of the nervous and cardiovascular system as well as in tumorigenesis through interaction with its established binding partners, such as vascular endothelial growth factor (VEGF) and semaphorin 3A (Sema3A). NRP1 is also expressed on immune cells and serves as a marker for murine Tregs. Although NRP1 contains domains homologous to ones found in some complement proteins, it has not been linked to the complement system. We demonstrate that binding of C4d to NRP1 expressing cells was dose-dependent and saturable, and had a KD value of 0.71 µM. Importantly, and in contrast to ILT4, NRP1 interacted with CSPs that were covalently bound to target surfaces in the course of complement activation, therefore representing a classical complement receptor. The binding site of CSPs was mapped to the b1 domain of the coagulation factor V/VIII homology domain of NRP1. Taken together, our results demonstrate a novel role for NRP1 as a receptor for CSPs deposited on surfaces during complement activation. Further work is required to elucidate the functional consequences of the NRP1-CSP interactions in immunity.

3.
Sci Rep ; 9(1): 7426, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092850

RESUMO

Invariant natural killer T (iNKT) cells are a specialized subset of T cells contributing to both, the innate and adaptive immune responses. In contrast to conventional T lymphocytes they recognize lipid antigens. The aim of the project is to establish a novel model system, to study iNKT-TCR - ligand interaction. An iNKT reporter cell line (JE6-1REP-iNKT) was engineered by introducing the human iNKT-TCR into a human leukemic T cell line carrying an NF-κB-driven fluorescent transcriptional reporter construct. Antigen presenting BWSTIM cells expressing human CD1d and CD80 were generated. Reporter induction in JE6-1REP-iNKT cells was assessed by flow cytometry. CRISPR/Cas9 was used for ß2M knock out in JE6-1REP-iNKT cells to abrogate CD1d expression and thus excluding antigen self-presentation. Reporter cells were shown to specifically react with iNKT antigens presented via CD1d. Their sensitivity towards α-GalCer was comparable to a murine iNKT hybridoma cell line. In conclusion, we created a novel iNKT reporter platform which, compared to traditional iNKT cell assays, is characterized by a shorter turnaround time and lower costs. It thus facilitates the identification of antigenic structures that drive the activation of iNKT cells in health and disease.

4.
Oncotarget ; 9(25): 17608-17619, 2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29707134

RESUMO

Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.

5.
Oncotarget ; 8(39): 64892-64906, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-29029399

RESUMO

Blockade of the T cell coinhibitory molecules CTLA-4 and PD-1 has clinical utility to strengthen T cell responses. In addition to these immune checkpoints an ever-growing number of molecules has been implicated in generating coinhibitory signals in T cells. However, investigating coinhibitory molecules in primary human cells is complicated by the restricted expression and promiscuity of both coinhibitory receptors and their ligands. Here we have evaluated the potential of fluorescence-based transcriptional reporters based on the human Jurkat T cell line in conjunction with engineered T cell stimulator cell lines for investigating coinhibitory pathways. CTLA-4, PD-1, TIGIT, BTLA and 2B4 expressing reporter cells were generated and activated with T cell stimulator cells expressing cognate ligands of these molecules. All accessory molecules tested were functional in our reporter system. Engagement of CTLA-4, PD-1, BTLA and TIGIT by their ligands significantly inhibited T cell activation, whereas binding of 2B4 by CD48 resulted in enhanced responses. Mutational analysis revealed intracellular motifs that are responsible for BTLA mediated T cell inhibition and demonstrates potent reporter inhibition by CTLA-4 independent of cytoplasmic signaling motifs. Moreover, considerably higher IC50 values were measured for the CTLA-4 blocker Ipilimumab compared to the PD-1 antibody Nivolumab. Our findings show that coinhibitory pathways can be evaluated in Jurkat-based transcriptional reporters and yield novel insights on their function. Results obtained from this robust reductionist system can complement more time consuming and complex studies of such pathways in primary T cells.

6.
PLoS One ; 12(5): e0178220, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28542462

RESUMO

Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs), which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an E. coli strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter assays can be measured on standard flow cytometers and in contrast to established detection methods, like luciferase-based systems or Limulus Amebocyte Lysate tests, they do not require costly reagents.


Assuntos
Fenômenos Microbiológicos , NF-kappa B/metabolismo , Receptores Toll-Like/metabolismo , Linhagem Celular , Meios de Cultura , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Imunidade Inata , Ligantes , Monócitos/imunologia , Monócitos/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptor 4 Toll-Like/agonistas , Receptores Toll-Like/agonistas
7.
J Immunol ; 196(3): 1387-99, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26729804

RESUMO

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression.


Assuntos
Basigina/imunologia , Interleucina-2/biossíntese , Ativação Linfocitária/imunologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Separação Celular , Citometria de Fluxo , Humanos , Immunoblotting , Interleucina-2/imunologia , Células Jurkat , Espectrometria de Massas , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Transdução Genética
8.
J Immunol Methods ; 430: 10-20, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26780292

RESUMO

Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.


Assuntos
Ativação Linfocitária , NF-kappa B/análise , Fatores de Transcrição NFATC/análise , Fator de Transcrição AP-1/análise , Antígenos CD2/fisiologia , Antígenos CD28 , Complexo CD3/metabolismo , Genes Reporter , Humanos , Células Jurkat , Proteínas Luminescentes , NF-kappa B/genética , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
9.
EMBO J ; 34(3): 393-409, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25535246

RESUMO

THEMIS is critical for conventional T-cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr-phosphorylation-independent fashion. Rather, SHP1 and THEMIS engage with the N-SH3 and C-SH3 domains of GRB2, respectively, a configuration that allows GRB2-SH2 to recruit the complex onto LAT. Consistent with THEMIS-mediated recruitment of SHP to the TCR signalosome, THEMIS knock-down increased TCR-induced CD3-ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock-down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK-mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T-cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T-cell development and differentiation.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Animais , Complexo CD3/genética , Complexo CD3/metabolismo , Diferenciação Celular/genética , Sobrevivência Celular/genética , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Mutação , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteínas/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Domínios de Homologia de src
10.
J Immunol ; 193(6): 2718-32, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25127865

RESUMO

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions, which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting T cells, which guarantees operative T cell effector functions.


Assuntos
Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Receptor IGF Tipo 2/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Humanos , Células Jurkat , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteínas de Membrana Transportadoras/imunologia , Camundongos , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Receptor IGF Tipo 2/biossíntese , Receptor IGF Tipo 2/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia
11.
PLoS One ; 9(6): e97144, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24887021

RESUMO

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5ß1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of ß1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and ß1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - ß1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Quimiocina CXCL12/farmacologia , Diacilglicerol Quinase/metabolismo , Integrina beta1/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , Transporte Proteico/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
12.
Trends Immunol ; 35(7): 311-8, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24951034

RESUMO

T cell development from immature CD4(+)CD8(+) double-positive (DP) thymocytes to the mature CD4 or CD8 single-positive (SP) stage requires proper T cell receptor (TCR) signaling. The current working model of thymocyte development is that the strength of the TCR-mediated signal - from little-or-none, through intermediate, to strong - received by the immature cells determines whether they will undergo death by neglect, positive selection, or negative selection, respectively. In recent years, several developmentally regulated, stage-specifically expressed proteins and miRNAs have been found that act like fine-tuners for signal transduction and propagation downstream of the TCR. This allows them to govern thymocyte positive selection. Here, we summarize recent findings on these molecules and suggest new concepts of TCR positive-selection signaling.


Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Timócitos/imunologia , Animais , Sinalização do Cálcio , Diferenciação Celular , Seleção Clonal Mediada por Antígeno , Proteínas Fetais/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptor Cross-Talk
13.
J Immunol ; 192(2): 771-81, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24337748

RESUMO

GTPases act as important switches in many signaling events in cells. Although small and heterotrimeric G proteins are subjects of intensive studies, little is known about the large IFN-inducible GTPases. In this article, we show that the IFN-γ-inducible guanylate binding protein 1 (GBP-1) is a regulator of T cell activation. Silencing of GBP-1 leads to enhanced activation of early T cell Ag receptor/CD3 signaling molecules, including Lck, that is translated to higher IL-2 production. Mass spectrometry analyses showed that regulatory cytoskeletal proteins, like plastin-2 that bundles actin fibers and spectrin ß-chain, brain 1 that links the plasma membrane to the actin cytoskeleton, are binding partners of GBP-1. The spectrin cytoskeleton influences cell spreading and surface expression of TCR/CD3 and the leukocyte phosphatase CD45. We found higher cell spreading and enhanced surface expression of TCR/CD3 and CD45 in GBP-1 silenced T cells that explain their enhanced TCR/CD3 signaling. We conclude that GBP-1 is a downstream processor of IFN-γ via which T cells regulate cytoskeleton-dependent cell functions.


Assuntos
Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Complexo CD3/genética , Complexo CD3/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citoesqueleto/genética , Citosol/metabolismo , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Células Jurkat , Antígenos Comuns de Leucócito , Leucócitos/metabolismo , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Linfócitos T/metabolismo , Regulação para Cima/genética
14.
Nature ; 504(7480): 441-5, 2013 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-24226767

RESUMO

Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate 'positive selection', causing maturation of CD4- or CD8αß-expressing 'single-positive' thymocytes from CD4(+)CD8αß(+) 'double-positive' precursors. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in 'negative selection' by activation-induced apoptosis or 'agonist selection' of functionally differentiated self-antigen-experienced T cells. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens.


Assuntos
Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Animais , Apoptose , Autoantígenos/imunologia , Sinalização do Cálcio , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Timócitos/imunologia
15.
J Immunol ; 190(7): 3749-56, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23460737

RESUMO

Thymocyte-expressed molecule involved in selection (THEMIS) is a recently identified regulator of thymocyte positive selection. THEMIS's mechanism of action is unknown, and whether it has a role in TCR-proximal signaling is controversial. In this article, we show that THEMIS and the adapter molecule growth factor receptor-bound protein 2 (GRB2) associate constitutively through binding of a conserved PxRPxK motif within the proline-rich region 1 of THEMIS to the C-terminal SH3-domain of GRB2. This association is indispensable for THEMIS recruitment to the immunological synapse via the transmembrane adapter linker for activation of T cells (LAT) and for THEMIS phosphorylation by Lck and ZAP-70. Two major sites of tyrosine phosphorylation were mapped to a YY-motif close to proline-rich region 1. The YY-motif was crucial for GRB2 binding, suggesting that this region of THEMIS might control local phosphorylation-dependent conformational changes important for THEMIS function. Finally, THEMIS binding to GRB2 was required for thymocyte development. Our data firmly assign THEMIS to the TCR-proximal signaling cascade as a participant in the LAT signalosome and suggest that the THEMIS-GRB2 complex might be involved in shaping the nature of Ras signaling, thereby governing thymic selection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Adaptadora GRB2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Timócitos/metabolismo , Sequência de Aminoácidos , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Proteína Adaptadora GRB2/química , Humanos , Sinapses Imunológicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Dados de Sequência Molecular , Nectinas , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Proteína-Tirosina Quinase ZAP-70/metabolismo
16.
Sci Signal ; 6(263): ra13, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23423439

RESUMO

The lymphocyte-specific Src family protein tyrosine kinase p56(Lck) (Lck) is essential for T cell development and activation and, hence, for adaptive immune responses. The mechanism by which Lck activity is directed toward specific substrates in response to T cell receptor (TCR) activation remains elusive. We used fluorescence lifetime imaging microscopy to assess the activation-dependent spatiotemporal changes in the conformation of Lck in live human T cells. Kinetic analysis of the fluorescence lifetime of Lck biosensors enabled the direct visualization of the dynamic local opening of 20% of the total amount of Lck proteins after activation of T cells with antibody against CD3 or by superantigen-loaded antigen-presenting cells. Parallel biochemical analysis of TCR complexes revealed that the conformational changes in Lck correlated with the induction of Lck enzymatic activity. These data show the dynamic, local activation through conformational change of Lck at sites of TCR engagement.


Assuntos
Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfócitos T/imunologia , Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Microscopia de Fluorescência , Conformação Proteica
17.
J Biol Chem ; 286(9): 7535-47, 2011 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-21189249

RESUMO

Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive "signaling waves" revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas/metabolismo , Proteômica , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Jurkat , Camundongos , Camundongos Mutantes , Fatores de Transcrição NFATC/metabolismo , Fosfoproteínas/genética , Fosforilação/imunologia , Proteínas/genética , Proteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Transcrição AP-1/metabolismo , Tirosina/metabolismo
18.
Immunity ; 32(6): 766-77, 2010 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-20541955

RESUMO

T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to approximately 40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-zeta phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Ativação Enzimática/imunologia , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Microscopia Confocal , Receptores de Antígenos de Linfócitos T/metabolismo
19.
Methods Enzymol ; 472: 133-51, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20580963

RESUMO

Our understanding of complex biological systems is based on high-quality proteomics tools for the parallelized detection and quantification of protein interactions. Current screening platforms, however, rely on measuring protein interactions in rather artificial systems, rendering the results difficult to confer on the in vivo situation. We describe here a detailed protocol for the design and the construction of a system to detect and quantify interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in living cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput, making it applicable as a screening tool. The proof-of-concept is demonstrated for the interaction between CD4, a major coreceptor in T-cell signaling, and Lck, a protein tyrosine kinase essential for early T-cell signaling.


Assuntos
Técnicas de Cultura de Células , Membrana Celular/metabolismo , Mapeamento de Interação de Proteínas , Animais , Antígenos CD4/metabolismo , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Membrana Celular/química , Células Cultivadas , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Mapeamento de Interação de Proteínas/instrumentação , Mapeamento de Interação de Proteínas/métodos , Propriedades de Superfície
20.
Mol Immunol ; 47(11-12): 2094-102, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20462637

RESUMO

Forkhead box protein 3 (Foxp3) is indispensable for the development of CD4(+)CD25(+) regulatory T cells (Tregs). Here we analyzed three prominent evolutionary conserved regions (ECRs) upstream of the transcription start site of the human FOXP3 gene. We show that ECR2 and ECR3 fragments derived from positions -1.3 to -2.0 kb and -5.0 to -6.0 kb, respectively, display basal transcriptional activity. Reporter constructs derived from ECR1, located between -0.6 and +0.23 kb and thus the most proximal ECR in respect of transcription initiation, remained almost inactive. However, ECR1 was transactivated by the NF-kappaB subunit p65 in HEK 293 cells. In Jurkat and primary T cells, in addition to p65, a second stimulus delivered by either T-cell receptor stimulation or addition of PMA was needed. Co-expression of I kappaB alpha inhibited p65-mediated FOXP3 proximal promoter transactivation, and the NF-kappaB inhibitor curcumin reduced Foxp3 neoexpression in IL-2/CD3/CD28/TGF-beta stimulated PBMCs. Moreover, proximal FOXP3 promoter transactivation was inhibited by Foxp3 and the SP transcription factor family member SP3. Thus, the human proximal FOXP3 promoter is controlled by activation through the TCR involving PKC and the NF-kappaB subunit p65 and by inhibition through a negative feedback loop and SP3.


Assuntos
Fatores de Transcrição Forkhead/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp3/fisiologia , Fator de Transcrição RelA/fisiologia , Ativação Transcricional , Linhagem Celular , Curcumina/farmacologia , Retroalimentação Fisiológica , Humanos , Linfócitos T Reguladores/metabolismo
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