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1.
Elife ; 82019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31663508

RESUMO

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen-presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5 µm3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates.

2.
Bioinformatics ; 33(14): i217-i224, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28881992

RESUMO

Motivation: Efforts to model how signaling and regulatory networks work in cells have largely either not considered spatial organization or have used compartmental models with minimal spatial resolution. Fluorescence microscopy provides the ability to monitor the spatiotemporal distribution of many molecules during signaling events, but as of yet no methods have been described for large scale image analysis to learn a complex protein regulatory network. Here we present and evaluate methods for identifying how changes in concentration in one cell region influence concentration of other proteins in other regions. Results: Using 3D confocal microscope movies of GFP-tagged T cells undergoing costimulation, we learned models containing putative causal relationships among 12 proteins involved in T cell signaling. The models included both relationships consistent with current knowledge and novel predictions deserving further exploration. Further, when these models were applied to the initial frames of movies of T cells that had been only partially stimulated, they predicted the localization of proteins at later times with statistically significant accuracy. The methods, consisting of spatiotemporal alignment, automated region identification, and causal inference, are anticipated to be applicable to a number of biological systems. Availability and implementation: The source code and data are available as a Reproducible Research Archive at http://murphylab.cbd.cmu.edu/software/2017_TcellCausalModels/. Contact: murphy@cmu.edu.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Transdução de Sinais , Software , Algoritmos , Humanos , Proteínas/análise , Linfócitos T/metabolismo
3.
Methods Mol Biol ; 1584: 409-421, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28255716

RESUMO

Three-dimensional live cell imaging of the interaction of T cells with antigen-presenting cells (APCs) visualizes the subcellular distributions of signaling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signaling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signaling as it occurs inside live cells with resolution in time and space across thousands of cells.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Sinapses Imunológicas/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Sinapses Imunológicas/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência/métodos
4.
Elife ; 62017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28112644

RESUMO

Notch is a critical regulator of T cell differentiation and is activated through proteolytic cleavage in response to ligand engagement. Using murine myelin-reactive CD4 T cells, we demonstrate that proximal T cell signaling modulates Notch activation by a spatiotemporally constrained mechanism. The protein kinase PKCθ is a critical mediator of signaling by the T cell antigen receptor and the principal costimulatory receptor CD28. PKCθ selectively inactivates the negative regulator of F-actin generation, Coronin 1A, at the center of the T cell interface with the antigen presenting cell (APC). This allows for effective generation of the large actin-based lamellum required for recruitment of the Notch-processing membrane metalloproteinase ADAM10. Such enhancement of Notch activation is critical for efficient T cell proliferation and Th17 differentiation. We reveal a novel mechanism that, through modulation of the cytoskeleton, controls Notch activation at the T cell:APC interface thereby linking T cell receptor and Notch signaling pathways.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteína Quinase C-theta/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Antígenos CD28/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo
5.
Sci Signal ; 9(424): rs3, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27095595

RESUMO

Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems.


Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Biologia Computacional/métodos , Linfócitos T/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Fatores de Despolimerização de Actina/genética , Animais , Western Blotting , Antígenos CD28/genética , Antígenos CD28/metabolismo , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Sinapses Imunológicas/metabolismo , Cinética , Ativação Linfocitária , Camundongos Transgênicos , Microscopia de Fluorescência , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Imagem com Lapso de Tempo/métodos , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética
6.
Sci Transl Med ; 8(321): 321ra7, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26764158

RESUMO

X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with the exuberant expansion of activated CD8(+) T cells after Epstein-Barr virus (EBV) infection. XLP-1 is caused by defects in signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), an adaptor protein that modulates T cell receptor (TCR)-induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death (RICD) and diminished TCR-induced inhibition of diacylglycerol kinase α (DGKα), leading to increased diacylglycerol metabolism and decreased signaling through Ras and PKCθ (protein kinase Cθ). We show that down-regulation of DGKα activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD via induction of the proapoptotic proteins NUR77 and NOR1. Pharmacological inhibition of DGKα prevents the excessive CD8(+) T cell expansion and interferon-γ production that occur in SAP-deficient mice after lymphocytic choriomeningitis virus infection without impairing lytic activity. Collectively, these data highlight DGKα as a viable therapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Morte Celular/efeitos dos fármacos , Citocinas/biossíntese , Diacilglicerol Quinase/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/metabolismo , Ativação Linfocitária , Contagem de Linfócitos , Transtornos Linfoproliferativos/tratamento farmacológico , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/deficiência , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/metabolismo , Tiazóis/farmacologia , Proteínas ras/metabolismo
7.
PLoS One ; 10(8): e0133299, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26237050

RESUMO

Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches.


Assuntos
Actinas/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia
8.
PLoS One ; 10(8): e0133231, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26237588

RESUMO

Dynamic subcellular distributions of signaling system components are critical regulators of cellular signal transduction through their control of molecular interactions. Understanding how signaling activity depends on such distributions and the cellular structures driving them is required for comprehensive insight into signal transduction. In the activation of primary murine T cells by antigen presenting cells (APC) signaling intermediates associate with various subcellular structures, prominently a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell. While actin dynamics are well established as general regulators of cellular organization, their role in controlling signaling organization in primary T cell:APC couples and the specific cellular structures driving it is unresolved. Using modest interference with actin dynamics with a low concentration of Jasplakinolide as corroborated by costimulation blockade we show that T cell actin preferentially controls lamellal signaling localization and activity leading downstream to calcium signaling. Lamellal localization repeatedly related to efficient T cell function. This suggests that the transient lamellal actin matrix regulates T cell signaling associations that facilitate T cell activation.


Assuntos
Actinas/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Depsipeptídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
9.
Eur J Immunol ; 44(12): 3522-31, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25209945

RESUMO

Changes in immune function during the course of systemic lupus erythematosus (SLE) are well characterized. Class-switched antinuclear antibodies are the hallmark of SLE, and T/B-cell interactions are thus critical. However, changes in immune function contributing to disease susceptibility are unknown. Here, we have analyzed primary T and B cells from a mouse model of SLE prior to the onset of disease. To allow cognate T-cell activation with low affinity, we have developed a lower potency peptide ligand for the OTII TCR. T- and B-cell couples formed less frequently and retained their polarity less efficiently preferentially in response to low-affinity stimulation in SLE-prone mice. This matched decreased recruitment of actin and Vav1 and an enhanced PKCΘ recruitment to the cellular interface in T cells. The induction of the GC B-cell marker GL7 was increased in T/B cell couples from SLE-prone mice when the T-cell numbers were limited. However, the overall gene expression changes were marginal. Taken together, the enhanced cell-couple transience may allow a more efficient sampling of a large number of T/B cell couples, preferentially in response to limiting stimuli, therefore enhancing the immune reactivity in the development of SLE.


Assuntos
Linfócitos B/imunologia , Comunicação Celular/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Linfócitos B/patologia , Feminino , Regulação da Expressão Gênica/imunologia , Centro Germinativo/imunologia , Centro Germinativo/patologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Proteína Quinase C-épsilon/imunologia , Proteínas Proto-Oncogênicas c-vav/imunologia , Linfócitos T/patologia
11.
Immunol Rev ; 256(1): 133-47, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24117818

RESUMO

T cells are activated through interaction with antigen-presenting cells (APCs). During activation, receptors and signaling intermediates accumulate in diverse spatiotemporal distributions. These distributions control the probability of signaling interactions and thus govern information flow through the signaling system. Spatiotemporally resolved system-scale investigation of signaling can extract the regulatory information thus encoded, allowing unique insight into the control of T-cell function. Substantial technical challenges exist, and these are briefly discussed herein. While much of the work assessing T-cell spatiotemporal organization uses planar APC substitutes, we focus here on B-cell APCs with often stark differences. Spatiotemporal signaling distributions are driven by cell biologically distinct structures, a large protein assembly at the interface center, a large invagination, the actin-supported interface periphery as extended by smaller individual lamella, and a newly discovered whole-interface actin-driven lamellum. The more than 60 elements of T-cell activation studied to date are dynamically distributed between these structures, generating a complex organization of the signaling system. Signal initiation and core signaling prefer the interface center, while signal amplification is localized in the transient lamellum. Actin dynamics control signaling distributions through regulation of the underlying structures and drive a highly undulating T-cell/APC interface that imposes substantial constraints on T-cell organization. We suggest that the regulation of actin dynamics, by controlling signaling distributions and membrane topology, is an important rheostat of T-cell signaling.


Assuntos
Actinas/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Comunicação Celular , Humanos , Ativação Linfocitária
12.
Nat Immunol ; 14(8): 858-66, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23793062

RESUMO

Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea-mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell-specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/imunologia , Antígenos CD28/imunologia , Proteínas de Transporte/imunologia , Guanilato Ciclase/imunologia , Proteína Quinase C/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/genética , Citometria de Fluxo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Análise de Sequência de DNA , Organismos Livres de Patógenos Específicos
13.
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
15.
J Cell Sci ; 125(Pt 22): 5302-14, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22956543

RESUMO

The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4(+) and CD8(+) T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3(B)) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3(B) domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux.


Assuntos
Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Humanos , Sinapses Imunológicas/metabolismo , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Transporte Proteico/imunologia , Proteínas Proto-Oncogênicas c-vav/química , Domínios de Homologia de src
16.
PLoS One ; 6(11): e27227, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22096541

RESUMO

Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP(2)) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP(2) biology. First, PIP(2) is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP(2) is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP(2) distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP(2)-generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP(2) turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP(2) blockade, yielded an increase in PIP(2) levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP(2). It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP(2) in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP(2) on T cell activation.


Assuntos
Ativação Linfocitária/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Linfócitos T/metabolismo , Actinas , Animais , Células Cultivadas , Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 4,5-Difosfato/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo
17.
Sci Signal ; 4(193): ra66, 2011 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-21971040

RESUMO

During T cell activation by antigen-presenting cells (APCs), the diverse spatiotemporal organization of components of T cell signaling pathways modulates the efficiency of activation. Here, we found that loss of the tyrosine kinase interleukin-2 (IL-2)-inducible T cell kinase (Itk) in mice altered the spatiotemporal distributions of 14 of 16 sensors of T cell signaling molecules in the region of the interface between the T cell and the APC, which reduced the segregation of signaling intermediates into distinct spatiotemporal patterns. Activation of the Rho family guanosine triphosphatase Cdc42 at the center of the cell-cell interface was impaired, although the total cellular amount of active Cdc42 remained intact. The defect in Cdc42 localization resulted in impaired actin accumulation at the T cell-APC interface in Itk-deficient T cells. Reconstitution of cells with active Cdc42 that was specifically directed to the center of the interface restored actin accumulation in Itk-deficient T cells. Itk also controlled the central localization of the guanine nucleotide exchange factor SLAT [Switch-associated protein 70 (SWAP-70)-like adaptor of T cells], which may contribute to the activation of Cdc42 at the center of the interface. Together, these data illustrate how control of the spatiotemporal organization of T cell signaling controls critical aspects of T cell function.


Assuntos
Ativação Linfocitária/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Actinas/genética , Actinas/imunologia , Actinas/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Fatores de Troca do Nucleotídeo Guanina , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/imunologia , Proteína cdc42 de Ligação ao GTP/metabolismo
18.
J Immunol ; 186(12): 6839-47, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21543646

RESUMO

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell-APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.


Assuntos
Complexo CD3/metabolismo , Sinapses Imunológicas , Fosfatidilinositóis/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Aminoácidos Básicos , Animais , Sítios de Ligação/imunologia , Complexo CD3/química , Complexo CD3/imunologia , Linhagem Celular , Humanos , Ativação Linfocitária/imunologia , Camundongos , Fosfatidilinositóis/imunologia , Ligação Proteica/imunologia , Transdução de Sinais/imunologia , Transfecção
19.
Sci Signal ; 3(132): pe24, 2010 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-20664063

RESUMO

Protein kinase C (PKC-theta), one of many PKC isoforms expressed in T cells, is important for the activation of mature effector T cells. During T cell activation, PKC-theta is recruited to the interface between the T cell and the activating cellular interaction partner, the antigen-presenting cell or a synthetic substitute thereof. New evidence establishes that PKC-theta function differs in regulatory T cells, a T cell subset that suppresses the function of effector T cells. In regulatory T cells, PKC-theta inhibits their function and, intriguingly, is sequestered from the activating cellular interface. This finding raises several questions of general interest. Does PKC-theta function overlap with that of other PKC family members? What are the functionally critical distinctions in the similar signaling systems of effector and regulatory T cells? Does the divergent localization of PKC-theta in regulatory T cells drive function?


Assuntos
Inibidores Enzimáticos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Humanos , Ativação Linfocitária/efeitos dos fármacos , Modelos Biológicos , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/fisiologia , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/fisiologia
20.
Proc Natl Acad Sci U S A ; 107(26): 11912-7, 2010 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-20547841

RESUMO

Cytolytic effectors polarize toward target cells for effective killing and IFN-gamma secretion. The spatiotemporal features of this polarization and their importance for cytolysis have not been resolved. In cytotoxic T cells and natural killer (NK) cells, transient polarization was consistently associated with effective killing. Polarization was regulated by Cdc42, a small Rho family GTPase universally critical for cytoskeletal dynamics. Transient accumulation of active Cdc42 at the cytolytic effector/target cell interface and focus of such accumulation on the interface center were closely related to cytolysis. Surprisingly, however, the intensity of Cdc42 activation was not. We interfered with Cdc42 activation in NK cells such that sustained polarization in long lasting nonkilling cell couples was selectively blocked. Thus the proportion of the NK cell population displaying transient polarization was increased. As a consequence, cytolytic responder frequency and IFN-gamma production were enhanced upon such interference with Cdc42 activation. These data support the notion that transience in polarization is critical for cytolytic effector function, likely by preventing cytolytic effectors from becoming trapped in nonproductive target cell interactions.


Assuntos
Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Citotoxicidade Imunológica , Técnicas In Vitro , Interferon gama/biossíntese , Camundongos , Camundongos Transgênicos , Modelos Imunológicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo
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