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1.
Cells ; 12(21)2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37947636

RESUMO

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to a persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3, and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct, with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.


Assuntos
Neoplasias , Linfócitos T , Camundongos , Animais , Antígeno CTLA-4/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias/metabolismo , Imunoterapia
2.
bioRxiv ; 2023 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-37503045

RESUMO

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3 and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and a biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

3.
Nature ; 614(7948): 416-418, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36725942
4.
Commun Biol ; 5(1): 9, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013519

RESUMO

Tumors generate an immune-suppressive environment that prevents effective killing of tumor cells by CD8+ cytotoxic T cells (CTL). It remains largely unclear upon which cell type and at which stage of the anti-tumor response mediators of suppression act. We have combined an in vivo tumor model with a matching in vitro reconstruction of the tumor microenvironment based on tumor spheroids to identify suppressors of anti-tumor immunity that directly act on interaction between CTL and tumor cells and to determine mechanisms of action. An adenosine 2A receptor antagonist, as enhanced by blockade of TIM3, slowed tumor growth in vivo. Engagement of the adenosine 2A receptor and TIM3 reduced tumor cell killing in spheroids, impaired CTL cytoskeletal polarization ex vivo and in vitro and inhibited CTL infiltration into tumors and spheroids. With this role in CTL killing, blocking A2AR and TIM3 may complement therapies that enhance T cell priming, e.g. anti-PD-1 and anti-CTLA-4.


Assuntos
Morte Celular , Citoesqueleto/fisiologia , Citosol/fisiologia , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor A2A de Adenosina/genética , Agonistas do Receptor A2 de Adenosina/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptor A2A de Adenosina/metabolismo
5.
Cells ; 10(2)2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671236

RESUMO

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIß inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Sinapses Imunológicas/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia
6.
Sci Signal ; 13(649)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934075

RESUMO

The killing of tumor cells by CD8+ T cells is suppressed by the tumor microenvironment, and increased expression of inhibitory receptors, including programmed cell death protein-1 (PD-1), is associated with tumor-mediated suppression of T cells. To find cellular defects triggered by tumor exposure and associated PD-1 signaling, we established an ex vivo imaging approach to investigate the response of antigen-specific, activated effector CD8+ tumor-infiltrating lymphocytes (TILs) after interaction with target tumor cells. Although TIL-tumor cell couples readily formed, couple stability deteriorated within minutes. This was associated with impaired F-actin clearing from the center of the cellular interface, reduced Ca2+ signaling, increased TIL locomotion, and impaired tumor cell killing. The interaction of CD8+ T lymphocytes with tumor cell spheroids in vitro induced a similar phenotype, supporting a critical role of direct T cell-tumor cell contact. Diminished engagement of PD-1 within the tumor, but not acute ex vivo blockade, partially restored cell couple maintenance and killing. PD-1 thus contributes to the suppression of TIL function by inducing a state of impaired subcellular organization.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Experimentais/imunologia , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia/métodos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais/genética , Microambiente Tumoral/imunologia
7.
Bio Protoc ; 10(21): e3806, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33659460

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 interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4+ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist peptide-loaded APCs were incubated with TCR transgenic CD4+ T cells for 4.5 min before fixation and antibody staining. Fixed cell couples were imaged using a 100x 1.4 NA objective on a Leica SP8 AOBS confocal laser scanning microscope. LAT clustered in multiple supramolecular complexes and their number and size distributions were determined. Using this protocol, cSMAC properties in its cellular context at the interface between a T cell and an APC could be quantified.

8.
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.


Cells receive dozens of signals at different times and in different places. Integrating incoming information and deciding how to respond is no easy task. Signaling molecules on the cell surface pass messages inwards using chemical messengers that interact in complicated networks within the cell. One way to unravel the complexity of these networks is to look at specific groups of signaling molecules in test tubes to see how they interact. But the interior of a living cell is a very different environment. Molecules inside cells are tightly packed and, under certain conditions, they interact with each other by the thousands. They form structures known as 'supramolecular complexes', which changes their behavior. One such supramolecular complex is the 'central supramolecular activation cluster', or cSMAC for short. It forms under the surface of immune cells called T cells when they are getting ready to fight an infection. Under the microscope, the cSMAC looks like the bullseye of a dartboard, forming a crowd of signaling molecules at the center of the interface between the T cell and another cell. Its exact role is not clear, but evidence suggests it helps to start and stop the signals that switch T cells on. The cSMAC contains two key protein adaptors called LAT and SLP-76 that help to hold the structure together. So, to find out what the cSMAC does, Clark et al. genetically modified these adaptors to gain control over when the cSMAC forms. Clark et al. examined mouse T cells using super-resolution microscopy and electron microscopy, watching as other immune cells delivered the signal to switch on. As the T cells started to activate, the composition of the cSMAC changed. In the first two minutes after the cells started activating, the cSMAC included a large number of different components. This made T cell activation more efficient, possibly because the supramolecular complex was helping the network of signals to interact. Later, the cSMAC started to lose many of these components. Separating components may have helped to stop the activation signals. Understanding how T cells activate could lead to the possibility of turning them on or off in immune-related diseases. But these findings are not just relevant to immune cells. Other cells also use supramolecular complexes to control their signaling. Investigating how these complexes change over time could help us to understand how other cell types make decisions.


Assuntos
Células Apresentadoras de Antígenos/fisiologia , Comunicação Celular , Interleucina-2/metabolismo , Linfócitos T/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos CD28/metabolismo , Células Cultivadas , Proteína Adaptadora GRB2/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo
9.
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
10.
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
11.
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
12.
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
13.
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
14.
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
15.
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
16.
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
18.
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
19.
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
20.
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
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