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1.
PLoS Comput Biol ; 18(3): e1009883, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35303007

RESUMO

The human immune system consists of a highly intelligent network of billions of independent, self-organized cells that interact with each other. Machine learning (ML) is an artificial intelligence (AI) tool that automatically processes huge amounts of image data. Immunotherapies have revolutionized the treatment of blood cancer. Specifically, one such therapy involves engineering immune cells to express chimeric antigen receptors (CAR), which combine tumor antigen specificity with immune cell activation in a single receptor. To improve their efficacy and expand their applicability to solid tumors, scientists optimize different CARs with different modifications. However, predicting and ranking the efficacy of different "off-the-shelf" immune products (e.g., CAR or Bispecific T-cell Engager [BiTE]) and selection of clinical responders are challenging in clinical practice. Meanwhile, identifying the optimal CAR construct for a researcher to further develop a potential clinical application is limited by the current, time-consuming, costly, and labor-intensive conventional tools used to evaluate efficacy. Particularly, more than 30 years of immunological synapse (IS) research data demonstrate that T cell efficacy is not only controlled by the specificity and avidity of the tumor antigen and T cell interaction, but also it depends on a collective process, involving multiple adhesion and regulatory molecules, as well as tumor microenvironment, spatially and temporally organized at the IS formed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The optimal function of cytotoxic lymphocytes (including CTL and NK) depends on IS quality. Recognizing the inadequacy of conventional tools and the importance of IS in immune cell functions, we investigate a new strategy for assessing CAR-T efficacy by quantifying CAR IS quality using the glass-support planar lipid bilayer system combined with ML-based data analysis. Previous studies in our group show that CAR-T IS quality correlates with antitumor activities in vitro and in vivo. However, current manually quantified IS quality data analysis is time-consuming and labor-intensive with low accuracy, reproducibility, and repeatability. In this study, we develop a novel ML-based method to quantify thousands of CAR cell IS images with enhanced accuracy and speed. Specifically, we used artificial neural networks (ANN) to incorporate object detection into segmentation. The proposed ANN model extracts the most useful information to differentiate different IS datasets. The network output is flexible and produces bounding boxes, instance segmentation, contour outlines (borders), intensities of the borders, and segmentations without borders. Based on requirements, one or a combination of this information is used in statistical analysis. The ML-based automated algorithm quantified CAR-T IS data correlates with the clinical responder and non-responder treated with Kappa-CAR-T cells directly from patients. The results suggest that CAR cell IS quality can be used as a potential composite biomarker and correlates with antitumor activities in patients, which is sufficiently discriminative to further test the CAR IS quality as a clinical biomarker to predict response to CAR immunotherapy in cancer. For translational research, the method developed here can also provide guidelines for designing and optimizing numerous CAR constructs for potential clinical development. Trial Registration: ClinicalTrials.gov NCT00881920.


Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Antígenos de Neoplasias/metabolismo , Inteligência Artificial , Biomarcadores/metabolismo , Humanos , Sinapses Imunológicas/metabolismo , Aprendizado de Máquina , Neoplasias/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Reprodutibilidade dos Testes , Microambiente Tumoral
2.
Biophys J ; 121(7): 1246-1265, 2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35196513

RESUMO

Cytotoxic T lymphocytes (T cells) and natural killer cells form a tight contact, the immunological synapse (IS), with target cells, where they release their lytic granules containing perforin/granzyme and cytokine-containing vesicles. During this process the cell repolarizes and moves the microtubule organizing center (MTOC) toward the IS. In the first part of our work we developed a computational model for the molecular-motor-driven motion of the microtubule cytoskeleton during T cell polarization and analyzed the effects of cortical-sliding and capture-shrinkage mechanisms. Here we use this model to analyze the dynamics of the MTOC repositioning in situations in which 1) the IS is in an arbitrary position with respect to the initial position of the MTOC and 2) the T cell has two IS at two arbitrary positions. In the case of one IS, we found that the initial position determines which mechanism is dominant and that the time of repositioning does not rise monotonously with the MTOC-IS distance. In the case of two IS, we observe several scenarios that have also been reported experimentally: the MTOC alternates stochastically (but with a well-defined average transition time) between the two IS; it wiggles in between the two IS without transiting to one of the two; or it is at some point pulled to one of the two IS and stays there. Our model allows one to predict which scenario emerges in dependency of the mechanisms in action and the number of dyneins present. We report that the presence of capture-shrinkage mechanism in at least one IS is necessary to assure the transitions in every cell configuration. Moreover, the frequency of transitions does not decrease with the distance between the two IS and is the highest when both mechanisms are present in both IS.


Assuntos
Sinapses Imunológicas , Centro Organizador dos Microtúbulos , Dineínas/metabolismo , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais , Ativação Linfocitária , Centro Organizador dos Microtúbulos/metabolismo
3.
Nat Commun ; 13(1): 1029, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35210420

RESUMO

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAP). We utilize synaptobrevin2-mRFP knock-in mice to isolate fusogenic cytotoxic granules in an unbiased manner and visualize them alone or in degranulating CTLs. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets.


Assuntos
Sinapses Imunológicas , Linfócitos T Citotóxicos , Animais , Membrana Celular , Grânulos Citoplasmáticos/metabolismo , Sinapses Imunológicas/metabolismo , Camundongos
4.
Lab Chip ; 22(5): 908-920, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35098952

RESUMO

Analyzing cell-cell interaction is essential to investigate how immune cells function. Elegant designs have been demonstrated to study lymphocytes and their interaction partners. However, these devices have been targeting cells of similar dimensions. T lymphocytes are smaller, more deformable, and more sensitive to pressure than many cells. This work aims to fill the gap of a method for pairing cells with different dimensions. The developed method uses hydrodynamic flow focusing in the z-direction for on-site modulation of effective channel height to capture smaller cells as single cells. Due to immune cells' sensitivity to pressure, the proposed method provides a stable system without any change in flow conditions at the analysis area throughout experiments. Paired live cells have their activities analyzed with calcium imaging at the immunological synapse formed under a controlled environment. The method is demonstrated with primary human T lymphocytes, acute myeloid leukemia (AML) cell lines, and primary AML blasts.


Assuntos
Sinapses Imunológicas , Leucemia Mieloide Aguda , Comunicação Celular , Humanos , Dispositivos Lab-On-A-Chip , Linfócitos T
5.
Cell ; 185(4): 585-602.e29, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35051368

RESUMO

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Magnésio/metabolismo , Animais , Infecções Bacterianas/imunologia , Restrição Calórica , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Células HEK293 , Humanos , Memória Imunológica , Sinapses Imunológicas/metabolismo , Imunoterapia , Ativação Linfocitária/imunologia , Sistema de Sinalização das MAP Quinases , Magnésio/administração & dosagem , Masculino , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo
6.
Elife ; 112022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35050850

RESUMO

T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events.


Assuntos
Antígenos CD28 , Membrana Celular , Transdução de Sinais/genética , Nexinas de Classificação , Animais , Antígenos CD28/genética , Antígenos CD28/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Sinapses Imunológicas/genética , Sinapses Imunológicas/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Células Jurkat , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo
7.
Nat Commun ; 12(1): 7036, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34857745

RESUMO

The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8+ T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sinapses Imunológicas/metabolismo , Optogenética/métodos , Proteômica/métodos , Receptores de Superfície Celular/imunologia , Anticorpos/química , Células Apresentadoras de Antígenos/citologia , Linfócitos B/imunologia , Linfócitos B/patologia , Produtos Biológicos/química , Linfócitos T CD8-Positivos/citologia , Comunicação Celular , Linhagem Celular Tumoral , Cromatografia Líquida , Expressão Gênica , Células HL-60 , Humanos , Ligantes , Luz , Ativação Linfocitária , Optogenética/instrumentação , Medicina de Precisão/instrumentação , Medicina de Precisão/métodos , Ligação Proteica , Proteômica/instrumentação , Receptores de Superfície Celular/genética , Transdução de Sinais , Oxigênio Singlete/química , Oxigênio Singlete/metabolismo , Bibliotecas de Moléculas Pequenas/química , Espectrometria de Massas em Tandem , Vírion/química
8.
Int J Mol Sci ; 22(21)2021 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-34768945

RESUMO

CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.


Assuntos
Sinalização do Cálcio/imunologia , Sinapses Imunológicas/metabolismo , Proteína ORAI1/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Imunidade Adaptativa , Proteína 1 Homóloga a Discs-Large/antagonistas & inibidores , Proteína 1 Homóloga a Discs-Large/genética , Proteína 1 Homóloga a Discs-Large/metabolismo , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Sinapses Imunológicas/genética , Sinapses Imunológicas/imunologia , Células Jurkat , Cinética , Ativação Linfocitária , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Proteína ORAI1/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
9.
Front Immunol ; 12: 721722, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34707605

RESUMO

Under physiological conditions, CD8+ T cells need to recognize low numbers of antigenic pMHC class I complexes in the presence of a surplus of non-stimulatory, self pMHC class I on the surface of the APC. Non-stimulatory pMHC have been shown to enhance CD8+ T cell responses to low amounts of antigenic pMHC, in a phenomenon called co-agonism, but the physiological significance and molecular mechanism of this phenomenon are still poorly understood. Our data show that co-agonist pMHC class I complexes recruit CD8-bound Lck to the immune synapse to modulate CD8+ T cell signaling pathways, resulting in enhanced CD8+ T cell effector functions and proliferation, both in vitro and in vivo. Moreover, co-agonism can boost T cell proliferation through an extrinsic mechanism, with co-agonism primed CD8+ T cells enhancing Akt pathway activation and proliferation in neighboring CD8+ T cells primed with low amounts of antigen.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Sinapses Imunológicas/metabolismo , Camundongos , Fosforilação , Ligação Proteica , Transporte Proteico , Percepção de Quorum , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Nano Lett ; 21(21): 9247-9255, 2021 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-34709845

RESUMO

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. Synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. It is hence important to determine the 3D topography of the immunological synapse at high precision. Current methods provide only rather coarse images of the protein distribution within the synapse. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows three-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and nonactivating bilayers, respectively.


Assuntos
Sinapses Imunológicas , Imagem Individual de Molécula , Sinapses Imunológicas/metabolismo , Microscopia de Fluorescência/métodos , Receptores de Antígenos de Linfócitos T , Imagem Individual de Molécula/métodos , Linfócitos T
11.
Commun Biol ; 4(1): 1151, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34608260

RESUMO

Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells' lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.


Assuntos
Aspergillus fumigatus/citologia , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais/citologia , Microscopia de Fluorescência/métodos , Imagem Óptica/instrumentação
12.
Front Immunol ; 12: 687367, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394081

RESUMO

The essential microelement zinc plays immunoregulatory roles via its ability to influence signaling pathways. Zinc deficiency impairs overall immune function and resultantly increases susceptibility to infection. Thus, zinc is considered as an immune-boosting supplement for populations with hypozincemia at high-risk for infection. Besides its role as a structural cofactor of many proteins, zinc also acts as an intracellular messenger in immune cell signaling. T-cell activation instructs zinc influx from extracellular and subcellular sources through the Zip6 and Zip8 zinc transporters, respectively. Increased cytoplasmic zinc participates in the regulation of T-cell responses by modifying activation signaling. However, the mechanism underlying the activation-dependent movement of zinc ions by Zip transporters in T cells remains elusive. Here, we demonstrate that Zip6, one of the most abundantly expressed Zip transporters in T cells, is mainly localized to lipid rafts in human T cells and is recruited into the immunological synapse in response to TCR stimulation. This was demonstrated through confocal imaging of the interaction between CD4+ T cells and antigen-presenting cells. Further, immunoprecipitation assays show that TCR triggering induces tyrosine phosphorylation of Zip6, which has at least three putative tyrosine motifs in its long cytoplasmic region, and this phosphorylation is coupled with its physical interaction with Zap70. Silencing Zip6 reduces zinc influx from extracellular sources and suppresses T-cell responses, suggesting an interaction between Zip6-mediated zinc influx and TCR activation. These results provide new insights into the mechanism through which Zip6-mediated zinc influx occurs in a TCR activation-dependent manner in human CD4+ T cells.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sinapses Imunológicas/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte de Cátions/genética , Humanos , Sinapses Imunológicas/imunologia , Células Jurkat , Ativação Linfocitária , Microdomínios da Membrana/imunologia , Proteínas de Neoplasias/genética , Fosforilação , Transdução de Sinais , Tirosina
13.
Front Immunol ; 12: 708908, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34421914

RESUMO

PI3K signalling is required for activation, differentiation, and trafficking of T cells. PI3Kδ, the dominant PI3K isoform in T cells, has been extensively characterised using PI3Kδ mutant mouse models and PI3K inhibitors. Furthermore, characterisation of patients with Activated PI3K Delta Syndrome (APDS) and mouse models with hyperactive PI3Kδ have shed light on how increased PI3Kδ activity affects T cell functions. An important function of PI3Kδ is that it acts downstream of TCR stimulation to activate the major T cell integrin, LFA-1, which controls transendothelial migration of T cells as well as their interaction with antigen-presenting cells. PI3Kδ also suppresses the cell surface expression of CD62L and CCR7 which controls the migration of T cells across high endothelial venules in the lymph nodes and S1PR1 which controls lymph node egress. Therefore, PI3Kδ can control both entry and exit of T cells from lymph nodes as well as the recruitment to and retention of T cells within inflamed tissues. This review will focus on the regulation of adhesion receptors by PI3Kδ and how this contributes to T cell trafficking and localisation. These findings are relevant for our understanding of how PI3Kδ inhibitors may affect T cell redistribution and function.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/fisiologia , Linfócitos T/fisiologia , Animais , Adesão Celular , Movimento Celular , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Sinapses Imunológicas/fisiologia , Integrinas/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Camundongos , Doenças da Imunodeficiência Primária/etiologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/fisiologia
14.
Front Immunol ; 12: 688918, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335592

RESUMO

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where ß actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.


Assuntos
Células Matadoras Naturais/imunologia , Citoesqueleto de Actina/imunologia , Movimento Celular , Humanos , Sinapses Imunológicas/imunologia , Células Matadoras Naturais/fisiologia , Mecanotransdução Celular , Nanoestruturas
15.
Cell Mol Immunol ; 18(11): 2472-2488, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34413489

RESUMO

Microglia shape the synaptic environment in health and disease, but synapses do not exist in a vacuum. Instead, pre- and postsynaptic terminals are surrounded by extracellular matrix (ECM), which together with glia comprise the four elements of the contemporary tetrapartite synapse model. While research in this area is still just beginning, accumulating evidence points toward a novel role for microglia in regulating the ECM during normal brain homeostasis, and such processes may, in turn, become dysfunctional in disease. As it relates to synapses, microglia are reported to modify the perisynaptic matrix, which is the diffuse matrix that surrounds dendritic and axonal terminals, as well as perineuronal nets (PNNs), specialized reticular formations of compact ECM that enwrap neuronal subsets and stabilize proximal synapses. The interconnected relationship between synapses and the ECM in which they are embedded suggests that alterations in one structure necessarily affect the dynamics of the other, and microglia may need to sculpt the matrix to modify the synapses within. Here, we provide an overview of the microglial regulation of synapses, perisynaptic matrix, and PNNs, propose candidate mechanisms by which these structures may be modified, and present the implications of such modifications in normal brain homeostasis and in disease.


Assuntos
Encéfalo/imunologia , Matriz Extracelular/metabolismo , Espaço Extracelular/metabolismo , Sinapses Imunológicas/imunologia , Microglia/imunologia , Animais , Humanos
16.
Elife ; 102021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34313220

RESUMO

T cells are activated by target cells via an intimate contact, termed immunological synapse (IS). Cellular mechanical properties, especially stiffness, are essential to regulate cell functions. However, T cell stiffness at a subcellular level at the IS still remains largely elusive. In this work, we established an atomic force microscopy (AFM)-based elasticity mapping method on whole T cells to obtain an overview of the stiffness with a resolution of ~60 nm. Using primary human CD4+ T cells, we show that when T cells form IS with stimulating antibody-coated surfaces, the lamellipodia are stiffer than the cell body. Upon IS formation, T cell stiffness is enhanced both at the lamellipodia and on the cell body. Chelation of intracellular Ca2+ abolishes IS-induced stiffening at the lamellipodia but has no influence on cell-body-stiffening, suggesting different regulatory mechanisms of IS-induced stiffening at the lamellipodia and the cell body.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Sinapses Imunológicas/metabolismo , Fenômenos Mecânicos , Elasticidade , Humanos , Microscopia de Força Atômica
17.
PLoS Pathog ; 17(7): e1009771, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34314469

RESUMO

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes.


Assuntos
Proteínas de Bactérias/metabolismo , Células Dendríticas/imunologia , Sinapses Imunológicas/imunologia , Receptores Acoplados a Proteínas G/imunologia , Linfócitos T/imunologia , Animais , Apresentação do Antígeno/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Salmonella/metabolismo , Salmonella enterica
18.
Cell Rep ; 36(1): 109318, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34233185

RESUMO

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.


Assuntos
Forma Celular , Imageamento Tridimensional , Células Matadoras Naturais/citologia , Linfócitos T/citologia , Complexo 2-3 de Proteínas Relacionadas à Actina/deficiência , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Adolescente , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Exocitose/efeitos dos fármacos , Humanos , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Masculino , Compostos Organosselênicos/farmacologia , Compostos de Organossilício/farmacologia , Análise de Célula Única , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tionas/farmacologia , Uracila/análogos & derivados , Uracila/farmacologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
19.
Trends Immunol ; 42(8): 649-653, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34226146

RESUMO

T cell asymmetry upon specific cell-cell interactions during mammalian immunological synapse (IS) contacts requires mammalian target of rapamycin complex (mTORC) activation and chaperones, such as the eukaryotic chaperonin containing TCP1 (CCT) for protein synthesis and folding. This mechanism can control cytoskeleton dynamics, and regulate mitochondrial fate, respiration, and metabolic rates, ultimately underlying cell reprogramming events that are relevant for CD4+ T cell functional outcomes.


Assuntos
Sinapses Imunológicas , Linfócitos T , Chaperonina com TCP-1/metabolismo , Citoesqueleto/metabolismo , Sinapses Imunológicas/metabolismo , Dobramento de Proteína , Linfócitos T/metabolismo
20.
J Autoimmun ; 123: 102702, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34311143

RESUMO

Programmed Cell Death 1 (PD-1) receptor and its ligands (PD-Ls) are essential to maintain peripheral immune tolerance and to avoid tissue damage. Consequently, altered gene or protein expression of this system of co-inhibitory molecules has been involved in the development of cancer and autoimmunity. Substantial progress has been achieved in the study of the PD-1/PD-Ls system in terms of regulatory mechanisms and therapy. However, the role of the PD-1/PD-Ls pathway in neuroinflammation has been less explored despite being a potential target of treatment for neurodegenerative diseases. Multiple Sclerosis (MS) is the most prevalent, chronic, inflammatory, and autoimmune disease of the central nervous system that leads to demyelination and axonal damage in young adults. Recent studies have highlighted the key role of the PD-1/PD-Ls pathway in inducing a neuroprotective response and restraining T cell activation and neurodegeneration in MS. In this review, we outline the molecular and cellular mechanisms regulating gene expression, protein synthesis and traffic of PD-1/PD-Ls as well as relevant processes that control PD-1/PD-Ls engagement in the immunological synapse between antigen-presenting cells and T cells. Also, we highlight the most recent findings regarding the role of the PD-1/PD-Ls pathway in MS and its murine model, experimental autoimmune encephalomyelitis (EAE), including the contribution of PD-1 expressing follicular helper T (TFH) cells in the pathogenesis of these diseases. In addition, we compare and contrast results found in MS and EAE with evidence reported in other autoimmune diseases and their experimental models, and review PD-1/PD-Ls-targeting therapeutic approaches.


Assuntos
Antígeno B7-H1/fisiologia , Esclerose Múltipla/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/fisiologia , Receptor de Morte Celular Programada 1/fisiologia , Animais , Antígeno B7-H1/química , Antígeno B7-H1/genética , Encéfalo/patologia , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica , Humanos , Sinapses Imunológicas , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/etiologia , Proteína 2 Ligante de Morte Celular Programada 1/química , Proteína 2 Ligante de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/genética , Transdução de Sinais/fisiologia , Células T Auxiliares Foliculares/imunologia
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