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1.
Nat Immunol ; 24(12): 2135-2149, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37932456

RESUMO

Current US Food and Drug Administration-approved chimeric antigen receptor (CAR) T cells harbor the T cell receptor (TCR)-derived ζ chain as an intracellular activation domain in addition to costimulatory domains. The functionality in a CAR format of the other chains of the TCR complex, namely CD3δ, CD3ε and CD3γ, instead of ζ, remains unknown. In the present study, we have systematically engineered new CD3 CARs, each containing only one of the CD3 intracellular domains. We found that CARs containing CD3δ, CD3ε or CD3γ cytoplasmic tails outperformed the conventional ζ CAR T cells in vivo. Transcriptomic and proteomic analysis revealed differences in activation potential, metabolism and stimulation-induced T cell dysfunctionality that mechanistically explain the enhanced anti-tumor performance. Furthermore, dimerization of the CARs improved their overall functionality. Using these CARs as minimalistic and synthetic surrogate TCRs, we have identified the phosphatase SHP-1 as a new interaction partner of CD3δ that binds the CD3δ-ITAM on phosphorylation of its C-terminal tyrosine. SHP-1 attenuates and restrains activation signals and might thus prevent exhaustion and dysfunction. These new insights into T cell activation could promote the rational redesign of synthetic antigen receptors to improve cancer immunotherapy.


Assuntos
Proteômica , Receptores de Antígenos de Linfócitos T , Complexo CD3 , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Membrana Celular/metabolismo , Ativação Linfocitária , Linfócitos T
2.
Nat Immunol ; 24(1): 174-185, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36564464

RESUMO

The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4-LCK and CD8-LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8+ T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor-LCK interactions as promising targets for immunomodulation.


Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Linfócitos T Citotóxicos , Camundongos , Animais , Linfócitos T Citotóxicos/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Antígenos CD4 , Transdução de Sinais , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos CD8/metabolismo
3.
Nat Immunol ; 21(8): 848-856, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32632291

RESUMO

Rational design of chimeric antigen receptors (CARs) with optimized anticancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen engagement triggers activation. We analyzed CAR-mediated antigen recognition via quantitative, single-molecule, live-cell imaging and found the sensitivity of CAR T cells toward antigen approximately 1,000-times reduced as compared to T cell antigen-receptor-mediated recognition of nominal peptide-major histocompatibility complexes. While CARs outperformed T cell antigen receptors with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-protein kinase ZAP-70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR designs, which presently limit the efficacy of CAR T cell therapies to target tumors with diminished antigen expression.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos Quiméricos/imunologia , Humanos
4.
Nat Immunol ; 19(8): 821-827, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30013143

RESUMO

The main function of T cells is to identify harmful antigens as quickly and precisely as possible. Super-resolution microscopy data have indicated that global clustering of T cell antigen receptors (TCRs) occurs before T cell activation. Such pre-activation clustering has been interpreted as representing a potential regulatory mechanism that fine tunes the T cell response. We found here that apparent TCR nanoclustering could be attributed to overcounting artifacts inherent to single-molecule-localization microscopy. Using complementary super-resolution approaches and statistical image analysis, we found no indication of global nanoclustering of TCRs on antigen-experienced CD4+ T cells under non-activating conditions. We also used extensive simulations of super-resolution images to provide quantitative limits for the degree of randomness of the TCR distribution. Together our results suggest that the distribution of TCRs on the plasma membrane is optimized for fast recognition of antigen in the first phase of T cell activation.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Membrana Celular/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Células Cultivadas , Senescência Celular , Simulação por Computador , Memória Imunológica , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Imagens de Fantasmas , Ligação Proteica , Agregação de Receptores , Receptores de Antígenos de Linfócitos T alfa-beta/genética
5.
Nat Immunol ; 19(5): 487-496, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29662172

RESUMO

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.


Assuntos
Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/imunologia , Complexo CD3/química , Complexo CD3/imunologia , Camundongos , Camundongos Transgênicos
6.
Immunity ; 54(1): 132-150.e9, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33271119

RESUMO

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Linfócitos T CD8-Positivos/imunologia , Glioma/imunologia , Glicoesfingolipídeos/metabolismo , Glicosiltransferases/metabolismo , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoterapia/métodos , Apresentação de Antígeno , Ácido Aspártico Endopeptidases/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioma/mortalidade , Glicoesfingolipídeos/imunologia , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ativação Linfocitária , Transdução de Sinais , Análise de Sobrevida , Evasão Tumoral
7.
Nat Immunol ; 17(12): 1352-1360, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27776107

RESUMO

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.


Assuntos
Actinas/metabolismo , Linfócitos B/imunologia , Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Síndromes de Imunodeficiência/genética , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Adolescente , Inibidores da Angiogênese/farmacologia , Linfócitos B/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/genética , Criança , Citotoxicidade Imunológica/genética , Análise Mutacional de DNA , Dineínas/metabolismo , Feminino , Células HEK293 , Humanos , Switching de Imunoglobulina/genética , Síndromes de Imunodeficiência/tratamento farmacológico , Células Jurkat , Células Matadoras Naturais/efeitos dos fármacos , Lenalidomida , Masculino , Mutação/genética , Linhagem , RNA Interferente Pequeno/genética , Linfócitos T/efeitos dos fármacos , Talidomida/análogos & derivados , Talidomida/farmacologia
8.
EMBO J ; 42(7): e113507, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36808636

RESUMO

T-cell antigen recognition is invariably affected by tensile forces as they are exerted on T-cell antigen receptors (TCRs) transiently binding antigenic peptide/MHC complexes. In this issue of The EMBO Journal, Pettmann and colleagues promote the concept that forces reduce the lifetime of more stable stimulatory TCR-pMHC interactions to a larger extent than that of less stable non-stimulatory TCR-pMHC interactions. The authors argue that forces impede rather than boost T-cell antigen discrimination, which is promoted by force-shielding within the immunological synapse through cell adhesion via CD2/CD58 and LFA-1/ICAM-1.


Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T , Receptores de Antígenos de Linfócitos T/metabolismo
9.
N Engl J Med ; 389(6): 527-539, 2023 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-37342957

RESUMO

BACKGROUND: Increasing evidence links genetic defects affecting actin-regulatory proteins to diseases with severe autoimmunity and autoinflammation, yet the underlying molecular mechanisms are poorly understood. Dedicator of cytokinesis 11 (DOCK11) activates the small Rho guanosine triphosphatase (GTPase) cell division cycle 42 (CDC42), a central regulator of actin cytoskeleton dynamics. The role of DOCK11 in human immune-cell function and disease remains unknown. METHODS: We conducted genetic, immunologic, and molecular assays in four patients from four unrelated families who presented with infections, early-onset severe immune dysregulation, normocytic anemia of variable severity associated with anisopoikilocytosis, and developmental delay. Functional assays were performed in patient-derived cells, as well as in mouse and zebrafish models. RESULTS: We identified rare, X-linked germline mutations in DOCK11 in the patients, leading to a loss of protein expression in two patients and impaired CDC42 activation in all four patients. Patient-derived T cells did not form filopodia and showed abnormal migration. In addition, the patient-derived T cells, as well as the T cells from Dock11-knockout mice, showed overt activation and production of proinflammatory cytokines that were associated with an increased degree of nuclear translocation of nuclear factor of activated T cell 1 (NFATc1). Anemia and aberrant erythrocyte morphologic features were recapitulated in a newly generated dock11-knockout zebrafish model, and anemia was amenable to rescue on ectopic expression of constitutively active CDC42. CONCLUSIONS: Germline hemizygous loss-of-function mutations affecting the actin regulator DOCK11 were shown to cause a previously unknown inborn error of hematopoiesis and immunity characterized by severe immune dysregulation and systemic inflammation, recurrent infections, and anemia. (Funded by the European Research Council and others.).


Assuntos
Actinas , Anemia , Fatores de Troca do Nucleotídeo Guanina , Inflamação , Animais , Humanos , Camundongos , Actinas/genética , Actinas/metabolismo , Anemia/etiologia , Anemia/genética , Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Hematopoese , Inflamação/etiologia , Inflamação/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
10.
Nat Immunol ; 14(3): 262-70, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23377202

RESUMO

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.


Assuntos
Complexo CD3/imunologia , Citocinas/biossíntese , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Complexo CD3/metabolismo , Linhagem Celular , Proliferação de Células , Células HEK293 , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo
12.
EMBO Rep ; 24(11): e57842, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37768718

RESUMO

Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non-stimulatory endogenous pMHCIIs is postulated to sensitize T-cells for the recognition of single antigens on the surface of dendritic cells and B-cells. When testing this premise with the use of advanced live cell microscopy, we observe pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR engagement of highly abundant endogenous pMHCIIs is low or non-existent and affects neither TCR engagement of rare agonist pMHCII in early and advanced synapses nor agonist-induced TCR-proximal signaling. Our findings highlight the capacity of single freely diffusing agonist pMHCIIs to elicit the full T-cell response in an autonomous and peptide-specific fashion with consequences for adaptive immunity and immunotherapeutic approaches.


Assuntos
Antígenos de Histocompatibilidade Classe II , Linfócitos T , Peptídeos/metabolismo , Antígenos , Receptores de Antígenos de Linfócitos T
13.
Nat Immunol ; 13(7): 674-80, 2012 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-22660579

RESUMO

The binding of T cell antigen receptors (TCRs) to specific complexes of peptide and major histocompatibility complex (pMHC) is typically of very low affinity, which necessitates the use of multimeric pMHC complexes to label T lymphocytes stably. We report here the development of pMHC complexes able to be crosslinked by ultraviolet irradiation; even as monomers, these efficiently and specifically stained cognate T cells. We also used this reagent to probe T cell activation and found that a covalently bound pMHC was more stimulatory than an agonist pMHC on lipid bilayers. This finding suggested that serial engagement of TCRs is dispensable for activation when a substantial fraction of TCRs are stably engaged. Finally, pMHC-bound TCRs were 'preferentially' transported into the central supramolecular activation cluster after activation, which suggested that ligand engagement enabled linkage of the TCR and its associated CD3 signaling molecules to the cytoskeleton.


Assuntos
Reagentes de Ligações Cruzadas/química , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/química , Linfócitos T/química , Animais , Complexo CD3/química , Complexo CD3/imunologia , Células Cultivadas , Corantes/química , Citoesqueleto/química , Citoesqueleto/imunologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia
14.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33468643

RESUMO

T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the minimum signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC molecules.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , DNA/imunologia , Complexo Principal de Histocompatibilidade/genética , Receptores de Antígenos de Linfócitos T/química , Animais , Células Apresentadoras de Antígenos/citologia , Linfócitos T CD4-Positivos/citologia , DNA/química , DNA/genética , Expressão Gênica , Ligantes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Ativação Linfocitária , Camundongos , Conformação de Ácido Nucleico , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Cultura Primária de Células , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Baço/citologia , Baço/imunologia
15.
Mol Ther ; 30(11): 3358-3378, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-35821635

RESUMO

Chimeric antigen receptor (CAR) T cells have revolutionized treatment of B cell malignancies. However, enhancing the efficacy of engineered T cells without compromising their safety is warranted. The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) inhibits release of cytolytic enzymes from cytotoxic T lymphocytes. Here, we examined the potency of EBAG9 silencing for the improvement of adoptive T cell therapy. MicroRNA (miRNA)-mediated EBAG9 downregulation in transplanted cytolytic CD8+ T cells (CTLs) from immunized mice improved their cytolytic competence in a tumor model. In tolerant female recipient mice that received organ transplants, a minor histocompatibility antigen was turned into a rejection antigen by Ebag9 deletion, indicating an immune checkpoint function for EBAG9. Considerably fewer EBAG9-silenced human CAR T cells were needed for tumor growth control in a xenotransplantation model. Transcriptome profiling did not reveal additional risks regarding genotoxicity or aberrant differentiation. A single-step retrovirus transduction process links CAR or TCR expression with miRNA-mediated EBAG9 downregulation. Despite higher cytolytic efficacy, release of cytokines associated with cytokine release syndrome remains unaffected. Collectively, EBAG9 silencing enhances effector capacity of TCR- and CAR-engineered T cells, results in improved tumor eradication, facilitates efficient manufacturing, and decreases the therapeutic dose.


Assuntos
Antígenos de Neoplasias , Imunoterapia Adotiva , Neoplasias , Animais , Feminino , Humanos , Camundongos , MicroRNAs/genética , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Citotóxicos , Inativação Gênica , Proteínas de Checkpoint Imunológico , Antígenos de Neoplasias/genética
16.
Nat Immunol ; 11(1): 90-6, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20010844

RESUMO

The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dual-color fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane-associated signal transduction.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Membrana Celular/ultraestrutura , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Ativação Linfocitária/imunologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Proteínas de Membrana/genética , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência/métodos , Modelos Biológicos , Fosfoproteínas/genética , Transporte Proteico , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/ultraestrutura , Transfecção
17.
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
18.
Nano Lett ; 21(1): 507-514, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33305952

RESUMO

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Here, we developed a traction force microscopy platform which allows for quantifying the pulls and pushes exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. We immobilized specific T-cell activating antibodies on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR, and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas CD45 is excluded from bead-microvilli contacts.


Assuntos
Ativação Linfocitária , Tração , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Linfócitos T
19.
Eur J Immunol ; 50(8): 1126-1141, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32222966

RESUMO

TIM-3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) has been proposed to bind TIM-3 and to regulate its function. Using a T cell reporter platform we confirmed CEACAM1-mediated inhibition, but CEACAM1 did not functionally engage TIM-3. TIM-3 and CEACAM1 coexpression was limited to a small subset of activated T cells. Moreover, results obtained in extensive binding studies were not in support of an interaction between TIM-3 and CEACAM1. Cytoplasmic sequences derived from TIM-3 induced inhibitory signaling in our human T cell reporter system. Our results indicate that TIM-3 functions are independent of CEACAM1 and that this receptor has the capability to promote inhibitory signaling pathways in human T cells.


Assuntos
Antígenos CD/fisiologia , Moléculas de Adesão Celular/fisiologia , Receptor Celular 2 do Vírus da Hepatite A/fisiologia , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Receptor Celular 2 do Vírus da Hepatite A/análise , Humanos , Células Jurkat , Ativação Linfocitária , Transdução de Sinais/fisiologia , Linfócitos T/imunologia
20.
J Autoimmun ; 119: 102610, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33621930

RESUMO

CD4+ T cell trafficking is a fundamental property of adaptive immunity. In this study, we uncover a novel role for histone deacetylase 1 (HDAC1) in controlling effector CD4+ T cell migration, thereby providing mechanistic insight into why a T cell-specific deletion of HDAC1 protects against experimental autoimmune encephalomyelitis (EAE). HDAC1-deficient CD4+ T cells downregulated genes associated with leukocyte extravasation. In vitro, HDAC1-deficient CD4+ T cells displayed aberrant morphology and migration on surfaces coated with integrin LFA-1 ligand ICAM-1 and showed an impaired ability to arrest on and to migrate across a monolayer of primary mouse brain microvascular endothelial cells under physiological flow. Moreover, HDAC1 deficiency reduced homing of CD4+ T cells into the intestinal epithelium and lamina propria preventing weight-loss, crypt damage and intestinal inflammation in adoptive CD4+ T cell transfer colitis. This correlated with reduced expression levels of LFA-1 integrin chains CD11a and CD18 as well as of selectin ligands CD43, CD44 and CD162 on transferred circulating HDAC1-deficient CD4+ T cells. Our data reveal that HDAC1 controls T cell-mediated autoimmunity via the regulation of CD4+ T cell trafficking into the CNS and intestinal tissues.


Assuntos
Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Quimiotaxia de Leucócito/imunologia , Histona Desacetilase 1/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Animais , Biomarcadores , Adesão Celular , Quimiotaxia de Leucócito/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/diagnóstico , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Células Endoteliais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Histona Desacetilase 1/genética , Imuno-Histoquímica , Inflamação/diagnóstico , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout
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