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12 pages, 2896 KiB

Open AccessArticle

Cooperative Stabilization of Close-Contact Zones Leads to Sensitivity and Selectivity in T-Cell Recognition

by Bartosz Różycki and Thomas R. Weikl

Cells 2021, 10(5), 1023; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10051023 - 26 Apr 2021

Cited by 4 | Viewed by 2006

Abstract

T cells are sensitive to 1 to 10 foreign-peptide-MHC complexes among a vast majority of self-peptide-MHC complexes, and discriminate selectively between peptide-MHC complexes that differ not much in their binding affinity to T-cell receptors (TCRs). Quantitative models that aim to explain this sensitivity [...] Read more.

T cells are sensitive to 1 to 10 foreign-peptide-MHC complexes among a vast majority of self-peptide-MHC complexes, and discriminate selectively between peptide-MHC complexes that differ not much in their binding affinity to T-cell receptors (TCRs). Quantitative models that aim to explain this sensitivity and selectivity largely focus on single TCR/peptide-MHC complexes, but T cell adhesion involves a multitude of different complexes. In this article, we demonstrate in a three-dimensional computational model of T-cell adhesion that the cooperative stabilization of close-contact zones is sensitive to one to three foreign-peptide-MHC complexes and occurs at a rather sharp threshold affinity of these complexes, which implies selectivity. In these close-contact zones with lateral extensions of hundred to several hundred nanometers, few TCR/foreign-peptide-MHC complexes and many TCR/self-peptide-MHC complexes are segregated from LFA-1/ICAM-1 complexes that form at larger membrane separations. Previous high-resolution microscopy experiments indicate that the sensitivity and selectivity in the formation of closed-contact zones reported here are relevant for T-cell recognition, because the stabilization of close-contact zones by foreign, agonist peptide-MHC complexes precedes T-cell signaling and activation in the experiments. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Early T Cell Signaling)

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13 pages, 1459 KiB

Open AccessArticle

Cooperative Interaction of Nck and Lck Orchestrates Optimal TCR Signaling

by Frederike A. Hartl, Jatuporn Ngoenkam, Esmeralda Beck-Garcia, Liz Cerqueira, Piyamaporn Wipa, Pussadee Paensuwan, Prapat Suriyaphol, Pankaj Mishra, Burkhart Schraven, Stefan Günther, Sutatip Pongcharoen, Wolfgang W. A. Schamel and Susana Minguet

Cells 2021, 10(4), 834; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10040834 - 7 Apr 2021

Cited by 5 | Viewed by 3966

Abstract

The T cell antigen receptor (TCR) is expressed on T cells, which orchestrate adaptive immune responses. It is composed of the ligand-binding clonotypic TCRαβ heterodimer and the non-covalently bound invariant signal-transducing CD3 complex. Among the CD3 subunits, the CD3ε cytoplasmic tail contains binding [...] Read more.

The T cell antigen receptor (TCR) is expressed on T cells, which orchestrate adaptive immune responses. It is composed of the ligand-binding clonotypic TCRαβ heterodimer and the non-covalently bound invariant signal-transducing CD3 complex. Among the CD3 subunits, the CD3ε cytoplasmic tail contains binding motifs for the Src family kinase, Lck, and the adaptor protein, Nck. Lck binds to a receptor kinase (RK) motif and Nck binds to a proline-rich sequence (PRS). Both motifs only become accessible upon ligand binding to the TCR and facilitate the recruitment of Lck and Nck independently of phosphorylation of the TCR. Mutations in each of these motifs cause defects in TCR signaling and T cell activation. Here, we investigated the role of Nck in proximal TCR signaling by silencing both Nck isoforms, Nck1 and Nck2. In the absence of Nck, TCR phosphorylation, ZAP70 recruitment, and ZAP70 phosphorylation was impaired. Mechanistically, this is explained by loss of Lck recruitment to the stimulated TCR in cells lacking Nck. Hence, our data uncover a previously unknown cooperative interaction between Lck and Nck to promote optimal TCR signaling. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Early T Cell Signaling)

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16 pages, 2576 KiB

Open AccessArticle

CD8 Co-Receptor Enhances T-Cell Activation without Any Effect on Initial Attachment

by Philippe Robert, Laurent Limozin, P. Anton van der Merwe and Pierre Bongrand

Cells 2021, 10(2), 429; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10020429 - 18 Feb 2021

Cited by 1 | Viewed by 1993

Abstract

The scanning of surrounding tissues by T lymphocytes to detect cognate antigens requires high speed, sensitivity and specificity. T-cell receptor (TCR) co-receptors such as CD8 increase detection performance, but the exact mechanism remains incompletely understood. Here, we used a laminar flow chamber to [...] Read more.

The scanning of surrounding tissues by T lymphocytes to detect cognate antigens requires high speed, sensitivity and specificity. T-cell receptor (TCR) co-receptors such as CD8 increase detection performance, but the exact mechanism remains incompletely understood. Here, we used a laminar flow chamber to measure at the single molecule level the kinetics of bond formation and rupture between TCR- transfected CD8+ and CD8− Jurkat cells and surfaces coated with five peptide-exposing major histocompatibility antigens (pMHCs) of varying activating power. We also used interference reflection microscopy to image the spreading of these cells dropped on pMHC-exposing surfaces. CD8 did not influence the TCR–pMHC interaction during the first few seconds following cell surface encounter, but it promoted the subsequent spreading responses, suggesting that CD8 was involved in early activation rather than binding. Further, the rate and extent of spreading, but not the lag between contact and spreading initiation, depended on the pMHC. Elucidating T-lymphocyte detection strategy may help unravel underlying signaling networks. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Early T Cell Signaling)

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24 pages, 14085 KiB

Open AccessArticle

A LAT-Based Signaling Complex in the Immunological Synapse as Determined with Live Cell Imaging Is Less Stable in T Cells with Regulatory Capability

by Yikui Li, Helen M. Tunbridge, Graham J. Britton, Elaine V. Hill, Parisa Sinai, Silvia Cirillo, Clare Thompson, Farnaz Fallah-Arani, Simon J. Dovedi, David C. Wraith and Christoph Wülfing

Cells 2021, 10(2), 418; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10020418 - 17 Feb 2021

Cited by 2 | Viewed by 2485

Abstract

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. [...] Read more.

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 IIIβ 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. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Early T Cell Signaling)

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18 pages, 3472 KiB

Open AccessArticle

Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism

by Juan José Saez, Stephanie Dogniaux, Massiullah Shafaq-Zadah, Ludger Johannes, Claire Hivroz and Andrés Ernesto Zucchetti

Cells 2021, 10(2), 359; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10020359 - 9 Feb 2021

Cited by 4 | Viewed by 3471

Abstract

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T cell activation the intracellular pool of LAT is recruited to [...] Read more.

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of four proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute to both pathways, are in our cellular context, specifically and respectively, involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point to the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Early T Cell Signaling)

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20 pages, 2367 KiB

Open AccessArticle

A Novel, LAT/Lck Double Deficient T Cell Subline J.CaM1.7 for Combined Analysis of Early TCR Signaling

by Inmaculada Vico-Barranco, Mikel M. Arbulo-Echevarria, Isabel Serrano-García, Alba Pérez-Linaza, José M. Miranda-Sayago, Arkadiusz Miazek, Isaac Narbona-Sánchez and Enrique Aguado

Cells 2021, 10(2), 343; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10020343 - 6 Feb 2021

Cited by 4 | Viewed by 3055

Abstract

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the [...] Read more.

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the LAT adaptor, respectively. LAT, linker for the activation of T cells, is a transmembrane adaptor protein that acts as a scaffold coupling the early signals coming from the TCR with downstream signaling pathways leading to cellular responses. The leukemic T cell line Jurkat and its derivative mutants J.CaM1.6 (Lck deficient) and J.CaM2 (LAT deficient) have been widely used to study the first signaling events upon TCR triggering. In this work, we describe the loss of LAT adaptor expression found in a subline of J.CaM1.6 cells and analyze cis-elements responsible for the LAT expression defect. This new cell subline, which we have called J.CaM1.7, can re-express LAT adaptor after Protein Kinase C (PKC) activation, which suggests that activation-induced LAT expression is not affected in this new cell subline. Contrary to J.CaM1.6 cells, re-expression of Lck in J.CaM1.7 cells was not sufficient to recover TCR-associated signals, and both LAT and Lck had to be introduced to recover activatory intracellular signals triggered after CD3 crosslinking. Overall, our work shows that the new LAT negative J.CaM1.7 cell subline could represent a new model to study the functions of the tyrosine kinase Lck and the LAT adaptor in TCR signaling, and their mutual interaction, which seems to constitute an essential early signaling event associated with the TCR/CD3 complex. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Early T Cell Signaling)

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12 pages, 4386 KiB

Open AccessFeature PaperArticle

A Multimodal Platform for Simultaneous T-cell Imaging, Defined Activation, and Mechanobiological Characterization

by Martin Fölser, Viktoria Motsch, René Platzer, Johannes B. Huppa and Gerhard J. Schütz

Cells 2021, 10(2), 235; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10020235 - 25 Jan 2021

Cited by 4 | Viewed by 3022

Abstract

T-cell antigen recognition is accompanied by extensive morphological rearrangements of the contact zone between the T-cell and the antigen-presenting cell (APC). This process involves binding of the T-cell receptor (TCR) complex to antigenic peptides presented via MHC on the APC surface, the interaction [...] Read more.

T-cell antigen recognition is accompanied by extensive morphological rearrangements of the contact zone between the T-cell and the antigen-presenting cell (APC). This process involves binding of the T-cell receptor (TCR) complex to antigenic peptides presented via MHC on the APC surface, the interaction of costimulatory and adhesion proteins, remodeling of the actin cytoskeleton, and the initiation of downstream signaling processes such as the release of intracellular calcium. However, multiparametric time-resolved analysis of these processes is hampered by the difficulty in recording the different readout modalities at high quality in parallel. In this study, we present a platform for simultaneous quantification of TCR distribution via total internal reflection fluorescence microscopy, of intracellular calcium levels, and of T-cell-exerted forces via atomic force microscopy (AFM). In our method, AFM cantilevers were used to bring single T-cells into contact with the activating surface. We designed the platform specifically to enable the study of T-cell triggering via functionalized fluid-supported lipid bilayers, which represent a widely accepted model system to stimulate T-cells in an antigen-specific manner. In this paper, we showcase the possibilities of this platform using primary transgenic T-cells triggered specifically via their cognate antigen presented by MHCII. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Early T Cell Signaling)

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Review

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13 pages, 946 KiB

Open AccessReview

Quantitative Methodologies to Dissect Immune Cell Mechanobiology

by Veronika Pfannenstill, Aurélien Barbotin, Huw Colin-York and Marco Fritzsche

Cells 2021, 10(4), 851; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10040851 - 9 Apr 2021

Cited by 2 | Viewed by 4242

Abstract

Mechanobiology seeks to understand how cells integrate their biomechanics into their function and behavior. Unravelling the mechanisms underlying these mechanobiological processes is particularly important for immune cells in the context of the dynamic and complex tissue microenvironment. However, it remains largely unknown how [...] Read more.

Mechanobiology seeks to understand how cells integrate their biomechanics into their function and behavior. Unravelling the mechanisms underlying these mechanobiological processes is particularly important for immune cells in the context of the dynamic and complex tissue microenvironment. However, it remains largely unknown how cellular mechanical force generation and mechanical properties are regulated and integrated by immune cells, primarily due to a profound lack of technologies with sufficient sensitivity to quantify immune cell mechanics. In this review, we discuss the biological significance of mechanics for immune cells across length and time scales, and highlight several experimental methodologies for quantifying the mechanics of immune cells. Finally, we discuss the importance of quantifying the appropriate mechanical readout to accelerate insights into the mechanobiology of the immune response. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Early T Cell Signaling)

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