Ligand-engaged TCR is triggered by Lck not associated with CD8 coreceptor

Casas, Javier; Brzostek, Joanna; Zarnitsyna, Veronika I.; Hong, Jinsung; Wei, Qianru; Hoerter, John A. H.; Fu, Guo; Ampudia, Jeanette; Zamoyska, Rose; Zhu, Cheng; Gascoigne, Nicholas R. J.

doi:10.1038/ncomms6624

Cited by 73 publications

(94 citation statements)

References 51 publications

(92 reference statements)

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“…Interestingly, although we found that pZap70 and pLat had a large fraction of molecules colocalized with CD3ζ, relatively few Lck molecules colocalized with CD3ζ (Fig. 4C), suggesting that transient Lck-CD3ζ interactions may be sufficient to initiate signaling at TCR signaling "hot spots" (26,38). More CD3ζ molecules colocalized with Lck at 5 min than at later time points (Fig.…”

Section: Tcr Engagement Leads To Receptor Clustering Independently Ofmentioning

confidence: 89%

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Pageon

Tabarin

Yamamoto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

201

235

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Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

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Section: Tcr Engagement Leads To Receptor Clustering Independently Ofmentioning

confidence: 89%

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Pageon

Tabarin

Yamamoto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

201

235

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“…The P a curve measured over a range of t c exhibits a two-stage pattern (Fig. 2C, red), similar to those observed in the studies of TCR/CD8 crosstalk [18][19][20] . Like the previous studies, this two-stage curve cannot be obtained by superposition of two single-stage curves each measured using the same batch of T cells contacted by RBCs coated with only one ligand species (Fig.…”

supporting

confidence: 61%

“…Interactions of two ligands (IgG1 and IgG2) to a common receptor (Fcγ receptor III B, FcγRIIIB) 16 or of two receptors (FcγRIIA and FcγRIIIB) to a common ligand (total IgG) 17 have been found to be concurrent and independent when the bond numbers are low enough for competition to be neglected, resulting in a total bond number that is equal to the sum of two bond species. By comparison, binding of a divalent ligand, pMHC or Thy-1, to the corresponding dual receptors, T cell receptor (TCR) and coreceptor CD8 [18][19][20] or integrin α 5 β 1 and Sydecan-4 21 , is cooperative and synergistic, resulting in an additional trimolecular bond species of greater number and longer duration than the sum of two bimolecular bond species separately formed between the ligand and the two receptors. Interestingly, the formation of the (TCR/CD8)-pMHC trimolecular bond exhibits a time delay and requires intracellular signaling via Src family kinases [18][19][20] whereas that of the (α 5 β 1 /Sydecan-4)-Thy-1 bond is elicited by pulling forces 21 .…”

mentioning

confidence: 99%

“…By comparison, binding of a divalent ligand, pMHC or Thy-1, to the corresponding dual receptors, T cell receptor (TCR) and coreceptor CD8 [18][19][20] or integrin α 5 β 1 and Sydecan-4 21 , is cooperative and synergistic, resulting in an additional trimolecular bond species of greater number and longer duration than the sum of two bimolecular bond species separately formed between the ligand and the two receptors. Interestingly, the formation of the (TCR/CD8)-pMHC trimolecular bond exhibits a time delay and requires intracellular signaling via Src family kinases [18][19][20] whereas that of the (α 5 β 1 /Sydecan-4)-Thy-1 bond is elicited by pulling forces 21 . The mechanically based methods have also been combined with fluorescence imaging to enable concurrent observations of receptor-ligand binding and intracellular signaling so induced, to allow for correlative analysis over time at the single-cell level [22][23][24] .…”

mentioning

confidence: 99%

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Dual Biomembrane Force Probe Enables Single-Cell Mechanical Analysis of Signal Crosstalk between Multiple Molecular Species

Chen

et al. 2018

Biophysical Journal

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Conventional approaches for studying receptor-mediated cell signaling, such as the western blot and flow cytometry, are limited in three aspects: 1) The perturbing preparation procedures often alter the molecules from their native state on the cell; 2) Long processing time before the final readout makes it difficult to capture transient signaling events (<1 min); 3) The experimental environments are force-free, therefore unable to visualize mechanical signals in real time. In contrast to these methods in biochemistry and cell biology that are usually population-averaged and non-real-time, here we introduce a novel single-cell based nanotool termed dual biomembrane force probe (dBFP). The dBFP provides precise controls and quantitative readouts in both mechanical and chemical terms, which is particularly suited for juxtacrine signaling and mechanosensing studies. Specifically, the dBFP allows us to analyze dual receptor crosstalk by quantifying the spatiotemporal requirements and functional consequences of the up-and down-stream signaling events. In this work, the utility and power of the dBFP has been demonstrated in four important dual receptor systems that play key roles in immunological synapse formation, shear-dependent thrombus formation, and agonist-driven blood clotting.Over the past decade, single-molecule biomechanical analyses on single cells have enabled studies of the inner workings of adhesion and signaling receptors one at a time 1 . In physiological conditions, however, multiple receptor species are present and often work cooperatively rather than independently. Understanding how multiple receptor species crosstalk to each other is essential for determining the mechanisms underlying a wide range of biological processes related to human health and diseases. One of the model receptor systems that exhibit such signaling crosstalk with other receptors is the integrin family. For example, integrin α L β 2 , or lymphocyte function-associated antigen-1 (LFA-1), crosstalks with chemokine receptors and antigen receptors via an "inside-out" signaling process 2 , which is essential for lymphocyte trafficking and immunological synapse formation 3 . The interaction between LFA-1 on a lymphocyte and its ligand, intercellular adhesion molecule-1 (ICAM-1) on an adjacent cell, can be upregulated by signals induced by binding of chemokines and antigens to their respective receptors on the same lymphocyte, manifesting enhanced cell adhesion 4,5 . As another example, upon a rapid shear force increase caused by blood flow perturbations, the function of integrin α IIb β 3 , or glycoprotein (GP) IIb-IIIa, on a platelet surface is rapidly upregulated by signals induced by ligand engagement with the mechanoreceptor GPIb 6,7 , which promotes platelet adhesion at the site of vascular injury as part of the hemostatic and thrombotic processes 8 . The synergistic binding of GPIb and GPIIb-IIIa enables efficient platelet recruitment

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“…We further analyzed the spatial colocalization between tension, TCR, CD8 (coreceptor), and lymphocyte-specific kinase (Lck), which is essential for T-cell activation (29,30). In contrast to the tension signal near the cell edge, TCRs were distributed uniformly across the cell-substrate interface (Fig.…”

Section: Tcr-ligand Tension Requires Coreceptor Engagement and Is Modmentioning

confidence: 99%

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity

Liu

Blanchfield

Pui‐Yan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

278

347

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T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12–19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically “filtered” to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

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Ligand-engaged TCR is triggered by Lck not associated with CD8 coreceptor

Cited by 73 publications

References 51 publications

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Dual Biomembrane Force Probe Enables Single-Cell Mechanical Analysis of Signal Crosstalk between Multiple Molecular Species

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity

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