scholarly journals Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor

2018 ◽  
Author(s):  
O. Sascha Yousefi ◽  
Matthias Günther ◽  
Maximilian Hörner ◽  
Julia Chalupsky ◽  
Maximilian Wess ◽  
...  
Keyword(s):  
T Cell ◽  
Ligand Binding ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Half Life ◽  
Cell Activation ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Experimental Systems ◽  
Kinetic Proofreading

AbstractThe pivotal task of the immune system is to distinguish between self and foreign antigens. The kinetic proofreading model (KPR) proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the ligand-TCR interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B to selectively control the dynamics of ligand binding to the TCR by light. Combining experiments with mathematical modeling we find that the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating the KPR hypothesis.One Sentence SummaryThe half-life of the ligand-T cell receptor complex determines T cell activation.

scholarly journals Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
O Sascha Yousefi ◽  
Matthias Günther ◽  
Maximilian Hörner ◽  
Julia Chalupsky ◽  
Maximilian Wess ◽  
...  
Keyword(s):  
T Cell ◽  
Ligand Binding ◽  
T Cell Receptor ◽  
Red Light ◽  
Cell Receptor ◽  
Unique Property ◽  
Phytochrome B ◽  
Tcr Signaling ◽  
Experimental Systems ◽  
Kinetic Proofreading

The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.

scholarly journals CD8β Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56lck Complexes

2001 ◽  
Vol 194 (10) ◽  
pp. 1485-1495 ◽  
Author(s):  
Alexandre Arcaro ◽  
Claude Grégoire ◽  
Talitha R. Bakker ◽  
Lucia Baldi ◽  
Martin Jordan ◽  
...  
Keyword(s):  
T Cell ◽  
Ligand Binding ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cell Receptor ◽  
High Affinity ◽  
Histocompatibility Complex ◽  
Peptide Derivative

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8β chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8αβ, but not CD8αα or soluble CD8αβ, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8β endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8β constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2Kd, and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8β, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56lck. In addition, the cytoplasmic portion of CD8β mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8αβ partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56lck in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

scholarly journals Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells

2017 ◽  
Vol 114 (30) ◽  
pp. E6117-E6126 ◽  
Author(s):  
Thomas C. J. Tan ◽  
John Knight ◽  
Thomas Sbarrato ◽  
Kate Dudek ◽  
Anne E. Willis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Ribosome Biogenesis ◽  
Cell Activation ◽  
Mrna Translation ◽  
Protein Translation ◽  
Cell Receptor ◽  
Tcr Signaling

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

scholarly journals Light-based tuning of ligand half-life supports kinetic proofreading model of T cell activation

2018 ◽  
Author(s):  
Doug Tischer ◽  
Orion D. Weiner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ligand Binding ◽  
T Cell Activation ◽  
Half Life ◽  
Cell Activation ◽  
Receptor Occupancy ◽  
Biophysical Parameters ◽  
Ligand Interaction ◽  
Kinetic Proofreading

AbstractT cells are thought to discriminate stimulatory versus non-stimulatory ligands by converting small changes in ligand binding half-life to large changes in cell activation. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, most notably the stability of the receptor-ligand interaction under load. Here we develop an optogenetic approach to specifically tune the binding half-life of a light-responsive ligand to a chimeric antigen receptor without changing other binding parameters. By simultaneously manipulating binding half-life while controlling for receptor occupancy, we find that signaling is strongly gated by ligand binding half-life. Our results provide direct evidence of kinetic proofreading in ligand discrimination by T cells.One Sentence SummaryDirect control of ligand binding half-life with light shows that lifetime, not occupancy, dominates T cell activation.

scholarly journals Dynamic palmitoylation events following T-cell receptor signaling

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Eliot Morrison ◽  
Tatjana Wegner ◽  
Andres Ernesto Zucchetti ◽  
Miguel Álvaro-Benito ◽  
Ashley Zheng ◽  
...  
Keyword(s):  
Signal Transduction ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Receptor Signaling ◽  
Tcr Signaling ◽  
T Cell Receptor Signaling ◽  
Cell Receptor Signaling

AbstractPalmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. The reversible nature of this modification makes it a prime candidate as a mechanism for regulating signal transduction in T-cell receptor signaling. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi and to localize to the immune synapse upon T cell activation.

scholarly journals Clustering of the ζ-Chain Can Initiate T Cell Receptor Signaling

2020 ◽  
Vol 21 (10) ◽  
pp. 3498 ◽  
Author(s):  
Yuanqing Ma ◽  
Yean J. Lim ◽  
Aleš Benda ◽  
Jieqiong Lou ◽  
Jesse Goyette ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Spatial Clustering ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
Tcr Signaling ◽  
T Cell Receptor Signaling ◽  
Cell Cytosol

T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

scholarly journals Sts-2 Is a Phosphatase That Negatively Regulates Zeta-associated Protein (ZAP)-70 and T Cell Receptor Signaling Pathways

2011 ◽  
Vol 286 (18) ◽  
pp. 15943-15954 ◽  
Author(s):  
Boris San Luis ◽  
Ben Sondgeroth ◽  
Nicolas Nassar ◽  
Nick Carpino
Keyword(s):  
T Cell ◽  
Signaling Pathways ◽  
Phosphatase Activity ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Homologous Proteins

T cell activity is controlled in large part by the T cell receptor (TCR). The TCR detects the presence of foreign pathogens and activates the T cell-mediated immune reaction. Numerous intracellular signaling pathways downstream of the TCR are involved in the process of T cell activation. Negative regulation of these pathways helps prevent excessive and deleterious T cell responses. Two homologous proteins, Sts-1 and Sts-2, have been shown to function as critical negative regulators of TCR signaling. The phosphoglycerate mutase-like domain of Sts-1 (Sts-1PGM) has a potent phosphatase activity that contributes to the suppression of TCR signaling. The function of Sts-2PGM as a phosphatase has been less clear, principally because its intrinsic enzyme activity has been difficult to detect. Here, we demonstrate that Sts-2 regulates the level of tyrosine phosphorylation on targets within T cells, among them the critical T cell tyrosine kinase Zap-70. Utilizing new phosphorylated substrates, we demonstrate that Sts-2PGM has clear, albeit weak, phosphatase activity. We further pinpoint Sts-2 residues Glu-481, Ser-552, and Ser-582 as specificity determinants, in that an Sts-2PGM triple mutant in which these three amino acids are altered to their counterparts in Sts-1PGM has substantially increased activity. Our results suggest that the phosphatase activities of both suppressor of TCR signaling homologues cooperate in a similar but independent fashion to help set the threshold for TCR-induced T cell activation.

scholarly journals Recruitment of Slp-76 to the Membrane and Glycolipid-Enriched Membrane Microdomains Replaces the Requirement for Linker for Activation of T Cells in T Cell Receptor Signaling

2000 ◽  
Vol 192 (7) ◽  
pp. 1047-1058 ◽  
Author(s):  
Nancy J. Boerth ◽  
Jeffrey J. Sadler ◽  
Daniel E. Bauer ◽  
James L. Clements ◽  
Shereen M. Gheith ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Promoter Activity ◽  
Cell Activation ◽  
Cell Receptor ◽  
Membrane Microdomains ◽  
Tcr Signaling

Two hematopoietic-specific adapters, src homology 2 domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224–244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cγ1 phosphorylation, extracellular signal–regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224–244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.

scholarly journals Clustering of CD3ζ is sufficient to initiate T cell receptor signaling

2020 ◽  
Author(s):  
Yuanqing Ma ◽  
Yean J Lim ◽  
Aleš Benda ◽  
Jesse Goyette ◽  
Katharina Gaus
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Spatial Clustering ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
Tcr Signaling ◽  
Biophysical Mechanism ◽  
Cell Cytosol

AbstractT cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of CD3ζ chains in a light controlled manner. We showed that spatial clustering of the CD3ζ intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of CD3ζ, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate CD3ζ phosphorylation upon CD3ζ clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed CD3ζ clusters at the plasma membrane. Taken together, our data suggest that clustering of the TCR can initialize proximal TCR signaling and thus constitute a biophysical mechanism of TCR triggering.

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