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

Yousefi, O. Sascha; Guenther, Matthias; Hörner, Maximilian; Chalupsky, Julia; Wess, Maximilian; Brandl, Simon M.; Smith, Robert W.; Fleck, Christian; Kunkel, Tim; Zurbriggen, Matías D.; Hoefer, Thomas; Weber, Wilfried; Schamel, Wolfgang W. A.

doi:10.1101/432740

Cited by 15 publications

(20 citation statements)

References 29 publications

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“…This is achieved by a few fractional steps (2.67) and a short proofreading time delay (2.8 s). This time delay is at the lower end of the range reported using soluble tetramers (8 s with 95 % CI: 3–19 s) (48) and consistent with the 4 s time delay between pMHC binding and LAT phosphorylation (49). The small number of steps is reasonable because, although the TCR undergoes a large number of biochemical modifications (2, 3), only steps that must be sequential contribute.…”

Section: Discussionsupporting

confidence: 80%

T cells exhibit unexpectedly low discriminatory power and can respond to ultra-low affinity peptide-MHC ligands

Pettmann

Abu-Shah

Kutuzov

et al. 2020

Preprint

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T cells use their T cell receptors (TCRs) to discriminate between peptide MHC (pMHC) ligands that bind with different affinities but precisely how different remains controversial. This is partly because the affinities of physiologically relevant interactions are often too weak to measure. Here, we introduce a surface plasmon resonance protocol to measure ultra-low TCR/pMHC affinities (KD ~ 1000 μM). Using naïve, memory, and blasted human CD8+ T cells we find that their discrimination power is unexpectedly low, in that they require a large >100-fold decrease in affinity to abolish responses. Interestingly, the discrimination power reduces further when antigen is presented in isolation on artificial surfaces but can be partially restored by adding ligands to CD2 or LFA-1. We were able to fit the kinetic proof-reading model to our data, yielding the first estimates for both the time delay (2.8 s) and number of biochemical steps (2.67). The fractional number of steps suggest that one of the proof-reading steps is not easily reversible.

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Section: Discussionsupporting

confidence: 80%

T cells exhibit unexpectedly low discriminatory power and can respond to ultra-low affinity peptide-MHC ligands

Pettmann

Abu-Shah

Kutuzov

et al. 2020

Preprint

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“…In T cells, binding of the TCR to peptides presented on major histocompatibility complexes (pMHCs) can induce phosphorylation of ITAMs by the Src-family kinase Lck leading to the recruitment and subsequent phosphorylation of ZAP70 at the TCR (9). The delay between pMHC binding and the activation of ZAP70 is thought to contribute to the kinetic proofreading chain that is critical for the ability of T cells to discriminate between short-lived self and longer-lived non-self pMHC interactions (19)(20)(21)(22)(23)(24)(25)(26). Critically, this mechanism requires that all signalling reactions within the chain are rapidly reversed upon pMHC unbinding so that short-lived self pMHC cannot exploit sustained TCR signals to short circuit the chain.…”

Section: Introductionmentioning

confidence: 99%

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

Goyette

Depoil

Yang

et al. 2020

Preprint

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Protein-protein binding domains are critical in signalling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain-containing proteins have tandem domains, which are thought to enhance binding affinity and specificity. However, a trade-off exists between long-lived binding and the ability to rapidly reverse signalling, which is a critical requirement of noise filtering mechanisms such as kinetic proofreading. Here, we use modelling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases when tandem domains bind by a kinetic, but not a static, avidity mode. We use surface plasmon resonance to show that ZAP70, a tandem SH2 domain-containing kinase, binds kinetically to biphosphorylated peptides from the T cell antigen receptor (TCR) and that the unbinding rate can be accelerated by the phosphatase CD45. An important functional prediction of regulated unbinding is that the intracellular ZAP70/TCR half-life in T cells will be correlated to the extracellular TCR/antigen half-life and we show that this is the case in both cell lines and primary T cells. The work highlights that binding by kinetic avidity breaks the trade-off between signal fidelity (requiring long half-life) and signal reversibility (requiring short half-life), which is a key requirement for T cell antigen discriminated mediated by kinetic proofreading. 1

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“…To overcome these experimental restrictions we have developed the opto-ligand-TCR system (Yousefi et al, 2019), by making use of the light-dependent interaction between phytochrome B (PhyB) and PhyB interacting factor (PIF) (Levskaya et al, 2009;Toettcher et al, 2013;Kolar and Weber, 2017).…”

Section: Controlmentioning

confidence: 99%

Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System

Yousefi¹,

Hörner²,

Wess³

et al. 2020

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T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a light-regulated TCR ligand. PhyB can be photoconverted between a binding (ON) and non-binding (OFF) conformation by 660 nm and 740 nm light illumination, respectively. PhyB ON is able to bind to a synthetic TCR, generated by fusing the PhyB interacting factor (PIF) to the TCRβ chain. Switching PhyB to the OFF conformation disrupts this interaction. Sufficiently long binding of PhyB tetramers to the PIF-TCR led to T cell activation as measured by calcium influx. Here, we describe protocols for how to generate the tetrameric ligand for our opto-ligand-TCR system, how to measure ligand-TCR binding by flow cytometry and how to quantify T cell activation via calcium influx.

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

Cited by 15 publications

References 29 publications

T cells exhibit unexpectedly low discriminatory power and can respond to ultra-low affinity peptide-MHC ligands

T cells exhibit unexpectedly low discriminatory power and can respond to ultra-low affinity peptide-MHC ligands

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System

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