Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαβ transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic ...tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRβ transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction.
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•TCRs can spontaneously switch from the resting to the active conformation.•Cholesterol binding to TCRβ prevents the switching to the active conformation.•Only in the active conformation can TCRs be phosphorylated.•The active conformation can signal without antigen.
The TCR can adopt an inactive, resting or an active, primed state. Schamel and colleagues show that the TCR is in equilibrium between these states. Peptide-MHC binding stabilizes the primed state that can be phosphorylated. Cholesterol binding stabilizes the resting state and thereby tunes the TCR activation threshold.
Most adaptive immune responses require the activation of specific T cells through the T cell antigen receptor (TCR)-CD3 complex. Here we show that cholesterol sulfate (CS), a naturally occurring ...analog of cholesterol, inhibits CD3 ITAM phosphorylation, a crucial first step in T cell activation. In biochemical studies, CS disrupted TCR multimers, apparently by displacing cholesterol, which is known to bind TCRβ. Moreover, CS-deficient mice showed heightened sensitivity to a self-antigen, whereas increasing CS content by intrathymic injection inhibited thymic selection, indicating that this molecule is an intrinsic regulator of thymocyte development. These results reveal a regulatory role for CS in TCR signaling and thymic selection, highlighting the importance of the membrane microenvironment in modulating cell surface receptor activation.
The Allostery Model of TCR Regulation Schamel, Wolfgang W A; Alarcon, Balbino; Höfer, Thomas ...
The Journal of immunology (1950),
01/2017, Volume:
198, Issue:
1
Journal Article
Peer reviewed
Open access
The activity of the αβ TCR is controlled by conformational switches. In the resting conformation, the TCR is not phosphorylated and is inactive. Binding of multivalent peptide-MHC to the TCR ...stabilizes the active conformation, leading to TCR signaling. These two conformations allow the TCRs to be allosterically regulated. We review recent data on heterotropic allostery where peptide-MHC and membrane cholesterol serve opposing functions as positive and negative allosteric regulators, respectively. In resting T cells cholesterol keeps TCRs in the resting conformation that otherwise would become spontaneously active. This regulation is well described by the classical Monod-Wyman-Changeux model of allostery. Moreover, the observation that TCRs assemble into nanoclusters might allow for homotropic allostery, in which individual TCRs could positively cooperate and thus enhance the sensitivity of T cell activation. This new view of TCR regulation will contribute to a better understanding of TCR functioning.
Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded ...photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelength‐specific, and dose‐ and space‐controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell‐compatible red/far‐red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics‐inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots.
An artificial extracellular matrix is described whose viscoelastic properties can be adjusted in a fully reversible manner by light in the red spectrum. The matrix relies on the light‐dependent dimerization of the cyanobacterial red light receptor Cph1. The matrix is applied to analyze how human mesenchymal stem cell mechanosignaling and T lymphocyte migration is affected by dynamic mechanical environments.
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.
Although memory T cells respond more vigorously to stimulation and they are more sensitive to low doses of antigen than naive T cells, the molecular basis of this increased sensitivity remains ...unclear. We have previously shown that the T cell receptor (TCR) exists as different-sized oligomers on the surface of resting T cells and that large oligomers are preferentially activated in response to low antigen doses. Through biochemistry and electron microscopy, we now showed that previously stimulated and memory T cells have more and larger TCR oligomers at the cell surface than their naive counterparts. Reconstitution of cells and mice with a point mutant of the CD3ζ subunit, which impairs TCR oligomer formation, demonstrated that the increased size of TCR oligomers was directly responsible for the increased sensitivity of antigen-experienced T cells. Thus, we propose that an “avidity maturation” mechanism underlies T cell antigenic memory.
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► Previously stimulated and memory T cells are enriched for oligomeric TCR complexes ► Oligomeric TCR complexes enrichment correlates with increased antigen sensitivity ► Genetic impairment of oligomeric TCRs formation reduces antigen sensitivity ► Oligomeric TCR formation depends on the transmembrane region of CD3?
Antigen binding to the B-cell antigen receptor (BCR) leads to receptor triggering and B-lymphocyte activation. Here, we have probed the molecular requirements for BCR triggering in primary murine B ...cells using a set of defined soluble haptenated peptides. Bi- and trivalent haptens activated the BCR, as measured by protein phosphorylation, Ca2+ influx, BCR down-modulation and CD69, CD86 and MHC class II up-regulation. In contrast, four distinct monovalent haptens were ineffective. Next, we used two different anti-idiotypic antibodies, which bind to the antigen-combining site of the BCR. Again, monovalent Fab fragments were ineffective, whereas bivalent antibodies could stimulate the BCR. These findings are compatible with ligand-induced clustering of monomeric BCRs or re-organization of BCR complexes within pre-formed BCR oligomers. Lastly, an increase in the valency of the haptenated peptides improved the activation potential, whereas variations in the distance between two haptens had no effect. This finding contributes to understand how the immune system can efficiently recognize structurally diverse antigens but still discriminate between foreign and self.
Summary
Despite the low affinity of the T‐cell antigen receptor (TCR) for its peptide/major histocompatibility complex (pMHC) ligand, T cells are very sensitive to their antigens. This paradox can be ...resolved if we consider that the TCR may be organized into pre‐existing oligomers or nanoclusters. Such structures could improve antigen recognition by increasing the functional affinity (avidity) of the TCR–pMHC interaction and by allowing cooperativity between individual TCRs. Up to approximately 20 TCRs become tightly apposed in these nanoclusters, often in a linear manner, and such structures could reflect a relatively generalized phenomenon: the non‐random concentration of membrane receptors in specific areas of the plasma membrane known as protein islands. The association of TCRs into nanoclusters can explain the enhanced kinetics of the pMHC–TCR interaction in two dimensional versus three dimensional systems, but also their existence calls for a revision of the TCR triggering models based on pMHC‐induced TCR clustering. Interestingly, the B‐cell receptor and the FcεRI have also been shown to form nanoclusters, suggesting that the formation of pre‐existing receptor oligomers could be widely used in the immune system.
Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. ...Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch ('Blue-OFF'), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering.
The ratio between κ and λ light chain (LC)-expressing B cells varies considerably between species. We recently identified Kinase D-interacting substrate of 220 kDa (Kidins220) as an interaction ...partner of the BCR.
ablation of Kidins220 in B cells resulted in a marked reduction of λLC-expressing B cells. Kidins220 knockout B cells fail to open and recombine the genes of the
locus, even in genetic scenarios where the
genes cannot be rearranged or where the κLC confers autoreactivity.
gene recombination and expression in Kidins220-deficient B cells is normal. Kidins220 regulates the development of λLC B cells by enhancing the survival of developing B cells and thereby extending the time-window in which the
locus opens and the genes are rearranged and transcribed. Further, our data suggest that Kidins220 guarantees optimal pre-BCR and BCR signaling to induce
locus opening and gene recombination during B cell development and receptor editing.
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