20 research outputs found
A unifying mathematical framework for experimental TCR-pMHC kinetic constants
Receptor binding and triggering are central in Immunology as T cells activated through their T cell receptors (TCR) by protein antigens orchestrate immune responses. In order to understand receptor-ligand interactions, many groups working with different experimental techniques and assays have generated a vast body of knowledge during the last decades. However, in recent years a type of assays, referred to as two-dimensional or membrane-to-membrane, has questioned our current understanding of the role of different kinetic constants (for instance, on- versus off-rate constants) on TCR-ligand interaction and subsequent T cell activation. Here we present a general mathematical framework that provides a unifying umbrella to relate fundamental and effective (or experimentally determined) kinetic constants, as well as describe and compare state-of-the-art experimental methods. Our framework is able to predict the correlations between functional output, such as 1/EC50, and effective kinetic constants for a range of different experimental assays (in two and three dimensions). Furthermore, our approach can be applied beyond Immunology, and serve as a âtranslation methodâ for the biochemical characterization of receptor-ligand interactions
Measuring diffusion and binding kinetics by contact area FRAP.
The immunological synapse is a stable intercellular structure that specializes in substance and signal transfer from one immune cell to another. Its formation is regulated in part by the diffusion of adhesion and signaling molecules into, and their binding of countermolecules in the contact area. The stability of immunological synapses allows receptor-ligand interactions to approximate chemical equilibrium despite other dynamic aspects. We have developed a mathematical model that describes the coupled reaction-diffusion process in an established immunological synapse. In this study, we extend a previously described contact area fluorescence recovery after photobleaching (FRAP) experiment to test the validity of the model. The receptor binding activity and lateral mobility of fluorescently labeled, lipid-anchored ligands in the bilayer resulted in their accumulation, as revealed by a much higher fluorescence intensity inside the contact area than outside. After complete photobleaching of the synapse, fluorescence recovery requires ligands to dissociate and rebind, and to diffuse in and out of the contact area. Such a FRAP time course consequently provides information on reaction and diffusion, which can be extracted by fitting the model solution to the data. Surprisingly, reverse rates in the two-dimensional contact area were at least 100-fold slower than in three-dimensional solution. As previously reported in immunological synapses, a significant nonrecoverable fraction of fluorescence was observed with one of two systems studied, suggesting some ligands either dissociated or diffused much more slowly compared with other ligands in the same synapse. The combined theory and experiment thus provides a new method for in situ measurements of kinetic rates, diffusion coefficients, and nonrecoverable fractions of interacting molecules in immunological synapses and other stable cell-bilayer junctions
Ligand engaged TCR is triggered by active Lck not associated with coreceptor
The earliest molecular events in T cell recognition have not yet been fully described, and
the initial TCR triggering mechanism remains a subject of controversy. Using
TIRF/FRET microscopy we observed a two-stage interaction between TCR, CD8, and
MHCp. There is an early (within seconds) interaction between CD3ζ and the coreceptor
CD8 that is independent of the binding of CD8 to MHC, but that requires CD8
association with Lck. Later (several minutes) CD3ζ-CD8 interactions require CD8-MHC
binding. Lck can be found free or bound to the coreceptor. In mature CD8 T cells we find
that the activated population of Lck is not coreceptor-associated. This work indicates that
the initial TCR triggering event is induced by free Lck
Compositional modelling of immune response and virus transmission dynamics
Transmission models for infectious diseases are typically formulated in terms of dynamics between individuals or groups with processes such as disease progression or recovery for each individual captured phenomenologically, without reference to underlying biological processes. Furthermore, the construction of these models is often monolithic: they do not allow one to readily modify the processes involved or include the new ones, or to combine models at different scales. We show how to construct a simple model of immune response to a respiratory virus and a model of transmission using an easily modifiable set of rules allowing further refining and merging the two models together. The immune response model reproduces the expected response curve of PCR testing for COVID-19 and implies a long-tailed distribution of infectiousness reflective of individual heterogeneity. This immune response model, when combined with a transmission model, reproduces the previously reported shift in the population distribution of viral loads along an epidemic trajectory. This article is part of the theme issue âTechnical challenges of modelling real-life epidemics and examples of overcoming theseâ
Flow-enhanced adhesion regulated by a selectin interdomain hinge
© 2006 The AuthorsThe electronic version of this article is the complete one and can be found online at: http://www.jcb.org/cgi/doi/10.1083/jcb.200606056DOI: 10.1083/jcb.200606056L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs fl ow-enhanced rolling by prolonging the lifetimes of L-selectinâligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the fl exibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation
Memory in receptorâligand-mediated cell adhesion
Single-molecule biomechanical measurements, such as the force to unfold a protein domain or the lifetime of a receptorâligand bond, are inherently stochastic, thereby requiring a large number of data for statistical analysis. Sequentially repeated tests are generally used to obtain a data ensemble, implicitly assuming that the test sequence consists of independent and identically distributed (i.i.d.) random variables, i.e., a Bernoulli sequence. We tested this assumption by using data from the micropipette adhesion frequency assay that generates sequences of two random outcomes: adhesion and no adhesion. Analysis of distributions of consecutive adhesion events revealed violation of the i.i.d. assumption, depending on the receptorâligand systems studied. These include Markov sequences with positive (T cell receptor interacting with antigen peptide bound to a major histocompatibility complex) or negative (homotypic interaction between C-cadherins) feedbacks, where adhesion probability in the next test was increased or decreased, respectively, by adhesion in the immediate past test. These molecular interactions mediate cell adhesion and cell signaling. The ability to ârememberâ the previous adhesion event may represent a mechanism by which the cell regulates adhesion and signaling
Vaccination reshapes the virus-specific T cell repertoire in unexposed adults
We examined how baseline CD4+ TÂ cell repertoire and precursor states impact responses to pathogen infection in humans using primary immunization with yellow fever virus (YFV) vaccine. YFV-specific TÂ cells in unexposed individuals were identified by peptide-MHC tetramer staining and tracked pre- and post-vaccination by tetramers and TCR sequencing. A substantial number of YFV-reactive TÂ cells expressed memory phenotype markers and contained expanded clones in the absence of exposure to YFV. After vaccination, pre-existing YFV-specific TÂ cell populations with low clonal diversity underwent limited expansion, but rare populations with a reservoir of unexpanded TCRs generated robust responses. These altered dynamics reorganized the immunodominance hierarchy and resulted in an overall increase in higher avidity TÂ cells. Thus, instead of further increasing the representation of dominant clones, YFV vaccination recruits rare and more responsive TÂ cells. Our findings illustrate the impact of vaccines in prioritizing TÂ cell responses and reveal repertoire reorganization as a key component of effective vaccination