High-speed measurements of TCR-proximal signaling [electronic resource] : implications for ligand discrimination

In chapter 1 of this dissertation, we review the role of T cells in the immune system, and describe the signaling pathways triggered by the T cell receptor for antigen (TCR). In chapter 2 we discuss the process of ligand discrimination by the TCR and we consider several different theoretical frameworks that have been proposed to explain the high degree of specificity and sensitivity of this process. In particular, we focus on the kinetic proofreading model (McKeithan, 1995). We outline some of the predictions of this model that are supported by experimental evidence in the literature. In chapter 3 we consider a set of quantitative predictions of the proofreading model that have been difficult to test experimentally. We show that the mathematical description of the kinetic proofreading model predicts that the signaling onset delay will be only weakly dependent on the density of peptide-MHC (pMHC) and on the off-rate of the pMHC as long three conditions are met: 1) the proofreading step, and not the ligand binding step is rate-limiting, 2) the threshold number of fully modified receptors required to initiate signaling is low, and 3) the number of proofreading steps, n, is not trivially small (n> 2). We review experimental data that suggest that TCR signaling meets conditions 1 and 2. We then consider the predictions of proofreading schemes that meet condition 3. In chapter 4, we describe the novel experimental methods we used to test the quantitative predictions of the proofreading model regarding signaling kinetics. This is an area that has received comparably little experimental examination, in large part due to the difficulty of precisely controlling the timing of the exposure of T cells to their physiological ligand. To solve this problem, we use the recently-developed peptide photocaging technology (DeMond et al., 2006; Huse et al., 2007) to precisely initiate the exposure of T cells to peptide-MHC. We then measure the kinetics of signaling elicited by agonists of different potency. In chapter 5, we describe the results of a series of these experiments, and compare these results to the behavior predicted by the mathematical description of the proofreading model. We find that in agreement with the predictions of the model, the median onset delays for three different signaling processes are not significantly different when triggered by a strong agonist, MCC, or a weak agonist 102S. These experiments were carried out under conditions where the weak agonist triggers a response in a significantly smaller fraction of cells than the strong agonist, indicating that the signaling mechanism can discriminate between the two pMHCs at this ligand density. Finally we find that the magnitude of the signaling processes elicited by 102S relative to MCC is lower in the more TCR distal step than in the TCR proximal steps. This result is also consistent with the predictions of the kinetic proofreading model. In chapter 6 we discuss the implications of these results. We argue that, taken together, our data are consistent with the predictions of the kinetic proofreading model. Furthermore, the observed onset delay times (2 to 8 seconds), for the most TCR-proximal steps examined, would allow reasonable discrimination between MCC and 102S, based on the measured half-lives of interaction between the 5C.C7 TCR and these peptides in complex with the class II MHC molecule I-Ek. For the reminder of chapter 6 we discuss the differences between the proofreading scheme and other models for ligand discrimination that have been proposed. One of the most important features of the kinetic proofreading model is that a ligated receptor does not "know" which type of ligand it has bound. An individual receptor progresses through the proofreading process at one constant rate as long as it remains ligated, regardless of whether it has bound an agonist, antagonist, or null peptide. This is in contrast to all models in which the rate of signaling is dependent on the quality of the bound ligand. This type of mechanism could occur in a system where the receptor directly reads out ligand quality by a conformational change that is elicited to different extents by different ligands, independent of ligand affinity, as has been reported for some G-protein-coupled receptors (Vilardaga et al., 2003) .. Our data do not support a model in which the rate of signal transduction is strongly dependent on the quality of the bound ligand. These results will help to inform and constrain future quantitative models of the TCR ligand discrimination process. Lastly, in chapter 7, we present some of the experimental approaches that were less successful, but which might still yield important insights in the future.

Advisor: Davis Mark M. primary advisor. Thesis advisor
Crabtree Gerald R. advisor. Thesis advisor
Garcia K. Christopher advisor. Thesis advisor
Meyer Tobias advisor. Thesis advisor