ABSTRACT - PROJECT 2 T lymphocytes (T cells) play a key role in orchestrating an adaptive immune response to infectious pathogens as well as cancer cells. T cells express T cell antigen receptors (TCRs) that respond specifically to MHC- associated antigenic peptides (pMHC) derived from pathogens or mutant self-proteins of cancer cells. Upon TCR engagement with such agonist pMHC, intracellular signaling ensues, ultimately leading to new gene transcription programs required for T cell activation. In the homeostatic state in vivo, naïve T cells require shorter duration TCR engagement with self-pMHC to produce tonic signaling events that are required for their survival and homoeostasis. However, these tonic signals do not lead to cell activation as that would result in immunopathology. The half-life differences between ligands that induce tonic signals and agonists are not large. Kinetic proofreading is considered to be the conceptual framework for understanding the fine specificity with which the TCR signaling pathway discriminates between ligands. In spite of much progress in understanding membrane-proximal TCR signaling and ligand discrimination, how the tonic survival signals qualitatively or quantitatively differ from activation signals is not completely known. Based on preliminary data, we hypothesize that TCR signaling events resulting from interactions with self-pMHC and agonist-pMHC differ because of feedback regulatory mechanisms superimposed on kinetic proofreading both proximally and distally from the TCR. We propose to determine the mechanisms underlying such feedback regulation and their impact on ligand discrimination by bringing together computational modeling, biochemistry, mouse models, and single molecule experiments in live cells and reconstituted systems. We will focus on two specific aims. In Aim 1, we will define negative feedback loops and where they act to regulate ligand discrimination. Our preliminary modeling studies have predicted that that negative feedback, proximal but not distal to the TCR, is important for dampening noise and inappropriate responses to self-pMHC. We will explore the involvement of 3 proximal negative feedback loops. Synergistic computational and experimental studies are expected to identify the sources, nodes of action, and impact of these negative feedback loops on ligand discrimination. In Aim 2, we will determine the mechanisms underlying the formation of the LAT condensate and its role in positive regulatory feedback. Our modeling studies suggest that positive feedback regulation distal from the receptor, but still responsive to TCR-pMHC dwell time, is important for a robust response to stimulation by agonists. Our preliminary experimental data reveal that LAT, a key regulator of TCR signaling, forms discrete condensates in response to individual TCR-pMHC binding events. By combining statistical physics-based modeling with experiments, we will dissect the mechanism of LAT condensation nucleation ...