The role of positive and negative regulation on ligand discrimination by the TCR signaling pathway

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 and its dependence on TCR-pMHC
binding dwell time, and the role of LAT condensation in mediating positive feedback regulation via SOS-catalyzed
Ras activation. The work proposed in this project bridges studies to be conducted in Projects 1, 3 and 4.

Grant Number: 5P01AI091580-14
NIH Institute/Center: NIH
Principal Investigator: Arup Chakraborty