ABSTRACT – PROJECT 4 T cells demonstrate remarkable sensitivity to activating TCR signals triggered by agonist pMHC recognition. These signals elicit T cell activation as well as differentiation in effector T cell subsets to coordinate immune protection to different pathogens, but the molecular basis for specific and dependable discrimination and mechanistic regulation of T cell commitment (kinetic proofreading) are not understood. How the discrimination between activating TCR signals triggered by agonist pMHC recognition versus self-pMHC encounters occurs at a mechanistic level will be investigated in Projects 1-3, with focus on the proximal kinase network, proofreading mechanisms through feedback loops, and LAT condensation characteristics. How the TCR pathway converts incoming signals as a function of agonist pMHC recognition or self-pMHC encounters and processes this information into instructive Effector Kinase Signals that control T cell activation and differentiation will be investigated in Project 4. In Project 4, we will test our hypothesis that - downstream of the LAT signalosome - Effector Kinase Signals triggered by two Ras Guanine nucleotide Exchange Factors (RasGEFs), SOS1 and RasGRP1 regulate controlled and balanced T cell activation and differentiation. Specifically, we propose that agonist pMHC recognition triggers the collective output of multiple RasGEF-Effector Kinase Signals that drive T cell activation and proliferation in a failsafe and efficient manner. Unique nuances in the RasGEF-Effector Kinase effector signals further influence development into effector T cell subsets, like regulatory T cells. By contrast, we propose that contacts with self-pMHC do not trigger the same RasGEF signals, but instead, convert self-pMHC induced sub-threshold, tonic signals into unique RasGEF signals that promote T cell survival and homeostasis. In Aim 1, we will Investigate RasGEF substrates and partner molecules in canonical and non-canonical pathways. In Aim 2, we will assess tonic RasGRP1-mTOR signals and in vivo relevance in T cells. In Aim 3, we will establish the functional significance of digital SOS1-Ras-ERK signaling in T cells and regulated immunity. In Aim 4, we will decipher non-canonical SOS1-P38 signaling in regulatory T cells and tumor immunity. Our past and ongoing, collaborative efforts and seminal discoveries on two Ras Guanine nucleotide Exchange Factors (RasGEFs), SOS and RasGRP, their canonical signals to Ras/MAPK pathways, their unexpected non-canonical signals to mTOR and P38- kinase pathways, and innovative technologies form the foundation for the four Aims of Project 4 in this revised P01 renewal. The Kuriyan-, Groves-, Salomon-, and Roose- teams are poised to address the major questions in T cell biology described in Project 4. Together, these studies will provide critical results how the TCR pathway converts incoming signals (Projects 1-3) into instructive signals (Project 4) that control T cell activation and d...