OVERALL SUMMARY T lymphocytes utilized T cell receptors (TCRs) to distinguish self versus non-self through recognition of sparse antigenic peptides bound to MHC molecules (pMHC) arrayed on antigen presenting cells (APC). Through remarkable specificity and digital sensitivity, T lymphocytes can destroy host cells altered by viruses, other infectious pathogens or cancerous transformations while leaving normal cellular counterparts intact. Until recently, it was unclear how TCR discrimination was achieved, given a lack of somatic mutations of TCR genes to boost receptor-ligand affinity unlike with B cell receptors. Contrary to conventional ligand associations exemplified by antigen-antibody interactions, however, it is now evident that physical force plays a crucial role in non-equilibrium TCR-based T cell activation. Here we investigate the overarching hypothesis that lineage receptors that recognize pMHC ligands, namely TCRs and preTCRs, function as mechanosensors, transducing biomechanical forces to impact thymocyte development as well as T cell antigen recognition and activation. Both TCRs and preTCRs utilize force to induce different receptor conformers associated with energized and non- energized states. Project 1 shall elucidate biophysical features driving TCR mechanosensing using paired single molecule and single cell measurements via optical tweezers (OT) to determine non-equilibrium dynamics and parameterization of energy landscapes under force. In turn, CD8 T cell responses such as antigen-specific in vitro triggering sensitivity and in vivo cellular proliferation, effector and memory T cell development will be assessed using TCR retrogenic mice. RNAseq analysis of various populations and single cells shall define the connection between force-dependent transcriptomes and physical load on TCR-pMHC bonds. Project 2 shall perform comparable OT biophysical studies on preTCRs and pMHC interactions using high throughput next generation sequencing (NGS) of DN3, DN4, DP large and DP small subsets to determine TCR repertoire changes in MHC-sufficient and MHC-deficient animals in vitro and in vivo. By determining chain clonotypes that are selected or disallowed during thymocyte developmental progression upon interaction with specific single- chain pMHC ligands, coupled RNAseq analysis of thymocytes expressing those preTCRs, OT profiling, Molecular Dynamics (MD) and NMR and X-ray crystallography structural studies, the rules governing early thymic selection by pMHC shall be defined. Distinctions among and TCR lineages with respect to mechanical force shall be similarly analyzed and compared. Project 3 shall develop cutting-edge NMR methods to reveal allosteric mechanisms of preTCR and TCR receptors upon pMHC ligation, characterizing major and minor state structures and kinetics of interconversion aided by the MD Core to enhance atomistic detailing. An Administrative Core (A), a Protein Production Core (B) and a MD Core ...