Project Summary/Abstract The long-term goal of this proposal is to develop new tools to study how T cells defend against pathogens and eradicate cancerous cells within our bodies. ~106-108 unique T cell clones circulate in the adult human body seeking evidence of foreign peptide fragments on the surface of other cells. Once the T cell encounters a target cell with foreign or mutant peptides, it initiates activation mechanisms that unleash a potent immune response. The very first step in T cell activation involves recognition between the T cell receptor (TCR) and the short peptides (8-11 amino acids) presented by the major histocompatibility complex (pMHC) protein. Because T cells are highly migratory and antigen recognition occurs when the T cell physically contacts a target cell, it is no surprise that the TCR-pMHC complex experiences molecular forces that influence antigen potency. Indeed, our lab and others have shown that TCR activation relies on pN force transmission to its cognate pMHC. Current screening technologies to identify antigenic peptides measure affinity without regard to mechanical force and are thus poor predictors of antigen potency. To address this problem, we will develop mechano-ID to specifically tag T cells based on the magnitude of mechanical forces transmitted through the TCR-pMHC complex. Mechano- ID integrates advances in proximity tagging with molecular tension probes pioneered by the PI. The fundamental principle behind this proposal is the concept that a well-defined TCR force unfolds a DNA hairpin, exposing a cryptic binding site that recruits enzymes. These enzymes, in turn, will generate reactive species, such phenoxy radicals that covalently tag proteins within a ~20 nm radius. Thus, mechano-ID detects energized biomechanical forces and nearby interactome rather than static protein-protein interactions. Preliminary data shows the feasibility of mechano-ID applied to primary T cells. In Aim 1, we will optimize mechano-ID to enhance its yield and specificity for force-induced tagging of TCR-pMHC mechanical events. Parameters such as reagent concentrations, timing, and the mechanical stability of the nucleic acid probes will be investigated. The assay will be integrated into a spherical bead platform to boost yield. In Aim 2, we will demonstrate mechano-ID for screening of T cell-antigen specific interactions. The altered peptide library of the SIINFEKL pMHC antigen will be tested to establish a direct correlation between antigen potency and mechano-ID signal. Chimeric antigen receptor (CAR) T cells will tested to further demonstrate mechano-ID in the area of cancer immunotherapy. The outcome of this proposal is an innovative method that allows one to tag cells based on mechanical phenotypes –thus opening the door to linking genotype to the mechanotype and enabling the field “mechanomics”. Finally, we note that mechano-ID transcends T cell biology and will be broadly useful in the study of virtually any mechanotra...