Abstract Recognition of antigen is the first critical step required in triggering T cell survival, expansion, development of effector functions, and transition to memory. In a joint project between the Evavold and Williams labs, we will determine how T cell receptor (TCR) and peptide:MHC (pMHC) affinity, bond lifetimes, and force magnitude define T cell phenotype. The proposed work is therefore impactful as it begins to delineate the role each of these parameters play in the rich T cell biology associated with TCR signal strength. To date, the concept of TCR strength of signal has generally been described with three-dimensional SPR affinity in mind and measured via representative surface markers, pMHC tetramers, and functional readouts. In contrast to these methods, our studies depend on the analysis of TCR and pMHC interactions at the surface of the cell membrane using novel assays that define the in-situ two-dimensional contact that occurs between T cells and APCs during antigen recognition. The preliminary work has discovered that bond lifetime and level of force as opposed to affinity provides the major driving force for phenotypic fate. The three specific aims will redefine the concept of TCR strength of signal, dissect affinity from bond lifetime, and determine outcomes of low affinity TCRs during infection. Currently, the TCR affinity and bond lifetime for pMHC are unknown for many T cell responses, limiting our knowledge on how T cell mediated responses are triggered and how affinity and bond lifetime are translated into TCR strength of signal and effector phenotypes. Therefore, our work will prove insightful by addressing the discrepancy between affinity based predictions and actual T cell functional outcomes.