CD8+ T cells play a critical role in adaptive immune defense and have recently become powerful therapeutic tools. Their primary function is to kill infected or malignant cells which they accomplish by expanding their numbers exponentially, differentiating to acquire effector functions, and then dying off once the threat is eliminated. The mechanisms that control this highly orchestrated process are incompletely understood. Over the last decade, modeling studies have discovered that these events are controlled by several independent and preset ‘timers’ that are set in motion upon antigen recognition. These timers control initiation of cell division, extent of proliferation, differentiation and death programming. Further elucidating the molecules and pathways that regulate these cellular events is necessary to fully realize the potential of T cell-based immunotherapies. However, to date, a major limitation of this model is that it relies on the use of mice from a single genetic background. We know that genetic variation is one of the largest drivers of immune response heterogeneity, therefore we hypothesize that in genetically diverse populations, such as humans, there remain unknown regulators of immune cell fate timers. In this application, we propose to leverage the Diversity Outbred (DO) model, a high diversity, multi-parental population of mice, to gain mechanistic insights into CD8+ T cell behavior. In Aim 1, we will identify the breadth of genetic variation in CD8+ T cell activation and differentiation by screening a cohort of DO mice for a battery of phenotypes associated with CD8+ T cell activation, proliferation and differentiation. To do this we will perform extensive flow cytometric analysis in naîve samples and perform detailed in vitro stimulation experiments to which we can apply mathematical modeling techniques to obtain definitive parameters for activation, proliferation and differentiation. Collectively, we will measure ~100 data points related to these processes per individual. In Aim 2, we will identify and validate specific gene loci that are linked to observed CD8+ T cell phenotypes and timer parameters. To complete this goal, we will genotype each mouse and then perform Quantitative Trait Locus mapping (QTL) using identified variable parameters. Additionally, we will collect samples from CD8+ T cells before and after T cell receptor (TCR)-mediated activation to perform RNA sequencing so that we can link functional traits and related gene loci with genome-wide gene expression patterns. In this way we will gain an understanding of the genes and gene networks that underpin observed functional variation. Importantly, identified loci will be validated using a small number of inbred Collaborative Cross strains, which share the same multi-parental population as the DO mice. At the completion of these studies, we will gain a more complete understanding of the molecular machinery, or timers, that regulate CD8+ T cell function. We expect our findings to pave the way for advances in diagnostics, personalized medicine and cell-based immunotherapies.