Project Summary T cell development, activation, and differentiation are all dependent on TCR signaling, along with important contributions from costimulatory and cytokine receptor pathways. Many lines of evidence indicate that variations in TCR signal strength have a major impact on the outcome of these processes. For mature CD8 T cells, TCR signal strength determines proliferative capacity and effector versus memory potential following infection. We assert that there is a growing need to develop a more comprehensive understanding of the pathways regulating distinct T cell functions and differentiation processes. Moreover, manipulation of T cell responses underlies many therapeutic strategies currently in practice and under development. As this field has progressed, it has become clear there is a need for more nuanced manipulation of T cell signaling pathways. To achieve clinical efficacy, we need to understand how to tune T cell responses. The field presently lacks an understanding of how differences in TCR stimulation strength produce distinct gene expression patterns that steer T cell responses. We now have a body of data supporting a key role for the TEC kinase ITK in tuning T-cell signaling. ITK is not required for all TCR signaling; instead, in its absence, TCR signaling is significantly reduced. From these studies, the clear function of ITK has been difficult to discern, as some aspects of T cell activation appeared normal in the absence of ITK, whereas other T cell functions were greatly impaired. Our data now provide a framework to understand these apparent discrepancies, and suggest that variations in antigen density and in TCR affinity dictate the magnitude of ITK activity modulating subsequent gene expression responses. This proposal will study the contribution of specific ITK activation mechanisms (Aim 1) and costimulatory interactions (Aim 2) to understand how ITK mediates tunable responses to TCR stimulation. Biochemical and structural biology insights are directly transferred into primary T cell experiments to elucidate the molecular mechanisms controlling ITK regulation. We will also study the role of ITK regulation in human disease (Aim 3). Genetic mutations that activate ITK have been recently identified in patients with autoimmunity and there is evidence that the ITK-SYK fusion tyrosine kinase interacts with normal ITK in promoting oncogenesis in peripheral T cell lymphomas. The results of these studies will provide us with critical information about the nature of ITK regulation and how ITK activity influences T cell activation in response to TCR signal strength.