Project Abstract/Summary. T cells are unique in their requirement for two activation signals to become functional. This separation of powers between the T cell receptor (TCR) and co-receptors, such as CD28, allows exquisite regulation of T cell responses. In recent years, T cell co-receptors have emerged as valuable targets of immunotherapy for autoimmune diseases and cancer. In autoimmunity blocking activating co-receptors can reduce the destructive effects of T cells on normal tissue, while in cancer blocking inhibitory co-receptors can activate a T cell response against malignant cells. However, co-receptor targeted immunotherapy often fails to produce desired results. Moreover, there remain large gaps in knowledge of how co-receptors coordinate the myriad cellular changes that allow T-cells to gain functional properties. The project proposed in this grant application will explore a novel mechanism by which T cell co-receptors coordinate changes in RNA maturation important for T-cells to generate sufficient numbers and make key molecules that allow them to kill other cells. These killer properties underlie the ability of T cells to contain infectious diseases and control tumor growth or to damage healthy tissue in autoimmune diseases. Preliminary studies provide evidence that CD28 signaling coordinates many changes in alternative splicing of newly made RNAs through effects on the RNA binding protein ARS2. Alternative splicing allows one gene to code for several different proteins by changing how the RNA produced from that gene is assembled. We find that in activated T cells CD28-ARS2 dependent alternative splicing of the mRNA coding for metabolic enzyme pyruvate kinase favors production of an isoform, known as PKM2, with known proliferation promoting properties. Proposed studies seek to 1) determine if alternative splicing to PKM2 induces changes in how T cells use nutrients to fuel proliferation, 2) examine how ARS2 regulates alternative splicing in T cells, and 3) establish CD28 regulated changes in alternative splicing as potential modulators of immunotherapy. The long-term goals of these studies are to understand how RNA binding proteins and RNA maturation shapes gene expression during the process of T-cell activation and to determine if such mechanisms of gene regulation can be therapeutically targeted to alter T-cell function in patients with cancer or autoimmune diseases.