Project Summary/Abstract Cytokine signaling is essential to the initiation of the immune response against microbial infection and cancer. The immune system is composed of two mechanisms of defense defined as the innate and adaptive immune systems, both of which critically rely on cytokine signaling to function. The innate immune system acts early during an infection or cancer and includes the type I IFN response. The adaptive immune system becomes fully active after approximately seven days. Although this response is delayed relative to the innate system, the time is needed to mount a T-cell response that is potent and specific. Interleukin-2 and interferon gamma are examples of cytokines that shape the response of the adaptive immune system. There are dozens of other cytokine families that each play an important role in the immune system including hematopoiesis, inflammation, apoptosis as well as many others. Understanding how cytokines signal, the genes they induce, and their functions are critical to understanding human health and disease. Recent examples of engineered cytokines demonstrate that tuning of cytokine signaling can drastically alter a cytokine’s response and may offer promising new therapeutic approaches. In this proposal, we aim to use protein engineering technologies to fill the large gaps in knowledge of cytokine signaling which may reveal new targets and approaches for therapeutic intervention. The paradigm of cytokine signaling is that cytokines drive the dimerization of cytokine receptors. Janus kinases (JAKs) are believed to be constitutively bound to the cytokine receptors. Upon receptor dimerization, the JAKs cross phosphorylate each other as well as the receptors. Signal transducers and activators of transcription (STATs) bind to the phosphorylated receptors, are then phosphorylated, dimerize, and translocate to the nucleus to elicit gene and functional responses. Recent tool development in our lab provides a streamlined approach to characterize protein-protein interactions which occur intracellularly, challenge assumptions in the field, and provide an opportunity to understand how every step in cytokine signaling contributes to cytokine signaling. We aim to show how altering these interactions tune cytokine signaling and response gene and functional signature.