ABSTRACT: Immunoregulatory cytokines engage transmembrane signaling receptors in order to mediate a wide range of functions including leukocyte proliferation, differentiation, and expansion through JAK/STAT activation. Most immunoregulatory cytokines possess both redundant and distinct activities that are critical to normal immune homeostasis, but this functional pleiotropy presents a major problem for the effective use of these cytokines as immunotherapeutic cancer drugs. Cytokine pleiotropy is a consequence of different cytokine receptors being expressed on multiple different counterbalancing cell types that serves to neutralize anti-tumor actions and lead to systemic toxicity. During the prior term of this award, we gained an appreciation for the extracellular structural architectures of a spectrum of different immune cytokine complexes, including those of IL-2, IL-10, IL-12, IL-21, IL-22, IL-23, IL-27, IFN, and IFN. In this renewal application, with these structural templates in hand, in Aim 1 we focus our studies on the cytokines IL-2, IFN, and IL-12. These immune master regulators engage different but overlapping branches of the immune system, and share the issues of pleiotropy and toxicity that, if uncoupled, could lead to powerful cancer immunotherapeutic agents. We propose to “tune” signaling through structure-based cytokine engineering to attempt to create variants with decoupled pleiotropy, cell subset preferences, enhanced anti-tumor efficacies, and reduced toxicity – both alone and in combination. In Aim 2, we wish to understand the mechanistic basis for how tuned cytokines can differentially activate signaling inside the cells. Based on a recent breakthrough in our 20-year quest to solve the full-length JAK structure, we continue to pursue structural information on how cytokine binding to their receptors activates Janus Kinase (JAK) molecules, by reconstituting and imaging activated JAK homo- and heterodimers bound to both cytokine and intracellular JAKs and STATs. In this fashion, by combining structural biology, protein engineering, cell signaling, and in vivo tumor studies, we propose to obtain a complete molecular snapshot of cytokine receptor signaling from the initial engagement of ligand through the activation of intracellular signaling cascades and leverage this information for the engineering of cancer immunotherapeutics.