Immunoregulatory cytokines engage transmembrane signaling receptors in order to mediate a wide range of functions including leukocyte proliferation, differentiation, and expansion. 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, targeted use of these cytokines as drugs. Cytokine pleiotropy is a consequence of a small number of shared receptors, such as common gamma chain and gp130, engaging many different cytokines, which then activate overlapping intracellular signaling pathways through a limited set of JAK and STAT proteins. During the prior term of this award, we gained an appreciation for the extracellular structural architectures of a spectrum of different cytokine complexes with shared receptors, including those of IL-1, IL-2, IL-4, IL-6, IL-13, IL-17, IL-23, and IFN, which exhibited an astonishing diversity of heterodimeric signaling geometries. In this renewal application, we focus our studies on the pleiotropic cytokines IL-2 and IL-15, to ask how extracellular structures of the receptor-cytokine complexes influence transmembrane signaling, intracellular activation of JAK and STAT, and subsequent in vivo function. We wish to determine if the binding chemistry and geometry of the IL-2 and IL-15 receptor complex subunits plays a role in modulating signaling specificity, and whether "tuning" signaling through structure-based cytokine engineering of receptor interactions is a viable means of developing novel immunotherapeutics with enhanced efficacy, cell subset preferences, and reduced toxicity. The overall goals of this highly collaborative proposal are:
Aim 1 - to determine the biophysical basis for the functional redundancy and specificity exhibited by two "natural surrogate" ?c cytokines, IL-2 and IL-15, that act through shared signaling receptors (IL-2R? and ?c) but private alpha-receptors;
Aim 2 - to utilize structure-based protein engineering to attempt to create IL- 2 variants with diverse signaling properties and T cell subset preferences, that may be more effective immunotherapeutics as assessed by collaborators in a variety of in vivo disease models;
and Aim 3 - to determine the mechanistic basis of antibody potentiation of IL-2 activity towards distinct T cell subsets, as well as discover new potentiating antibodies that could remodel the conformation of wild-type IL-2 and alter its biological activity. Finally, in Aim 4, we continue to pursue structural information on how cytokine receptor intracellular segments engage Janus Kinase (JAK) molecules, by reconstituting an entire full-length cytokine receptor transmembrane complex, bound to both cytokine and intracellular JAK for imaging by crystallography and electron microscopy. In this fashion, by combining structure (X-ray crystallography, Electron Microscopy, and NMR), protein engineering, signaling, and in vivo studies, we propose to obtain a complete molecular snapshot of shared cytokine receptor signaling from the initial engagement of ligand through the activation of intracellular signaling cascades.
Immunoregulatory cytokines and their receptors are essential to the normal functioning of the immune system, and dysfunction of this network underlies numerous human disease conditions such as autoimmunity and cancer. Yet, it remains unclear how the structural aspects of cytokine receptor engagement impacts function, and whether molecular approaches are a viable means of developing effective immunotherapeutics. We propose to use the techniques of structural biology and protein engineering to visualize, and manipulate, how binding of cytokines, such as IL-2 and IL-15, to their cellular receptors is translated into signaling outcomes relevant to immune disease. We intend to test these engineered cytokines in immune disease models in vivo with translational collaborators. These studies will give us insight into basic receptor signaling mechanisms, as well as facilitate the development of immunotherapeutic drugs.