Rewiring cytokine receptors for customized cellular responses

Abstract

Engineered cell-based therapies are a rapidly advancing technology that have much potential in treating a wide range of diseases. However, it still remains challenging to customize cellular responses to the extracellular environment to maximize cells’ therapeutic efficacy. Here, I propose a platform for synthetic receptor design based on cytokine receptors. Cytokine receptors use the simple, compact Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, providing many opportunities for customizing responses when repurposed as synthetic receptors. We investigated the modularity of cytokine receptors as a synthetic platform by reprogramming type I and type III interferon (IFN) receptors to phosphorylate alternative STATs and novel substrates that can trigger user-defined responses. Our findings demonstrate that cytokine receptors can be modified to sense other molecules, such as erythropoietin (EPO), and activate different substrates without disrupting receptor activity, including a phosphorylated tyrosine tag (pYtag) in the form of an immune tyrosine activation motif (ITAM). Activation of this pYtag triggers the recruitment of a cognate tandem-SH2 domain that can be conjugated to a fluorophore for detection. We show that this IFN-pYtag system can be used to monitor IFN signaling dynamics at a high spatiotemporal resolution. We further developed the IFN biosensor system design to trigger cleavage by a protease, which can potentially induce the nuclear localization of user-specified transcription factors for customized biological functions. The tools and approaches developed in this thesis could be used to design synthetic cell signaling pathways for a broad range of therapeutic applications.