Multiplex IP-FCM reveals network-scale protein-protein interaction differences between pro-immunogenic and pro-tolerogenic TCR signaling (P1130)

Abstract

Abstract T cell receptor signaling involves interactions of various kinases, phosphatases, ubiquitin ligases and adaptor proteins. However, the network-scale protein-protein interactions (PPIs) that mediate signaling, as well as the key differences between pro-immunogenic (agonist) and pro-tolerogenic (antagonist) signals, remain unclear. We have developed a novel method to examine large networks of signaling PPIs, multiplex immunoprecipitation measured by flow cytometry (mIP-FCM). Using multiple classes of Luminex® microspheres, we created a 96-well-formatted matrix array capable of measuring 484 PPIs in a single overnight assay. The array is focused on the most proximal signaling proteins associated with the TCR/CD3 complex. As a model system, we have used Jurkat cells that express the public LC13 TCR, stimulated by antigen presenting cells loaded with either LC13 agonist (FLRGRAYGL) or antagonist (FLRGRFYGL) peptide. Time-course studies have revealed both quantitative and kinetic differences between the two types of signal. We present our latest results mapping the PPI network that mediates TCR signaling. Our results have significantly impacted our understanding of the difference between pro-immunogenic and pro-tolerogenic TCR signals.