Developing a novel multiplexed high throughput flow cytometry based immune assay to screen kinase modulators of primary T cell activation

Abstract

Abstract Modulating TCR engagement and signaling using biologics, small molecules or genetic engineering is highly relevant to many therapeutic areas. TCR signal initiation is mediated by cytosolic tyrosine kinases, leading to signal amplification through a network of serine-threonine kinases. Genetic defects, mutations and other mechanisms leading to increased kinase activity and enhanced T cell activation (TCA) is involved in many autoimmune pathologies, making kinases attractive targets for the direct inhibition of TCA and treatment of autoimmune disease. The development of drugs and therapies regulating TCR activity require assays to profile T cell function and cell health. To address this need, we developed a novel multiplexed TCA assay based on high throughput flow cytometry technology with advanced computational algorithms for data analysis. The assay simultaneously measures T cell phenotype, time-dependent expression of T cell activation markers, cell proliferation and viability and quantitates secreted cytokines from a single 10 ml sample using a 96 or 384-well plate format. To illustrate the value of the TCA assay in drug discovery, we performed a phenotypic screen using a 152 kinase inhibitor (KI) small molecule library for their ability to inhibit human primary TCA. Using the Profile Mapping algorithm in our ForeCyt software, we integrated 11 TCA metrics to identify multiple inhibition pathways. The Profile Map tool was used to group KI based on their inhibition phenotypes, irrespective of their known targets. These results illustrate the use of Intellicyt’s technology to deliver solutions for drug and biologics discovery.