Identification of Mediators of T-cell Receptor Signaling <em>via</em> the Screening of Chemical Inhibitor Libraries

Abstract

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

A Dual Inhibitor of Cdc7/Cdk9 Potently Suppresses T Cell Activation

“…We have recently reported a method of small molecule library screening designed to identify potential new targets in the TCR signaling pathway (24, 25). CD3/CD28 stimulation induces strong TCR signaling in primary thymocytes, resulting in caspase-3 activation and cell death, mimicking negative selection (28).…”

“…Active caspase-3 staining was carried out using an active caspase-3 staining kit from BD Pharmingen (#550480). The staining procedures were carried out as detailed previously (24, 25).…”

“…In this study, we investigated Cdc7/Cdk9 inhibitor PHA-767491 as a hit from a small molecule screen designed to identify new modulators of T cell development and activation (24, 25). We show that PHA-767491 and other Cdc7 and Cdk9 inhibitors potently suppress T cell activation, proliferation, and effector functions.…”