Development of a screening strategy for new modulators of T cell receptor signaling and T cell activation

Abstract

Activation of the T cell receptor (TCR) leads to the generation of a network of signaling events critical to the developmental decision making and activation of T cells. Various experimental approaches continue to identify new signaling molecules, adaptor proteins, and other regulators of TCR signaling. We propose a screening strategy for the identification of small molecules affecting TCR signaling based on the uncoupling of TCR stimulation from cellular responses in developing thymocytes. We demonstrate that this strategy successfully identifies inhibitors of kinases already shown to act downstream of TCR engagement, as well as new inhibitors. The proposed strategy is easily scalable for high throughput screening and will contribute to the identification of new druggable targets in T cell activation.

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.…”

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

“…The inhibitors, diluted to nontoxic concentrations, were used in the assay for TCR-stimulation-induced apoptosis in thymocytes 5,17 . The stimulation was carried out using anti-CD3/CD28 beads for 18 h, and the cells were subsequently stained for caspase-3 activation in the CD4 + and CD8 + DP thymocyte population (Figure 2).…”

“…The screening assay presented here is able to successfully identify compounds that inhibit canonical molecules of the TCR signaling pathway, as well as potential, novel compounds with inhibitory effects on TCR signaling. For example, we identified inhibitors of GSK3β and Hsp90 as new compounds affecting T-cell responses 17 . The assay is able to distinguish the inhibitors that interfere with signal transduction, due to a reduction in the apoptotic response, from the TCR-independent effects of the inhibitors on cellular toxicity.…”