Identification of Small-Molecule Inducers of FOXP3 in Human T Cells Using High-Throughput Flow Cytometry

Abstract

The increasing use of disease-relevant human primary cells in cellular target assays in drug discovery and phenotypic screening is driving the requirement for more sensitive high-throughput technologies that derive maximum information from fewer and fewer cells. It is widely recognized that heterogeneous primary cell populations are more suited to high-content single-cell analysis techniques such as flow cytometry. A robust flow cytometry assay was developed to identify compounds that up-regulate FOXP3 in primary human T cells. Over 70,000 small-molecule compounds were screened in single shot and several thousand hits were followed up at full dose response. Several compounds were identified that had a profound effect on FOXP3 expression in T cells; one in particular had an EC50 of <100 nM. These novel tool compounds can be used for dissecting signaling pathways upstream of FOXP3 and provide a molecular target that may lead to therapeutic intervention for autoimmune and inflammatory disease. However, a good lead starting point was not found. In order to increase chances of identifying lead molecules, greater compound structural diversity is required, meaning significantly larger compound libraries containing millions of compounds. The high cost and limited availability of human tissues pose challenges for attempts to screen large compound libraries.