A chemical biology-based approach to the study of FOXP3 and Regulatory T Cell Fate - ABSTRACT Pharmacological modulation of regulatory T cell numbers and function has significant potential in the context of autoimmune and inflammatory disease. However, the overlapping and pleotropic nature of immune-related signaling pathways associated with currently known methods to expand functional regulatory T cells makes it challenging to increase regulatory T cell populations, without having intolerable immunosuppressive or cytotoxic effects. Agents reported to date can result in the expansion of additional cell types that can hinder the effect of Tregs or produce nonfunctional Tregs. Here, we have conducted a high throughput flow cytometry screen to address this issue and have identified multiple Treg-inducing small molecules, including A205804, as novel inducers of FOXP3 expression. A205804 induces FOXP3 expression in human and mouse CD4+ T cells and Tregs induced to differentiate using this compound maintain suppressive activity. Mechanistic studies revealed that A204804’s ability to regulate Treg differentiation occurs independently of its reported impact on endothelial cells (i.e., functional inhibition of ICAM-1 expression) or NF-kB signaling and also functions independent of known mechanisms related to IL-2 or TGF- signaling. To achieve the overall objective of this proposal, the following three specific aims will be successfully completed: 1A. Identify the relevant biomolecular target and associated mechanism of action for A205804 in the context of its ability to induce functional human and mouse CD4 Treg differentiation. 1B. Optimize the pharmacokinetic properties of A205804. 2A. Determine selectivity, human activity and validate induction of suppressive Treg function for three novel mechanism of action small molecule scaffolds (SR0723, SR0767, SR0320). 2B. Determine potential cross-reactivity with identified target of A205804 and identify relevant biomolecular target(s) for small molecules demonstrated to function independently of the A205804 mechanism. 3. Determine the ability of A205804 and at least one novel MOA compound to selectively induce CD4 Treg differentiation, impact total CD4 Treg numbers and positively impact remyelination in the cuprizone demyelination/remyelination model. Successful completion of the research objectives described in this proposal will address a key knowledge gap in our understanding of the biology and application of naïve CD4 progenitor T cells, which is relevant to human biology and diverse human disease contexts. Specifically, by identifying actionable targets for the selective induction of Treg differentiation, results from the proposed research activities will facilitate future preclinical studies and efforts aimed at developing stem cell-based therapies with applications across diverse forms of inflammatory and auto-immune related human diseases.
