A novel small molecule enhances the release of extracellular vesicles with immunostimulatory potency

Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, University of California San Diego researchers sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. The researchers previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca²⁺ elevation enhances EV release, they screened 80 hit compounds and identified ethyl 2-(benzo[c][1,2,5]thiadiazole-4-sulfonamido)-4,5-dimethylthiophene-3-carboxylate (hereafter designated as 634) as a Ca²⁺ influx inducer. 634 enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from 634-treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca²⁺ elevation in immunostimulatory EV release, the researchers performed structure-activity relationship (SAR) studies of 634. The analogues that retained the ability to induce Ca²⁺ influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca²⁺ influx. The levels of Ca²⁺ induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, this study presents that a small molecule, 634, enhances the release of EVs with immunostimulatory potency via induction of Ca²⁺ influx. This agent is a novel tool for EV-based immune studies and vaccine development.