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, we sought small-molecule compounds
that increase immunostimulatory EVs released by antigen-presenting
dendritic cells (DCs) for enhancement of vaccine immunogenicity. We
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, we screened 80 hit compounds and identified compound 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, we 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, our 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.