Extracellular Vesicles from Wharton's Jelly Mesenchymal Stem Cells Suppress CD4 Expressing T Cells Through Transforming Growth Factor Beta and Adenosine Signaling in a Canine Model

Mesenchymal stem cells (MSCs) are widely investigated as potential therapeutic agents due to their potent immunomodulatory capacity. Although specific mechanisms by which MSC acts on immune cells are emerging, many questions remain, including the potential of extracellular vesicles (EVs) to mediate biological activities. Canine MSCs are of interest for both veterinary and comparative models of disease and have been shown to suppress CD4(pos) T cell proliferation. The aim of this study was to determine whether EV isolated from canine Wharton's jelly-derived MSC (WJ-MSC EV) suppresses CD4(pos) T cell proliferation using biochemical mechanisms previously ascribed to soluble mediators [transforming growth factor beta (TGF-beta) and adenosine]. WJ-MSC EV exhibited mode of 125 nm diameter, low buoyant density (1.1 g/mL), and expression of EV proteins Alix and TSG101. Functionally, EVs inhibited CD4(pos) T cell proliferation in a dose-dependent manner, which was absent in EV-depleted samples and EVs from non-MSC fibroblasts. EV suppression of CD4(pos) T cell proliferation was inhibited by a TGF-beta RI antagonist, neutralizing antibodies to TGF-beta, or A2A adenosine receptor blockade. TGF-beta was present on EVs as latent complexes most likely tethered to EV membrane by betaglycan. These data demonstrate that canine WJ-MSC EV utilizes TGF-beta and adenosine signaling to suppress proliferation of CD4(pos) T cell and will enable further investigation into mechanisms of immune cell modulation, as well as refinement of WJ-MSC and their EVs for therapeutic application.