CD24 regulates extracellular vesicle release via an aSMase/PI3K/mTORC2/ROCK/actin pathway in B lymphocytes

CD24 is a glycophosphatidylinositol-linked protein that regulates B cell development. We previously reported that stimulation of CD24 on donor B cells promotes the transport of functional receptors to recipient B cells via extracellular vesicles (EVs). However, the mechanisms regulating EV formation in response to CD24 are unknown. Using bioinformatics, we found a connection between CD24 and the PI3K/AKT and mTOR signaling pathways. To determine if PI3K or mTOR regulates EV release, we made use of our co-culture model, whereby donor B cells carrying the B cell receptor (BCR, IgM) release EVs labeled with palmitoylated GFP upon CD24 stimulation are incubated with recipient B cells that lack IgM and express palmitoylated tdTomato. Using flow cytometry, we are able to follow the transfer of EVs carrying lipid-associated GFP and surface IgM from donor to recipient cells. Using chemical and genetic inhibition, we found that a PI3K/mTORC2/ROCK/actin pathway regulates EV release. We also found that acid sphingomyelinase (aSMase) activates PI3K to induce EV release. Lastly, through live cell imaging, we found that ROCK is required for inducing the membrane dynamics associated with EV release. Overall, our data suggest that these EVs are ectosomes budded from the plasma membrane and not intracellularly-derived exosomes. Importantly, we have uncovered a novel pathway regulating ectosome release.