The role and regulation of extracellular vesicles released by B cells in response to CD24

Phan, Hong Dien (2024) The role and regulation of extracellular vesicles released by B cells in response to CD24. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Extracellular vesicles (EVs) are membrane-encapsulated nanosized particles that carry bioactive cargo, including proteins, nucleic acids, and lipids. EVs are secreted by most living cells, including B lymphocytes (B cells). B cells are antibody-producing immune cells that develop in the bone marrow, where different cell surface receptors regulate their maturation. One of the earliest surface proteins expressed in developing B cells is called CD24, a glycophosphatidylinositol (GPI)-linked protein localized to lipid rafts on the cell plasma membrane. Past research in the Christian lab showed that engagement of CD24 on the immature murine WEHI-231 B lymphoma cell line could cause the release of bioactive EVs. Following in the footsteps of the first discovery, this work employed a model system where donor cells expressing palmitoylated GFP (WEHI-231-GFP) were co-cultured, after stimulation, with recipient cells lacking either IgM (WEHI-303 murine B cells) or CD24 (CD24 knock-out mouse bone marrow B cells). The study found that EVs traffic lipid and membrane proteins between B cells in response to stimulation of either CD24 or IgM on the donor cells. Importantly, this study found that EV-mediated transfer of CD24 or BCR may affect B cell development by inducing apoptosis in recipient bystander cells. The following study aimed to determine how CD24 regulates the release of EVs. Bioinformatic analysis showed that CD24 expression is linked to the PI3K/AKT and mTOR signaling pathways. Using chemical and genetic inhibition, I found that an aSMase/PI3K/mTORC2/ROCK/actin pathway regulates EV release. Lastly, through live cell imaging, the study confirmed that ROCK is required for inducing the membrane dynamics required for EV release, presumably by regulating tethering of the actin cytoskeleton to the plasma membrane. The information obtained from this study and other research indicate that EVs induced by CD24 stimulation are ectosomes that budded off from the plasma membrane rather than exosomes that originated from multivesicular bodies. Significantly, these data have uncovered a novel pathway regulating ectosome release that has not been reported in any cell types. This research topic provides a window into the diverse function of CD24 as well as increasing our knowledge of how CD24 regulates EV release in cell-to-cell communication.

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