Mesenchymal stromal cell-derived extracellular vesicles as modulators of B Cell activation

Mesenchymal stromal cells (MSCs) are multipotent cells with low immunogenicity and the capacity to differentiate into multiple lineages. Initially, their therapeutic potential was attributed to their ability to engraft and differentiate to repair damaged tissues. However, subsequent studies revealed that most of their effects are mediated through paracrine mechanisms involving the secretion of bioactive factors collectively termed the MSC secretome. Among these, extracellular vesicles (EVs), membrane-bound particles containing proteins, nucleic acids, and lipids, have emerged as key mediators of MSC-driven immunomodulation. Pro-inflammatory “licensing” of MSCs, achieved by stimulation with cytokines such as IFN-γ and TNF-α, enhances their immunomodulatory potency and reduces donor-to-donor and tissue-source variability. EVs released from licensed MSCs exhibit distinct molecular cargos and frequently display superior immunoregulatory effects compared with those from unlicensed MSCs. B cells, traditionally recognized for their role in adaptive immunity, also participate in innate-like responses and immune regulation through subsets such as regulatory B cells. Dysregulated B cell activation contributes to chronic inflammation, impaired healing, and autoimmunity, leading to the development of therapies that deplete this cell population. However, many of these approaches show limited efficacy and, in some cases, have resulted in secondary immune disorders or worsening of the condition. This underscores the need for therapies that modulate rather than deplete B cells. To date, most studies investigating the immunomodulatory potential of MSC-EVs have focused on phagocytic cells such as macrophages and dendritic cells, while their effects on B cells remain poorly understood. Existing research reports conflicting findings regarding the influence of MSC-EVs on B cells, likely due to methodological differences in both B cell activation protocols and EV treatment conditions. For instance, variations in B cell stimulation methods, EV dosages, and the timing or parameters used to assess phenotypic changes can significantly alter observed effects. These inconsistencies are further compounded by variability in EV isolation methodologies. EVs are most frequently isolated using ultracentrifugation (UC), which can co-isolate soluble proteins and confound biological outcomes. More refined approaches, such as density gradient UC or size-exclusion chromatography (SEC), yield purer and more intact EV populations. However, density gradient UC is labor-intensive technique and less scalable for large volume processing compared to SEC. Notably, combining tangential flow filtration (TFF) with SEC has been proposed as a promising strategy for the scalable production of pure EVs suitable for clinical applications. Despite this, studies employing such combined approaches are limited, hindering the clinical translation of MSC-EV-based therapies. This thesis investigates how pro-inflammatory licensing alters the immunomodulatory capacity of murine MSC-EVs and how these vesicles influence allogeneic B cell activation under distinct activation contexts. Using the clonal murine bone-marrow MSC line D1 ORL UVA, a licensing protocol with IFN-γ and TNF-α was optimized in EVdepleted culture conditions. EVs were isolated using TFF followed by SEC and characterized according to MISEV 2023 guidelines. PalmtdTomato-expressing MSCs were generated to enable visualization of EV uptake by B cells, without the artifacts typically introduced by lipophilic dyes. The effects of MSC-EV dose and licensing status on B cell activation markers were assessed under T cell–like (anti-IgM + CD40L or CD40L alone) and T cell–independent (CpG ODN) stimulation. Their influence on CpG-induced B cell proliferation was also examined. Finally, the capacity of EVs from licensed and unlicensed MSCs to modulate antigen presentation by CD40L-activated B cells was evaluated using an antigen-restricted OT-II model. MSC-EVs binding and uptake over time was observed to be dependent on the activation state of B cells. MSC-EVs inhibited B cell activation in a dose dependent manner, regardless of the activation method used, and reduced the proliferation index of T cells co-cultured with EV-treated B cells. Conversely, extremely high doses of EVs appeared to enhance B cell activation. Furthermore, pro-inflammatory licensed MSCs possessed a superior capacity to modulate B cell activation induced by CpG, compared with EVs from unlicensed MSCs and to reduce B cell antigen-presenting capacity of CD40L activated B cells. Finally, MSC-EV–mediated modulation was influenced by the B cell activation mechanism. Specifically, MSC-EVs primarily downregulated CD69 and CD86 on CpG-activated B cells, while modulating MHCII, CD80, and CD86 on anti-IgM plus CD40L or CD40L-activated B cells, with CD69 largely unaffected. By addressing key methodological gaps—including the combined use of TFF and SEC for scalable high-purity EV isolation, standardized quantification approaches, avoidance of lipophilic dyes, and evaluation of dose-,timing-, and activation stimulation-dependent effects—this work aims to advance understanding of MSC-EV–mediated immunoregulation and contribute to the development of standardized, cell-free MSC based immunotherapies.

Funder

European Research Council
Higher Education Agency

Publisher

University of Galway

Rights

CC BY-NC-ND

cbn

Collections

University of Galway Theses (PhD Theses)