Privileged or Not?: Immunologic Properties of Allogenic Endothelial Progenitor Cell-Derived Extracellular Vesicles

Introduction: Extracellular vesicles (EV) from allogeneic progenitor cells are considered safe for treatment of ischemic myocardium based on the assumption of immune-privilege. However no studies have rigorously examined the local immune response following pooled-donor EV therapy. Understanding the specific immune reaction to endothelial progenitor cell-derived EVs (EEVs) has critical implications for clinical translation to treatment of acute myocardial infarction (MI). Aim: We aim to characterize the immune cells that respond acutely to injected intramyocardial delivery of pooled donor EEVs using in-depth flow cytometric (FCM) analysis. Methods: EEVs were isolated from pooled, primary bone-marrow EPCs from male Wistar rats. Uptake of DiI labeled EEVs by rat endothelial cells, cardiac fibroblasts, and myoblasts was assessed in vitro . In vivo immune response to EEVs in rats was studied after intramyocardial injection of PBS and PBS-EV. Left ventricular (LV) tissue digested at D1 and D6 was analyzed by 9-Color FCM for detection and differentiation of T, B, and NK cells, neutrophils, and monocytes. Local immune cell infiltration was assessed by H&E staining at D5 post-injection. Results: All cells tested had robust EEV internalization of DiI stain. H&E showed immune cell infiltration near the injection site, but no gross difference in EEV vs. PBS at D5. In-depth FCM of the LV at D1 and D6 post injection showed marked decrease in lymphocyte infiltration, but increased percentage of NK, B and CD8+ T cells from D1 to D6. EEVs did not significantly change the percentage of lymphocyte infiltration vs. PBS at D1 or D6. Conclusions: FCM can be successfully used for in-depth characterization of cardiac immune cell-types in rat models of allogeneic myocardial therapy. Allogeneic EEV therapy demonstrates robust, rapid internalization into the major cardiac cell types and elicit minimal additional innate or acquired immune response compared to saline injection.