Introduction: The cellular events involved in the generation of an auto and allo-reactive immune response in recipients of islet transplantation are not well understood. In both physiological and pathological conditions, cells release a variety of signals, including extracellular vesicles (EVs). These nano-sized, membrane bound vesicles are known to present antigen to the immune system in other inflammatory diseases and mouse models of diabetes. Previous work in our laboratory has identified EVs produced by human islets. This raises the question of whether human islet-derived EVs are capable of and/or responsible for eliciting an immune responses following islet transplantation. Methods: Multi-organ donor pancreases were retrieved under expressed research consent for isolation using standard protocols. Islet conditioned media (ICM) was collected and frozen at -80 [deg] after 24 hrs of culture. ICM samples were analysed by Nanoparticle Tracking Analysis (NTA). Additionally, EVs were purified by ultracentrifugation at 200 000 g [times] 90 min. Peripheral blood mononuclear cells (PBMCs) were isolated from normal volunteers by Ficoll centrifugation. Purified EVs were labelledand co-cultured with PBMCs (5*10⁵). EV internalization and resulting cytokine production were analyzed using flow cytometry and ImageStream®^x. ANOVA was used to compare controls and EV-stimulated samples. Results:We demonstrate that islet-derived EVs can be characterized using the NTA technology. The majority of EVs are between 100 and 300 nm in size, representing a heterogeneous population. We observe selective internalization of islet-derived EVs by CD14+ monocytes using flow cytometry and ImageStream®^x. Twenty-one percent of CD14+ monocytes were found to be EV+. Human islet-derived EVs contain innate stimuli that can induce production of pro-inflammatory cytokines. EV stimulation triggers an 11% increase in TNFα productionand a 30% increase in IL-6 production in monocytes compared to controls (p<0.05). Conclusions: Islet-derived EVs are selectively internalized by CD14+ monocytes, and trigger cytokine production. Further analyses must be conducted to determine the antigen presentation pathway of islet-derived EVs. Advances in this field may help to better understand the mechanisms by which islets and immune cells cross-talk to generate islet-reactive immune responses in diabetes and islet transplantation.

CITATION INFORMATION: Rutman A, Negi S, Hasilo C, Gasparrini M, Tchervenkov J, Paraskevas S. Human Islet-Derived Extracellular Vesicles Stimulate CD14+ Monocytes Resulting in Proinflammatory Cytokine Production. Am J Transplant. 2017;17 (suppl 3).

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