Platelet-derived EVs peaked at day 21, consistent with the increase in platelet-derived EVs recently shown during refrigerated, short-term storage of whole blood.32The proportion of platelet-derived EVs returned to baseline at day 42, potentially as a result of their faster degradation during storage relative to RBC-derived EVs. and EV-activated monocytes. Contrary to our hypothesis, the data demonstrate that EVs isolated from RBC models increase the potency of APCs and boost mitogen-driven T-cell proliferative responses. == Introduction == Extracellular vesicles (EVs) can be released from leukocytes, platelets, endothelial cells, and cells of other tissues under physiological or pathological conditions in response to activation, stress, necrosis, or apoptosis1-3and can be found in body fluids.4,5Three groups of EVs have been described according to their size and mechanism of generation: microvesicles are large cell membrane-derived particles in the range of 200 to 1200 nm.1,6Exosomes, with an approximate size of 30 to 150 nm, are byproducts of exocytosis.5Apoptotic bodies (50-500 nm) are the last group of EVs that are released from apoptotic cells.5 EVs may play immunosuppressive or immunostimulatory functions.7,8It has been shown that C-phosphate-G (CPG)-stimulated B cells from HIV patients produce lower quantities of immunoglobulin G in the presence of EVs from the same patients.9Platelet-derived EVs have been demonstrated to bias macrophages to an antiinflammatory response and secretion of transforming growth factor-.6However, exosomes DL-AP3 bearing autoimmune antigens are immunostimulatory in a NOD mouse model of diabetes, leading to production of DL-AP3 proinflammatory cytokines and proliferation of T cells.10 In this article we examine the role of EVs in potentially mediating an immune modulatory effect associated with blood transfusion. It is believed that transfusion of fresh blood may carry less risk of adverse reactions compared with aged blood, attributed to a red blood cell (RBC) storage DL-AP3 lesion, which has been described as physical and chemical changes of RBCs during the time of storage.11-13Morphological changes to RBCs in stored packed-RBC units are accompanied by shedding and release of EVs from RBCs or from residual platelets and leukocytes in the bag.14-16The overall balance of physical and chemical changes in stored blood may contribute to immunomodulation and potential adverse DL-AP3 effects in patients who have received older blood, and EVs may be key mediators of immune modulation in transfusion recipients.7,12,13,17,18 EVs express different markers on their surface depending on their cell of origin, and they may contain RNA, DNA, and proteins.5,19Increased generation of some EV subtypes has been associated with increased risk of specific diseases, and EVs may serve as useful diagnostic biomarkers in the future.1,20-22The cellular source of EVs and the immunomodulatory role of EVs generated during the storage of human RBC units are not well understood.7,23,24Here, we tracked the quantity and cell of origin for EVs found in RBC models DL-AP3 throughout the standard storage period. Furthermore, we hypothesized that RBC-EVs would suppress T-cell immune responses, and we tested whether EVs could modulate T-cell responses and whether antigen-presenting cells (APCs) participated in EV-driven modulation of the immune response. == Methods == == Study samples == Six leukoreduced packed RBC units were received from Blood Centers of the Pacific. Peripheral blood mononuclear cells (PBMCs) from 6 donors were Sema3b recovered from the leukoreduction chamber after platelet apheresis. PBMCs were purified and stored in liquid nitrogen. Supplemental Table 1 (available on theBloodWeb site) provides more detail on packed cell preparation and apheresis technology. Written consent was obtained from the healthy blood donors in accordance with the Declaration of Helsinki, and the samples were de-identified. The study protocols were approved by the University of California, San Francisco Committees on Human and Animal Research. == Storage of packed RBC models and purification of EVs == Packed RBC units were split into 35-mL aliquots in replicate 180-mL transfer bags and stored at 4C. EVs were isolated using differential centrifugation with an initial velocity of 1500gto individual supernatant, followed by spinning the supernatant at 13 000gfor preparation of platelet free supernatant. Three mL of platelet free supernatant was added to 32 mL phosphate-buffered saline and spun for 1 hour at 100 000g. EV pellets were resuspended in 1 mL RPMI and stored at 80C. Fractionation of EVs to small and large EVs was performed with 0.22-m centrifugal filters for 2 minutes at 750g (Millipore). Large EVs (microvesicles) were recovered from the top of the filters by washing, and the flow-through (small EVs) was termed exosomes. == Characterization of EVs == Fluorochrome-conjugated monoclonal antibodies were used to determine the origin of EVs (RBCs [CD235a-FITC], platelets [CD41a-PerCP/Cy5.5], lymphocytes [CD3-PerCP/Cy5.5, CD19-Alexa/700, and CD16-V450], monocytes [CD14-APC/Cy7], and endothelial cells [CD142-PE] [Biolegend])..