SUMMARY The innate and adaptive immune response against allografts is the main impediment to successful transplantation. Heart transplantation is the best option for selected pediatric and adult patients with end-stage heart failure, however, the threat of rejection remains high, despite improvements in immunosuppressive therapies, conditioning regimens and medical care. Importantly, currently used interventions are not donor-specific and therefore may cause increased risk of infections and cancer. A deeper understanding of the basis of allo-recognition is needed for the development of novel donor-specific therapies to treat graft rejection minimizing the harmful side effects. Recent studies have challenged the dogma that initiation or re-activation of anti-donor immunity in graft- draining secondary lymphoid tissues (SLTs) depends mainly on donor Ag-presenting cells (APCs) mobilized from the grafts. Increasing evidence and our preliminary studies indicate that heart and non-vascularized skin allografts release extracellular vesicles (EVs) carrying donor-Ag that traffic to the SLTs where the graft EVs stimulate donor- reactive B cells and T cells. Despite increasing information during the past 5 years on the role of graft EVs on elicitation of anti-donor immunity and their potential use as biomarkers in transplantation, the mechanisms in vivo by which graft EVs interact with recipient’s immune cells in graft-draining SLTs and promote anti-donor immunity remain largely unknown. The family of EVs encompasses vesicles with different biogenesis, size and composition that includes exosomes and microvesicles. Although growing evidence indicates that EVs represent a mechanism by which cells horizontally transfer proteins, mRNAs, non-coding RNAs and lipids, the function of EVs in vivo remains an enigma. Therefore, we propose to investigate the mechanisms in vivo by which graft EVs initiate or re-activate in graft-draining SLTs, the innate and adaptive immune responses that lead to rejection of allografts. We will analyze these mechanisms in mouse experimental models of cardiac and non-vascularized skin allografts. We hypothesize that “graft EVs constitute a cell-free platform that by multiple mechanisms initiates or re-activates the anti-donor immune response in the recipient’s SLTs”. This application will investigate these mechanisms in situ and in vivo using transplant models in mice and a translational model in humanized mice. We will test our hypothesis in the following aims: Aim 1 will investigate the origin of graft EVs and their effects on recipient APCs in graft-draining SLTs, Aim 2 will analyze the mechanisms by which graft EVs generate anti-donor B cell immunity in SLTs and Aim 3 will analyze how graft EVs elicit anti-donor T cell immunity in SLTs and its relevance to transplantation in humans. Our long-term goal is to understand how graft EVs function in vivo to provide new grounds for development of EV-based therapies and disease markers o...