Proteomic Profiling of Extracellular Vesicles during Acute Cellular Rejection

Abstract

Purpose Non-invasive tests for the diagnosis of acute cellular rejection (ACR) represents an unmet need in the surveillance of cardiac allograft recipients. Extracellular vesicles (EVs) are sub-micron, membrane-enclosed particles released into the circulation upon tissue injury or stress, and T-cell derived EVs have been shown to play a role in ACR pathophysiology. The molecular cargo of EVs are thought to reflect the state of their cell of origin and are therefore considered a potential source of biomarkers. The aim of this study was to profile immune-related proteins in isolated EVs from cardiac transplantation patients to identify novel biomarkers for ACR. Methods Plasma was collected serially from 8 cardiac allograft recipients before, during and after biopsy-proven ACR grade ≥2R (2 [25%] women, mean age 45.2 ±21.2). EVs were isolated from plasma using acoustic seed trapping. The levels of 92 low-abundant immune-related proteins were assayed in plasma and EV lysates using a panel of proximity extension assays. Proteins that were dysregulated during ACR were identified by repeated measures ANOVA, adjusting for multiple comparisons using a false discovery rate approach. The presence of EV proteins on leukocyte-, endothelial- and platelet-derived EVs was confirmed by co-staining of isolated EVs with antibodies specific for CD16, CD31 and CD41a, respectively, and analyzed by flow cytometry. Results 48 proteins were defined as detected (i.e. above the limit of detection in >50% of samples) in the EV lysates. The levels of EV-proteins were overall poorly correlated with those in plasma: only 16.7 % of EV-proteins showed a significant correlation between plasma and EV lysates. The mean levels of 20 EV-proteins were numerically elevated during ACR. One of these, TNF-related weak inducer of apoptosis (TWEAK), was significantly increased (q Conclusion The EV proteome is perturbed during ACR and differs substantially from that in corresponding plasma samples. The analysis of isolated EVs can provide additional diagnostic and pathophysiological information over plasma alone. TWEAK is a vesicle-associated protein with potential utility as a biomarker for ACR.