Chapter 6 164 after transplantation but were unable to detect dd-EVs above pre-transplantation signals in KTRs who underwent a ‘for-cause’ biopsy. Mechanistically, it is currently unknown whether the lower concentrations of ddEVs during allograft dysfunction is a consequence of decreased production of EVs by the allograft, increased consumption of dd-EVs by recipient immune cells, or a combination of both 24. Donor exosomes have been shown to be involved in donor antigen presentation to recipient alloreactive T cells in lymphoid organs by the recipient dendritic cells in a phenomenon known as cross-dressing 35, 37-39. As Habertheuer et al. suggested, this mechanism of T cell activation may suppress production of exosomes by the transplanted tissue even before there is targeted injury to the allograft 24. At any rate, the detection of dd-EVs can be associated with stable allograft function – which is in contrast to the detection of e.g. donorderived cell-free DNA, which has been observed to increase in concentration as a consequence of allograft damage 40. In conclusion, our calibrated IFCM-based methodology to directly detect and characterize plasma-derived EV subsets is applicable in patient human plasma samples. We believe that this methodology – after validation of markers of interest – could boost the EV biomarker research in a variety of clinical contexts, of which monitoring of kidney transplant integrity appears especially promising. MATERIALS & METHODS Clinical sample selection To analyze distinct donor-derived EVs (dd-EVs) in KTR plasma samples, 36 donorKTR couples were selected on the basis of an HLA-A3 mismatch between donors (HLA-A3+) and KTRs (HLA-A3-). KTRs had not received a previous HLA-A3+ graft. All donor-KTR couples participated in an observational study which aimed to identify minimally invasive biomarkers for the diagnosis of acute kidney transplant rejection, and was approved by the institutional review board of the Erasmus MC (Medical Ethical Review Board number 2018-035); details of this study are described elsewhere 40. All patients provided written informed consent. Blood samples from living donors were obtained before donation and collected as part of our ongoing Biobank program (Medical Ethical Review Board number 2010-022). These donors served as healthy controls. The study was conducted in accordance with the principles of the Declaration of Helsinki.
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