Background.Memory T cells are important mediators of transplant rejection but are not routinely measured before or after kidney transplantation. The aims of this study were as follows: (1) validate... Show moreBackground.Memory T cells are important mediators of transplant rejection but are not routinely measured before or after kidney transplantation. The aims of this study were as follows: (1) validate whether pretransplant donor-reactive memory T cells are reliable predictors of acute rejection (AR) (2) determine whether donor-reactive memory T cells can distinguish AR from other causes of transplant dysfunction. Methods.Samples from 103 consecutive kidney transplant recipients (2018-2019) were obtained pretransplantation and at time of for-cause biopsy sampling within 6 mo of transplantation. The number of donor-reactive interferon gamma (IFN-gamma) and interleukin (IL)-21-producing memory T cells was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay. Results.Of the 63 patients who underwent a biopsy, 25 had a biopsy-proven acute rejection (BPAR; 22 aTCMR and 3 aAMR), 19 had a presumed rejection, and 19 had no rejection. Receiver operating characteristic analysis showed that the pretransplant IFN-gamma ELISPOT assay distinguished between patients who later developed BPAR and patients who remained rejection-free (area under the curve [AUC] 0.73; sensitivity 96% and specificity 41%). Both the IFN-gamma and IL-21 assays were able to discriminate BPAR from other causes of transplant dysfunction (AUC 0.81; sensitivity 87% and specificity 76% and AUC 0.81; sensitivity 93% and specificity 68%, respectively). Conclusions.This study validates that a high number of donor-reactive memory T cells before transplantation is associated with the development of AR after transplantation. Furthermore, it demonstrates that the IFN-gamma and IL-21 ELISPOT assays are able to discriminate between patients with AR and patients without AR at the time of biopsy sampling. Show less
Background: Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. The characterization of EVs... Show moreBackground: Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. The characterization of EVs released by kidney grafts during normothermic machine perfusion (NMP) may present a promising avenue to assess graft status before transplantation. Methods: We phenotyped and determined the concentrations of EVs in the perfusate of 8 discarded expanded-criteria donor human Kidneys during 6h of NMP. Perfusate samples were taken at 0/60/180/360min and examined with nanoparticle tracking analysis and imaging flow cytometry (IFCM). Using IFCM, EVs were identified by their expression of common EV markers CD9, CD63, and CD81 (tetraspanins) in combination with either platelet endothelial cell adhesion molecule (CD31), pan-leukocyte protein (CD45), or carboxyfluorescein succiminidyl ester (CFSE) fluorescence. Results: Nanoparticle tracking analysis measurements revealed the release of nanoparticles <400nm into the perfusate during NMP. With IFCM, tetraspanin protein signatures of the released nanoparticles were characterized, and the majority (similar to 75%) of CFSE+ EVs were found to be CD81+, whereas similar to 16% were CD9+ and similar to 8% CD63+. Correlation analysis of concentrations of identified EV subsets with crude donor characteristics and NMP viability characteristics revealed significant correlations with cold ischemia time, donor age, and renal flow. Conclusions: Our findings demonstrate that discarded expanded-criteria donor kidney grafts release distinct EV subsets during NMP. Because these subsets correlate with well-established indicators of transplant outcome, EVs might represent new potential candidates for assessment of kidney graft quality. Show less
Boer, K.; Baan, C.C.; Donk, N. van; Jonge, E. de; Clahsen-van Groningen, M.C.; Hesselink, D.A.; Schaik, R.H.N. van 2018
