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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 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 6 h of NMP. Perfusate samples were taken at 0/60/180/360 min 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 <400 nm into the perfusate during NMP. With IFCM, tetraspanin protein signatures of the released nanoparticles were characterized, and the majority (~75%) of CFSE+ EVs were found to be CD81+, whereas ~16% were CD9+ and ~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.
BackgroundThe gap between demand and supply of kidneys for transplantation necessitates the use of kidneys from extended criteria donors. Transplantation of these donor kidneys is associated with inferior results, reflected by an increased risk of delayed graft function. Inferior results might be explained by the higher immunogenicity of extended criteria donor kidneys. Normothermic machine perfusion (NMP) could be used as a platform to assess the quality and function of donor kidneys. In addition, it could be useful to evaluate and possibly alter the immunological response of donor kidneys. In this study, we first evaluated whether complement was activated during NMP of porcine and human discarded kidneys. Second, we examined the relationship between complement activation and pro-inflammatory cytokines during NMP. Third, we assessed the effect of complement activation on renal function and injury during NMP of porcine kidneys. Lastly, we examined local complement C3d deposition in human renal biopsies after NMP.MethodsNMP with a blood-based perfusion was performed with both porcine and discarded human kidneys for 4 and 6 h, respectively. Perfusate samples were taken every hour to assess complement activation, pro-inflammatory cytokines and renal function. Biopsies were taken to assess histological injury and complement deposition.ResultsComplement activation products C3a, C3d, and soluble C5b-9 (sC5b-9) were found in perfusate samples taken during NMP of both porcine and human kidneys. In addition, complement perfusate levels positively correlated with the cytokine perfusate levels of IL-6, IL-8, and TNF during NMP of porcine kidneys. Porcine kidneys with high sC5b-9 perfusate levels had significantly lower creatinine clearance after 4 h of NMP. In line with these findings, high complement perfusate levels were seen during NMP of human discarded kidneys. In addition, kidneys retrieved from brain-dead donors had significantly higher complement perfusate levels during NMP than kidneys retrieved from donors after circulatory death.ConclusionNormothermic kidney machine perfusion induces complement activation in porcine and human kidneys, which is associated with the release of pro-inflammatory cytokines and in porcine kidneys with lower creatinine clearance. Complement inhibition during NMP might be a promising strategy to reduce renal graft injury and improve graft function prior to transplantation.
Background: Extracellular Vesicles (EVs) represent stable, tissue specific nano-sized particles that reflect the conditional state of their tissue of origin.Here, the dynamic release and phenotype of kidney EVs was characterized and quantified during Normothermic Machine Perfusion (NMP) of Expanded-Criteria Donor (ECD) kidneys to examine whether EVs could function as a potential biomarker for assessing kidney quality before transplantation. Materials & Methods: Eight discarded ECD kidneys (~13 ± 5 hours of cold ischemia, age 68 ± 7 (mean ± standard deviation), all male) were perfused in a closed system at 37 0 C for 6 hours. Perfusates were taken before and at 1, 3 and 6 hours of NMP and examined with Nanoparticle Tracking Analysis (NTA) and Imaging Flow Cytometry (IFCM). For IFCM, perfusates were stained with the tetraspanins CD9, CD63 or CD81 (general EV markers), or a mix of these three markers in combination with CFDA-SE (a non-fluorescent molecule that acquires fluorescent properties after cleavage by intravesicular esterases) to identify, quantify and characterize EVs. Results: Analysis of perfusates with NTA revealed that the majority of nanoparticles present in the perfusates are <300 nm. For CFSE and the mix of tetraspanin double-positive EVs, we observed a ~700/ 740/ 560 fold increase compared to EV levels before perfusion at 1, 3 and 6 hours of NMP, respectively. Analysis of EV concentrations with crude donor characteristics (e.g. age, cold ischemia time (CIT), kidney weight) and NMP viability characteristics (renal flow, renal flow resistance, urine production) revealed that doublepositive EV are negatively correlated with CIT whilst positive correlations were found with donor age after the first hour of NMP. Furthermore, tetraspanin CD81 was found to represent the majority (~75%) of the excreted doublepositive EV (CD9: ~16%/ CD63 ~8%) (Figure). Conclusion: EVs <300 nm are released by ECD kidneys during NMP with highest excretion levels during the first hour of perfusion. Tetraspanin CD81 is predominantly present on these EVs, and EV concentrations were shown to be correlated with well-established indicators of kidney quality such as donor age and CIT. The characterization of the excreted EVs as well as their correlation with clinical parameters provide a starting point to study their role as potential biomarkers of kidney quality.
Introduction:We have demonstrated the therapeutic potential of human polyclonal and antigen-specific CAR-modified CD8 + Tregs cell therapy to prevent allogeneic human skin transplantation rejection and xenogeneic GvHD in humanized NSG mice (Bézie et al., Front Immunol.2017, Blood Adv.2019). However, their potential has never been evaluated in a clinical trial. We are thus preparing the launch of a phase I/IIa human clinical trial using polyclonal CD8 + Tregs cell therapy in living donors kidney transplant patients. Method: CD8 + cells were isolated from blood of healthy volunteers and patients with kidney failure by Clinimacs System then CD8+CD4-CD45RClow/-CD56 -T cells were sorted by MACSQuant Tyto cell sorter. Cells were stimulated with anti-CD3 and CD28 mAbs every week and cultured in presence of rapamycin, IL-2 and IL-15. Cytotoxicity against allogeneic PBMCs was assessed by Annexin V/DAPI staining and suppression capacity was assessed in vitro on syngeneic CD4 + T cells proliferation in response to allogeneic APCs and in vivo on 1.5Gy-irradiated NSG mice coinjected with human PBMCs. Results: We developed a new GMP-compatible cell manufacturing process. First, we determined a new method of isolation of CD8 + Tregs from peripheral blood using a safe closed system. Next, we identified the optimal clinically compatible culture medium, refined cytokines doses and stimulation methods to preserve a high proliferation rate, phenotypic profile and suppressive function of CD8 + Tregs. Using this process, we were able to efficiently expand CD8 + Tregs from peripheral blood of patients with renal failure, with high purity, while preserving their regulatory function. We confirmed the absence of cytotoxicity. In addition, we showed that CD8 + Tregs were phenotypically and functionally stable for 4h after conditioning, which is important for the logistic delay between cell harvest and patient infusion. Finally, we showed that CD8 + Tregs persist for more than 90 days in NSG mice without inducing any signs of xenogeneic GVHD, and could withstand combined immunosuppressive drug therapy administered to the patient. Conclusion: We designed a clinically compatible manufacturing process to isolate and culture CD8 + Tregs from renal failure patients that preserves their function and patient safety.
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