Novel approaches in complement profiling; application in kidney transplantation

DiscussionIn the Eurotransplant region, ten-years-death-censored graft survival is around 75-80% forpatients receiving a kidney from a living donor and 65-70% for patients receiving a kidneyfrom a deceased donor.1 As the complement system plays an important role in the cause ofacute and chronic kidney allograft failure, we explored in this thesis the pathomechanisms ofthe complement system in kidney transplant recipients, focusing on both systemic and renalcellular complement activation. We showed the importance of novel complement assays todetermine the complement profi le, screen for the optimum choice of complement inhibitionand monitoring patients upon treatment.PROTEINURIA-DRIVEN, ALTERNATIVE PATHWAY ACTIVATION IN KIDNEY TRANSPLANTRECIPIENTSUrinary complement markers and kidney transplantationIn Chapter 2 of this thesis, we have shown the presence of properdin and soluble (s)C5b-9 in the urine of kidney transplant recipients (KTR), and hypothesized that this could indicateinvolvement of the alternative complement pathway (AP) in chronic allograft failure. Inclinical practice, surveillance of allograft health after transplantation relies on invasive biopsyprocedures that enable pathologic assessment of graft failure and rejection.2–6 In addition,current noninvasive methods to assess graft function and rejection are primarily based on serumcreatinine and urinary protein measurements.7 These markers are unspecifi c for rejection andtherefore numerous urinary and serum biomarkers have been evaluated as potential alternativerejection biomarkers.8–12 Both clinical and experimental work showed a critical contribution ofthe alternative pathway (AP) in the pathogenesis of several renal diseases. Literature has shownthat properdin is an important mediator in proteinuria induced tubular epithelial damage.13,14 Wethus hypothesized that properdin and/or the membrane attack complex; C5b-9, are present inthe urine of kidney transplant patients with proteinuria,15,16 and may have an important role inthe proinfl ammatory and pro-fi brotic eff ects that directly contribute to chronic tubulo-interstitialdamage and graft loss. Next to showing the presence of properdin and sC5b-9 in the urine of KTR,we furthermore assessed the potential of urinary properdin and sC5b-9 as novel biomarkers forchronic allograft failure. Indeed, we could show that graft survival is reduced in kidney transplantpatients in whom properdin and sC5b-9 are detectable in urine. This association was independentof proteinuria and kidney function. Also in patients without overt proteinuria, we showed thatproperdin, sC5b-9 or both properdin and sC5b-9, were associated with worse graft survival.Interestingly, the prevalence of both death censored and overall graft failure were signifi cantlyhigher in the patients with the combination of both properdin and sC5b-9 present in the urine.More importantly, not only the presence of properdin and sC5b-9 was signifi cantly associatedwith graft failure, but both factors were robustly associated with graft failure when analyzedas continuous parameters, pointing toward a dose-dependent eff ect. It could be possible thatlocally produced properdin or fi ltered properdin with other small complement componentscause intratubular complement activation, that leads to progressive transplant failure even inthe absence of manifest proteinuria.17 It is generally believed that small amounts of proteinuria,General discussion and future perspectives8577501-L-bw-LammertsProcessed on: 18-5-2022 PDF page: 202202defined as proteinuria <0.5g/24h, are harmless in KTR.18,19 According to the American Society ofTransplantation guidelines, it is the persistent proteinuria of >0.5g/24h for at least 3-6 months thatis considered significant. However low-grade proteinuria, that is often referred to as ‘subclinicalproteinuria’, might be less harmless than originally described.20 Also within the low-gradeproteinuria KTR group, it has already been shown that with every 0.1g/24h increase in proteinuriathe risk for graft loss rises with 25%.21 Our observation that adjustment for proteinuria, definedas >0.5 g/24h did not alter the prospective association of properdin and sC5b-9 measurements,supports our hypothesis that AP complement activation could be an important driving forceof chronic graft failure. Urinary properdin and sC5b-9 might be novel noninvasive biomarkersthat could add important supplemental information for treatable complement mediated injuryprocesses when compared to proteinuria alone.Alternative pathway complement activation on proximal tubular epithelial cellsIn recent years a debate has emerged on whether properdin, as the single positive regulator ofthe AP, also functions as a pattern recognition molecule by direct binding to specific surfaces andthereby forming an initiation site of AP activation. Epithelial cells in the renal tubules, that barelyexpress complement regulatory proteins, may be particularly susceptible for properdin patternrecognition, and this could be followed by complement activation during proteinuria after kidneytransplantation, leading to allograft failure. Our group revealed that, under proteinuric conditions,properdin binds to heparan sulphate proteoglycans on proximal tubular epithelial cells (PTECs).These studies also revealed that the properdin binding to HSPGs occurs via the heparan sulphateglycosaminoglycan side chains and that the binding of properdin to PTECs was significantlydifferent from binding to endothelial cells.22,23 However, Harboe et al. showed that properdinbinding to endothelial cells is dependent on the initial binding of C3b.24 We thus questionedwhether prior deposition of C3b is also necessary for properdin binding to PTEC, as this wouldessentially direct therapeutic approaches targeting complement activation in the tubules.In Chapter 3 we investigated the mechanism of properdin mediated complement activation onthe tubular epithelium. We identified and characterized molecular structures on PTECs that areable to interact with properdin. We demonstrated that both serum-derived and recombinantproperdin bind to PTECs and showed that binding of properdin to PTECs is independent of priorC3b deposition. Syndecan-1 is one of the most important heparan sulfate proteoglycans, andby utilizing syndecan-1 knockout PTECs, we showed that properdin binding largely dependson syndecan-1. This binding can be dose-dependently inhibited by Salp20 in vitro, but not withthe C3 inhibitor Compstatin, showing direct binding of properdin to the tubular surface. Onthe contrary, subsequent C3 activation via the AP was effectively blocked by Compstatin andalso Salp20. Heparin(oids) and Salp20 could also block the binding of properdin to C3b. Salp20mediated blocking of both the binding of C3b and HSPGs to properdin suggests that C3b, HSPGsand Salp20 share the same binding epitope on properdin. However, others have shown that theepitopes for sulfated glycoconjugates and C3b on properdin are positioned very close to eachother, but are not the same. These studies showed that C3b and HSPG binding is dependent ontrombospondin type I repeats (TSR 4 & 5) in the properdin monomer, but that trypsinisation ofproperdin resulted in a cleaved TSR5 domain, abolishing C3b binding while still allowing sulfatedChapter 8577501-L-bw-LammertsProcessed on: 18-5-2022 PDF page: 203203glycoconjugates to bind.25,26 In addition, van den Bos et al. recently showed that C3b binding toproperdin occurs mainly via the TSR5 domain.27 Yet, Salp20 blocks both C3b and HSPGs bindingto properdin. All in all, data suggests that the binding site for C3b and glycosaminoglycans onproperdin is probably diff erent, although very close, insinuating glycosaminoglycan binding toproperdin via TSR4 and C3b via TSR5.AP activation is involved in many proteinuric renal diseases, as described in the introduction ofthis thesis. The dual eff ects of Salp20 could be a useful way to eff ectively block alternative pathwayactivation in proteinuric diseases. The applicability of Salp20 has already been demonstrated inpre-clinical setting by reducing AP activation and subsequent injury in elastase induced aorticaneurysms and Ovalbumine-induced asthma.28 When aiming at going towards an ultimateintroduction of Salp20 in clinical practice, the immunogenicity of Salp20 has to be addressedafter which the overall clinical safety and effi cacy of this strong inhibitor should be tested in futuretrials.ANTIBODY-MEDIATED COMPLEMENT ACTIVATION IN KIDNEY TRANSPLANT RECIPIENTSPrimary renal endothelial cells to study antibody-mediated complement activationAs endothelial cells (ECs) are the main players in vascular homeostasis and infl ammation, and arealigned in the inner layer of the blood vessel, they are also a direct alloimmune target in kidneytransplantation. In order to be able to study ECs from the vasculature of the kidney, we developedin a renal EC isolation technique. In Chapter 4 we showed that primary renal microvascular andglomerular EC can realistically be derived from numerous donors with a wide variety of bloodgroup and human leukocyte antigen (HLA)-types. We developed a straightforward procedure toisolate machine perfusion derived primary renal endothelial cells (MP-PRECs) from the perfusionfl uid of human donor kidneys by a combination of negative selection of monocytes/macrophages,positive selection by CD31 Dynabeads and propagation in endothelial specifi c culture medium.At the site of the endothelium, the immunogenic reactions are mediated by both innate andadaptive immune mechanisms. HLA antibodies can recognize and bind to endothelial cells andsubsequently activate the classical route of the complement cascade. As an additional result ofcomplement activation by these donor specifi c antibodies (DSAs), the cellular immune responseis stimulated by directly activating antigen-presenting cells, including monocytes and T-cells.29–31The recognition of the detrimental role of HLA antibodies was one of the most importantadvances in transplantation medicine.32,33 The alloimmune response mediated by HLA antibodiesplays a key role in the failure of kidney allografts.33 However, there is a wide spectrum of graft injuryrelated to these antibodies, ranging from fl orid rejection to no recognizable damage.34,35 Thesevariations in pathogenic potential of anti-HLA DSAs highlight the need to carefully characterizethe pathogenicity of DSAs and their interaction with the donor endothelium after transplantationin a nuanced manner. A major advantage of isolating MP-PREC is that the HLA typing and bloodgroup of the EC donor is known, saving time and costs before cross-matching can start. CD31Dynabeads were used to select the endothelial cells from the perfusate, as CD31 is known to bea general endothelial cell marker.36 For further endothelial phenotyping, also Tie2/TEK and CD34were selected as general endothelium-resticted markers.36,37 vWF was selected as a macrovascularGeneral discussion and future perspectives8577501-L-bw-LammertsProcessed on: 18-5-2022 PDF page: 204204marker, VEGFR-2 as a glomerular and peritubular microvascular marker, PV-1 as a peritubularmicrovascular marker, and podoplanin was selected as a lymphatic endothelial marker.38–42 TheMP-PRECs were positive for endothelial cell markers from these different vascular compartmentsof the kidney; they were positive for CD31, von Willebrand Factor, CD34, VEGFR-2, Tie2/TEK,and PV-1, all to variable extents. HLA class I was constitutively expressed, and HLA class II eitherconstitutively expressed or could be induced by IFN-γ. These differences in EC markers betweendifferent donors could be due to alterations in culture composition, indicating endothelial celldedifferentiation over time or, alternatively, overgrowth of MP-PRECs from a specific segmentover time.43,44 We also found that the distribution of vWF, VEGFR-2, PV-1, and HLA-DR within oneculture varied for each donor. This likely reflects cultured MP-PRECs obtained from different renalmicrovasculature structures, having an arterial, glomerular, peritubular, or venous origin.36,39–41The MP-PREC biobank could therefore be a rich source for creating different immortalized renalendothelial cells, which differ in phenotype. However, it is important to realize that the usefulnessof the MP-PRECs could be limited by shifts in their in vivo gene expression signature. This couldbe due to the loss of micro-environmental cues and by the early onset of senescence.45,46 In thefuture, we should select the MP-PRECs based on their gene expression profile, in the context ofthe application of interest. In line with other modified cell lines, the immortalized cell lines shouldthen be validated thoroughly.47As explained in the introduction of this thesis, due to the lack of proper and reliablesources of renal EC, the ability to properly investigate (antibody-mediated) rejection at thesite of the endothelium is still limited.48–51 Consequently, antibody-mediated rejection (ABMR)remains a diagnostic and therapeutic challenge. In Chapter 4 we additionally showed potentialdiagnostic value of a renal endothelial biobank in a kidney specific endothelial cross match test.This allows us to study the pathogenetic role of various types of DSAs (anti-HLA I, anti-HLA II, andanti-endothelial antibodies on complement mediated endothelial cell injury and/or death. Wepostulate that the MP-PRECs form an attractive basis for the development of assays to assess thepathogenicity of HLA antibodies and detect non-HLA antibodies against the renal endothelium.HLA and non-HLA antibodies in kidney transplantationApproximately 30% of the patients on the waiting list for a kidney transplant in the Netherlandsare sensitized against alloantigens. Allogenic sensitization is defined as a cumulative panelreactive antibodies (PRA) percentage >0%.52 Sensitizing events leading to the formation ofantibodies against HLA are pregnancies, blood transfusions, and previous transplantations.53Serum of patients on the waiting list is regularly screened for antibodies by the HLA antibodyscreening assays and with the classic complement dependent cytotoxicity (CDC) cross match.Sensitized transplant candidates may experience prolonged waiting times,54 as the presence ofthese pre-transplant HLA antibodies leads to exclusion of donors with the antigens to which theantibodies are directed. In addition to this, patients can form antibodies against targets otherthan HLA.55 In Chapter 5 we utilized the primary renal endothelial cells (MP-PRECs) described inChapter 4 to perform EC crossmatch studies. In Chapter 5, we describe the accelerated rejectionof a blood group compatible, living related donor kidney, in the absence of DSA pre- or post-Chapter 8577501-L-bw-LammertsProcessed on: 18-5-2022 PDF page: 205205transplantation. The biopsy taken during the rejection episode showed features of C4d negativeABMR including extensive hemorrhagic areas and loss of renal vascular ECs. Anti-endothelial cellantibodies (AECAs) were identifi ed in crossmatch assays using MP-PRECs from various donors.Two years later, after desensitization for presumed AECA with plasmapheresis and rituximab, asuccessful second transplantation was performed. The success of the desensitization procedurecould be monitored with our MP-PREC cross-match assay. The results suggest that the patient hadantibodies against ECs and that eff ective reduction of complement mediated renal endothelialcytotoxicity upon treatment before transplantation can be monitored. Multiple cases of acuteallograft dysfunction with histological signs of ABMR but without (detectable) circulating DSAshave been described.56–58 This phenomenon has partly been addressed in a cohort study of 935transplantations.59 In this cohort, 208 patients who underwent a biopsy for a specifi c indicationor patients who underwent protocol biopsies met the histological criteria of ABMR the accordingto Banff 2015 and 2017 classifi cation.4,5 However, it was found that 59% of the patients hadno detectable DSA at time of biopsy. Unlike for HLA-antibodies, it not known yet if there is adistinct relation with (classical) sensitizing events and non-HLA-antibody formation.60 Also, theseantibodies are also known to occur in healthy individuals.61,62 It could very well be that due tograft injury induced by ischemia reperfusion injury and DSA binding to the donor endothelium orchronic infl ammation results in the exposure of cryptic antigens and formation of neoantigens.63,64This suggests that, as sensitizing mechanism, non-HLA antibodies can be considered to arise denovo early post-transplantation or following acute rejection, or as autoantibodies that bind selfantigens.63,65 As the allograft vascular endothelium represents the fi rst contact surface betweenthe recipient’s immune system and the donor organ, a signifi cant proportion of non-HLAantibodies that are associated with allograft rejection are directed against antigens expressedby endothelial cells. However, also antigens expressed on the underlying basement membraneand the extracellular matrix of the endothelial cells that may only be expressed under certaincircumstances related to allograft injury, may be targeted.60,66Current immunological risk stratifi cation for patients awaiting a kidney transplant isbased on the detection of circulating HLA antibodies reacting with lymphocytes. Althoughlymphocytes have HLA antigen expression in common with the graft, they have a diff erentialrepertoire of cell surface antigens as compared to endothelial cells. A well-known example isthe absence of blood group antigens on lymphocytes while these are present on endothelialcells. Unfortunately, despite the aforementioned increasing evidence for these antibodies to beinvolved in rejection, screening for non-HLA anti-endothelial antibodies (AECA) has not yet beenimplemented in clinical practice. Our report illustrates the need for further studies evaluating indepth the mechanisms leading to hyperacute, acute and late rejection caused by AECAs.In Chapter 6 we review the literature and report current non-HLA antibodies assaysand provide an overview of non-HLA crossmatch assays developed for use in solid organtransplantation, either in a research setting or commercially. The term non-HLA antibody intransplantation covers auto-reactive and allo-reactive antibodies, specifi c for targets other thanHLA. However as said, the nature of these antibodies is still largely unknown, and to date noGeneral discussion and future perspectives8577501-L-bw-LammertsProcessed on: 18-5-2022 PDF page: 206206widely accepted assays exist. Immune triggers that lead to non-HLA antibody formation andpathogenicity are complex and poorly understood.57,60,67 The ability of non-HLA antibodies tomediate allograft injury may depend upon their affinity and strength (titer), target specificity,density of the target antigen, and synergy with donor-specific HLA antibodies. The discovery ofnew targets against which non-HLA antibodies develop, is ongoing.68–70 Although several non-HLAantibody specificities have been identified, the current available assays are limited by identifyingonly the known non-HLA antibodies.71,72 It seems likely that not all relevant antigens are includedin the current available assays, underlining the need for solid in vitro crossmatch assays that aidin studying these differences.72 Efforts to develop reliable and sensitive diagnostic non-HLAantibody tests are continuously made. This is essential, considering the technical difficulties ofnon-HLA antibody assays and the large variation in reported incidences of antibodies, resulting inhighly heterogenic study outcomes. However, despite the heterogenic study designs and partlyconflicting results, the clinical relevance of non-HLA on graft survival should not be neglected. Inorder to be able to fully elucidate on the clinical relevance of non-HLA antibodies, future studiesshould harmonize and validate the existing non-HLA assays, in addition to a rigorous step-by-stepscientific process to identify and test for new and relevant non-HLA antibodies.Profiling systemic and renal complement activation in patients with renal transplantationand antibody-mediated rejectionDespite the fact that the histopathological heterogeneity of ABMR is increasingly acknowledgedand reflected in diagnostic standards, pathomechanistic models to comprehensively addressthis heterogeneity are missing.2,73 To be more precise, the role of complement activation in thedevelopment of ABMR is insufficiently understood. In Chapter 7 we analyzed systemic andlocal complement activation in kidney transplant recipients with ABMR, cellular rejection, andwithout rejection. Compared to the patients experiencing cellular rejection or without rejection,plasma complement activation was not increased in ABMR patients. On a local level, only C4dand C3d, but not C5b-9 depositions, were more pronounced in the biopsies from patients withABMR. In the in vitro renal EC assay similar to the one described in Chapter 4 and Chapter 5,we replicated these findings and showed classical complement pathway activation by C4d andC3d deposition, but minor C5b-9 deposition. The kidney endothelium appeared to be effectivelyprotected against C5b-9 activation, although this protection does not seem to prevent ABMR. Inorder to be able to investigate the prognostic value of local C3d for graft failure in ABMR, it wouldbe interesting to expand the number of biopsies and to extend the follow up period. Others havealready indicated that C3d might be valuable in KTR with rejection though this has not beenthoroughly studied to date.74 Initial analysis on our small ABMR cohort is promising, showingperitubular C3d deposition linked to graft failure.The presence of complement regulatory proteins like CD46, CD55 or CD59 on theendothelial cell membrane might account for the absence of C5b-9 in ABMR patients.75 To testthis hypothesis, we investigated the presence of CD59 in the biopsies of ABMR patients anddeposition of CD59 on endothelial cells in vitro. We found a constitutive expression of CD59 invivo and in pre-transplant biopsies in vitro on renal ECs. Surprisingly, post-transplantation no CD59was found in ABMR biopsies. This finding needs further exploration, but could possibly be due toChapter 8577501-L-bw-LammertsProcessed on: 18-5-2022 PDF page: 207207internalization of CD59/C9 complexes.76 Earliest research on immunohistopathology of ABMR byFeucht et al. in 1993 revealed that proximal depositions of complement activation products C4dand C3d were present in a kidney biopsy with ABMR, yet no convincingly terminal complementactivation was found. Based on the fi ndings by Feucht et al. and confi rmations by followingresearch, C4d was established as a robust and sensitive marker for ABMR. Guidelines started tobe developed stating that C4d needed to be present for diagnosis of ABMR. In addition to that,the C5 complement inhibitor eculizumab, that proved to be eff ective in complement drivendiseases like aHUS, started to be investigated in trials with patients with for ABMR.77–79 Some casestudies describe impressive therapeutic eff ectiveness in ABMR with eculizumab.80–82 However,in recent years more studies describe cases in which patients are highly suspected of havingABMR based on the morphology of the rejected transplant, but no C4d staining is present. Thediagnosis C4d-negative ABMR emerged and also the most recent Banff diagnostic criteria wasadapted. In addition to that, Nishi et al. reported the absence of co-deposition of C4d and C5b-9in peritubular capillaries in kidney transplant recipients with acute rejection.83 Moreover, studiesshowing disappointing results with treatment with eculizumab started to emerge.84,85 Thesefacts place a question mark behind the universal role of complement in ABMR. The results fromChapter 7 indicate that terminal complement pathway activation is probably not central in thepathogenesis of ABMR. Future studies should, therefore, examine whether complement regulationindeed plays an important role in the lack of involvement of C5b-9 during in ABMR. A possibleexplanation that needs further exploration would be the development of accommodation, anadaptation of the graft resulting in resistance against the acute pathogenic eff ects of DSAs andthe fi xation of complement,86,87 that is primarily recognized in AB0-incompatible transplantation.Biopsies from AB0-incompatible transplants regularly show C4d deposition without other signsof rejection, suggesting regulatory mechanisms to prevent terminal complement activation.87 InAB0-incompatible transplantation, the endothelium appears to become resistant to blood groupantibody-induced complement-mediated lysis by upregulating its expression of cytoprotectiveproteins, including complement regulatory proteins CD55 and CD59.88,89 Our data on CD59 in vivoand on glomerular endothelial cells in vitro might point to a similar involvement of complementregulation in ABMR. Other processes that potentially limit host EC injury by complement, couldbe clearance of complement by ECs, (with or without the antibody)90 or exocytosis/endocytosis,as is described for C5b-9 on neutrophils.91In addition, to the accommodation theory, our data presented in Chapter 7 could alsosuggest that complement independent mechanisms might account for the heterogeneouspicture in ABMR, and thus explain why patients with ABMR do not always benefi t from complementinhibition.72,77,78,92,93 Complement-independent pathomechanisms via Fc-receptors (FcR) could beinvolved in ABMR; a mechanism referred to as antibody-dependent cellular cytotoxicity (ADCC).Accumulating evidence suggests that natural killer (NK) cells are important mediators of ADCCin ABMR. Experimental models as well as analysis of clinical biopsies have shown that NK cells areincreased in ABMR and that their activation status is increased when patients have DSA.31,94–97 Inaddition, NK cell depleted mice show a reduced incidence of acute ABMR, however these micestill develop chronic ABMR.98,99 Taken together, multiple parallel mechanisms of EC activationcould contribute to the development of acute ABMR and chronic ABMR.General discussion and future perspectives8577501-L-bw-LammertsProcessed on: 18-5-2022 PDF page: 208208Future perspectivesIn this thesis we focused on the potential mechanism of tubular complement activation in theproteinuric setting and we showed the potential of heparins and Salp20 in inhibiting properdinbinding to proximal tubular epithelial cells, which might result in reducing alternative pathwaymediated renal tubular injury. Since the reduction of proteinuria is a major therapeutic goal inreducing the risk for progression of renal injury, it is very interesting that Salp20 can inhibit boththe binding of C3b and HSPG to properdin. This bimodal inhibiting effect should be furtherexplored for developing an AP-blocking agent for proteinuric patients. The focus should be onconstructing non-immunogenic analogues and on determining the exact properdin-bindingepitope. The latter could be tested in vitro and in experimental (animal) models for their APinhibitingpotential. With regard to heparins, it is interesting to study whether non-anticoagulantheparins or related glycomimetics can inhibit complement activation on tubular cells and reduceinjury , as for instance in experimental proteinuria models.The results presented in this thesis support the interpretation that C5b-9 plays a relativelyminor role in most cases of ABMR compared to proximal complement components, consistentwith the failure of the C5 inhibitor Eculizumab to prevent and resolve ABMR. We also presenteddata suggesting that CD59 expression on renal endothelial cells may play an important rolein limiting C5b-9 formation in ABMR. After AB0-incompatible transplantation, endothelial cellsappear to become more resistant against antibody-induced complement-mediated cell lysisthrough upregulation in the expression of cytoprotective proteins like complement regulatoryproteins CD55 and CD59. These findings support the concept that the capacity of donororgan cells to regulate complement can influence the susceptibility to antibody-mediated andcomplement-dependent allograft injury.Whilst there are differences in the characteristics of HLA- and AB0-antibodies, and desensitizationtherapy in HLA-incompatible transplantation seems to be less effective when compared to AB0-incompatible transplantation, the hypothesis of the occurrence of some form of accommodationin the context of HLA-DSAs explaining the variance of damage associated with circulating HLAatibodies is intriguing. As we also developed a robust method to isolate primary renal endothelialcells, it will be interesting to perform experiments utilizing these renal endothelial cells, in whichwe expose endothelial cells to AB0i and HLAi serum and measure complement regulation anddamage markers.In addition to complement regulation, ADCC as a complement-independent mechanismmight be an important mediator in the development of ABMR without the presence of C5b-9.Apparently complement activation is not detrimental in all cases and it is important to definethe conditions for complement-dependent and complement-independent antibody-mediatedrejection. Quality and quantity of involved antibodies might be pivotal in this context. From crossmatching comparison studies it appears that it is both the nature of the heterogenous mixturesof antibodies and the particular set of antigens on the target cell that mainly determines theeffectiveness of antibody-antigen binding and thus further immune system activation. The renalChapter 8577501-L-bw-LammertsProcessed on: 18-5-2022 PDF page: 209209endothelial cell bank (Chapter 6), covering the majority of known and probably also unknownHLA and non-HLA polymorphisms, could help us in unraveling the pathogenic eff ects of variousantibodies and their regulation on a cellular level. In the near future, further development of theMP-PREC crossmatch assay into routinely applicable assays is necessary. In order to achieve this,the primary cells need to be immortalized and characterized in depth. Additionally, the MP-PRECsmight also facilitate the investigation of a broad array of non-HLA polymorphisms. As patientsmay have a mixture of HLA and non HLA antibodies immortalized primary ECs need to be madedevoid of endogenous HLA expression.The EC crossmatch test could add important value to the recent immunological risk markersand could, after thorough validation, also clinically be relevant to predict or diagnose rejection.Integration of the more traditional risk markers like (pe-) transplant antibody profi le, recipientage and HLA mismatches with other, newer, risk markers may result in a more personalized andcomplete risk profi le. This risk profi le can then be used for more precise donor-recipient matching,posttransplant surveillance and assess personalized immunosuppression adequacy (Figure 1).In addition, the MP-PRECs might aid in further in vitro studies to understand the mechanismsleading to hyperacute, acute and late rejection caused by HLA and non-HLA antibodies on anendothelial level.

Dissertation: http://hdl.handle.net/11370/5b145c0f-7b27-4f7f-892b-aaba77dbf9d4