Rotterdam: Main port for organ transplantation research in the Netherlands

Transplant Immunology 31 (2014) 200–206

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Transplant Immunology journal homepage: www.elsevier.com/locate/trim

Rotterdam: Main port for organ transplantation research in the Netherlands Jaap Kwekkeboom a,⁎,1, Luc J.W. van der Laan b,1, Michiel G.H. Betjes c, Olivier C. Manintveld d, Rogier A.S. Hoek e, Karlien Cransberg f, Ron W.F. de Bruin b, Frank J.M.F. Dor b, Jeroen de Jonge b, Patrick P.C. Boor a, Rogier van Gent a, Nicole M. van Besouw c, Karin Boer c, Nicolle H.R. Litjens c, Dennis A. Hesselink c, Martin J. Hoogduijn c, Emma Massey c, Ajda T. Rowshani c, Jacqueline van de Wetering c, Huib de Jong f, Rudi W. Hendriks e, Herold J. Metselaar a, Teun van Gelder c,g, Willem Weimar c, Jan N.M. IJzermans b, Carla C. Baan c a

Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Centre, Rotterdam, The Netherlands Department of Surgery, Erasmus MC-University Medical Centre, Rotterdam, The Netherlands Department of Internal Medicine, Erasmus MC-University Medical Centre, Rotterdam, The Netherlands d Department of Cardiology, Erasmus MC-University Medical Centre, Rotterdam, The Netherlands e Department of Pulmonary Diseases, Erasmus MC-University Medical Centre, Rotterdam, The Netherlands f Department of Pediatric Nephrology, Erasmus MC-University Medical Centre, Rotterdam, The Netherlands g Department of Clinical Pharmacology, Erasmus MC-University Medical Centre, Rotterdam, The Netherlands b c

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Available online 18 September 2014 Keywords: Living donation MicroRNA Dietary restriction Mesenchymal stromal cell Regulatory T cell Immunosuppressive drug

a b s t r a c t This overview describes the full spectrum of current pre-clinical and clinical kidney-, liver-, heart- and lung transplantation research performed in Erasmus MC – University Medical Centre in Rotterdam, The Netherlands. An update is provided on the development of a large living donor kidney transplantation program and on optimization of kidney allocation, including the implementation of a domino kidney-donation program. Our current research efforts to optimize immunosuppressive regimens and find novel targets for immunosuppressive therapy, our recent studies on prevention of ischemia-reperfusion-induced graft injury, our newest findings on stimulation of tissue regeneration, our novel approaches to prevent rejection and viral infection, and our latest insights in the regulation of allograft rejection, are summarized. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The Erasmus University Medical Centre (Erasmus MC) in Rotterdam is the largest organ transplantation center in the Netherlands. Each year approximately 200 kidney transplantations, 60 liver transplantations, 20 heart transplantations, and 20 lung transplantations are performed. Supported by improved surgical techniques and post-transplantation clinical care, the patient- and graft-survival rates have improved significantly during the last three decades (Fig. 1). Unfortunately, the issue of donor organ shortage is yet to be resolved. While previously early graft dysfunction and acute rejection were of prime concern, the main current clinical problems are related to chronic graft damage and long-term patient survival. The half-life of

⁎ Corresponding author at: Erasmus MC-University Medical Centre, Department of Gastroenterology and Hepatology, P.O. Box 2040, Room Na-1009, 3000 CA Rotterdam, The Netherlands. Tel.: +31 10 7034776. E-mail address: [email protected] (J. Kwekkeboom). 1 These authors contributed equally.

http://dx.doi.org/10.1016/j.trim.2014.09.005 0966-3274/© 2014 Elsevier B.V. All rights reserved.

heart, lung and liver grafts and that of kidneys from deceased donors is about 10 years. The long-term complications after organ transplantation are caused by early determinants of the transplantation procedure such as ischemia–reperfusion injury (IRI) and delayed graft function, as well as events shortly after transplantation like acute rejection and recurrence of original disease, and by non-adherence to immunosuppressive medication or over-immunosuppression. To find solutions for all these issues, and thereby improve long-term graft and patient survival, an interlinked approach between clinic and laboratory and between different specializations is required to gain better understanding of the underlying processes. In addition, solutions for the current organ shortage are required. This overview of current research on organ transplantation in Rotterdam covers the full spectrum of our pre-clinical and clinical research programs, and includes the development of a successful living donor kidney transplantation program, optimization of graft allocation, prevention of ischemia–reperfusion induced graft injury, transplantation immunology, optimization of immunosuppression, and ethical and psychosocial studies. We believe that an integrated approach

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Fig. 1. Patient survival after heart transplantation in Erasmus MC according to time period (1984–1999 versus 2000–2013). Kaplan Meier curves for patient survival after heart transplantation. Patients transplanted after 2000 have a significantly better 10 year survival rate than patients transplanted in 1984–1999 (p = 0.02).

covering the whole spectrum of these subjects will provide future directions to solve the current problems in organ transplantation. 2. Optimizing living kidney donation 2.1. Establishment of a surgical protocol to optimize living kidney donation The Erasmus MC has the largest living kidney donor transplantation program in Europe, performing about 150 procedures annually. Until 1998 donor nephrectomies were performed via lumbotomy, an approach characterized by a lengthy hospital stay with discomfort and pain, and a long recovery before returning to work. With the introduction of minimally invasive techniques for live donation we optimized the surgical approach for living kidney donation. We explored both mini-incision and laparoscopic donor nephrectomy (LDN) in a randomized controlled trial (RCT). LDN was demonstrated to be safe, limit discomfort, shorten length of hospital stay, and enable faster recovery [1]. However, for surgeons starting a living program the laparoscopic technique was also found to have a moderate learning curve. Therefore, the hand-assistance approach was added to LDN in 2002 to benefit from the advantages of open surgery. A RCT was performed comparing handassisted retro-peritoneoscopic (HARP) versus LDN. HARP was proven to be safer, easier to learn and have comparable results to LDN [2,3], albeit with some inconvenience for surgeons during longer procedures [4]. The introduction of robotic surgery enabled evaluation of potential benefits of robot-assisted donor nephrectomy. In 2009 we found that the robotic approach with 3D vision, which offered better range of movement of instruments and ergonomics, had similar donor outcomes compared to 2D LDN. As the 3D facilities were highly appreciated by surgeons, we are currently evaluating 3D vision laparoscopy with conventional 2D-laparoscopy. 2.2. Overcoming barriers in living kidney donation Immunogenetic factors that influence the access to renal transplantation are ABO blood type and HLA type in combination with panel reactive antibodies [5,6]. Alternative living donation programs have been developed in order to circumvent donor-specific HLA antibodies or ABO incompatibility. The first alternative program, developed in 2004, was the national donor-exchange program, which facilitated transplantation of incompatible couples [5]. Second, we developed the domino-donation program, a combination of the unspecified donation

program and the donor-exchange program which further optimizes the chances for transplantation [7,8]. Between 2000 and 2013, the domino donation program of Erasmus MC enabled 135 kidney transplantations with 63 unspecified donors, an area that Erasmus MC has pioneered over the past 15 years [9,10]. Unspecified donors have made a sizeable contribution to our program and a total of 106 donors yielded 178 transplantations. Instead of circumventing immunological barriers, desensitization protocols can be employed to enable direct donation of ABO or HLA incompatible kidneys from living donors. The ABO-incompatible program was introduced in 2006 and includes on average 10 patients per year. Recently, we have evaluated the outcome of the ABOincompatible program and found similar renal graft survival compared to ABO-compatible transplantations, despite an early high rate of acute humoral rejection [11]. However, about 40% of complementdependent cytotoxicity-cross match positive donor-patient combinations cannot benefit from this alternative program. Therefore, we have recently introduced a national HLA densensitization program, which is currently recruiting patients.

2.3. Ethical and psychological aspects of living kidney donation As the mainstay of living kidney donation is donor safety and convenience on short, as well as long term, we are currently investigating the long-term physiological and psychological effects of live kidney donation. The search for a living donor is a pertinent issue that endstage renal disease patients struggle with. To meet the need for greater support, we have developed two highly successful home-based education programs for pre-dialysis and dialysis patients, respectively. These programs support shared, informed decision-making, and have been effective in increasing living donation rates. Justification of living donation can only be maintained if donor safety can be guaranteed. To this end, we are currently conducting retrospective and prospective studies on psychological outcomes of living donors. Together with our international collaborators within ELPAT, the Ethical Legal and Psychosocial Aspects of organ Transplantation section of ESOT, we are also expanding the knowledge and developing professional standards for psychological screening of living donors. Once patients receive a transplant, they are confronted with a strict, lifelong lifestyle and medication regime [12]. How to best support selfmanagement and medication adherence and thus improve transplant outcomes, is another crucial area that we focus on [13]. Finally, given

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the scarcity of organs, some patients travel abroad for transplantation. Erasmus MC leads an international research project on organ tourism and trafficking of persons for the purpose of organ removal subsidized by the European Union (www.hottproject.com). 3. Prevention of graft damage An important factor determining transplantation outcome is damage to the graft during procurement, cold storage, and after reperfusion in the patient. Moreover, the quality of grafts can be compromised due to unfavorable characteristics of the donor. With the increase of donor organ shortage there has been a move toward expanded criteria donor (ECD) donation and donation after circulatory death (DCD) as additional sources for donor organ grafts, which is however accompanied by complications after transplantation. We explore novel therapeutic strategies to overcome these problems and investigate whether biomarkers can predict the quality of donor organs. 3.1. Organ preservation by machine perfusion The current standard for organ preservation is simple cold storage. However, preservation time is limited because of the risk of early allograft dysfunction, which remains a serious concern because it is associated with higher morbidity and early re-transplantation rates, affecting long-term survival. In addition, simple cold storage is not well suited for “less than ideal” organs (e.g. from DCD, aged livers or steatotic livers), highlighting the need for better organ preservation methods. One such strategy is Hypothermic Oxygenated machine Perfusion (HOPE). Specially designed perfusion machines of two Dutch manufacturers generate a controlled recirculating flow that allows complete perfusion of the liver, and maintain oxygen supply to the cold organ. HOPE supports its capacity to synthesize ATP and results in less reactive oxygen species. This may attenuate reperfusion injury, especially in marginal liver grafts. During the short period of 1–2 h that is required to establish the effect of HOPE, the quality of the liver graft can be tested, before actual implantation of the organ, using biomarkers such as microRNAs (miRNAs) [14,15]. In the near future, increasing the temperature of the machine perfusion from hypothermia to (sub) normothermia will allow functional testing of initially rejected liver grafts, and aims for ex-vivo organ reconditioning using pharmacotherapeutic interventions. 3.2. MicroRNAs as graft quality biomarkers MiRNAs are small non-coding RNAs which regulate expression of protein-coding genes. The recent discovery that miRNAs are present in body fluids has generated enormous interest in their use as potential non-invasive biomarkers. We have observed that liver graft injury is associated with release of hepatocyte-derived miRNAs into the circulation. MiRNAs represent promising candidates as early, stable and sensitive biomarkers of hepatic injury after liver transplantation. Further research is now focussing on using miRNA profiles in liver- and kidney graft preservation solutions, urine and bile, to determine graft quality prior to transplantation and as potential prognostic markers for complications after transplantation [14–17]. In kidney transplantation, we aim to find miRNA profiles that may identify kidneys that could benefit from conditioning, e.g. machine perfusion or ischemic conditioning. 3.3. Ischemic conditioning and dietary restriction to minimize ischemia reperfusion injury Organ grafts from ECD and DCD donors are highly sensitive to ischemia-reperfusion induced injury (IRI). Current transplantation preservation procedures using cold storage and different preservation fluids cannot fully protect organ IRI, and new strategies to limit IRI,

such as ischemic conditioning and dietary restriction, are being studied. Brief interruptions of arterial blood flow before the onset of reperfusion, called ischemic pre-conditioning, can protect experimental animal organs from IRI. Interestingly, brief interruptions of the arterial blood flow at the onset of reperfusion (ischemic post-conditioning) are equally protective in experimental animals. A literature review demonstrates that ischemic post-conditioning improves outcome after renal IRI in animal models [18]. We were the first to assess feasibility and safety of ischemic post-conditioning in human DCD kidney transplantation, but we did not find a significant effect on kidney graft function [19]. Ongoing clinical trials in kidney-, liver-, and heart transplantation will show if there is potential for conditioning strategies to be implemented in future transplantation procedures. Apart from pre- and post-ischemic strategies, tissue injury can also be controlled by food restriction. We have shown that preoperative restriction of food intake, as well as fasting, provides protection against renal and hepatic IRI in animal studies [20]. Protection is associated with up-regulation of cyto-protective and antioxidant genes, and reduced organ injury and inflammation [21]. In addition, developmental arrest of T and B-cells, changes in lymphocyte distribution, and complement activity, may also be involved in this protection [22]. These preclinical data have led to the design and performance of two clinical studies on dietary restriction before kidney transplantation. Further studies on dietary interventions in our mouse models will reveal on a molecular and phenotypical level the consequences of food restriction on tissue injury, and this will aid improving its clinical application [23–25]. 3.4. Stem cell strategies to promote tissue regeneration Mesenchymal stem cells, also called mesenchymal stromal cells (MSC), are residing in virtually all organs and tissues and play an important role to tissue homeostasis and regeneration after tissue injury. No evidence is found that MSC are mobilized into circulation in transplant recipients [26], but MSC are mobilized from the liver graft during graft preservation [27]. These MSC have self-renewal and pluripotent differentiation capacities. Recent evidence suggests that trophic factors produced by MSC play an important role in these regenerative responses. Moreover we found that resident human liver MSC secrete factors that promote liver regeneration in mice after partial hepatectomy and under immunosuppression [28]. 4. Immunosuppression, drug monitoring, pharmacogenetics, and novel immunosuppressive drugs 4.1. Optimization of immunosuppressive regimens Erasmus MC has participated in many multicenter trials investigating the efficacy and safety of calcineurin inhibitor (CNI) and early glucocorticoid withdrawal in the setting of IL-2-receptor blockade in kidney transplant recipients [29]. In liver transplantation, we participated in multicenter trials comparing the effects of different immunosuppressive regimens on renal function and post-transplant HCV-induced liver fibrosis [30,31]. The current golden standard of immunosuppressive drug treatment after kidney transplantation, i.e., a combination therapy of tacrolimus and mycophenolate mofetil (MMF), is effective but fraught with side effects. Optimizing therapeutic drug monitoring (TDM) and understanding the genetic basis of an individual's response to these agents have been the topics of extensive research at our center in recent years. Following our observation that inter-individual differences in tacrolimus pharmacokinetics are associated with genetic variation in the CYP3A5 gene, we started a randomized-controlled trial to determine the added value of genetics-based tacrolimus dosing [32]. Recently, the importance of intra-individual (as opposed to interindividual) variability in tacrolimus exposure for long-term kidney transplant outcome was recognized [33]. Our focus has been on optimizing early exposure and minimizing toxicity of tacrolimus. We

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showed that the tacrolimus target concentrations that are currently aimed for early after renal transplantation may be too high, and that lowering the target range may result in less toxicity without compromising efficacy [34]. In addition, we demonstrated that a single-nucleotide polymorphism (SNP) in the cytochrome P450 (CYP) 3A5 gene (6986A N G) is associated with tacrolimus dose requirement. Patients expressing CYP3A5 need an approximately 50% higher tacrolimus dose compared with non-expressers [35]. To determine whether a genotype-based approach to tacrolimus dosing improves patient outcome, we recently started a randomized-controlled trial in livingdonor kidney transplantation. A clinical trial which compared concentration-controlled MMF dosing to treatment with a fixed-dose showed that dosing renal transplant recipients to mycophenolate mofetil (MPA) plasma concentrations does improve clinical outcome [36]. Further personalisation of MPA therapy using TDM and pharmacogenetics is an active research interest. SNPs in the metabolizing enzyme UGT1A9 were found to associate with MPA exposure and rejection risk [37]. Likewise, hematologic MPA toxicity was found to have a genetic basis [38]. 4.2. Novel immunosuppressive drugs In kidney transplantation, novel immunosuppressive drugs, such as the Jak3 inhibitor CP-690,550 (Tofacitinib) and the PKCθ inhibitor sotrastaurin or AEB071, were studied in phase 1 and 2 trials [39–41]. At present, the effects of induction treatment with rabbit antilymphocyte globulin (rATG) on T-cell repopulation are being studied. We found that repopulation of memory T-cells and regulatory T-cells is primarily the result of homeostatic proliferation and not of thymopoiesis [42]. In addition, treatment of glucocorticoid-resistant acute rejection with alemtuzumab targeting the CD52 molecule, and the mechanisms of rejection occurring during co-stimulation CD28-CD80/86 blockade with belatacept therapy after kidney transplantation are being studied [43]. In the latter study, the effect of belatacept on memory T-lymphocytes and the interaction between T and B-lymphocytes is being studied and compared with tacrolimus-based immunosuppression. Our first results show that, in addition to CD28-negative T-cells, also a subset of CD28positive T-cells escapes from co-stimulatory blockade. These findings may explain the reported higher rejection incidences found in belatacept therapy. High dose intravenous immunoglobulins (IVIg) are increasingly being used for treatment of auto-immune diseases. Interestingly, in contrast to current immunosuppressive drugs, even life-long antiinflammatory IVIg therapy has no adverse effects, and high-dose IVIg treatment ameliorates both antibody responses and cellular immune responses [44]. We found that high dose IVIg therapy can prevent liver graft rejection [45]. For these reasons, IVIg could be an interesting alternative for current immunosuppressive drugs used after organ transplantation, but implementation in transplantation is impaired by its high costs and by the need for intravenous administration. Therefore, we are studying which immunosuppressive pathways are stimulated by IVIg therapy, in order to identify molecular and cellular targets for development of novel immunosuppressive compounds [44,46]. 5. Transplant immunology and virology Life-long immunosuppressive drug treatment, which is required to prevent acute and chronic rejection after organ transplantation, is accompanied by severe adverse effects such as infections, cancer, and nephrotoxicity. Organ transplantation research in Rotterdam has a long-standing tradition of studying immunological and virological mechanisms involved in graft rejection and damage ex vivo, using cells and tissues derived from human transplant recipients. This type of research aims at finding biomarkers that can guide immunosuppression, identifying unknown mechanisms that contribute to or regulate

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graft rejection, exploring innovative cellular therapies, and preventing viral infections after transplantation. 5.1. Alloreactivity and its regulation Our current research lines in this field focus on: A. Premature T-cell aging. Loss of renal function is associated with T-cell defects including severe depletion of naïve T cells, expansion of terminally differentiated memory T-cells, and a pronounced influence of cytomegalovirus-seropositivity on T-cell function and phenotype. These defects are similar to those observed in elderly. We showed that assessment of T-cell ageing as well as the differentiation status of circulating T-cells prior to kidney transplantation can facilitate identification of kidney transplant recipients at risk for developing acute rejection, which we are currently validating in a prospective study [47–49]. B. Novel mechanisms involved in allograft rejection. Renal tubular epithelial cells (TEC) are part of the renal microenvironment and probably play a pivotal role in the pathogenesis of renal allograft rejection. By studying the interactions between renal TEC and immune cells, and the effects of immunosuppressive drugs on T cells and NK cells after TEC encounter, we uncovered novel concepts which are important for the development of new drugs targeting graft injury [50,51]. The contribution of monocytic cells to organ graft rejection has been attracting increasing attention. We demonstrated a shift in circulating monocytes toward pro-inflammatory CD16+ monocytes after kidney transplantation compared to healthy individuals [52]. Current studies on graft-infiltrating cells in human heart allografts revealed that IL-17 producing CD4 + T-cells home into cardiac grafts and contribute to acute rejection. Furthermore, we found that IL-17 expression is associated with acute and chronic rejection (Bronchchiolitis Obliterans Syndrome) after lung transplantation. Follicular T-helper cells stimulate differentiation of B-cells into plasmablasts that can produce donor-specific alloantibodies which are detrimental for allografts. Our recent studies reveal that follicular T-helper cells of kidney transplant recipients are well able to stimulate B-cell differentiation into plasmablasts, indicating that current immunosuppressive regimens do not inhibit T-cell driven B cell differentiation. C. Immunological tolerance to liver grafts. Liver grafts are unique among organ grafts in their capacity to induce immunological tolerance. In about 40% of stable liver graft recipients immunosuppressive therapy can be gradually withdrawn without the occurrence of graft rejection. We are developing novel assays to quantify donor-specific T-cell responses after liver transplantation and aim to use these in clinical studies to identify liver transplant recipients in which immunosuppressive medication can be safely minimized or completely withdrawn [53]. In addition, we investigate whether and how donor-derived immune cells, such as dendritic cells and NK cells, that migrate from liver grafts into the recipients, influence liver graft acceptance [54–56], and are currently studying the influence of viral infections after liver transplantation on T-cell exhaustion and donor-specific T-cell immunity. D. CD4 + FOXP3 + regulatory T cells (Treg). Our extensive research using cells and tissues from transplant recipients has shown that the anti-donor immune response after transplantation is controlled by CD4 + FOXP3 + Treg to allow organ grafts to survive [42,57], while impaired regulation by Treg is associated with rejection [58]. We also found that immunosuppressive drugs, including CNI, do not hamper the function of these suppressor T cells [57,59]. Currently, we are dissecting the relative contributions of thymus-derived Treg and peripherallygenerated Treg in regulation of human heart allograft rejection

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by measuring the methylation status of the FOXP3 gene in biopsies. Our data indicate that peripherally generated Tregs have the potential to inhibit graft rejection, but thymus-derived peripheral Treg do not. E. Allograft reactivity in young children and adolescents. Renal allograft survival is different for various age groups. The youngest children have the best survival, higher than all other age groups including adults, and is in contrast to the survival in adolescents which is worst of all age groups. We study the hypothesis that during the youngest years of life the immune system is immature, reflected by a state of hypo-immune reactivity. In contrast, the immune system is activated during adolescence possibly as a consequence of the pubertal hormone storm. One of the involved hormones may be growth hormone, which is excreted at high levels during adolescence. Using our immunological assays, alloreactivity and regulatory activity toward the transplanted kidney are being evaluated in relation to the age of the patients and their endocrine profile. 5.2. Cellular therapy to prevent graft rejection and viral infections As life-long treatment with immunosuppressive drugs is associated with severe adverse effects, the identified regulatory mechanisms stimulated interest in cellular therapy with Treg and other cells with immune-regulatory potential as an alternative for immunosuppressive drugs. Cells may have fewer side effects than drugs as they interact with host cells rather than non-selectively blocking signal transduction pathways. Today application of Tregs and MSC as anti-inflammatory mediators seems most feasible. Recently, we developed a technique that enables isolation of antigen-specific Treg [60], and we are developing a protocol in which cells of donor origin are used to drive antigenspecific Treg expansion ex vivo to obtain sufficient donor-specific Treg for therapeutic purposes. MSC interact with a plethora of immune cells, including T and B cells, via soluble factors and cell surface proteins. For example, MSC inhibit the proliferation of alloreactive CD8 + CD28 − T cells [61]. Recently, we demonstrated that MSC from kidney disease patients are capable of inhibiting alloreactivity in vitro, indicating that it is possible to use MSC of kidney transplant patients in an autologous manner [62]. In vivo, they act via yet unknown mechanisms to reset the immune system, and may therefore be effective in preventing chronic rejection later after transplantation, but also as induction therapy [63]. We observed that MSC induce Treg, which may propagate the anti-inflammatory the effects of MSC [64]. In addition, we recently discovered that human MSC can inhibit virus replication, including hepatitis viruses, by secretion of soluble factors with distinct antiviral properties from interferons. 5.3. Recurrence and reactivation of viruses after transplantation In the early postoperative period the complexity of the pathophysiology of systemic inflammation is not determined by a single parameter. Therefore we study whether combining several parameters can produce diagnostic scores that accurately enable the diagnosis of microbial infection, its likelihood, invasiveness and severity immediately after lung transplantation. In addition to this approach, the immune responses of transplant recipients toward viral infections are studied to better understand the anti-viral defense mechanisms of immunosuppressed transplant patients. Patients on immunosuppressive therapy are at risk to develop varicella zoster virus (VZV), hepatitis C virus (HCV), and hepatitis E (HEV) infections. Primary infection with VZV results in lifelong infection of the neurons in sensory ganglia which may reactivate resulting in herpes zoster infection. The incidence rate of herpes zoster after lung transplantation is significantly higher than in the age-matched healthy population. We found that the numbers of VZV-reactive memory T-cells are significantly decreased in kidney transplant recipients [65]. However, our recent data show that, despite

immunosuppression, numbers of circulating VZV-reactive CD4 + and CD8+ memory T-cells, as well as VZV-specific IgG-producing B-cells, increase after herpes zoster in kidney transplant recipients. Therefore, we advocate boosting the adaptive immune response against VZV by prophylactic VZV vaccination before transplantation, in order to limit the incidence of herpes zoster after organ transplantation. Long-term outcomes of liver transplantation in HCV-infected patients are severely compromised by the universal virological recurrence in the liver graft. Previously, we have reported that MPA has direct antiviral activity on HCV replication [66], and we are currently extending these findings using new in vitro and in vivo models [67]. Likewise, HEV replication is differentially affected by immunosuppressive drugs [68], which should be considered when selecting immunosuppressive therapies for organ transplant recipients who are infected with HEV. 6. Challenges for the future Organ transplantation has become the treatment of choice for patients with end-stage organ failure. However, as a result of this success organ shortage has become a tremendous problem. Consequently, there is an urgent need for innovative solutions to tackle insufficient organ supply. We aim to maximize and optimize the kidney donor pool through education and innovation: education to raise awareness and eliminate barriers to living donation; and continuing innovation to develop and expand alternative living donation programs, for example the inclusion of compatible pairs in kidney exchange and promotion of unspecified donation. Main priorities are 1. to continue developing innovative ways to support patients in discussing and finding a living donor, 2. to promote pre-emptive transplantation, 3. to support optimal self-management after transplantation, 4. to continue expanding alternative living donation programs, and 5. to provide justification of these programs through long term donor follow-up. Improved techniques and novel interventions to prevent graft damage during storage and after transplantation are required in order to enable better outcomes of transplantation of organs from expanded criteria and circulatory death donors. Novel strategies to promote tissue regeneration may further increase the possibilities to use sub-optimal organs for transplantation. Past predictions that induction of immunological allograft tolerance, introduction of specific as well as personalized immunosuppression, and implementation of biomarkers that predict whether patients are at risk of rejection, were just around the corner, have been proven incorrect. Predicting graft- and patient-outcome after transplantation is complex, and creative research driven by ‘thinking out-of-the-box’ is required to find biomarker(s) that predict outcomes of transplantation. Over the past few decades our knowledge about the responses of the human immune system to allografts has increased considerably. We have identified cell populations, genes and proteins involved in alloreactivity and its regulation. However, how they behave in molecular and cellular networks that drive or regulate alloreactivity is poorly understood. Novel techniques are now available to study the epigenome, genome, transcriptome, proteome, metabolome and phenotype of immune cells involved in allo-reactivity more comprehensively. With the help of digital platforms, integrated approaches will enable elucidation of core molecular pathways involved in regulation of immunity to allografts, allowing the development of novel targeted immunosuppressive compounds. In addition, studying immune-modulatory pathways activated by ‘old’ anti-inflammatory drugs with unknown mechanism-of-action, such as IVIg, may also enable identification of biologically active compounds suitable for immunosuppressive therapy after organ transplantation. Finally, cellular therapy to prevent graft rejection may allow minimization or withdrawal of immunosuppressive drug treatment. In conclusion, innovative ways to solve the organ donor shortage problem, safer immunosuppression, and integrated approaches to

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