Transplantation is the treatment of choice for patients with end-stage organ failure (1, 2) as it improves their survival and quality of life (3). Nevertheless, long waiting lists, side effects of immunosuppressive medication, and limited graft survival still perpetuate the dream of tolerance. Initial enthusiasm was fueled in the 1950s by the observation of Medawar and Billingham that dizygotic twins of free martin cattle readily accept skin grafts from each other (4). They attributed this phenomenon to the coexistence of red blood cells from their siblings (mixed chimerism) as a result of a common placental circuit (5). Since fraternal erythrocytes also persisted in adult animals, they concluded that stem cells had to be exchanged which constantly give rise to short-lived red blood cells. Based on this observation, they demonstrated for the first time that tolerance to alloantigens can actively be acquired in neonatal mice by the intrauterine injection of allogeneic cells (6). Since then, a myriad of mouse studies established bone marrow transplantation as promising approach to achieve donor-specific transplantation tolerance (7). In the clinical setting, the usability of this approach was corroborated by anecdotal cases in which patients developed immunological tolerance to a renal allograft after having previously received a hematopoietic stem cell transplant from the same donor for a hematological disorder (8). Despite this knowledge, the establishment of tolerance inducing protocols as a clinical routine is still long in coming.

Three groups in the US from Harvard University, Stanford University and the association between the Universities of Louisville and Northwestern have pioneered the first steps toward clinical implementation. All three groups have elaborated distinct approaches with individual assets and drawbacks and so far, nearly 70 patients have been enrolled (9–11). Here, we discuss the current state of affairs of chimerism-based tolerance, what we have learned so far and which new challenges we have to face.

Continuous suppression of the immune system puts transplant recipients at risk of increased morbity and mortality through cardiovascular disease, de novo diabetes, dyslipidemia, and malignancies. By now, death with a functioning graft has become a leading cause of graft loss (2). Some of these side effects result from impaired immune surveillance while others constitute drug-specific toxicities of the immunosuppressive medication. Cyclosporine was initially celebrated as “wonder drug,” until it was realized that it is fairly toxic at higher doses (12). In the light of this, a substantial number of non-renal organ transplant recipients develop renal failure due to calcineurin inhibitor-toxicity (13). Apart from that, immunosuppressive drugs are ineffective in preventing late graft loss from chronic rejection (14) which is why long-term graft survival has improved only marginally over the last decades (2).

Since tolerance is expected to provide remedy, the search for the “Holy Grail” of transplantation has never ceased. To assess whether tolerance actually meets these high expectations, tolerant kidney transplant recipients have recently been compared to a matched cohort receiving conventional immunosuppression. The tolerant group experienced significantly longer initial hospital stays and more frequent readmissions leading to three times higher costs during the first year in comparison to conventional transplant recipients. In turn, tolerant patients required significantly less treatment for hypertension and none of them developed new-onset diabetes, dyslipidemia, or malignancy. In this survey, the continuous costs for medications of conventional patients exceeded those of tolerant patients after 10 years (15, 16). The sample size was small but this preliminary study emphasized the potential benefit of tolerance inducing protocols. Another group estimated the expected lifetime savings through tolerance induction for a 40-year old patient receiving a kidney from a human leukocyte antigen (HLA)-matched living donor to approximately 92.000$ (17). Besides, it should be taken into account that innovative treatment options become less expensive if employed as clinical routine, especially if they are also applicable to other medical fields. In this regard, mixed chimerism becomes increasingly attractive as treatment option for autoimmune disorders (18, 19).

In addition, tolerant patients evidently enjoy a higher quality of life (16), which is not only a matter of convenience but also correlates with reduced morbidity and mortality (20). The discomfort resulting from the immunosuppressive therapy increases the probability of non-adherence which in further consequence leads to decreased donor organ survival. Considering that kidney transplant recipients take a median of 15 capsules a day (21), it appears not surprising that non-adherence is estimated to occur roughly in a third of all transplant recipients (2).

Tolerance inducing protocols are currently measured against kidney transplant recipients receiving conventional immunosuppressive therapy. One-year graft survival rates of over 90% and half-lives of 16 years set the bar fairly high (22, 23). Innovative strategies aiming to improve patient and organ survival will further increase the high demands for tolerance inducing strategies. New algorithms have been developed to allocate best-quality organs to those recipients expected with the highest survival (24) and paired living kidney donation programs optimize allograft survival (25). Furthermore, the long-lasting supremacy of calcineurin inhibitors has recently been challenged by the advent of costimulation blockers in the clinical setting. Belatacept was associated with enhanced overall patient and graft survival in subsets of patients, improved kidney allograft function and avoided major side effects of calcineurin inhibitors (26).

In summary, tolerance is still of great value, particularly in the longer run, but the question remains whether it can be achieved at an acceptable prize in terms of safety. Tolerance would certainly obviate the common risks of immunosuppressive medication but at the same time expose patients to other serious hazards.

When it comes to clinical translation, patient safety takes the highest priority. Therefore, the conditioning necessary to achieve allogeneic bone marrow engraftment is a matter of great concern. In contrast to conventional kidney allograft recipients, patients receiving hematopoietic stem cell transplantation for the purpose of tolerance induction are additionally exposed to a considerable risk of developing GvHD. Approximately 15% of patients undergoing hematopoietic stem cell transplantation for hematological diseases succumb to GvHD (27). The incidence and severity of acute GvHD can directly be correlated with the degree of HLA mismatch (28). Since tolerance is particularly desirable for HLA-mismatched recipients, the prevention and treatment of GvHD is a delicate issue. The occurrence of GvHD is highly dependent on the recipient conditioning and the composition of the allograft. Therefore, we will discuss which efforts the individual groups have made to minimize the risk of GvHD in the clinical pilot trials of chimerism-based tolerance.

These first pilot trials provide the conceptual framework for the further development of chimerism-based approaches as clinically viable treatment option. In order to refine tolerance inducing strategies, it will be important to understand the underlying mechanisms establishing and maintaining tolerance in greater detail. While the circumstances seem fairly evident in the event of full chimerism, the tolerization of pre-existing recipient T cells in mixed and transient chimeras with an intact T cell repertoire still remains incompletely understood. The group from Stanford conducted mechanistic studies in the murine setting. They found that repeated doses of TLI cause severe DNA damage which drives large numbers of lymphocytes into apoptosis. CD8⁺ dendritic cells are less sensitive to irradiation and possess specialized receptors (Tim-4, DEC205) that recognize products of dying cells. Activated CD8⁺dendritic cells activate the immunomodulatory enzyme indoleamine 2,3-dioxygenase and trigger host NKT cells to produce IL-4. Under these conditions, the interaction of IL-4-producing NKT cells with myeloid-derived suppressor cells and Tregs contributes to the induction of tolerance. Myeloid-derived suppressor cells thwart alloreactive T cell responses through the release of Arginase-1 which deprives local T cells from the essential amino acid l-Arginine and increases the production of superoxide (93). Furthermore, myeloid-derived suppressor cells exhibit an increased surface expression of PDL1 and prompt the residual T cell pool including Tregs to express PD1 (94, 95). The interaction of PDL1 with PD1 attenuates effector T cell functions while promoting the induction, maintenance, and function of Tregs (96). Expression of PD1 on Tregs was also linked to enhanced secretion of IL-10 which preserved the allograft and chimerism (97).

The group from Boston started to investigate the mechanisms sustaining tolerance in patients that had lost chimerism. They found that both circulating and intragraft Foxp3⁺ Tregs were increased in tolerant patients while alloreactive T cells were decreased (98). The increase of peripheral Tregs in the blood resulted from proliferation, thymic emigration, and in one patient from conversion of conventional T cells (99). In order to track alloreactive T cells, they defined a genetic fingerprint of T cells responding to donor cells in vitro before transplantation using high-throughput sequencing. Those clones were specifically reduced in tolerant patients but remained unchanged in stable kidney recipients on immunosuppressive therapy (100). Since these patients exhibit transient chimerism, it is likely that donor-reactive T cells were deleted by peripheral mechanisms.

Recently, the group exploited a cohort of tolerant non-human primates that had accumulated over the years to investigate the underlying tolerance mechanisms emerging through transient chimerism in more detail. Tolerant recipients lost anti-donor CD8 T cell response while a considerable number of CD4 T cells proliferated against donor cells in vitro. A substantial fraction of these responding CD4 T cells constituted Foxp3 Tregs which were induced from conventional T cells in a TGF-β-dependent manner. The suppressive function of these induced Tregs was contact dependent and inhibiting their induction through TGF-β blockade restored CD8 T cell proliferation in vitro (101).

As different as the individual approaches may be, they all agree on the pivotal role of Tregs, suggesting that steering the differentiation of naïve CD4 T cells toward regulation is a critical step for the induction of tolerance. The differentiation of naïve CD4 T cells into various T helper subsets is guided by the surrounding cytokine milieu during T cell receptor stimulation. If CD4 T cells commit to a distinct helper lineage, they start to express certain transcription factors that determine their subsequent mode of action such as immunity, tolerance, autoimmunity, or allergy. Therefore, the specific blockade of immunogenic cytokines could be envisioned as suitable approach to drive naïve CD4 T cells into the corner of regulation. This strategy has already been proven successful for the treatment of several inflammatory diseases (102) and it will be interesting to see whether this success will continue in the field of transplantation.

IL-6 is probably the most crucial factor guiding the differentiation of naïve CD4 T cells either toward regulation or inflammation. Inhibition of IL-6 signaling with Tocilizumab is already an established treatment option for autoimmune diseases and currently gains traction in the field of transplantation. Coadministration of Tocilizumab to standard GvHD prophylaxis revealed promising potential in a phase I/II trial (32) and is also tested in several transplant trials (103). In the experimental setting, blocking IL-6 displayed a beneficial effect on the induction of chimerism and tolerance in non-human primates and mice (104, 105).

Alternatively, Treg induction could be promoted by establishing the appropriate cytokine environment. The most vital cytokine for the induction and maintenance of Tregs is IL-2. Nonetheless, current maintenance therapies targeting calcineurin inhibit IL-2 secretion and may, therefore, not be suitable for tolerance-inducing strategies (106). The addition of IL-2 has recently been shown to restore the survival and function of Tregs under calcineurin inhibition and to improve allograft survival (107). Harnessing IL-2 to tip the balance between immunity and regulation is, however, a delicate issue as it is essential for both. The compilation and tissue distribution of the IL-2 receptor probably accounts for its pleiotropic effects. IL-2 preferentially binds to its high-affinity receptor which is constitutively expressed on Tregs and rapidly upregulated on conventional T cells and NK cells upon activation, while resting NK cells and memory CD8 T cells constitutively carry the low-affinity receptor (108). Low doses of recombinant IL-2 have been shown to promote tolerance to islet (109) and skin allografts (110) while high doses of IL-2 abrogated kidney allograft tolerance in non-human primates (111). IL-2/α-IL2 complexes accelerated bone marrow rejection in mice independent of their dose implying that the effect of IL-2 also varies with the transplanted tissue (112).

The first clinical pilot trials underpinned the difficulties to achieve durable mixed chimerism over MHC barriers. This issue has often been disregarded in the murine setting where the number of memory T cells is rather low (5–10% of all T cells). Murine models can be improved by enriching recipients with alloantigen primed memory T cells (113) or by using old mice with an increased pool of memory T cells (114). In the human setting, the amount of memory T cells can compromise up to 50% of all circulating T cells (115) from which 1–10% can recognize intact allogeneic MHC molecules through direct allorecognition (116), even in “non-sensitized” recipients through the mechanism of heterologous immunity. Memory T cells are a robust hurdle for tolerance induction due to their lower activation threshold, vigorous effector functions, and their resistance to common immunosuppressive drugs. The presence of memory T cells pre-transplantation has been associated with an increased risk for acute rejection of kidney transplants (117). Pre-exisitng alloreactive memory T cells are most likely generated through recognition of commensal bacteria or environmental antigens (heterologous immunity). Apart from this, memory T cells can occur during homeostatic proliferation following cell depletion or in lymphopenic hosts. Homeostatic proliferation and heterologous immunity have both been shown to impede tolerance induction in mice (118, 119).

Cell-depleting agents enrich memory T cells as they preferentially affect naïve and regulatory T cells (120). T cells with effector/memory phenotype are detectable after Alemtuzumab or ATG induction therapy (120, 121). Moreover, it should be kept in mind that innate immunity which is responsible for the clearance of antibody-coated cells becomes activated (122). Therefore, it would be preferable to block alloreactive cells rather than depleting them. So far CTLA4-Ig (belatacept) is the only costimulation blocker available for clinical use. Besides its availability, CTLA4-Ig also caught attention through its ability to promote chimerism and tolerance in mice (82) and non-human primates (83). Unfortunately, also modern blocking agents have difficulties to keep tab on memory T cells. Terminally effector memory CD4 and CD8 T cells lose CD28 expression and, thus, become resistant to costimulation blockade through CTLA4-Ig (123). Terminal effector T cells are resistant to costimulation blockade even before they lose CD28 expression and their pretransplant frequency has recently been shown to predict episodes of allograft rejection in belatacept-treated patients (124, 125). Increased numbers of CD28⁻ CD4 and CD8 memory T cells have been associated with a poor outcome in renal transplant recipients (126). IL-15 could restore the proliferation of alloreactive CD28⁻ memory CD8 T cells in vitro (127). Accordingly, strategies are warranted to block IL-15 signaling under costimulation blockade. Blocking the β-chain (CD122) of the IL-15 receptor has recently been shown to synergize with CTLA4-Ig to prolong allograft survival (128).

Alternative strategies include the use of Alefacept which is a fusion protein consisting of extracellular LFA3 domain and human IgG1. It interacts with CD2 which is upregulated on CD45RO⁺ effector/memory T cells. In this way, Alefacept preferentially targets memory T cells but spares the remaining T cell pool (129). Pre-transplant Alefacept synergized with CTLA4-Ig by targeting CD8⁺ CD28⁻ effector/memory T cells (130). Moreover, blocking the integrins LFA-1 and VLA-4 prolonged skin allograft survival in a mouse model of costimulation blockade-resistant rejection mediated by memory CD8 T cells (113, 131).

One of the fundamental challenges of tolerance-inducing protocols is to identify those patients in which the protocol was successful and which can, thus, safely be weaned off immunosuppression. Stable multi-lineage chimerism has long been regarded as the most robust predictor for tolerance and donor T cell engraftment in particular as a critical parameter (132). However, the prediction of tolerance becomes more sophisticated in the absence of durable chimerism. Accordingly, there is a high demand for other markers that reliably identify tolerant patients.

The optimal biomarker should allow repeated and non-traumatic measurements that faithfully reflect intragraft processes in real-time with a high precision at affordable prices. The demands are certainly high but advancing biotechnological methods offer unknown opportunities. In the effort to find a reliable biomarker for tolerance, conventional transplant recipients retaining normal kidney function over an extended period of time after withdrawal of all immunosuppressive medication (operationally tolerant) have been compared to healthy subjects or immunosuppressed kidney recipients. Tolerant patients exhibited a specific increase in blood CD4⁺ CD45RA⁻ Foxp3^high memory Tregs in comparison to stable kidney recipients and healthy controls. These Tregs exhibited an increased surface expression of GITR and CD39 together with a decreased demethylation of the Treg-specific demethylated region (133). Since only a minor fraction of the whole Treg pool circulates within the blood, the accumulation of Tregs within the graft drew more interest. Indeed, operationally tolerant patients exhibited an increased proportion of Foxp3 Tregs inside their allografts (134). Besides, dissecting the non-regulatory T cell compartment emerged as useful tool to assess alloreactivity. Analyzing the recipients T cell receptor repertoire by high-throughput sequencing becomes increasing popular on that account. Once a genetic fingerprint of donor-reactive T cell clones has been defined, they can easily be tracked over time. The group from Boston could confirm that a reduction of the alloreactive T cell receptor repertoire correlates with tolerance while an increase is associated with rejection (135).

Whole genome microarray-based transcriptional profiling studies additionally revealed B cells as important markers of kidney tolerance (136, 137). These studies led to the disclosure of a distinct B cell signature in operationally tolerant patients and in patients rendered tolerant through hematopoietic stem cell transplantation (138). This specific B cell profile has recently been deployed to identify a cohort of immunosuppressed patients with improved renal allograft function (139). The circumstance that the B cell signature of tolerant patients was conspicuously similar to those of healthy controls raised the question whether these profiles would merely reflect the absence of immunosuppression. Re-evaluation of the data set revealed that common immunosuppressants bias the expression of the investigated genes. Nonetheless, a new gene expression profile could be created that was independent of drug-effects (140). In an alternative approach, tolerant patients could be differentiated from stable patients on the basis of elevated miR-142-3p levels in peripheral blood mononuclear cells. Besides, miR-142-3p expression was stable over time and not affected by immunosuppression (141). The fact that miR-142-3p mainly originated from B cells further substantiated their contribution for tolerance.

In line with this B cell profile, tolerant patients also displayed an increased proportion of naïve (CD20⁺ CD24^low CD38^low) and transitional B cells (CD20⁺ CD24^high CD38^high) in comparison to immunosuppressed kidney recipients (138). In this respect again, it could not be excluded that the redistribution of B cell subsets might result from the absence of immunosuppression. Patients on azathioprine or prednisone namely feature lower numbers of transitional B cells in comparison to patients off drugs (140). On the other hand, the lack of transitional B cells was associated with kidney allograft rejection (142).

No biomarker has emerged yet that has been sufficiently validated to be used for clinical decision making in the routine setting, but progress achieved so far is encouraging and will likely yield success in the future.

After an extended period of hibernation, the dream of tolerance is gaining increasing attention again. The Holy Grail seems to be in one’s reach but “he who finds the Grail must face the final challenges—three devices of such lethal cunning” (143). In the event of transplantation tolerance, these final challenges would be to reduce host toxicity, to avoid GvHD, and to improve clinical efficacy. Improving tolerance-inducing strategies is a cumbersome and tedious process but the increasing number of patients undergoing such protocols shows that we are heading into the right direction. Besides, novel approaches such as costimulation blockade, Treg therapy, pro-apoptotic molecules, or immunomodulatory antibodies hold out encouraging prospects for the future. Further important steps toward clinical applicability will be to find appropriate biomarkers that reliably predict tolerance.

On the shady side, the burning desire for eternal organ life sometimes makes us forget the involved risks as can be seen by the two recent cases of GvHD. As Goetz von Berlichingen already said in Goethe’s drama: “Where there is bright light, there is also deep shadow.” Throughout the course of history progress has often claimed victims but it remains questionable whether this can be justified for the field of (living donation kidney) transplantation. Be that as it may, the rapidly advancing field of immunology will hopefully allow us to achieve tolerance by safer means. In this regard, the field of tumor immunology emerged as major ally. The mechanisms of how tumors evade host immunity provide vital insights for the design of innovative strategies and could guide future directions (144).

Developing tolerance-inducing strategies has always been and continues to be a highly elaborate process. Therefore, it still remains difficult to assess when tolerance-inducing protocols will reach clinical maturity but there is hope that they will not remain a laboratory solution forever.

BM, NG and TW wrote the article and MM revised the manuscript. BM and MM designed the figures and the table.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.