Operational tolerance after liver transplantation☆

Article Outline

• Abstract
• 1. Introduction
  • 1.1. Definition
• 2. Group A: immunosuppressive drugs only
  • 2.1. Pittsburgh – the original work
  • 2.2. London
  • 2.3. Columbus–Madison
  • 2.4. Kyoto
  • 2.5. Murcia
  • 2.6. Rome
  • 2.7. Brussels
  • 2.8. Taiwan
  • 2.9. Sydney
• 3. Group B: molecule-based tolerogenic protocols
  • 3.1. Pittsburgh – the tolerogenic protocol
  • 3.2. New Orleans
  • 3.3. Safed–London (Ontario)–Winnipeg
• 4. Group C: cell-based tolerogenic protocols
  • 4.1. Innsbruck–Vienna
  • 4.2. Gothenburgh
  • 4.3. Ghent–Brussels
  • 4.4. Miami
• 5. Group D: previous bone marrow transplantation for hematologic diseases
  • 5.1. Innsbruck
  • 5.2. Zurich
  • 5.3. Chapel Hill
  • 5.4. The European consortium for COT after LT
• 6. How does the mechanism for COT occur?
• 7. What is the ‘gold standard’ to induce COT after LT?
• 8. Conclusions
• 9. Future perspectives
• Acknowledgements
• References
• Copyright

The achievement of an immunosuppression (IS)-free state after transplantation represents the ultimate goal of any immunosuppressive regimen. While clinical operational tolerance (COT) remains the exception after other types of solid organ transplantation, several cases of COT have been described after liver transplantation (LT). Overall, the experience gained so far worldwide demonstrates that COT can be achieved safely in one quarter of selected individuals, irrespective of the immunological background of donor and recipient, patient age, indication for LT, study endpoint, length of the weaning period and of pre/post-weaning follow-up, presence or not of chimerism. However, most transplant physicians still believe that the achievement of COT is still out of reach for the majority of LT recipients because of the potential risk for transplant survival, the non-randomized nature of most of the studies reported so far, and the selective nature of the patients enrolled in such studies, making them non-representative of the whole population of LT recipients. Despite these concerns, the present article demonstrates that this attitude is potentially no longer justified, given the growing evidence that a permanent and stable IS-free state can be achieved in a proportion of individuals who have received a LT for non-immune mediated liver diseases.

Abbreviations: COT, clinical operational tolerance, SOT, solid organ transplantation, IS, immunosuppression, LT, liver transplantation, GVHD, graft-versus-host disease, UDCA, ursodeoxycholic acid, MSC, mesenchymal stem cells, DBMC, donor bone marrow cells

Keywords: Liver transplantation, Immunosuppression, Clinical operational tolerance

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1. Introduction 

The clinical era of transplantation began on December 23rd 1954, when Dr. Joseph Murray and co-workers performed the first successful renal transplant on the Herrick twins [1]. As a result of the genetic identity between the brothers, Richard Herrick did not receive any immunosuppression (IS) after the operation, thus representing the very first case of clinical operational tolerance (COT) in solid organ transplantation (SOT). In the same year, Billingham, Brent and Medawar first introduced the term transplantation tolerance, with the report of skin graft acceptance in mice that had received neonatal injections of donor mononuclear cells [2]. More than 50 years later, COT remains an extremely difficult goal to achieve in the majority of transplant recipients. However, in liver transplantation (LT) there is a growing body of evidence that COT can be achieved safely in a proportion of recipients. In this article, we will summarize and comment on all of the cases of COT described after LT reported to date, and will demonstrate that the achievement of an IS-free state – namely, COT – is definitely possible and safe after LT. The manuscript will emphasise the clinical perspectives, and will touch only briefly on the immunological mechanisms relevant to the understanding of the IS-free state achieved in the different studies described herein where, despite improved knowledge, understanding of the immune mechanisms underlying the phenomenon remains inadequate.

The cases of COT discussed will be divided into four different groups and sorted timewise according to the weaning strategy adopted. The first group identifies cases in which no tolerogenic molecule- or cell-based protocols were used, whereas the second and third groups will include cases in which tolerogenic molecule- and cell-based regimens were implemented, respectively. The cases of COT that developed after bone marrow transplantation will be included in the last group.

1.1. Definition 

The authors define COT as the condition whereby a liver transplant retains function and lacks histological signs of rejection in the absence of any IS. The LT recipient in question is an immunocompetent host capable of responding to other immune challenges, including infections [3].

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2. Group A: immunosuppressive drugs only 

2.1. Pittsburgh – the original work 

The very first cases of COT after LT were documented by Starzl and colleagues in the early 1990s [4], [5], [6], [7], [8], [9], [10]. Based on the finding that 11 LT recipients had been IS-free as a consequence of non-compliance or post-transplant lymphoproliferative disorders, the authors designed a prospective trial in which IS was intentionally withdrawn in patients experiencing IS-derived chronic toxicity. Out of 95 patients enrolled, 28 (29%, Table 1) were successfully weaned off IS after a mean time from enrollment of 2.2 years; currently, the LT recipients in this group have been off IS for an average period of 10.8 years.

Table 1. Up to December 2008, 100 LT recipients – out of 461 in whom the weaning off IS has been attempted, and so accounting for a success rate of 22% – have been reported to be IS-free for at least 1 year. However, the total number of patients in whom COT has been achieved and described is 163 (see footnote b).

Legend: NA, not available; FU, follow up; PTLD, post transplant lymphoproliferative disorder; UDCA, ursodeoxycholic acid; GVHD, graft-versus-host disease; IS, immunosuppression; COT, clinical operational tolerance.

Most individuals (22/28, 79%) were transplanted before the age of 18, and patients with autoimmune diseases – namely, primary biliary cirrhosis and autoimmune hepatitis – were excluded from the studies from 1997 on due to the risk for disease recurrence.

In the attempt to identify a fingerprint of tolerance in IS-free LT recipients that might enable selection of patients who may be prone to develop COT, the authors proposed a set of immunologic assays studying cytokine gene polymorphism and subsets of dendritic cells [11], [12], [13], [14]. These parameters require validation in further randomized trials.

2.2. London 

Girlanda et al. recently published the 10-year follow-up of a single arm trial in which 18 patients suffering from IS-derived chronic toxicity were enrolled and weaned off IS [15], [16]. The weaning protocol was initially successful in 5 patients (28%), but only 2 of them (11%) remained completely off IS in the long term. The remaining three resumed IS for a variety of reasons; late acute rejection (1×), re-transplantation for chronic rejection (1×), and kidney transplantation for secondary end-stage renal failure (1×). Non-immune-mediated liver disorders, fewer donor-recipient HLA mismatches and no previous acute rejection were identified as parameters predictive of successful weaning, while HCV-related cirrhosis is an absolute contraindication to IS withdrawal. The London experience demonstrated that COT may be extremely difficult to maintain in the long term, as the immunologic balance between the host and the donor may be lost at any time for numerous unforeseeable reasons.

2.3. Columbus–Madison 

In the attempt to define immunological parameters that identify potentially tolerant patients, Burlingham and colleagues utilized the human-to-mouse trans-vivo delayed-type hypersensitivity assay [17], [18]. One of the three IS-free individuals analysed in the first study was a LT recipient and had been IS-free for 3 years, without experiencing any sign of rejection.

By observing that allograft acceptors failed to exhibit donor-reactive delayed type hypersensitivity responses when recipient leukocytes were challenged with donor antigen, although they frequently develop donor-reactive alloantibodies, the authors demonstrated that this pattern of immune responses is not due to an absence of allosensitization, but to the development of an immune mechanism that actively inhibits antidonor delayed-type (i.e., cell-mediated) immune responses. In doing so, they emphasized the fundamental concept of immune regulation, rather than immune-suppression [19].

2.4. Kyoto 

Tanaka and co-workers documented their initial experience on COT in a large population of 63 pediatric recipients who received a liver graft from a living donor [20], [21]. COT was achieved in 24/63 (38%) individuals, after a mean period of 23.5 (range 3–69) months. A further 23 individuals were at various stages of weaning at the time of data collection. The remaining 16 developed rejection after a mean period of 9.5 (1–63) months from initiation of tapering of IS, but the resumption of maintenance IS or the introduction of additional steroid bolus resolved the rejection.

In 2007, the same group reported on the immunological and pathological aspects of COT, as observed in a larger population of 87 IS-free pediatric LT patients, inclusive of the above mentioned 24 tolerant individuals [22]. The authors demonstrated that non-deletional tolerance mechanisms take place in patients off IS, as demonstrated by the finding that potentially reactive T cells to donor-antigens remain physically in the immune repertoire, but specifically suppressed by unknown mechanisms [22], [23], [24]. Noteworthy, tolerant patients, albeit showing normal liver function, exhibited a decrease in size and an increase in number of bile ducts, as well as a higher degree of fibrosis, when compared with patients on maintenance IS. This finding reveals that: (1) standard liver function tests are not reliable markers of COT; (2) liver pathology is essential in the monitoring of patients off IS; (3) the risk for chronic rejection could be significantly higher in such patients, on the long run.

2.5. Murcia 

Nine patients were included in a prospective IS weaning protocol the aim of which was to investigate the role of liver endothelial cell chimerism in the onset of COT [25]. Three LT recipients were completely weaned off IS, while deterioration of graft function occurred in the remaining six. Liver endothelial cell chimerism was studied in five patients, and was found to be unrelated to successful weaning.

2.6. Rome 

In 2006, Tisone and colleagues published the results at 4 years of a prospective trial, in which the withdrawal of IS was attempted in 24 LT patients with established HCV recurrence [26], [27]. Based on the finding that the progression of HCV disease recurrence after LT is strongly determined by the power of the IS adopted, (i.e., the stronger the IS, the higher the risk for a faster and more dramatic progression), the authors speculated that the onset of an IS-free state could slow down disease progression.

The working hypothesis was confirmed by the data obtained. In fact, maintenance IS could be withdrawn safely in 8/34 (27.2%) patients, and the reconstitution of immune surveillance mechanisms was followed by a striking slowdown of HCV disease progression, as indicated by a combination of histological, biochemical and virological findings. Weaned patients showed either stabilisation or improvement of necro-inflammatory changes in the graft, resulting in a marked reduction of the fibrosis progression rate, and improvement of liver function tests. In contrast, the majority of patients unable to achieve an IS-free state underwent deterioration of histological and biochemical parameters despite continuation of IS. Noteworthy, the average time needed to wean off IS was only a few months, and the occurrence of an episode of acute rejection in intolerant individuals was not harmful to patients.

After 6.5 years, the clinical results of this study remain satisfactory [28]. However, the impact of the IS-free state on the progression of HCV recurrence is not as pronounced as was found at the 4 year time point, as demonstrated by the presence of similar histological and biochemical profiles in both groups at 6.5 years. Despite this finding, tolerant individuals noted a better quality of life, as demonstrated by higher individual satisfaction, lower number of medications required, and reduced IS-related morbidity found in patients off IS. It is noteworthy that quality of life is currently regarded as an important measure of outcome after SOT, and is used in conjuction with rejection rates to determine therapeutic choices [29].

2.7. Brussels 

Reding and colleagues reported on a pediatric patient who had IS withdrawn following the onset of lymphoproliferative disorder [30]. This patient was enrolled in an observational study in which the authors intended to monitor the cytokine fluctuation in the serum of 40 pediatric LT recipients, the objective of the study being to identify patients who are rejecting or accepting their liver graft. Increased interleukin-10 blood levels at 2h after reperfusion, on days 1 and 4 after LT as compared with baseline, and decreased tumor necrosis factor alpha and interferon gamma blood levels at the same time intervals, characterized the cytokine profile of the IS-free recipient.

2.8. Taiwan 

Hsu and colleagues described the cytokine and serum protein profile of 1 tolerant LT patient of their series, in the attempt to identify reliable and non-invasive markers of tolerance [31]. They detected significantly high serum concentrations of haptoglobulin, transthyretin and alpha1-antitrypsin, as well as undetectable levels of interleukin 2.

2.9. Sydney 

Complete hematopoietic chimerism and tolerance of a liver allograft harvested from an O RhD-positive deceased male donor was described in an O RhD-negative 9-year-old girl, with no evidence of graft-versus-host disease 17 months after LT [32]. The onset of COT was preceded by a period of severe hemolysis due to the production of antibodies by residual recipient derived B lymphocytes against engrafted erythroid cells from the donor, and suggestive of the development of chimerism by engraftment of the recipient marrow from passenger hematopoietic stem cells within the transplanted liver. The hemolysis was resistant to standard steroid treatment but resolved after the gradual withdrawal of all IS. The patient is reported to be very well with normal graft function and no rejection, 5 years after the transplant.

As the patient experienced a profound lymphopenia of unknown origin, perioperatively and in the subsequent months after LT, the authors speculate that these phenomena may have contributed to the engraftment of donor hematopoietic stem cells. This speculation is consistent with the clonal-deletion theory formulated by Starzl, for which a key event before the engraftment is the dramatic reduction of immune competent cells induced by the lympho-depleting regimen [33].

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3. Group B: molecule-based tolerogenic protocols 

3.1. Pittsburgh – the tolerogenic protocol 

In 2003, Starzl and colleagues published the results of a trial in which they administered ab initio an immunosuppressive protocol expected to be tolerogenic, to 82 adult kidney, liver, pancreas and intestinal transplant recipients [33]. The hypothesis on which this protocol was designed was that the need for continuous high dose IS can be avoided in most cases with the use of a strong lymphocyte-depleting regimen prior to engraftment, followed by the administration of low dose tacrolimus monotherapy. The goal of the induction treatment was the removal of clones of immune cells that could elicit graft rejection before contact with foreign donor antigens; in this situation the repletion of immune cells through homeostatic expansion triggered as a result of the leukocyte depletion would occur once the new antigens are in place after transplantation. In addition, the minimization of maintenance IS aimed to reduce further the anti-donor response with just enough treatment to prevent irreversible immune damage to the graft, but not with such heavy treatment that the donor-specific clonal exhaustion-deletion process is precluded.

After an 18-month follow-up, 1-year patient and graft survival rates were 95% and 82%, IS-related morbidity was virtually eliminated, and 48/72 surviving recipients receiving spaced doses of tacrolimus monotherapy. These results were described as ground-breaking, as 25/39 (64%) renal, 12/17 (70%) liver, 5/12 (42%) pancreas, as well as 6/11 (54%) intestinal transplant recipients, were on spaced doses at the time of manuscript preparation. Even if the finding that no patient could be weaned off IS represents a matter of concern and questions the working hypothesis, the striking reduction of the daily dose of IS should be regarded itself as an outstanding achievement for two reasons: it was obtained after transplantation of organs (namely, kidney, pancreas and intestine) considered highly immunogenic; it led to a significantly reduction of IS-related morbidity. It is important to note that other protocols based on a similar strategy, i.e. leukocyte depletion followed by low-dose IS has been described previously for renal transplant recipients [34], [35], [36], [37], [38], [39].

3.2. New Orleans 

Inspired by the Pittsburgh experience, Eason and co-workers attempted the complete weaning of IS in eighteen patients who had not experienced any acute rejection and had reached at least 6 months following LT [40]. These patients received rabbit anti-thymocyte globulin for induction, in combination with mofetil mycophenolate and tacrolimus, followed by maintenance tacrolimus mono-therapy. No steroids were ever administered.

IS could be withdrawn completely in only one patient, whereas three patients rejected after an initial response, and 11 patients developed acute rejection after tapering of IS. The fate of the remaining three recipients is difficult to ascertain. The authors concluded from their limited experience that IS withdrawal after LT is harmful, rather than beneficial, to patients.

The lesson to learn from the Pittsburgh and New Orleans trials is that the working hypothesis is either erroneous, or incomplete. In fact, several central and peripheral mechanisms other than exhaustion and deletion might be involved in the induction of tolerance, including intra-thymic clonal deletion of precursor T cells expressing T-cell receptor, dendritic cells, peripheral clonal deletion of allogenic T cells, anergy of allogenic T cells, cytokine deviation, cellular regulation of T cells, etc. In addition, in most situations, it seems that leukocyte depletion is not accompanied by permanent and complete deletion of alloaggressive donor-reactive cells, and the establishment of a regulatory network is required to maintain tolerance. Moreover, a number of laboratory studies analysing samples from recipients treated with leukocyte depleting agents have shown that antigen experienced or memory T cells are less susceptible to depletion and may be resistant to suppression by some immunosuppressive drugs [41], [42]. Thus in some recipients, residual memory T cells may abrogate the potential benefit of induction with a leukocyte depleting agent.

3.3. Safed–London (Ontario)–Winnipeg 

Assy and colleagues published the results of a controlled trial on total IS withdrawal after LT [43], [44]. They randomized 26 stable LT patients to receive 15mg/kg of ursodeoxycholic acid (UDCA) or placebo, followed by the sequential weaning of IS. The rationale of such a strategy was the presumed evidence that UDCA reduces the incidence and severity of acute rejection in the liver allograft, by attenuating the expression of major histocompatibility complex class molecules in biliary epithelia and central vein endothelia, during the early phase of LT [45].

Results did not confirm the working hypothesis, as 15/26 individuals (58%) developed an episode of acute rejection within 12–21 weeks from tapering of IS. All of the rejection episodes responded to the resumption of IS and none of the patients developed chronic rejection. Only two patients (8%) were IS-free 1 year from the study entry. Although the fate of the remaining nine individuals who did not experience rejection remains unclear, it is presumed that they were still under the weaning protocol at the time of publication.

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4. Group C: cell-based tolerogenic protocols 

4.1. Innsbruck–Vienna 

Gadner and colleagues reported on the case of a 4-month-old girl with familial hemophagocytic lymphohistiocytosis, an inherited disorder whose only curative treatment is allogeneic stem cell transplantation [46]. The little girl underwent stem cell transplantation from her haploidentical mother, 2 months after receiving a living-related LT from the same donor for acute hepatic failure of unknown origin. The diagnosis of familial hemophagocytic lymphohistiocytosis could only be made after the surgery, from the histology of the explanted liver and from bone marrow biopsies. Myeloid engraftment occurred on day +10, but full chimerism was documented only on day +57. At that time, IS was discontinued. Four months after IS withdrawal, the patient was reported to be doing fine with normal graft function and histology.

4.2. Gothenburgh 

The group from the Queen Silvia Children’s Hospital reported on the case of a 1-year-old patient who became tolerant 4 weeks after living donor LT performed for a veno-occlusive liver disease complicating stem cell transplantation [47]. The infant girl was diagnosed with acute myeloid leukemia and treated with first-line chemotherapy followed by consolidation with stem cell transplantation. The onset of severe veno-occlusive disease resulted in an emergency LT where the liver segments 2 and 3 were donated by her HLA-identical father, who also was the source of stem cells. IS consisted of prednisolone and tacrolimus. Prednisone was rapidly tapered and withdrawn by the end of the first week, while tacrolimus was stopped by the end of the first postoperative month. This strategy was adopted following the above reported previous Austrian experience in LT and further experience in lung transplantation.

4.3. Ghent–Brussels 

Donckier and colleagues developed a protocol combining non-myeloablative conditioning and donor stem cell infusion [48], [49], [50]. Such a protocol was investigated in three patients suffering from multifocal hepatocarcinoma (n=2) or cholangiocarcinoma (n=1), who were otherwise ineligible for LT according to the local guidelines and without any other potentially curative options. They all received a right lobe living donor graft, as well as purified CD34⁺ stem cells harvested from the same donors. IS and conditioning consisted of steroids, rapamycin and antithymocyte globulin; stem cells were infused on day 7.

Patient #1 had IS withdrawn on post-LT day 18, developed acute rejection on day 108 requiring additional steroid therapy, followed by progressive weaning for 6 days. Thereafter, he remained off IS without any signs of rejection until his death on day 561 from tumor recurrence. Patient #2 was weaned off IS on day 23 but developed acute rejection on day 80, which was followed by progressive weaning for 10 days. Thereafter, he remained off IS until he died from tumor recurrence on day 356. Patient #3 could not be weaned off IS until day 213 because of major biliary problems requiring a switch from sirolimus to tacrolimus. One month later, he developed acute rejection and tacrolimus was resumed leading to prompt resolution of the clinical picture. The patient is still alive and under tacrolimus monotherapy, 498 days after LT. In all three patients, there was no detection of circulating cells from donor origin at any time point during follow-up, thus ruling out a role for micro/macrochimerism as the mechanism for COT in these particular cases.

Apart from concerns related to early tumor recurrences requiring a change in protocol that precludes the inclusion of patients with extended cancer indications, this protocol is unique in that the two different principles of nonmyeloablative conditioning (Pittsburgh, New Orleans) and stem cell-related immune modulation were used.

4.4. Miami 

Tzakis and co-workers hypothesized that the perioperative donor bone marrow cell (DBMC) infusion might favour the onset of COT after LT [51]. The conceptual framework underlying the working hypothesis was that the infusion of bone marrow cells prolongs allograft survival by still incompletely understood mechanisms [52], [53]. One hundred and four individuals enrolled in this study were stratified into two groups: Group 1 received DBMC infusions at the time of transplantation; Group 2 did not receive any DBMC infusion. Maintenance IS was tapered over a 3-year time period.

Results from this study were discouraging in the context of added benefit from DBMC infusion, as the incidences of acute rejection and COT were similar in both groups. In addition, no significant differences were observed in chimerism measured throughout the study. Overall, the authors classified their results as negative, as the working hypothesis was not confirmed. However, the robust evidence [54], [55] that has accumulated in the last decade of the immunomodulatory properties of stem cells and other progenitor cells like bone marrow-derived mesenchymal stem cells [MSC, which show in vitro, the same immunologic characteristics described in the whole bone marrow infusion used by the Pittsburgh and Miami groups [52]], suggests that their working hypothesis might have been correct, although the clinical translation may not yet have been optimised. In fact, because MSC are a rare cell population present in the bone marrow microenvironment, and because it has been demonstrated that the immunodmodulatory effects exerted by MSC are dose-dependent – i.e., the higher the number of cells, the stronger the inhibition of the immune cells [55] – it could be argued that the number of MSC infused to patients in the Miami protocol was insufficient. Noteworthy, a similar strategy has been successfully implemented by Sachs and co-workers in five patients with end-stage renal disease who received combined bone marrow and kidney transplants from living related donors, with the use of a nonmyeloablative preparative regimen [56]. In four of them, it was possible to intentionally discontinue all immunosuppressive therapy 9–14 months after the transplant, and renal function has remained stable for up to 5.3 years since transplantation.

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5. Group D: previous bone marrow transplantation for hematologic diseases 

5.1. Innsbruck 

Margreiter and colleagues reported the case of a 7.5-year-old patient who received a deceased-donor LT for skin- and liver-based chronic GVHD, occurring 7 years after an allogeneic bone marrow transplantation performed for sideroblastic anemia [57]. The donor was an HLA-identical female cousin. After the operation, the pre-existing chronic GVHD of the skin disappeared, and the immunosuppressive therapy – consisting of azathioprine, prednisone and cyclosporine – was gradually tapered and finally withdrawn 71 months after LT. Two and a half years after discontinuation of all IS, the patient was in excellent condition with neither signs of chronic GVHD nor rejection of the liver graft. No further information is provided, and the presence or absence of donor chimerism was not investigated.

5.2. Zurich 

Clavien and colleagues reported a case of COT obtained in a recipient of a living-related LT who was suffering from an advanced hilar cholangiocarcinoma [58]. Thirteen years earlier, the patient had received a bone marrow transplant from the same donor – namely, his HLA-identical sibling – for acute lymphatic leukemia. In this case, the concept of COT was combined with the potential eradication of microscopic tumor disease and downstaging of the main tumor mass, through a neoadjuvant chemo-irradiation protocol (namely, external beam radiation and intravenous 5-fluorouracil), followed by radical tumor excision through a complete hepatectomy, in order to offer the best possible chance for a longterm cure. The removal of post-LT maintenance IS was justified by the presence of full chimerism in the recipient and aimed to eliminate a major risk factor for recurrence after transplantation (namely, IS itself). A control liver biopsy at 1 year from the LT showed no rejection, while imaging at 21 months showed no evidence of tumor recurrence. Twenty-five months after LT, the patient was doing well with normal graft function.

5.3. Chapel Hill 

Andreoni and colleagues described the case of a living donor LT for HCV-related cirrhosis in a 42-year-old woman who had undergone bone marrow transplantation at the age of fifteen for aplastic anemia [59]. The donor was in both cases the patient’s HLA-identical sister. Before LT, analysis of tetranucleotide markers on chromosome 3, 7 and 12 showed complete engraftment of the transplanted bone marrow. Postoperatively, after 6 months of IS to provide protection from GVHD, the IS was progressively tapered and eventually stopped at 1 year. Twenty-four months after LT, the patient was reported to be doing well with normal graft function and histology.

5.4. The European consortium for COT after LT 

Dr. Sanchez-Fueyo conceived and led a European consortium with the goal to define a clinically applicable assay of COT that would be able to identify a cohort of LT patients who might be candidates for IS withdrawal [60], [61]. Seventeen tolerant patients from four European centers were enrolled and compared with 79 controls. The authors studied the transcriptional patterns in the peripheral blood by employing oligonucleotide microarrays and quantitative real-time polymerase chain reaction, and discovered and validated several gene signatures comprising a modest number of genes capable of identifying tolerant and non-tolerant recipients with high accuracy. Multiple peripheral blood lymphocyte subsets contributed to the tolerance-associated transcriptional patterns, although natural killer cells and gamma–delta T cell receptor-positive T cells exerted the predominant influence. These latter findings are consistent with data previously reported by the Kyoto group in IS-free children who had received a living donor LT [23]. The data obtained from transcriptional profiling of the peripheral blood of IS-free LT recipients suggest that there is a molecular signature of tolerance that can be employed to identify liver transplant recipients who can be weaned off IS and that innate immune cells are likely to play a major role in the maintenance of COT after LT.

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6. How does the mechanism for COT occur? 

Thus far, our efforts to understand the mechanisms underlying the phenomenon of COT have been mostly in vain. We know that the liver has immunomodulatory properties and that LT recipients are more prone to develop COT than recipients of other types of solid organ allografts. However, we do not know how the cells and mechanisms involved cooperate in order to induce and maintain COT, why COT occurs and when the chances for COT to develop are at their highest. Production of donor-strain soluble MHC antigen by the transplanted liver, induction of donor-derived microchimerism by stem cells transferred with the graft, mass-effect attributed to passenger leukocytes originating from the donor and many other mechanisms have been proposed, but none of these theories has been able to explain this fascinating clinical phenomenon so far.

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7. What is the ‘gold standard’ to induce COT after LT? 

This article of the current literature demonstrates that strategies that have been investigated to date with the objective of achieving a permanent IS-free state are numerous and very heterogeneous in terms of concept, immunological background and rationale, patient age, underlying indication for LT, endpoint, type of LT (deceased versus living donor), length of the weaning period, length of follow-up, presence or not of donor chimerism, full or partial chimerism, tolerogenic tools adopted, etc. However, the worldwide experience reported in the English literature to date, could be summarized as follows: a permanent and stable IS-free state can be achieved in some patients who have received a LT for hepatic-based non-immunological diseases, and those patients who do not become tolerant and develop rejection are not exposed to a higher risk for graft loss or death. Moreover, recent data suggest that it may be possible to use transcriptional profiling to identify LT transplant recipients who may be susceptible to the development of an IS-free state.

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8. Conclusions 

Effective COT has been reported more frequently in LT recipients than after transplantation of any other organ. In some cases COT has been documented as an accidental finding, but in the last decade it has been obtained intentionally in selected patients, in a number of clinical trials. The present paper reviews 163 cases of COT and demonstrates that the belief that COT cannot be achieved without exposing the recipient to an increased and unacceptable risk of graft loss is no longer justified and is perhaps outdated in the field of LT. The evidence that a permanent and stable IS-free state can be safely attempted and sometimes achieved in patients who have received a LT for treatment of non-immune mediated liver diseases, is incontrovertible. Notably, patients who develop acute rejection during the protocols designed to discontinue IS, are not exposed to further risks of graft loss once maintenance IS is resumed. Consequently, the belief that weaning off IS after LT is harmful in terms of an increased risk of graft loss is not substantiated by the published data.

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9. Future perspectives 

The failure of tolerogenic molecule-based protocols means that the molecular strategies investigated to date may be sub-optimal. The pathways of the immune response triggered by the engraftment of an allogeneic organ, may be too numerous to be controlled by just one or few compounds. In the stem cell era, the field of SOT has just started to address its interest towards progenitor cells which show impressive immunomodulatory properties. As stem cells have been used to treat conditions characterized by immunologic dysregulation such as Crohn’s disease and graft-versus-host-disease after allogeneic haematopoietic stem cell transplantation, we may speculate that the same immunomodulatory properties might be potentially useful for the prevention or treatment of SOT rejection, as well as for the induction of COT, as demonstrated by the cases reported by Gothenburgh, and the Austrian and Belgian consortiums [45], [46], [47], [48], [49]. Moreover, as stem cells are capable of promoting tissue repair, harnessing both the immunomodulatory capabilities of such cells and their ability for tissue repair provides an exciting opportunity for further research in the field of SOT [54].

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Acknowledgements 

The authors would like to address a special thank to Robert J. Stratta and Peiman Hematti for critical review. Gratitude is expressed by GO to Giuseppe Tisone and Jan Lerut for education, and to Alberto Sanchez-Fueyo for inspiration.

Giuseppe Orlando is recipient of the Marie Curie International Outgoing Fellowship POIF-GA-2008-221850, financed by the European Commission under the 7th Framework Program for Research and Development.

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