operational tolerance after liver transplantation

www.elsevier.com/locate/jhep

Journal of Hepatology 50 (2009) 1247–1257

Special article

Operational tolerance after liver transplantationq

Giuseppe Orlando1,2,*, Shay Soker2, Kathryn Wood1

1Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, Headington, Oxford OX3 9DU, UK2Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USA

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 experiencegained 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 fornon-immune mediated liver diseases.

� 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: Liver transplantation; Immunosuppression; Clinical operational tolerance

1. Introduction

The clinical era of transplantation began on Decem-ber 23rd 1954, when Dr. Joseph Murray and co-workersperformed the first successful renal transplant on theHerrick twins [1]. As a result of the genetic identitybetween the brothers, Richard Herrick did not receiveany immunosuppression (IS) after the operation, thus

0168-8278/$36.00 � 2009 European Association for the Study of the Liver.

doi:10.1016/j.jhep.2009.03.006

Associate Editor: P.-A. Clavienq The authors who have taken part in this study declared that they do

not have anything to disclose regarding funding from industry orconflict of interest with respect to this manuscript.

* Corresponding author. Tel.: +44 505 272 5593; fax: +44 505 2723518.

E-mail address: [email protected] (G. Orlando).Abbreviations: COT, clinical operational tolerance; SOT, solid or-

gan transplantation; IS, immunosuppression; LT, liver transplanta-tion; GVHD, graft-versus-host disease; UDCA, ursodeoxycholic acid;MSC, mesenchymal stem cells; DBMC, donor bone marrow cells.

representing the very first case of clinical operational tol-erance (COT) in solid organ transplantation (SOT). Inthe same year, Billingham, Brent and Medawar firstintroduced the term transplantation tolerance, with thereport of skin graft acceptance in mice that had receivedneonatal injections of donor mononuclear cells [2].More than 50 years later, COT remains an extremelydifficult goal to achieve in the majority of transplantrecipients. However, in liver transplantation (LT) thereis a growing body of evidence that COT can be achievedsafely in a proportion of recipients. In this article, wewill summarize and comment on all of the cases ofCOT described after LT reported to date, and will dem-onstrate 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 mech-anisms relevant to the understanding of the IS-free stateachieved in the different studies described herein where,despite improved knowledge, understanding of the

Published by Elsevier B.V. All rights reserved.

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1248 G. Orlando et al. / Journal of Hepatology 50 (2009) 1247–1257

immune mechanisms underlying the phenomenonremains inadequate.

The cases of COT discussed will be divided into fourdifferent groups and sorted timewise according to theweaning strategy adopted. The first group identifiescases in which no tolerogenic molecule- or cell-basedprotocols were used, whereas the second and thirdgroups will include cases in which tolerogenic molecule-and cell-based regimens were implemented, respectively.The cases of COT that developed after bone marrowtransplantation will be included in the last group.

1.1. Definition

The authors define COT as the condition whereby aliver transplant retains function and lacks histologicalsigns of rejection in the absence of any IS. The LT reci-pient in question is an immunocompetent host capableof responding to other immune challenges, includinginfections [3].

2. Group A: immunosuppressive drugs only

2.1. Pittsburgh – the original work

The very first cases of COT after LT were docu-mented by Starzl and colleagues in the early 1990s [4–10]. Based on the finding that 11 LT recipients had beenIS-free as a consequence of non-compliance or post-transplant lymphoproliferative disorders, the authorsdesigned a prospective trial in which IS was intentionallywithdrawn in patients experiencing IS-derived chronictoxicity. Out of 95 patients enrolled, 28 (29%, Table 1)were successfully weaned off IS after a mean time fromenrollment of 2.2 years; currently, the LT recipients inthis group have been off IS for an average period of10.8 years.

Most individuals (22/28, 79%) were transplantedbefore the age of 18, and patients with autoimmune dis-eases – namely, primary biliary cirrhosis and autoim-mune hepatitis – were excluded from the studies from1997 on due to the risk for disease recurrence.

In the attempt to identify a fingerprint of tolerance inIS-free LT recipients that might enable selection ofpatients who may be prone to develop COT, the authorsproposed a set of immunologic assays studying cytokinegene polymorphism and subsets of dendritic cells [11–14]. These parameters require validation in further ran-domized trials.

2.2. London

Girlanda et al. recently published the 10-year follow-up of a single arm trial in which 18 patients sufferingfrom IS-derived chronic toxicity were enrolled and

weaned off IS [15,16]. The weaning protocol was initiallysuccessful in 5 patients (28%), but only 2 of them (11%)remained completely off IS in the long term. The remain-ing three resumed IS for a variety of reasons; late acuterejection (1�), re-transplantation for chronic rejection(1�), and kidney transplantation for secondary end-stage renal failure (1�). Non-immune-mediated liverdisorders, fewer donor-recipient HLA mismatches andno previous acute rejection were identified as parameterspredictive of successful weaning, while HCV-related cir-rhosis is an absolute contraindication to IS withdrawal.The London experience demonstrated that COT may beextremely difficult to maintain in the long term, as theimmunologic balance between the host and the donormay be lost at any time for numerous unforeseeablereasons.

2.3. Columbus–Madison

In the attempt to define immunological parametersthat identify potentially tolerant patients, Burlinghamand colleagues utilized the human-to-mouse trans-vivodelayed-type hypersensitivity assay [17,18]. One of thethree IS-free individuals analysed in the first study wasa LT recipient and had been IS-free for 3 years, withoutexperiencing any sign of rejection.

By observing that allograft acceptors failed to exhibitdonor-reactive delayed type hypersensitivity responseswhen recipient leukocytes were challenged with donorantigen, although they frequently develop donor-reac-tive alloantibodies, the authors demonstrated that thispattern of immune responses is not due to an absenceof allosensitization, but to the development of animmune mechanism that actively inhibits antidonordelayed-type (i.e., cell-mediated) immune responses. Indoing so, they emphasized the fundamental concept ofimmune regulation, rather than immune-suppression[19].

2.4. Kyoto

Tanaka and co-workers documented their initialexperience on COT in a large population of 63 pediatricrecipients 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 fur-ther 23 individuals were at various stages of weaning atthe time of data collection. The remaining 16 developedrejection after a mean period of 9.5 (1–63) months frominitiation of tapering of IS, but the resumption of main-tenance IS or the introduction of additional steroidbolus resolved the rejection.

In 2007, the same group reported on the immunolog-ical and pathological aspects of COT, as observed in alarger population of 87 IS-free pediatric LT patients,inclusive of the above mentioned 24 tolerant individuals

Table 1Up 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).

Center Study type No

of pts

Mean

age at

time of

weaning

(years)

Rationale

of IS

withdrawal

Inclusion

criteria

Mean f–u

time between

LT and IS

withdrawal

(months)

Accidental

finding

Intention-

to-treat

Weaning

time

Patient off IS

(success rate)a

F–U time

from IS

withdrawal

(months)

Trials in which no tolerogenic molecules were tested, or stem cell-based protocol were used to favor the onset of COT

Pittsburgh

[4–14]

Prospective,

single arm

95 11.8 Chronic

IS-related

toxicity

P60-Month

follow up,

IS-related

chronic

toxicity,

24 months

without

any episode of

acute rejection,

compliance to

treatment

70 Yes Yes 5.9 Month 28 (29%) 180

London

[15,16]

Prospective,

single arm

18 47.7 Chronic

IS-related

toxicity

P60-Month

follow up,

at least

one IS-related

side effect

60 Yes No Months 2 (11%) 120

Columbus–

Madison

[17,18]

Observational 1 Not

specified

Accidental

finding

– 36 Yes No Not specified 1 36

Kyoto [20–24] Mixed,

single arm

63 1.1 Chronic

IS-related

toxicity.

PTLD

P24-Month

follow up,

no previous

acute rejection,

normal graft

function

P24 No Yes 3–6 Months 24 (38%) 87b 24

Murcia [25] Prospective,

single arm

9 53.1 Not specified P24-Month

follow up,

normal graft

function

P60 No Yes <10 Months 3 (33%) 20

Rome [26–28] Prospective,

controlled

34 62.2 Impact of

IS on the

natural

history

of HCV

disease

recurrence

P12-Month

follow up,

established HCV

disease

recurrence,

normal graft

function,

compliance

to treatment

63.5 No Yes 3.6 Months 8 (24%) 45

Brussels [30] Prospective,

placebo-

controlled,

randomized

1 2 PTLD First single

transplant, non

autoimmune

diseases, age

<15 years

2 No Yes Not specified 1 55

Kaohsiung [31] Observational 1 Not

specified

PTLD – 60 No Yes Not specified 1 60

Sidney [32] Observational 1 9 GVHD – 14 No Yes 3 Months 1 46

Trials in which tolerogenic non-stem cell-based protocols were tested

Pittsburgh

tolerogenic

protocol [33]

Prospective,

single arm

82 Not specified

(adult

population)

Clonal-deletion

theory

First transplant Ab initio No Yes Months 0 (0%) 15

New

Orleans [40]

Prospective,

single arm

18 Not specified

(adult population)

Clonal-deletion

theory

P6-Months

follow up,

no previous

acute rejection,

first transplant

P6 No Yes Months 1 (6%) 12

Safed–London

(Ontario)–

Winnipeg

[41,42]

Prospective,

controlled

26 53 UDCA

immuno-

modulation

Rejection-free

for P24 Months

56 Months Yes No Months 2 (8%) 12

Trials in which the onset of tolerance was induced by the infusion donor-derived stem/progenitor cells

Innsbruck–

Vienna

[46]

Observational 1 0.5 Treatment

of underlying

disease

– 2 No Yes Months 1 4

Gothenburg

[47]

Observational 1 1 Stem cell

immuno-

modulation

– Ab initio No Yes 1 Month 1 60

(continued on next page)

G. Orlando et al. / Journal of Hepatology 50 (2009) 1247–1257 1249

Table 1 (continued)

Center Study type No of pts Mean

age at

time of

weaning

(years)

Rationale

of IS

withdrawal

Inclusion

criteria

Mean f–u

time between

LT and IS

withdrawal

(months)

Accidental

finding

Intention-

to-treat

Weaning

time

Patient off IS

(success rate)a

F–U time

from IS

withdrawal

(months)

Gent–Brussels

[48–50]

Prospective,

single arm

3 54 Stem cell

immuno-

modulation

Liver cancer

not amenable

to surgery or

transplantation,

no other

treatment

option, living

donor available

Ab initio No Yes Months 3 (100%),

but 2 patients

died after

561 and 356

days for

tumour

recurrence

543, 356,

498 days,

respectively

Miami [51] Prospective,

controlled

104 Not specified

(adult)

Bone marrow

mesenchymal

stem cells

immune-

modulation

P36-month

follow up, no

previous acute

rejection, normal

graft function,

no autoimmune

diseases

P12 No Yes 3 Years 20 (19%) 26

Trials in which the onset of tolerance was preceded by a bone marrow transplant performed for hematologic diseases which induced the onset of full

chimerism in the recipient

Innsbruck [57] Observational 1 7.5 Not

specified

– 71 No Yes Not specified 1 30

Zurich [58] Prospective 1 41 Bone

marrow-induced

full

chimerism

– Ab initio No Yes Months 1 12

Chapel Hill [59] Observational 1 42 Bone

marrow-induced

full chimerism

– 6 Months No Yes 6 Months 1 24

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.a For obvious reasons, in the case of 1-patient report the success rate has not been calculated.b The University of Kyoto reported on 87 pediatric living-donor LT who had achieved the complete withdrawal of IS since the beginning of the LT program, and so accounting for 15.0% of all the

patients transplanted at that center [22]; 24 of them had already been described in a previous paper illustrating the results of a prospective trial in which the weaning of IS was attempted in 63

individuals [21].


[22]. The authors demonstrated that non-deletional tol-erance mechanisms take place in patients off IS, asdemonstrated by the finding that potentially reactiveT cells to donor-antigens remain physically in theimmune repertoire, but specifically suppressed byunknown mechanisms [22–24]. Noteworthy, tolerantpatients, albeit showing normal liver function, exhib-ited a decrease in size and an increase in number of bileducts, as well as a higher degree of fibrosis, when com-pared with patients on maintenance IS. This findingreveals that: (1) standard liver function tests are notreliable markers of COT; (2) liver pathology is essentialin the monitoring of patients off IS; (3) the risk forchronic rejection could be significantly higher in suchpatients, on the long run.

2.5. Murcia

Nine patients were included in a prospective IS wean-ing protocol the aim of which was to investigate the roleof liver endothelial cell chimerism in the onset of COT[25]. Three LT recipients were completely weaned offIS, while deterioration of graft function occurred inthe remaining six. Liver endothelial cell chimerism wasstudied in five patients, and was found to be unrelatedto successful weaning.

2.6. Rome

In 2006, Tisone and colleagues published the resultsat 4 years of a prospective trial, in which the withdrawalof IS was attempted in 24 LT patients with establishedHCV recurrence [26,27]. Based on the finding that theprogression of HCV disease recurrence after LT isstrongly determined by the power of the IS adopted,(i.e., the stronger the IS, the higher the risk for a fasterand more dramatic progression), the authors speculatedthat the onset of an IS-free state could slow down dis-ease progression.

The working hypothesis was confirmed by the dataobtained. In fact, maintenance IS could be withdrawnsafely in 8/34 (27.2%) patients, and the reconstitutionof immune surveillance mechanisms was followed by astriking slowdown of HCV disease progression, as indi-cated by a combination of histological, biochemical andvirological findings. Weaned patients showed either sta-bilisation or improvement of necro-inflammatorychanges in the graft, resulting in a marked reductionof the fibrosis progression rate, and improvement ofliver function tests. In contrast, the majority of patientsunable to achieve an IS-free state underwent deteriora-tion of histological and biochemical parameters despitecontinuation of IS. Noteworthy, the average time


needed to wean off IS was only a few months, and theoccurrence of an episode of acute rejection in intolerantindividuals was not harmful to patients.

After 6.5 years, the clinical results of this study remainsatisfactory [28]. However, the impact of the IS-free stateon the progression of HCV recurrence is not as pro-nounced as was found at the 4 year time point, as demon-strated by the presence of similar histological andbiochemical profiles in both groups at 6.5 years. Despitethis finding, tolerant individuals noted a better qualityof life, as demonstrated by higher individual satisfaction,lower number of medications required, and reduced IS-related morbidity found in patients off IS. It is noteworthythat quality of life is currently regarded as an importantmeasure of outcome after SOT, and is used in conjuctionwith rejection rates to determine therapeutic choices[29].

2.7. Brussels

Reding and colleagues reported on a pediatric patientwho had IS withdrawn following the onset of lympho-proliferative disorder [30]. This patient was enrolled inan observational study in which the authors intendedto monitor the cytokine fluctuation in the serum of 40pediatric LT recipients, the objective of the study beingto identify patients who are rejecting or accepting theirliver graft. Increased interleukin-10 blood levels at 2 hafter reperfusion, on days 1 and 4 after LT as comparedwith baseline, and decreased tumor necrosis factor alphaand interferon gamma blood levels at the same timeintervals, characterized the cytokine profile of the IS-free recipient.

2.8. Taiwan

Hsu and colleagues described the cytokine and serumprotein profile of 1 tolerant LT patient of their series, inthe attempt to identify reliable and non-invasive mark-ers of tolerance [31]. They detected significantly highserum concentrations of haptoglobulin, transthyretinand alpha1-antitrypsin, as well as undetectable levelsof interleukin 2.

2.9. Sydney

Complete hematopoietic chimerism and tolerance ofa liver allograft harvested from an O RhD-positivedeceased male donor was described in an O RhD-nega-tive 9-year-old girl, with no evidence of graft-versus-hostdisease 17 months after LT [32]. The onset of COT waspreceded by a period of severe hemolysis due to the pro-duction of antibodies by residual recipient derived Blymphocytes against engrafted erythroid cells from thedonor, and suggestive of the development of chimerismby engraftment of the recipient marrow from passenger

hematopoietic stem cells within the transplanted liver.The hemolysis was resistant to standard steroid treat-ment but resolved after the gradual withdrawal of allIS. The patient is reported to be very well with normalgraft function and no rejection, 5 years after thetransplant.

As the patient experienced a profound lymphopeniaof unknown origin, perioperatively and in the subse-quent months after LT, the authors speculate that thesephenomena may have contributed to the engraftment ofdonor hematopoietic stem cells. This speculation is con-sistent with the clonal-deletion theory formulated byStarzl, for which a key event before the engraftment isthe dramatic reduction of immune competent cellsinduced by the lympho-depleting regimen [33].

3. Group B: molecule-based tolerogenic protocols

3.1. Pittsburgh – the tolerogenic protocol

In 2003, Starzl and colleagues published the results of atrial in which they administered ab initio an immunosup-pressive protocol expected to be tolerogenic, to 82 adultkidney, liver, pancreas and intestinal transplant recipients[33]. The hypothesis on which this protocol was designedwas that the need for continuous high dose IS can beavoided in most cases with the use of a strong lympho-cyte-depleting regimen prior to engraftment, followedby the administration of low dose tacrolimus mono-therapy. The goal of the induction treatment was theremoval of clones of immune cells that could elicit graftrejection before contact with foreign donor antigens; inthis situation the repletion of immune cells throughhomeostatic expansion triggered as a result of the leuko-cyte depletion would occur once the new antigens are inplace after transplantation. In addition, the minimizationof maintenance IS aimed to reduce further the anti-donorresponse with just enough treatment to prevent irrevers-ible immune damage to the graft, but not with such heavytreatment that the donor-specific clonal exhaustion-dele-tion process is precluded.

After an 18-month follow-up, 1-year patient andgraft survival rates were 95% and 82%, IS-related mor-bidity was virtually eliminated, and 48/72 survivingrecipients receiving spaced doses of tacrolimus mono-therapy. These results were described as ground-break-ing, as 25/39 (64%) renal, 12/17 (70%) liver, 5/12(42%) pancreas, as well as 6/11 (54%) intestinal trans-plant recipients, were on spaced doses at the time ofmanuscript preparation. Even if the finding that nopatient could be weaned off IS represents a matter ofconcern and questions the working hypothesis, the strik-ing reduction of the daily dose of IS should be regardeditself as an outstanding achievement for two reasons: itwas obtained after transplantation of organs (namely,


kidney, pancreas and intestine) considered highly immu-nogenic; it led to a significantly reduction of IS-relatedmorbidity. It is important to note that other protocolsbased on a similar strategy, i.e. leukocyte depletion fol-lowed by low-dose IS has been described previously forrenal transplant recipients [34–39].

3.2. New Orleans

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

IS could be withdrawn completely in only onepatient, whereas three patients rejected after an initialresponse, and 11 patients developed acute rejection aftertapering of IS. The fate of the remaining three recipientsis difficult to ascertain. The authors concluded fromtheir limited experience that IS withdrawal after LT isharmful, rather than beneficial, to patients.

The lesson to learn from the Pittsburgh and NewOrleans trials is that the working hypothesis is either erro-neous, or incomplete. In fact, several central and periph-eral mechanisms other than exhaustion and deletionmight be involved in the induction of tolerance, includingintra-thymic clonal deletion of precursor T cells express-ing T-cell receptor, dendritic cells, peripheral clonal dele-tion of allogenic T cells, anergy of allogenic T cells,cytokine deviation, cellular regulation of T cells, etc. Inaddition, in most situations, it seems that leukocyte deple-tion is not accompanied by permanent and complete dele-tion of alloaggressive donor-reactive cells, and theestablishment of a regulatory network is required tomaintain tolerance. Moreover, a number of laboratorystudies analysing samples from recipients treated withleukocyte depleting agents have shown that antigen expe-rienced or memory T cells are less susceptible to depletionand may be resistant to suppression by some immunosup-pressive drugs [41,42]. Thus in some recipients, residualmemory T cells may abrogate the potential benefit ofinduction with a leukocyte depleting agent.

3.3. Safed–London (Ontario)–Winnipeg

Assy and colleagues published the results of a con-trolled trial on total IS withdrawal after LT [43,44].They randomized 26 stable LT patients to receive15 mg/kg of ursodeoxycholic acid (UDCA) or placebo,followed by the sequential weaning of IS. The rationaleof such a strategy was the presumed evidence thatUDCA reduces the incidence and severity of acute rejec-tion in the liver allograft, by attenuating the expression

of major histocompatibility complex class molecules inbiliary epithelia and central vein endothelia, during theearly phase of LT [45].

Results did not confirm the working hypothesis, as15/26 individuals (58%) developed an episode of acuterejection within 12–21 weeks from tapering of IS. Allof the rejection episodes responded to the resumptionof IS and none of the patients developed chronic rejec-tion. Only two patients (8%) were IS-free 1 year fromthe study entry. Although the fate of the remaining nineindividuals who did not experience rejection remainsunclear, it is presumed that they were still under theweaning protocol at the time of publication.

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 lymphohis-tiocytosis, an inherited disorder whose only curativetreatment is allogeneic stem cell transplantation [46].The little girl underwent stem cell transplantation fromher haploidentical mother, 2 months after receiving a liv-ing-related LT from the same donor for acute hepaticfailure of unknown origin. The diagnosis of familialhemophagocytic lymphohistiocytosis could only bemade after the surgery, from the histology of the explant-ed liver and from bone marrow biopsies. Myeloidengraftment occurred on day +10, but full chimerismwas documented only on day +57. At that time, IS wasdiscontinued. Four months after IS withdrawal, thepatient was reported to be doing fine with normal graftfunction and histology.

4.2. Gothenburgh

The group from the Queen Silvia Children’s Hospitalreported on the case of a 1-year-old patient who becametolerant 4 weeks after living donor LT performed for aveno-occlusive liver disease complicating stem cell trans-plantation [47]. The infant girl was diagnosed with acutemyeloid leukemia and treated with first-line chemother-apy followed by consolidation with stem cell transplan-tation. The onset of severe veno-occlusive diseaseresulted in an emergency LT where the liver segments2 and 3 were donated by her HLA-identical father,who also was the source of stem cells. IS consisted ofprednisolone and tacrolimus. Prednisone was rapidlytapered and withdrawn by the end of the first week,while tacrolimus was stopped by the end of the firstpostoperative month. This strategy was adopted follow-ing the above reported previous Austrian experience inLT and further experience in lung transplantation.


4.3. Ghent–Brussels

Donckier and colleagues developed a protocol com-bining non-myeloablative conditioning and donor stemcell infusion [48–50]. Such a protocol was investigated inthree patients suffering from multifocal hepatocarcinoma(n = 2) or cholangiocarcinoma (n = 1), who were other-wise ineligible for LT according to the local guidelinesand without any other potentially curative options. Theyall received a right lobe living donor graft, as well as puri-fied CD34+ stem cells harvested from the same donors. ISand conditioning consisted of steroids, rapamycin andantithymocyte globulin; stem cells were infused on day 7.

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

Apart from concerns related to early tumor recur-rences requiring a change in protocol that precludesthe inclusion of patients with extended cancer indica-tions, this protocol is unique in that the two differentprinciples of nonmyeloablative conditioning (Pitts-burgh, New Orleans) and stem cell-related immunemodulation were used.

4.4. Miami

Tzakis and co-workers hypothesized that the periop-erative donor bone marrow cell (DBMC) infusion mightfavour the onset of COT after LT [51]. The conceptualframework underlying the working hypothesis was thatthe infusion of bone marrow cells prolongs allograft sur-vival by still incompletely understood mechanisms[52,53]. One hundred and four individuals enrolled inthis study were stratified into two groups: Group 1received DBMC infusions at the time of transplantation;Group 2 did not receive any DBMC infusion. Mainte-nance IS was tapered over a 3-year time period.

Results from this study were discouraging in the con-text of added benefit from DBMC infusion, as the inci-dences of acute rejection and COT were similar in both

groups. In addition, no significant differences wereobserved 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 thelast decade of the immunomodulatory properties of stemcells and other progenitor cells like bone marrow-derivedmesenchymal stem cells [MSC, which show in vitro, thesame immunologic characteristics described in the wholebone marrow infusion used by the Pittsburgh and Miamigroups [52]], suggests that their working hypothesismight have been correct, although the clinical translationmay not yet have been optimised. In fact, because MSCare a rare cell population present in the bone marrowmicroenvironment, and because it has been demon-strated that the immunodmodulatory effects exerted byMSC are dose-dependent – i.e., the higher the numberof cells, the stronger the inhibition of the immune cells[55] – it could be argued that the number of MSC infusedto patients in the Miami protocol was insufficient. Note-worthy, a similar strategy has been successfully imple-mented by Sachs and co-workers in five patients withend-stage renal disease who received combined bonemarrow and kidney transplants from living relateddonors, with the use of a nonmyeloablative preparativeregimen [56]. In four of them, it was possible to inten-tionally discontinue all immunosuppressive therapy 9–14 months after the transplant, and renal function hasremained stable for up to 5.3 years since transplantation.

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 forskin- and liver-based chronic GVHD, occurring 7 yearsafter an allogeneic bone marrow transplantation per-formed for sideroblastic anemia [57]. The donor was anHLA-identical female cousin. After the operation, thepre-existing chronic GVHD of the skin disappeared,and the immunosuppressive therapy – consisting of aza-thioprine, prednisone and cyclosporine – was graduallytapered and finally withdrawn 71 months after LT. Twoand a half years after discontinuation of all IS, the patientwas in excellent condition with neither signs of chronicGVHD nor rejection of the liver graft. No further infor-mation is provided, and the presence or absence of donorchimerism was not investigated.

5.2. Zurich

Clavien and colleagues reported a case of COTobtained in a recipient of a living-related LT who was


suffering from an advanced hilar cholangiocarcinoma[58]. Thirteen years earlier, the patient had received abone marrow transplant from the same donor – namely,his HLA-identical sibling – for acute lymphatic leuke-mia. In this case, the concept of COT was combinedwith the potential eradication of microscopic tumor dis-ease and downstaging of the main tumor mass, througha neoadjuvant chemo-irradiation protocol (namely,external beam radiation and intravenous 5-fluorouracil),followed by radical tumor excision through a completehepatectomy, in order to offer the best possible chancefor a longterm cure. The removal of post-LT mainte-nance IS was justified by the presence of full chimerismin the recipient and aimed to eliminate a major risk fac-tor for recurrence after transplantation (namely, ISitself). A control liver biopsy at 1 year from the LTshowed no rejection, while imaging at 21 monthsshowed no evidence of tumor recurrence. Twenty-fivemonths after LT, the patient was doing well with normalgraft function.

5.3. Chapel Hill

Andreoni and colleagues described the case of a livingdonor LT for HCV-related cirrhosis in a 42-year-oldwoman who had undergone bone marrow transplanta-tion at the age of fifteen for aplastic anemia [59]. Thedonor was in both cases the patient’s HLA-identical sis-ter. Before LT, analysis of tetranucleotide markers onchromosome 3, 7 and 12 showed complete engraftmentof the transplanted bone marrow. Postoperatively, after6 months of IS to provide protection from GVHD, theIS was progressively tapered and eventually stopped at1 year. Twenty-four months after LT, the patient wasreported to be doing well with normal graft functionand histology.

5.4. The European consortium for COT after LT

Dr. Sanchez-Fueyo conceived and led a Europeanconsortium with the goal to define a clinically applicableassay of COT that would be able to identify a cohort ofLT patients who might be candidates for IS withdrawal[60,61]. Seventeen tolerant patients from four Europeancenters were enrolled and compared with 79 controls.The authors studied the transcriptional patterns in theperipheral blood by employing oligonucleotide micro-arrays and quantitative real-time polymerase chain reac-tion, and discovered and validated several genesignatures comprising a modest number of genes capa-ble of identifying tolerant and non-tolerant recipientswith high accuracy. Multiple peripheral blood lympho-cyte subsets contributed to the tolerance-associatedtranscriptional patterns, although natural killer cellsand gamma–delta T cell receptor-positive T cells exerted

the predominant influence. These latter findings are con-sistent with data previously reported by the Kyotogroup in IS-free children who had received a livingdonor LT [23]. The data obtained from transcriptionalprofiling of the peripheral blood of IS-free LT recipientssuggest that there is a molecular signature of tolerancethat can be employed to identify liver transplant recipi-ents who can be weaned off IS and that innate immunecells are likely to play a major role in the maintenance ofCOT after LT.

6. How does the mechanism for COT occur?

Thus far, our efforts to understand the mechanismsunderlying the phenomenon of COT have been mostlyin vain. We know that the liver has immunomodula-tory properties and that LT recipients are more proneto develop COT than recipients of other types of solidorgan allografts. However, we do not know how thecells and mechanisms involved cooperate in order toinduce and maintain COT, why COT occurs and whenthe chances for COT to develop are at their highest.Production of donor-strain soluble MHC antigen bythe transplanted liver, induction of donor-derivedmicrochimerism by stem cells transferred with thegraft, mass-effect attributed to passenger leukocytesoriginating from the donor and many other mecha-nisms have been proposed, but none of these theorieshas been able to explain this fascinating clinical phe-nomenon so far.

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

This article of the current literature demonstratesthat strategies that have been investigated to date withthe objective of achieving a permanent IS-free stateare numerous and very heterogeneous in terms of con-cept, immunological background and rationale,patient age, underlying indication for LT, endpoint,type of LT (deceased versus living donor), length ofthe weaning period, length of follow-up, presence ornot of donor chimerism, full or partial chimerism, tol-erogenic tools adopted, etc. However, the worldwideexperience reported in the English literature to date,could be summarized as follows: a permanent and sta-ble IS-free state can be achieved in some patients whohave received a LT for hepatic-based non-immunolog-ical diseases, and those patients who do not becometolerant and develop rejection are not exposed to ahigher risk for graft loss or death. Moreover, recentdata suggest that it may be possible to use transcrip-tional profiling to identify LT transplant recipientswho may be susceptible to the development of an IS-free state.


8. Conclusions

Effective COT has been reported more frequently inLT recipients than after transplantation of any otherorgan. In some cases COT has been documented asan accidental finding, but in the last decade it has beenobtained intentionally in selected patients, in a numberof clinical trials. The present paper reviews 163 cases ofCOT and demonstrates that the belief that COT cannotbe achieved without exposing the recipient to anincreased and unacceptable risk of graft loss is nolonger justified and is perhaps outdated in the field ofLT. The evidence that a permanent and stable IS-freestate can be safely attempted and sometimes achievedin patients who have received a LT for treatment ofnon-immune mediated liver diseases, is incontrovert-ible. Notably, patients who develop acute rejection dur-ing the protocols designed to discontinue IS, are notexposed to further risks of graft loss once maintenanceIS is resumed. Consequently, the belief that weaning offIS after LT is harmful in terms of an increased risk ofgraft loss is not substantiated by the published data.

9. Future perspectives

The failure of tolerogenic molecule-based protocolsmeans that the molecular strategies investigated to datemay be sub-optimal. The pathways of the immuneresponse triggered by the engraftment of an allogeneicorgan, may be too numerous to be controlled by justone or few compounds. In the stem cell era, the fieldof SOT has just started to address its interest towardsprogenitor cells which show impressive immunomodula-tory properties. As stem cells have been used to treatconditions characterized by immunologic dysregulationsuch as Crohn’s disease and graft-versus-host-diseaseafter allogeneic haematopoietic stem cell transplanta-tion, we may speculate that the same immunomodula-tory properties might be potentially useful for theprevention or treatment of SOT rejection, as well asfor the induction of COT, as demonstrated by the casesreported by Gothenburgh, and the Austrian and Belgianconsortiums [45–49]. Moreover, as stem cells are capableof promoting tissue repair, harnessing both the immuno-modulatory capabilities of such cells and their ability fortissue repair provides an exciting opportunity for furtherresearch in the field of SOT [54].

Acknowledgements

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

Giuseppe Orlando is recipient of the Marie CurieInternational Outgoing Fellowship POIF-GA-2008-221850, financed by the European Commission underthe 7th Framework Program for Research andDevelopment.

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