Operational tolerance in liver transplantation: Shall we predict or promote?

Authors

  • Geoffrey W. McCaughan,

    Corresponding author
    1. Australian National Liver Transplantation Unit, Royal Prince Alfred Hospital, University of Sydney, Sydney, Australia
    • A. W. Morrow Gastroenterology and Liver Centre, Centenary Institute, University of Sydney, Sydney, Australia
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  • David G. Bowen,

    1. A. W. Morrow Gastroenterology and Liver Centre, Centenary Institute, University of Sydney, Sydney, Australia
    2. Australian National Liver Transplantation Unit, Royal Prince Alfred Hospital, University of Sydney, Sydney, Australia
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  • Patrick Bertolino

    1. A. W. Morrow Gastroenterology and Liver Centre, Centenary Institute, University of Sydney, Sydney, Australia
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  • See Article on Page 937

Address reprint requests to Geoffrey W. McCaughan, M.D., Ph.D., A. W. Morrow Gastroenterology and Liver Centre, Centenary Institute, Royal Prince Alfred Hospital, University of Sydney, Missenden Road, Camperdown, New South Wales 2050, Australia. Telephone: +61 2 9515 8578; FAX: +61 2 9515 8242; E-mail: g.mccaughan@centenary.usyd.edu.au

The liver is recognized to be the most tolerogenic organ. Liver transplants are accepted in most mouse and some rat strain combinations across complete major histocompatibility complex mismatches and in some outbred pigs.[1] Transplantation of the liver in some rat strains can actually prevent the rejection of skin, heart, and kidney allografts and can, in fact, turn off an ongoing rejection episode in those organs.[2] In humans, hyperacute rejection of the graft by antibody-mediated mechanisms is rare, and it is recognized that chronic allograft rejection is uncommon.[3] It is clear that tolerance in humans can be observed if bone marrow chimerism becomes established, but this is rare.[4]

More general observations indicate that some patients are observed to be tolerant many years after transplantation. On an intention-to-treat basis from the time of transplantation, only approximately 5% to 10% of adults end up in this situation.[5] However, if patients are highly selected by successful weaning before the withdrawal of immunosuppression, then approximately 20% to 40% of adults and up to 60% of children can be defined as tolerant.[7-11] An increased frequency of successful immunosuppression withdrawal is associated with an increasing length of time between the withdrawal of immunosuppression and transplantation as well as a lack of inflammation on prewithdrawal biopsy samples. This state is not true tolerance, but it has been labeled operational tolerance.[12]

These patients have been studied for gene signatures in peripheral blood leukocytes and within the allograft itself that may identify tolerance before immunosuppression withdrawal. The initial study from a European collaboration showed that 16 tolerant patients studied 11 years after transplantation could be characterized by an enrichment in peripheral blood leukocyte natural killer gene signatures together with an increased frequency of γδ T cells and regulatory T cells.[13] Follow-up studies confirmed this in tolerant patients before the withdrawal of immunosuppression.[14] A more recent study of hepatic gene transcripts indicated an enrichment of iron-associated genes, particularly hepcidin, together with an anti-inflammatory gene set.[15] In a pediatric population, a 3-gene signature that may define tolerance has recently been identified in peripheral blood leukocytes.[16] The 3 genes (ERBB2, SEN6, and FEMIC) are thought to be associated with natural killer cell function and predicted tolerance with a positive predictive value of 100% and a negative predictive value of 96%.

A publication in this journal now claims that the selection of patients for immunosuppression is even easier.[17] Once again, the criteria used to select these patients for a trial of immunosuppression withdrawal were very strict (>3 years after transplantation, no autoimmune or current viral disease, no rejection in the past 12 months, normal liver function tests, and at least 1 significant immunosuppression complication), and they resulted in 67 potential subjects. Thirty-one entered the study, but 7 were excluded because of inflammatory changes on biopsy, and this left 24 for the initiation of immunosuppression withdrawal. Quite extraordinarily, 15 patients had normal liver function tests at a median of 14 months after withdrawal. Operational tolerance was achieved in all 7 patients who had undergone transplantation more than 10 years before the start of immunosuppression withdrawal and whose lymphocytes displayed a low stimulation index to a non–antigen-specific mitogen.

Although this study[17] improves the prediction rate for the successful withdrawal of immunosuppression, it raises some key issues. First, it is unclear what proportion of the initial transplant cohort these patients actually represent. It is stated that 15 of 138 patients (11%) were tolerant, but the 138 patients did not include patients who had died after transplantation and those who were no longer being followed at the transplant center. Second, no liver biopsy data obtained either before withdrawal or from the last follow-up were presented for the tolerant patients. Some previous studies, although not all, have suggested that many of these patients have inflammatory or fibrotic changes in follow-up biopsy samples that can improve with the re-introduction of immunosuppression.[7, 18] Importantly, such changes may be present even in the setting of normal liver function tests. Third, the lymphocyte stimulation test was a global assessment rather than an allograft-specific assessment of lymphocyte function. The fact that the peripheral blood leukocytes of tolerant patients exhibited low stimulation index levels suggests some form of a global, nonspecific state of immune hyporesponsiveness rather that donor-specific tolerance. No data were provided about lymphocyte responses to other unrelated antigens such as tetanus toxoid. If these had also been reduced, the specificity of the tolerant state of these patients would have been questionable.

So where does this leave us? It does appear that some patients can be defined as operationally tolerant many years (>5 years) after transplantation. From the time of transplantation, this figure is likely to be approximately 5% of all patients, although it could be as high as 60% (or even 100%!) in a population selected for immunosuppression withdrawal by stringent criteria. It also seems that various gene signatures can be used to select some of these patients. Challenges occur in identifying similar patterns in cohorts from different centers, including international collaborations. A further crucial issue is the study of the timing of the onset of these signatures: how early do they develop?

The latter question takes us to the issue of tolerance induction versus maintenance. Experimentally, during the maintenance phase, liver transplant tolerance can be transferred by recipient splenic lymphocytes in animal models.[2] However, this is possible only after 70 to 100 days after transplantation. Recent studies have clarified that T regulatory cells (CD4+, CD25+, and forkhead box P3+) are associated with tolerance and can transfer the tolerant state.[19-21] This implies that effective regulatory responses occurring after transplantation take time to become established. The correlation with the timing of the development of operational tolerance in humans supports this concept. Is recognizing this important? The answer is a strong yes! The outcome of a recent analysis of immunosuppression minimization very early after liver transplantation suggests that this concept is not always understood. In that study, anti-thymocyte globulin was used in an attempt to induce a tolerogenic predisposition, and reduced-dose tacrolimus was administered with early tapering.[22] This approach failed with an allograft rejection rate of 52%. Furthermore, no patients were able to be withdrawn from immunosuppression.

This approach fails to take into account much of the experimental data on the process of the induction of liver transplant tolerance. It is known from experiments from a number of groups, including ours, that the induction of liver tolerance (versus rejection) in rats occurs in the first 1 to 2 weeks after transplantation.[1, 2] During this phase, tolerance is not due to immune regulation because tolerance cannot be transferred by recipient splenocytes. In fact, liver transplant tolerance during this phase is an active deletion process by which an actual allograft rejection episode does not lead to full-blown rejection but rather leads to tolerance.[1, 2] This process requires both donor leukocytes and donor liver parenchyma.[23] During the immediate posttransplant phase, donor leukocytes leave the allograft and activate alloreactive recipient T cells in host lymphoid tissues; this triggers their deletion.[24, 25] Concomitantly, naive or activated alloreactive recipient CD8 T cells entering the allograft from systemic lymphoid tissues are deleted[26, 27] without the development of cytolytic function via degradation in hepatocytes (suicidal emperipolesis) and Bim-dependent apoptosis.[28-30] These processes combine and lead to effective early deletion of the CD8 T cell allograft-specific response, and this results in the tolerance and initial acceptance of the liver allograft.

This leads us to 2 questions. First, if tolerance induction requires activation of the recipient's immune response, why would an ablative immunosuppression approach induce tolerance? The answer is that it would not; in fact, it could (and did) promote allograft rejection.[22] A corollary to this is whether we know exactly what our current immunosuppressive regimes do to the induction of tolerance. Experimentally, we know that corticosteroid therapy actually breaks tolerance in the rat model.[31] Theoretically, significant levels of immunosuppression during the earliest posttransplant phase (from day 1) should also prevent early recipient immune activation and hence prevent the induction of tolerance. We have previously argued that the best way to induce tolerance in humans might be to delay or minimize immunosuppression for the first 3 days after transplantation.[3, 5, 32] However, most transplant units, including our own, are reticent to do this without a better understanding of the nature and characterization of tolerance induction in humans. It should be noted that the low rate of tolerance in humans when it is analyzed on an intention-to-treat basis may be due to our practice of preventing rejection and tolerance at the same time during these early stages after transplantation. This leads to the second question: what is the link between the gene signatures that have been shown to correlate with tolerance in the long term and events during the induction phase? Furthermore, how early after liver transplantation do these signatures develop, and how soon can they be characterized? We simply do not know the answers to these questions, and until we do, accurate identification of potentially tolerant patients earlier than 5 to 10 years after transplantation will remain elusive. Thus, although many data have now emerged on the withdrawal of immunosuppression late after liver transplantation, it is likely that this timing is far too late for interventions that will actually alter the likelihood of achieving operational tolerance.[9] The differences in tolerance in the early and late phases after transplantation and the links between operational tolerance and early tolerance induction need to be understood (Table 1). If we can achieve this, then maybe we can promote tolerance in the earliest phases after transplantation instead of just predicting tolerance late.

Table 1. Induction and Maintenance Phases of Liver Transplant Tolerance
Induction PhaseMaintenance Phase
1. Occurs from day 1 to 2 weeks in experimental models1. Takes >100 days to establish in experimental models
2. Not transferable by splenocytes in experimental models2. Transferable by splenocytes in experimental models
3. Timing not known in humans but may be blocked by early immunosuppression3. Seen after >5 years in humans
4. Not associated with regulatory T cells4. Associated with regulatory T cells in experimental models and humans
5. Associated with immune activation and deletion rather than regulation5. Associated with immune regulation in experimental models and humans

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