Chi-Xian Zhang, Pei-Hao Wen and Yu-Ling Sun

Zhengzhou, China

Withdrawal of immunosuppression in liver transplantation and the mechanism of tolerance

Chi-Xian Zhang, Pei-Hao Wen and Yu-Ling Sun

Zhengzhou, China

BACKGROUND: Immunosuppression reagents have side effects and cause considerable long-term morbidity and mortality in patients after liver transplantation. Sufficient evidences showed that minimization or withdrawal of immunosuppression reagents does not deteriorate the recipient's immune response and physiological function and therefore, is feasible in some recipients of liver transplantation. However, the mechanisms are not clear. The present review was to update the current status of immunosuppression in liver transplantation and the mechanism of minimization or withdrawal of immunosuppression in liver recipients.

DATA SOURCES: We searched articles in English on minimization or withdrawal of immunosuppression in liver transplantation in PubMed. We focused on the basic mechanisms of immune tolerance in liver transplantation. Studies on immunosuppression minimization or withdrawal protocols and biomarker in tolerant recipients were also analyzed.

RESULTS: Minimization or withdrawal of immunosuppression can be achieved by the induction of immune tolerance, which may not be permanent and can be affected by various factors. However, accurately evaluating immune status post-transplant is a prerequisite to achieve individualized immunosuppression. Numerous mechanisms for immune tolerance have been found, including immunophenotypic shift of memory CD8+T cells and CD4+T cell subsets. Activation of the inflammasome through apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) in dendritic cells is associated with rejection after liver transplantation.

CONCLUSIONS: Minimization or withdrawal of immunosuppression can be achieved by the induction of immune tolerance via different mechanisms. This process could be affected by immunophenotypic shift of memory CD8+T cells and CD4+T cell subsets, which may be correlated with activation of the inflammasome through ASC in dendritic cells.

(Hepatobiliary Pancreat Dis Int 2015;14:470-476)

liver transplantation;

immunosuppression;

minimization;

withdrawal;

tolerance

Introduction

Liver transplantation is the only effective treatment for end-stage liver disease. However, rejection after transplantation results in short-term graft loss. Although immunosuppressive drugs prevent early graft rejection, long-term complications including infections, renal toxicity, neurotoxicity and bone marrow suppression are common.[1-4]To address the side effects of longterm immunosuppression, scientists developed strategies on minimization or withdrawal of immunosuppressive drugs in liver recipients without rejecting liver grafts while maintaining the normal immune response. In addition, physicians can provide individualized treatment plans with varying drug concentrations based on the patient's immune status. Despite these research advances in immunosuppression,[5-7]the failure in the process of withdraw of immunosupression happened and there were no biological markers for monitoring the immune status. Furthermore, the mechanism of operational tolerance was still not clear. The present review was to explore the application of minimization or withdrawal of immunosuppression in liver recipients and the possible mechanisms of immune tolerance in this population.

Clinical application of minimization or withdrawal of immunosuppression

Minimization or withdrawal of immunosuppressant afterliver transplantation is much easier in children because their immature immune systems allow reshaping of the immune response. In a study[8]performed at the University of Pittsburgh, 20% of children had successfully withdrawn from immunosuppression after liver transplantation, and 37% were in the process of withdrawal. In a pilot study reported by Feng et al,[9]60% of pediatric recipients of parental living donor liver transplants remained off immunosuppression therapy for at least 1 year with normal graft function and stable allograft histology. Because of the inherent tolerogenic property of the liver and careful scrutiny of accumulated clinical experience, immunosuppression weaning is feasible in almost 20% of selected liver transplant recipients.[10-12]Although minimization or withdrawal of immunosuppression can be achieved by the induction of immune tolerance, tolerance may not be permanent and can be affected by various factors.[9]It is therefore necessary to monitor the immune status of these patients. However, no reliable biological markers are currently available.

The timing and specific approach to minimization or withdrawal of immunosuppression are important factors influencing outcomes. In clinical practice, initiation of immunosuppressive minimization or withdrawal ranges from 3 months to 2 or even 5 years after transplantation. However, in most patients minimization of immunosuppression was initiated 1 year after the last rejection occurred.[13,14]The successful rate of earlier withdrawal (in 6 months after transplantation) is very low (1/18). However, this rate is much higher and the procedure is safer when minimization or withdrawal was initiated 1 year after the transplant.[15]The process of immunosuppressive minimization or withdrawal generally begins with glucocorticoid and extends to calcineurin inhibitor until complete withdrawal of immunosuppressive drugs.[16-18]One study on long-term outcomes of 600 living donor liver transplants for pediatric patients at a single center concluded that it is easier to induce immune tolerance and achieve a high success rate with minimization or withdrawal by prolonging maintenance immunosuppression rather than frequently decreasing the doses during minimization. Furthermore, graft rejection is typically mild and easy to control.[19]These data are summarized in Table.

After complete withdrawal of immunosuppression, patients who underwent living donor liver transplantation often experienced some degree of liver fibrosis accompanied by small bile duct hyperplasia and luminal narrowing; these complications often ameliorated after the use of immunosuppression via an unknown mechanism.[20]Egawa and colleagues[21]demonstrated that if minimization or withdrawal of immunosuppression results in rejection, original immunosuppressive therapy should be resumed.

Mechanisms of operative tolerance in liver transplantation

With or without immunosuppression, immune tolerance to self-major histocompatibility complex (MHC) antigens has been a limitation in organ transplantation, with no satisfactory method to induce tolerance in humans.[22]Although it is common to completely withdraw immunosuppressive drugs after liver transplantation because of the immunologically privileged status of the liver, immunologic tolerance induction in organ transplantation is not yet fully understood. Numerous mechanisms for immune tolerance have been found, including immune incompetence of antigen-specific T cells induced by antigen-presenting cells and suppression of memory cell reactions by the generation of regulatory T cells (Tregs).[23,24]Studies[25,26]have also found that soluble MHC-related molecules, Fas protein, leukocytes from the donor liver and hematopoietic chimerism are all involved in the formation of immune tolerance.

To achieve minimization or withdrawal of immunosuppression after liver transplantation, transplant centers have used a variety of methods to induce immune tolerance. In 2003, Starzl et al[27]successfully achieved minimization of immunosuppression by clearing lymphocytes before transplantation to induce immune tolerance. In this study, 82 patients awaiting for transplantation of the kidney, liver, pancreas or intestine were pretreatedwith a broadly reacting rabbit antithymocyte globulin in several hours. Post-transplant immunosuppression was restricted to tacrolimus unless additional drugs were needed to treat breakthrough rejection. After 4 months, patients on tacrolimus monotherapy were considered for dose-spacing to every other day or longer intervals. 95% (78/82) patients survived for 1 year or for 13-18 months. Graft survival rate was 89% (73/82) at 1 year and 88% (72/82) at 13-18 months. Of the 72 recipients with surviving grafts, 43 were on spaced doses of tacrolimus monotherapy: every other day (n=6), three times per week (n=11), twice per week (n=15), or once per week (n=11). Inspired by Starzl's method, other researchers used rabbit anti-thymocyte globulin to induce immune tolerance in transplant patients. These patients not only successfully achieved steroid withdrawal, but the dose of tacrolimus was also significantly reduced.[28]During early liver transplantation, ursodeoxycholic acid can reduce the expression of MHC-associated molecules on biliary and central vein epithelial cells. However, attempts to use ursodeoxycholic acid to induce immune tolerance were not encouraging.[14,29]Other researchers injected hematopoietic stem cells into three transplanted liver to induce hematopoietic chimerism.[30]In 2 cases, in vitro studies showed indices of immunological tolerance as assessed by specific hyporesponsiveness to donor alloantigens in mixed lymphocytes culture. In the third patient, acute rejection rapidly occurred after discontinuation of immunosuppression, and minimal immunosuppression has to be maintained in a long-term follow-up. In a child with familial hemophagocytic lymphohistiocytosis, infusion of hematopoietic stem cells from the donor mother before transplantation enabled the complete withdrawal of immunosuppression.[31]Similar results have been reported by a number of research institutions.[32-34]

Table. Studies in immunosuppression minimization or withdrawal after liver transplantation

Estimation of immune status and biological markers

Dose and concentration of immunosuppressants given after liver transplantation are closely related to the patient's immune status. Therefore, accurately evaluating immune status post-transplant is a prerequisite for the achievement of individualized immunosuppression. Currently, γδT cell phenotypes, natural killer cell receptors, precursor dendritic cell (DC) numbers, ratio of plasma cells to myeloid DCs, peripheral blood CD4+CD25+T cells, CD8+Tregs, chimerism, and low titers of donorspecific antibodies contribute to the induction of immune tolerance to achieve minimization or withdrawal of immunosuppression.[20,35-38]However, the relationship between these biological markers and dynamic changes in immune status is not clear, because these markers are found only in patients who have already established tolerance and cannot accurately predict the patient's immune status over time.

Normal liver function alone after successful reduction or withdrawal of immunosuppression cannot serve as a valid biological marker, because these patients often experience chronic liver fibrosis, requiring occasional liver biopsies. Additionally, the risk of chronic rejection in these patients is also significantly increased.[39]

Memory CD8+T lymphocytes and immune tolerance

Although minimization or withdrawal of immunosuppression in children and adults has been achieved, the presence of memory T cells can interfere with this stable immune status through different mechanisms.[40,41]Memory CD8+T cells are the main effector cells in rejection and are important target cells for immune tolerance in transplantation. The authors[40]found that the phenotypes of memory CD8+T cell subsets in recipients with stable immune status after liver transplantation change regularly with time. After reducing the dose and concentration of immunosuppressive drugs, the patients experienced immune rejection, and the phenotypes of memory CD8+T cell subsets also changed. However, with enhanced immunosuppressant therapy, the main proportion of CD8+cells were dominated by naive CD8+cells and then rapidly restored to the immunophenotypes of memory T cells after effective treatment. Namely, the memory CD8+T cell subsets were rapidly restored to their original phenotypes. These results demonstrated that immunophenotypic shift of memory CD8+T cells was closely related to the change of the immune status in the patients after liver transplantation. Monitoring the immunophenotypic shift of memory CD8+T cells is of great importance in the prediction for allograft rejection and treatment effectiveness after liver transplantation.[42]The mechanisms underlying rejection after minimization or withdrawal of immunosuppression are not clear. In view of the important role of CD8+memory T cells in immune rejection of the transplanted liver, understanding the differentiation and function of these cells is essential to the immunosuppressive treatment. Therefore, developing new methods to identify the role of memory CD8+T cells and their differential roles in rejection may be key issues for new immunosuppressive treatments in liver transplantation.

Additionally, one study[41]showed that immunosuppression has an important effect on the formation and function of memory T cells. Immunosuppression notonly effectively suppresses T cell function and reduces the secretion of cytokines associated with rejection but also has an important role in the generation and maintenance of memory T cells. An in vitro experiment[43]showed that most immunosuppressants can inhibit the production and function of effector cells, with tacrolimus showing the strongest effect, but this inhibitory effect can be reversed by the CD28 co-stimulatory pathway. However, the role of rapamycin is different. Although it can promote the generation of memory CD8+T cells, rapamycin fails to prevent the activation of memory CD8+T cells, and its ability to suppress the function of memory CD8+T cells function is also limited.[44]

CD4+T lymphocytes and immune tolerance

CD4+T cells represent a heterogeneous cell population.[45]A better understanding of CD4+T cell subsets in the immunology of rejection will be instrumental for the development of effective strategies to induce clinical transplantation tolerance. CD4+CD25+Foxp3+Tregs, a minor population of CD4+T cells, play an important role in the control of immunologic tolerance to foreign and alloantigens.[46]Tregs include two major groups: naturally occurring Tregs and adaptive Tregs. The former are produced in a central lymphoid organ and the latter are generated in the periphery after antigenic stimulation in a specific tolerogenic microenvironment.[47]

Over the past decade, compelling evidence has shown the importance of Tregs in the promotion of transplant tolerance in animal models and patients.[48-50]In 2002, Graca et al[51]reported that T cell suppression of graft rejection is an active process that operates beyond secondary lymphoid tissue, and involves the persistent presence of regulatory T cells at the site of the tolerated transplant in skin grafts. In 2008, Li et al[52]found that Foxp3+CD25+CD4+T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-beta expression and IL-4 production. Depletion of recipient CD25+CD4+T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3+Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4+and CD8+T cells in anti-CD25-mAb-treated recipients. Almost at the same time, the study from Pons et al[15]also demonstrated a significant increase in the number of peripheral Tregs in liver transplant recipients who developed tolerance versus those who did not. The mechanism of Tregs in establishing transplantation tolerance was associated with the suppression of all subsets of CD4+T-helper cells in inflammation and transplantation.[45]

In addition to the therapeutic role of Tregs in the induction of allograft tolerance, gene expression profiling examining Treg molecules (Foxp3 and CTLA4) may be useful for identifying transplant patients experiencing tolerance and transplant patients experiencing rejection,[53,54]but it should be pointed out that Foxp3 expression in humans, not like in mice, is not specific for cells with a regulatory phenotype and may be a consequence of activation status.[55]

Future research is needed to identify the mechanisms of different subsets of CD4+T cells in the complex of transplantation rejection and tolerance. The mechanistic studies will help the clinician to devise the strategies to achieve transplantation tolerance in clinical practice.

Other factors

Genetic polymorphism of cytokines and chemokines is closely related to rejection in liver transplantation and may also affect post-transplant immunosuppression.[56]In addition, donor-related and unrelated antibody levels, tissue-specific immune responses, and endothelium-related immune stimulation are closely related to immune tolerance.

Inflammation is a main process of rejection, and activation of the inflammasome in DCs is associated with rejection after transplantation.[57]The up-regulation of the inflammation-associated molecule apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is directly related to rejection in rat liver transplantation models.[58]Inflammasome activation involves nucleotide-binding oligomerization domain-like receptors in the cytoplasm to activate the linker protein containing a C-terminal caspase recruitment domain (ASC) and caspase-1, which cleaves IL-1β from its precursor.[59]The study[58]has confirmed that activation of the inflammasome in DCs is closely related to T cell immunity and plays an important role in transplantation tolerance. The inflammasome in DCs accelerates the proliferation of memory CD8+T cells and regulates their function through different signaling pathways. Memory CD8+T cells also inhibit DC activation and expression of the inflammasome.[60,61]

Summary

Minimization or withdrawal of immunosuppression can be achieved by the induction of immune tolerance, which may not be permanent and can be affected by various factors. The process of immunosuppressiveminimization or withdrawal generally begins with glucocorticoid, extends to calcineurin inhibitor and finally achieves complete withdrawal of immunosuppression. Numerous mechanisms for immune tolerance have been found, including immune incompetence of antigenspecific T cells induced by antigen-presenting cells and suppression of memory cell reactions by the generation of Tregs, soluble MHC-related molecules, Fas protein, leukocytes from the donor liver and hematopoietic chimerism. However, accurately evaluating immune status post-transplant is a prerequisite for the achievement of individualized immunosuppression. Immunophenotypic shift of memory CD8+T cells and CD4+T cell subsets is closely related to the change of the immune status. Activation of the inflammasome through ASC in DCs is associated with rejection after transplantation. However, the relationship between immunophenotypic shift of memory CD8+T cells and activation of the inflammasome through ASC is not clear. Future research is needed to identify the mechanisms of transplantation rejection and tolerance to facilitate the development of strategies to achieve transplantation tolerance in liver recipients.

Contributors: SYL proposed the study. ZCX and WPH performed the study and wrote the first draft. All authors have read and approved the final manuscript. SYL is the guarantor.

Funding: This study was supported by grants from the National Natural Science Foundation of China (81100304) and Science and Technology Bureau of Zhengzhou (131PLJRC658).

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Accepted after revision June 25, 2015

Life isn't about getting and having, it's about giving and being.

—Kevin Kruse

10.1016/S1499-3872(15)60411-8

Author Affiliations: Institute of Hepatobiliary and Pancreatic Diseases, Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhengzhou University, School of Medicine, Zhengzhou 450052, China (Zhang CX, Wen PH and Sun YL)

Yu-Ling Sun, MD, PhD, Institute of Hepatobiliary and Pancreatic Diseases, Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhengzhou University, School of Medicine, Zhengzhou 450052, China (Tel:+86-371-66862122; Fax: +86-371-66964927; Email: ylsun@zzu.edu.cn)

? 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

Published online September 9, 2015.

August 15, 2014