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Operational Tolerance after Liver Transplantation: First Report from India

Published:September 17, 2022DOI:https://doi.org/10.1016/j.jceh.2022.09.002
      Operational tolerance after liver transplantation is an ideal goal to avoid long-term morbidities associated with chronic immunosuppressive medication use. It is achievable in a highly selected group of post-transplant recipients but requires long-term follow-up and strict monitoring. We hereby report a post-transplant case who achieved spontaneous operational tolerance after inadvertent immunosuppression withdrawal.

      Keywords

      Abbreviations:

      DSA (donor-specific antibody), LT (liver transplantation)
      Over the last few decades, liver transplantation (LT) has become a lifesaving and effective treatment option for many irreversible liver diseases with ever improving outcomes, secondary to better understanding of graft pathophysiology and improved immunosuppression protocols. But overall quality of life and survival outcomes are still far from being optimal, especially for pediatric recipients due to need of lifelong immunosuppression and its attendant risks. In this regard, immunosuppression minimization or even withdrawal is now considered the ideal goal in post-transplant management. Multiple studies, mostly single-center reports, have now shown that around 20–60% patients can achieve operational tolerance (i.e. stable allograft function and histology in the absence of chronic immunosuppression).
      • Feng S.
      • Ekong U.D.
      • Lobritto S.J.
      • et al.
      Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants.
      • Feng S.
      • Demetris A.J.
      • Spain K.M.
      • et al.
      Five-year histological and serological follow-up of operationally tolerant pediatric liver transplant recipients enrolled in WISP-R.
      • Wozniak L.J.
      • Venick R.S.
      • Naini B.V.
      • et al.
      Operational tolerance is not always permanent: a 10-year prospective study in pediatric liver transplantation recipients.
      • Londono M.C.
      • Rimola A.
      • O'Grady J.
      • Sanchez-Fueyo A.
      Immunosuppression minimization vs. complete drug withdrawal in liver transplantation.
      • Benitez C.
      • Londono M.C.
      • Miquel R.
      • et al.
      Prospective multicenter clinical trial of immunosuppressive drug withdrawal in stable adult liver transplant recipients.
      • Tang Y.
      • Chen J.
      • Chen B.
      • Guo C.
      Clinical characteristics of immune tolerance after pediatric liver transplantation.
      • Feng S.
      • Bucuvalas J.C.
      • Mazariegos G.V.
      • et al.
      Efficacy and safety of immunosuppression withdrawal in pediatric liver transplant recipients: moving toward personalized management.
      We hereby describe a pediatric post-liver transplant patient who achieved operational tolerance after inadvertent immunosuppression withdrawal.

      Case report

      A female child, diagnosed as a case of biliary atresia in early infancy, underwent Kasai portoenterostomy at 93 days of age at an outside institution. It was successful with resolution of jaundice 3 months after surgery. But then, she started developing recurrent episodes of cholangitis (including late onset cholangitis) and later developed secondary biliary cirrhosis and progressive portal hypertension (including hepatopulmonary syndrome) with growth failure. She was listed for LT for the same and subsequently underwent living-related LT at 12.5 years of age with mother (blood group compatible) as the donor (using left lobe graft). Immunosuppression was given as per institutional protocol (intraoperative methylprednisolone followed by gradual tapering and shifting to oral prednisolone; along with tacrolimus and mycophenolate mofetil). Post-operative period was otherwise uneventful with no major complications. Graft function was maintained throughout the course except for a mild transient increase in alanine transaminase and aspartate transaminase levels at 2 months after LT (requiring short-term increase in oral immunosuppression). Steroids were later tapered and stopped at 6 months after LT, and child was then continued on tacrolimus monotherapy. She remained of regular follow-up for 27 months after LT but was then lost to follow-up. She again presented to the institute 6 years after last follow-up, having stopped immunosuppression completely for last 5 years on her own. She was evaluated in detail for the current graft status. Her biochemical profile was normal including normal hepatic transaminases (which was maintained for further 12 months at last follow-up), normal serum IgG levels, and negative autoimmune markers except anti-smooth muscle antibody (1:40) positive. Graft liver biopsy showed minimal acinar disarray; minimal portal tract inflammation comprising of lymphomononuclear cells with admixed neutrophils – no evidence of portal vein endothelitis or lymphocytic bile duct damage noted; minimal hepatocyte intracellular lipofuscin and few glycogenated nuclei; occasional foci of lobular inflammation; sinusoids prominent and dilated at few places [Figure 1(a, b)]. Masson's Trichrome stain showed maintained architecture with no increase in fibrosis [Figure 1(c, d)]. Immunohistochemistry showed CD45- and CD3-positive lymphocytes in portal tracts, with negative staining for C4d stain [Figure 2]. Details of histology as per standard format are available in Table 1. The donor-specific antibodies (DSA) and donor human leukocyte antigen (HLA) testing could not be done due to unfortunate death of the donor 6 years after LT due to unrelated causes. Recipient HLA testing showed A∗02-A∗11:B∗40-B∗44:DRB1∗10-DRB1∗15 status. Flow cytometry analysis for characterization of Treg and B cells was done in whole blood for the patient and then compared with a healthy control and another post-LT pediatric patient with acute cellular rejection [Table 2; also see supplementary file-1]. This showed that percent frequency of T-regulatory cells was higher in this index patient compared to the healthy control and the patient with acute cellular graft rejection.
      Figure 1
      Figure 1Maintained acinar architecture with unremarkable hepatocytes (A); portal tract with minimal inflammation (B); Masson's Trichrome stain showing maintained architecture with no increase in fibrosis (C & D).
      Figure 2
      Figure 2(A) and (B) highlights immunostaining for CD45 and CD3 positive lymphocytes in portal tracts; C4d staining with no significant deposition (C); CD34 stain highlighting portal vasculature (D).
      Table 1Description of Liver Biopsy Findings as per “Withdrawal of Immunosuppression in Pediatric Liver Transplant Recipients” (WISP-R) Study (Reference no. 2).
      Size/adequacyCriteriaNegative criteriaPositive criteria
      Liver tissue length – 1.5 cmNo central fibrosisNo bile duct damageNo cholangiolar proliferation or steatosis
      Eight portal tractsNo portal fibrosis – 0No significant bile duct lossPrimary portal inflammatory cell type– lymphocytes
      Intensity of inflammation – <1No interface activity, bile duct damage, venular inflammation, lobular ballooning, or spotty necrosisSecondary portal inflammatory cell type – neutrophils

      Tertiary portal inflammatory cell type– plasma cells
      No interface activity,

      Perivenular inflammation and lobular inflammation

      C4d score – 0
      RAI bile duct score – 0

      RAI venous inflammation score – 0
      RAI portal inflammation score – 1

      Primary cell type of lobular inflammation –lymphocytes

      Total RAI-1/9
      Table 2Flow Cytometry Analysis Comparing Index Patient vs. Patient With Acute Post-transplant Graft Rejection vs. a Healthy Control.
      PopulationGroups
      HC (healthy control)PTR (post-liver transplant rejection)PTT (post-liver transplant tolerance - index patient)
      Lymphocytes20.625.722.1
      Total T cells (CD3+)4450.961.3
      % of CD4+ T cells/total T cells43.313.750.3
      % of CD8+ T cells/total T cells56.786.349.7
      Treg cells (CD25+ CD127 low)/CD4+ T cells2.5718.822.7
      Memory treg/tregs32.423.125.8
      Naive tregs/tregs67.676.972.6
      B cells7.49.7320
      Translational B cells/B cells19.32.676.94
      Plasmablast/B cells35.674.120
      Naive B cells/B cells4.48458.04

      Discussion

      Spontaneous operational tolerance after LT is an ideal outcome considering the fact that it avoids long-term complications associated with chronic immunosuppressive medication use.
      • Feng S.
      • Ekong U.D.
      • Lobritto S.J.
      • et al.
      Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants.
      ,
      • Londono M.C.
      • Rimola A.
      • O'Grady J.
      • Sanchez-Fueyo A.
      Immunosuppression minimization vs. complete drug withdrawal in liver transplantation.
      This is especially relevant to pediatric population who receive liver grafts early in the lifetime and have to endure immunosuppression over many forthcoming decades. Thus, minimization and if possible, even withdrawal of these medications is currently the guiding principle of designing individualized immunosuppression protocols worldwide. If achieved, this immunosuppression-free state has been proven to reduce the metabolic syndrome risks and other attendant side-effects and also improve overall quality of life.
      • Benitez C.
      • Londono M.C.
      • Miquel R.
      • et al.
      Prospective multicenter clinical trial of immunosuppressive drug withdrawal in stable adult liver transplant recipients.
      ,
      • Feng S.
      • Bucuvalas J.C.
      • Mazariegos G.V.
      • et al.
      Efficacy and safety of immunosuppression withdrawal in pediatric liver transplant recipients: moving toward personalized management.
      ,
      • Mohammad S.
      • Sundaram S.S.
      • Mason K.
      • et al.
      Improvements in disease-specific health-related quality of life of pediatric liver transplant recipients during immunosuppression withdrawal.
      Previously, studies on tolerance were mainly focused on situations where medications were withheld either due to patient non-adherence or secondary to their contraindications or side-effects. Protocol-based immunosuppression withdrawal is now being increasingly practiced in expert centers in a highly selected group of patients. Inclusion criteria are these studies usually comprise of stable graft function over a prolonged time period on single immunosuppression (with stable dosage), no recent episodes of graft rejection and a non-viral or non-autoimmune etiology of liver disease at baseline.
      • Feng S.
      • Ekong U.D.
      • Lobritto S.J.
      • et al.
      Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants.
      ,
      • Wozniak L.J.
      • Venick R.S.
      • Naini B.V.
      • et al.
      Operational tolerance is not always permanent: a 10-year prospective study in pediatric liver transplantation recipients.
      ,
      • Benitez C.
      • Londono M.C.
      • Miquel R.
      • et al.
      Prospective multicenter clinical trial of immunosuppressive drug withdrawal in stable adult liver transplant recipients.
      • Tang Y.
      • Chen J.
      • Chen B.
      • Guo C.
      Clinical characteristics of immune tolerance after pediatric liver transplantation.
      • Feng S.
      • Bucuvalas J.C.
      • Mazariegos G.V.
      • et al.
      Efficacy and safety of immunosuppression withdrawal in pediatric liver transplant recipients: moving toward personalized management.
      ,
      • Orlando G.
      • Manzia T.
      • Baiocchi L.
      • Sanchez-Fueyo A.
      • Angelico M.
      • Tisone G.
      The Tor Vergata weaning off immunosuppression protocol in stable HCV liver transplant patients: the updated follow up at 78 months.
      Some studies have shown that if carefully selected, these operationally tolerant subjects can maintain stable allograft histology (with absence of increased inflammation or progressive fibrosis on long-term follow-up liver biopsies) in spite of apparently active humoral alloimmune responses (in form of positive de novo DSAs).
      • Feng S.
      • Demetris A.J.
      • Spain K.M.
      • et al.
      Five-year histological and serological follow-up of operationally tolerant pediatric liver transplant recipients enrolled in WISP-R.
      On the other hand, recent studies have this spontaneous operational tolerance is a dynamic and a non-permanent state, and thus, patients may lose tolerance on follow-up.
      • Wozniak L.J.
      • Venick R.S.
      • Naini B.V.
      • et al.
      Operational tolerance is not always permanent: a 10-year prospective study in pediatric liver transplantation recipients.
      Thus, long-term regular follow-up including laboratory monitoring and protocol-based surveillance biopsies are a must in such cases to rule out subclinical rejection.
      Although difficult to predict, some variables have been found to be associated with operational tolerance: pediatric age group, younger age, prolonged time interval after transplantation, male sex, nonimmune-mediated liver diseases, and long-term stable graft function among others.
      • Feng S.
      • Ekong U.D.
      • Lobritto S.J.
      • et al.
      Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants.
      ,
      • Wozniak L.J.
      • Venick R.S.
      • Naini B.V.
      • et al.
      Operational tolerance is not always permanent: a 10-year prospective study in pediatric liver transplantation recipients.
      ,
      • Benitez C.
      • Londono M.C.
      • Miquel R.
      • et al.
      Prospective multicenter clinical trial of immunosuppressive drug withdrawal in stable adult liver transplant recipients.
      ,
      • Feng S.
      • Bucuvalas J.
      Tolerance after liver transplantation: where are we?.
      ,
      • Talisetti A.
      • Hurwitz M.
      • Sarwal M.
      • et al.
      Analysis of clinical variables associated with tolerance in pediatric liver transplant recipients.
      In some studies, maternal donor status has been seen to promote long-term clinical immune tolerance.
      • Tang Y.
      • Chen J.
      • Chen B.
      • Guo C.
      Clinical characteristics of immune tolerance after pediatric liver transplantation.
      In our patient, pediatric age group with cholestatic etiology at baseline favored tolerance. Although we do not practice protocol-based immunosuppression withdrawal as of now in our institute, non-adherence on part of the subject provided us with an unique opportunity to study tolerance in detail.
      Tolerance may be affected by presence of specific immune cells especially T-regulatory cells, which can induce a tolerogenic environment. Higher frequencies of T-regulatory cells (especially CD4+ T cells with a CD5+CD25+CD38−/loCD45RA− cell surface marker) have been associated with tolerance as has been documented in previous studies and as was seen in our patient also.
      • Li Y.
      • Koshiba T.
      • Yoshizawa A.
      • et al.
      Analyses of peripheral blood mononuclear cells in operational tolerance after pediatric living donor liver transplantation.
      • Koshiba T.
      • Li Y.
      • Takemura M.
      • et al.
      Clinical, immunological, and pathological aspects of operational tolerance after pediatric living-donor liver transplantation.
      • Lau A.H.
      • Vitalone M.J.
      • Haas K.
      • et al.
      Mass cytometry reveals a distinct immunoprofile of operational tolerance in pediatric liver transplantation.
      Although presence of DSAs (either at baseline or de novo after LT) is usually indicative of an active humoral response, its presence in itself does not portend loss of tolerance as these may also be seen in long term survivors with stable graft function on immunosuppression and in those with operational tolerance on long-term follow-up.
      • Feng S.
      • Demetris A.J.
      • Spain K.M.
      • et al.
      Five-year histological and serological follow-up of operationally tolerant pediatric liver transplant recipients enrolled in WISP-R.
      ,
      • Wozniak L.J.
      • Venick R.S.
      • Naini B.V.
      • et al.
      Operational tolerance is not always permanent: a 10-year prospective study in pediatric liver transplantation recipients.
      ,
      • Feng S.
      • Bucuvalas J.C.
      • Mazariegos G.V.
      • et al.
      Efficacy and safety of immunosuppression withdrawal in pediatric liver transplant recipients: moving toward personalized management.
      To conclude, operational tolerance is an achievable target in selected group of liver transplant recipients to achieve the goal of a medication-free state. But strict individualized center-specific protocols are needed to implement immunosuppression withdrawal with need of long-term close monitoring and follow-up.

      CREDIT AUTHORSHIP CONTRIBUTION STATEMENT

      Vikrant Sood: Conceptualization, Methodology, Writing – review and editing; Bikrant Bihari Lal: Conceptualization, Methodology, Writing – original draft, Resources; Ashwini N.S: Methodology, Resources, Writing – review and editing; Rajeev Khanna: Writing – review and editing; Viniyendra Pamecha: Methodology, Writing – review and editing; Nirupama Trehanpati: Methodology, Resources; Seema Alam: Conceptualizing, Writing – review and editing.

      Conflicts of interest

      The authors have no conflicts of interest to report.

      Funding

      None.

      Appendix A. Supplementary data

      The following are the supplementary data to this article:

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