Abstract
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: operational tolerance, hepatic graft rejection, pediatric liver transplantation, immunosuppression withdrawal
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).1, 2, 3, 4, 5, 6, 7 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.
Maintained 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.
(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 1.
Description of Liver Biopsy Findings as per “Withdrawal of Immunosuppression in Pediatric Liver Transplant Recipients” (WISP-R) Study (Reference no. 2).
| Size/adequacy | Criteria | Negative criteria | Positive criteria |
|---|---|---|---|
| Liver tissue length – 1.5 cm | No central fibrosis | No bile duct damage | No cholangiolar proliferation or steatosis |
| Eight portal tracts | No portal fibrosis – 0 | No significant bile duct loss | Primary portal inflammatory cell type– lymphocytes |
| Intensity of inflammation – <1 | No interface activity, bile duct damage, venular inflammation, lobular ballooning, or spotty necrosis | Secondary 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 2.
Flow Cytometry Analysis Comparing Index Patient vs. Patient With Acute Post-transplant Graft Rejection vs. a Healthy Control.
| Population | Groups |
||
|---|---|---|---|
| HC (healthy control) | PTR (post-liver transplant rejection) | PTT (post-liver transplant tolerance - index patient) | |
| Lymphocytes | 20.6 | 25.7 | 22.1 |
| Total T cells (CD3+) | 44 | 50.9 | 61.3 |
| % of CD4+ T cells/total T cells | 43.3 | 13.7 | 50.3 |
| % of CD8+ T cells/total T cells | 56.7 | 86.3 | 49.7 |
| Treg cells (CD25+ CD127 low)/CD4+ T cells | 2.57 | 18.8 | 22.7 |
| Memory treg/tregs | 32.4 | 23.1 | 25.8 |
| Naive tregs/tregs | 67.6 | 76.9 | 72.6 |
| B cells | 7.4 | 9.73 | 20 |
| Translational B cells/B cells | 19.3 | 2.67 | 6.94 |
| Plasmablast/B cells | 35.6 | 74.1 | 20 |
| Naive B cells/B cells | 4.48 | 45 | 8.04 |
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.1,4 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.5,7,8
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.1,3,5, 6, 7,9 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).2 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.3 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.1,3,5,10,11 In some studies, maternal donor status has been seen to promote long-term clinical immune tolerance.6 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.12, 13, 14 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.2,3,7
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.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jceh.2022.09.002.
Appendix A. Supplementary data
The following are the supplementary data to this article:
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