Basics and Art of Immunosuppression in Liver Transplantation

Abstract

Liver transplantation is one of the most challenging areas in the medical field. Despite that, it has already been established as a standard treatment option, especially in decompensated cirrhosis and selected cases of hepatocellular carcinoma and acute liver failure. Complications due to graft rejection, including mortality and morbidity, have greatly improved over time due to better immunosuppressive agents and management protocols. Currently, immunosuppression in liver transplant patients makes use of the best possible combinations of effective agents to achieve optimal immunosuppression for long-term graft survival. Induction agents are no longer used routinely, and the aim is to provide minimal immunosuppression in the maintenance phase. Currently available immunosuppressive agents are mainly classified as biological and pharmacological agents. Though the protocols may vary among the centers and over time, the basics of effective use usually remain similar. Most protocols use the combination of multiple agents with different mechanisms of action to reduce the dose and minimize the side effects. Along with the improvement in operative and perioperative techniques, this art of immunosuppression has contributed to the recent progress made in the outcomes of liver transplants. In this review, we will discuss the various types of immunosuppressive agents currently in use, the different protocols of immunosuppression used, and the art of optimal use for achieving maximum immunosuppression without increasing toxicity. We will also discuss the practical aspects of various immunosuppression regimens, including drug monitoring, and briefly discuss the concepts of immunosuppression minimization and withdrawal.

Graphical abstract

Highlights

• •The long-term survival benefit of effective immunosuppressive agents, although it prolongs the graft survival but also comes with various metabolic and organ-specific side effects.
• •The art of immunosuppression is to achieve a balance between effective and optimal immunosuppression in the best possible combination to achieve the desired outcomes while avoiding side effects.
• •Strategies for immunotherapy minimization and withdrawal can change the overall quality of life with a reduction of long-term side effects, but come with the risk of rejection and graft failure.
• •Studies on the use of stem cells, regulatory dendritic cells (DCreg), and regulatory T cells (Treg) therapy to promote liver allograft tolerance, may eliminate the requirement of immune suppression in the future.
• •With the application of biomarkers of tolerance and artificial intelligence, a real paradigm shift in complex decision-making and personalized therapy can be expected.

Since the first successful liver transplantation (LT) by Thomas Starzl in 1967, there has been a tremendous improvement in understanding and achievements in this field.¹ Currently, liver transplantation (LT) is the only curative treatment in decompensated cirrhosis, unresectable cases of hepatocellular carcinoma (HCC) within the defined criteria, and certain cases of acute liver failure. The field of LT has further expanded to include different malignancies (for example, liver-only metastatic colorectal carcinoma) and metabolic conditions (for example, glycogen storage disease, maple syrup urine disease), which can prolong survival and even cure the underlying disease. Over the last fifty-five years, the outcome has greatly improved, with one-year survival over 90% at most of the transplant centers. Improvement in surgical techniques, perioperative care, and better immunosuppressive regimens are the important factors that made this achievement possible.2, 3, 4

As of other solid organ transplantations, the recipient’s immune system recognizes and tries to reject the allograft, leading to graft dysfunction and failure. Immunosuppressive agents are used with the aim of preventing the graft from this immune recognition and rejection process. Azathioprine and corticosteroids were the only IS agents available when liver transplantation was started. Over time, newer and better IS agents came into picture, leading to better graft survival and concurrently decreasing the side effects.5, 6, 7 (Figure 1). This long-term survival benefit of effective IS agents, although it prolongs graft survival, also comes with various metabolic and organ-specific side effects, including the development of opportunistic infections and malignancies. Hence, the immunosuppressive agents, dose, and duration should be planned not only by considering the graft and patient profile but also their specific side effects that determine morbidity and mortality in patients.^8,9

Figure 1.

The journey of major immunosuppressive agents currently in use.

Immunosuppressive regimens in solid organ transplant use various combinations of agents from different groups, i.e. calcineurin inhibitors (CNIs), corticosteroids, mammalian target of rapamycin (mTOR) inhibitors, antimetabolites, and biological agents.¹⁰ This combination protocol allows the use of these agents at lower possible doses without increasing the risk of graft rejection while reducing the toxicity of each agent. Hence, such regimens allow a delicate balance between optimal immunosuppression and toxicity as much as possible. Each regimen needs to be personalized, taking the patient's preoperative and perioperative risk profile into account while changing according to its efficacy, toxicity, and time from transplant.^11,12 In this article, we will discuss the basic concepts and practice of IS agents in liver transplantation. We will discuss, in short, the management of different types of graft rejection. We will also discuss in brief the concepts of IS minimization and withdrawal. Immunosuppression protocols in ABO-incompatible liver transplantation, which is a totally different scenario, will not be discussed here.

Pathophysiology and Immunobiology of Allograft Rejection

The immune system of our body is designed to combat foreign antigens, which is a strong barrier to the tolerance of the graft antigen. Although the liver has unique features as an immunoregulatory organ with the ability to promote an enhanced tolerogenic response in the allograft recipient compared to other organs, immunological rejection still remains a common and significant cause of mortality and morbidity. But, in comparison to other solid organ transplantation, the liver has lower rejection rates and a better recovery rate. Also, the immunosuppression used following liver transplantation is lower as compared to other organ transplants. Furthermore, acute cellular rejection episodes usually do not affect long-term liver graft survival.^13,14 As nonself-donor alloantigen remain throughout the life of the recipient, liver transplant recipients theoretically require lifelong immunosuppression.^13,15

Graft rejection is a multistep process that includes recognition of alloantigen, lymphocyte activation, clonal expansion, and finally graft inflammation. In general, the immune response elicited after LT is predominantly driven by T cells. The basic signals of graft rejection are discussed below.

• I.Antigen presentation – Signal 1

The alloantigen of the donor liver gets complexed with recipient major histocompatibility complex (MHC) proteins, which is initiated by surgical trauma and organ reperfusion injury. Presentation is done by an antigen-presenting cell (APC - like Kupffer cells and dendritic cells). The antigen, bound to an MHC molecule, binds to the T-cell receptor, which engages costimulatory receptors on T cells like CD28 and CD154. This is the first of three signals that are required for T-cell activation.¹⁶ This step can be aborted by antithymocyte globulin (ATG) and antilymphocyte globulin (ALG) (Figure 2).¹⁷

• II.Lymphocyte Activation (Co-stimulation) – Signal 2

Figure 2.

Basic pathways of the host immune response. Adapted from Ref.¹⁴

After the alloantigen presentation to the T-cell receptor in the presence of appropriate costimulation, internalization of the receptor complex occurs. Costimulation is the process in which a number of ligands on the APC bind to a variety of T-cell receptors, including CD28, CD154, CD2, CD11a, and CD54.^18,19 Once the T-cell receptor complex gets internalized, a number of downstream signals gets activated, including immunophillin, which then stimulates calcineurin and finally activates the nuclear factor of T-cell (NFAT). This activated NFAT translocates to the nucleus, where it drives interleukin (IL)-2 transcription. Cyclophilin and FK-binding protein are the two immunophillins that are targets of cyclosporine and tacrolimus, respectively (Figure 2). Both agents ultimately block calcineurin; hence they are called calcineurin inhibitors.20, 21, 22

• III.Clonal expansion – Signal 3

Newly transcripted IL-2 gets secreted by T cells and binds to IL-2 receptors (IL-2R) on the cell surface in an autocrine fashion. IL-2 has growth-promoting activity and plays a critical role in the proliferation and activation of effector T cells, i.e. it leads to clonal expansion and cell proliferation.¹³ Basiliximab and daclizumab are monoclonal antibodies against the IL-2 receptor, where they block T-cell expansion.²³ Sirolimus and everolimus, which bind to the downstream mechanistic target of rapamycin (mTOR), also act at this step.²⁴ Finally, the T-cell proliferation burst can be inhibited at the level of DNA synthesis by the antimetabolite drugs, i.e. azathioprine and mycophenolate mofetil (Figure 2).²⁵

• IV.Inflammation – The final common pathway

T-cell proliferation generates large numbers of effector T and B-cells in lymphoid organs. It leads to cell-mediated cytotoxicity with the release of cytokines, chemokines, and adhesion molecules. The secreted mediators recruit additional inflammatory cells, like neutrophils, to the graft tissue. The final result is the creation of an inflammatory milieu with additional toxic and vasoactive mediators, which, if untreated, can lead to graft loss.^13,26 Control of this step is possible with glucocorticoids and antilymphocyte antibodies.

Phase of Immunosuppression in Liver Transplantation

There are two phases in liver transplant immunosuppression therapy, i.e. the induction and maintenance phases. The induction phase or early IS therapy, constitutes the combination of multiple agents in a higher dose, followed by the maintenance phase, which usually lasts a lifetime.

Induction Phase

In this phase, we use a short course of potent IS agents to reduce the robust immune response of T lymphocytes against the grafted liver that occurs in the immediate posttransplant period. This therapy usually gets started intraoperatively and continues for the initial 30 days of transplantation.^5,11 Induction agents most commonly used include a triple-dose combination of CNIs (cyclosporine or tacrolimus), antimetabolites (MMF or Azathioprine), and corticosteroids. Antibody preparation, antithymocyte globulin (ATG), and interleukin 2 (IL-2) receptor antagonists, Basiliximab may be used as steroid or CNIs-sparing agents. CNIs and mTOR inhibitor drug levels are regularly monitored during this phase, along with liver biochemical tests and the patient’s clinical profile.^14,27

Maintenance Phase

This phase starts after the induction phase and continues as chronic IS and constitutes various combinations of CNIs, antiproliferative agents, mTOR inhibitors, and corticosteroids. Usually, steroids are stopped within the first three months’ posttransplantation, antimetabolites like MMF after 6 months to 2 years, and mTOR and/or CNIs usually get continued the whole life. Patients with autoimmune conditions like autoimmune hepatitis usually need long-term steroids at low doses (2.5–5 mg of prednisolone).^28,29 As the duration of transplantation increases, CNIs and mTOR inhibitors serum trough level measurement is less frequently monitored, and graft function is mainly monitored by liver biochemical tests, clinical profile, and other noninvasive (for example, ultrasonography and transient elastography) and invasive methods (liver biopsy) as indicated.

Usually, the choice of long-term IS agents and dose should be individualized based upon multiple factors, such as underlying preoperative disease, the presence of comorbidities, the development of drug-related side effects, and laboratory parameters.⁵

Classification of Immunosuppressive Drugs

Immunosuppressive agents used in liver transplants are classified into two groups, i.e. pharmacological agents and biological agents (Table 1).

• A.Pharmacological agents

Table 1.

Classification of Immunosuppressive Agents in Liver Transplantation.

	Class	Drug	Mechanism of action
Pharmacological agents	Corticosteroids		Inhibit cytokine transcription by antigen presenting cells, broad spectrum of effects
	Calcineurin inhibitors	Cyclosporin, tacrolimus	Inhibits calcineurin via cyclophilin and FKB-12, blocking IL2 transcription
	Antimetabolites	Mycophenolate mofetil (MMF) azathioprine	Inhibition of purine and DNA synthesis and prevention of T cell proliferation
	m-TOR inhibitors	Sirolimus, everolimus	Inhibition of signal 3 transduction and prevention of T cell proliferation
Biological agents		Anti-CD3 (monoclonal): OKT3	Anti-CD3-specific antibody causing T-cell depletion
	T cell depleting agents	ATG/ALG: horse and rabbit	Interference with signal 1, antibody preparation directed against lymphocytes
		Anti-CD52 campath-1H (alemtuzumab):	Depletion of thymocytes, T cell, B cells, monocytes
	Non-T cell depleting agents	Anti–IL-2 receptors, basiliximab, daclizumab	Blocks IL2 engagement and resultant lymphocyte proliferation
		Belatacept	CTLA-4 homolog competing with CD28 for CD80/86 binding, inhibiting T-cell costimulation

Abbreviations: FKBP12 - FK-binding protein-12; IL2 - interleukin 2; mTOR - mechanistic/mammalian target of rapamycin; ATG - antithymocyte globulin; ALG - antilymphocyte globulin.

Adapted from Refs ^14,30,31

The pharmacological agents act by inhibiting cytokine release (calcineurin inhibitors, corticosteroids) or by inhibiting the cell cycle anti-metabolites and mammalian targets of rapamycin (mTOR) inhibitors.

• 1.Calcineurin inhibitors

Cyclosporine and tacrolimus are the two available agents in this group of immunosuppressants. The landscape of immunosuppression in LT changed after the landmark paper by the US Multicenter FK506 Liver Study Group in 1994, which compared cyclosporine (CSA) and tacrolimus (TAC) for immunosuppression after LT.²⁰ The discovery of these drugs has significantly improved the graft and patient survival post-LT. Cyclosporine binds to cyclophilin, which interferes with calcineurin's dephosphorylation of nuclear factor of activated T cells (NFAT), thus preventing its translocation into the nucleus and proinflammatory cytokine production.¹⁴ On the other hand, tacrolimus binds to the FK506 binding protein (FKBP12) and leads to calcineurin inhibition. The binding inhibits IL-2 gene transcription, preventing T cell activation and proliferation. Tacrolimus is considered to be 100 times more potent on a molar level than cyclosporine.³²

CNIs are metabolized by the cytochrome P450 system, which makes these drugs potential for multiple drug interactions and requires drug level monitoring (Table 2, Table 3). Not only this, but foods that alter p-glycoprotein levels also have an effect on CNI absorption. In patients with chronic hepatitis C infection, drug interactions with direct-acting antiviral agents (DAA) should be checked when treatment is needed after LT. CSA and TAC have similar side effect profiles, including neurotoxicity, renal dysfunction, metabolic syndrome, and vasculopathy. Diabetes and neurotoxicity are more common with TAC, whereas hypertension, hyperlipidemia, and endothelial dysfunction leading to cardiovascular disease are more common with CSA use. Gingival hyperplasia and hypertrichosis are typically caused by cyclosporine alone. The risk of opportunistic infections and malignancies is increased by both TAC and CSA. CNIs produce dose-dependent afferent renal arteriolar vasoconstriction that is often reversible. However, long-standing ischemic glomerular and tubular injuries can occur in 20% of patients on chronic CNI therapy, leading to the development of chronic renal dysfunction.^11,33

Table 2.

Drugs That Alter the Trough Level of Calcineurin Inhibitors.

Drugs that increase CNI levels	Macrolides: clarithromycin, erythromycin, azithromycin Antifungals: azole drugs Calcium channel blockers: verapamil, diltiazem, nifedipine Others: cisapride, metoclopramide, amiodarone, danazol, cimetidine, protease inhibitors
Drugs that decrease CNI levels	Antibiotics: rifabutin, rifampin Anticonvulsants: carbamazepine, phenobarbital, phenytoin, fosphenytoin

Abbreviations: CNIs – calcineurin inhibitors.

Table 3.

Pharmacological Agents in Liver Transplantation.

Drug	Dose	Half life (Hours)	Therapeutic range	Side effects
Tacrolimus	0.1–0.15 mg/kg/daily divided in 2 doses, 12 h apart	2–36	General range: 5–12 ng/mL	Nephrotoxicity; neurotoxicity; glucose intolerance; alopecia; diarrhea, hypertension, hyperlipidemia, hyperkalemia
Cyclosporin	10–15 mg/kg/daily, divided in 2 doses, 12 h apart	5–8	General range: 100–250 ng/mL	Nephrotoxicity; hypertension; hyperlipidemia; hyperuricemia; hirsutism; acne; gingival hyperplasia
MMF	500–1000 mg twice daily	11–12	Therapeutic monitoring not recommended	Myelosuppression, gastrointestinal side effects, viral infections (CMV, HSV), spontaneous abortions in pregnant women
Azathioprine T- and	1–2.5 mg/kg PO daily	variable, ∼2	Therapeutic monitoring not recommended	Leukopenia, thrombocytopenia; gastrointestinal disturbances, pancreatitis; hepatotoxicity
Everolimus	Start with 0.25–0.5 mg twice daily, titrate to target level	30	General range: 3–8 ng/mL	Hyperlipidemia, myelosuppression, proteinuria, poor wound healing, pneumonitis, skin rash
Sirolimus	Start with 1–2 mg PO daily and up-titrate to target level	62	5–10 ng/mL	Hyperlipidemia, metabolic syndrome, myelosuppression, proteinuria, poor wound healing, pneumonitis, skin rash
Corticosteroids	5 mg–10 mg/kg induction followed by tapering dose	2.5–3.5	Therapeutic monitoring not recommended	Diabetes, metabolic syndrome, hypertension, obesity, osteoporosis, avascular necrosis, growth retardation, cushingoid features, psychosis, poor wound healing, adrenal suppression, cataract

Abbreviations: MMF – mycophenolate mofetil, CMV – cytomegalo virus, HSV – herpes simplex virus.

Adapted from Refs ^14,30,31

Because of their excellent allograft protective effects, CNIs, especially tacrolimus, have been included as the backbone in induction and maintenance immunosuppression protocols in most LT centers worldwide. Tacrolimus is preferred over cyclosporine because of its greater potency and improved cardiovascular adverse side effect profile.³⁴ Tacrolimus is started at a low oral dose (0.1–0.15 mg/kg daily in two divided doses 12 h apart) on the first day post-LT, and the dose is titrated to achieve an adequate trough level. The usual trough targets for TAC are 6–10 ng/mL in the initial three months and then 5–6 ng/mL after that. Extended-release (TAC-ER) and prolonged-release (TAC-PR) once-daily formulations of TAC have been shown to have better adherence and similar efficacy to conventional TAC.35, 36, 37

Cyclosporine is not the CNI of choice for transplant recipients, but in special cases like neurotoxicity and TAC-related liver injury, there might be a need to switch from tacrolimus to cyclosporine. When it has to be used, the recommended dose is 10–15 mg/kg per day, divided into 2 doses, but may be started at lower doses with a gradual increase to achieve the target level. The target trough levels (C0 and C2) are 200–250 ng/mL in the initial three months and then 150 ng/mL.^10,14,38

• 2.Antimetabolites

Antimetabolites are usually added to CNIs and the initial dose of steroids as part of the standard maintenance IS. Azathioprine (AZA), mycophenolate mofetil (MMF), and mycophenolate sodium are the main agents used in post-LT immunosuppression. They are reversible purine synthesis inhibitors with antiproliferative activity against T-cells and B-cells. They block the signal 3 of cell activation, with MMF having additional immunomodulatory action.

Azathioprine

Azathioprine (AZA) is an antimetabolite with potent immunosuppressive action and one of the first immunosuppressive agents used in the field of solid organ transplantation. But it is inferior to MMF in the LT setting, with studies showing a higher incidence of acute cellular rejection with AZA. Nowadays, azathioprine is used when other agents are not tolerated or affordable. The major adverse side effects of azathioprine are related to bone marrow suppression, its hematologic consequences, and hepatotoxicity.^32,39

Mycophenolate Mofetil

MMF was introduced to the field of LT in the 1990s as an immunosuppressive agent. The lack of potential nephrotoxicity made it useful for patients with renal dysfunction who need to decrease CNI doses. MMF is also a useful agent in combination with CNI in immunosuppressive regimens where corticosteroid withdrawal is desirable. MMF has already replaced azathioprine as the most commonly used antimetabolite agent in solid organ transplantation.⁴⁰ Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil (MMF), and the enteric-coated formulation is mycophenolate sodium (EC-MPS). It inhibits the de novo synthesis of guanosine nucleotides by blocking inosine-5′-monophosphate dehydrogenase (IMPDH), thereby suppressing T-cell proliferation. Therapeutic drug monitoring is not recommended for MPAs as their bioavailability is high. The average daily dose for MMF is 500 mg to 1 g twice daily (360–720 mg twice daily for EC-MPS).⁴¹

The major adverse side effects of MPA are hematologic and gastrointestinal (GI). Bone marrow suppression is usually dose-dependent and responds to dose reduction. Nausea, vomiting, and abdominal discomfort are common complaints in patients taking MPA derivatives. The risks of opportunistic infections is also increased with MMF.²⁵ Diarrhea is a common, dose-limiting side effect. Dividing the dose into four-times-a-day schedule may be helpful. Serious adverse GI effects such as inflammatory bowel disease-like colitis and graft-versus-host disease-like enteropathy related to MPA have also been reported. Gastrointestinal symptoms usually resolve with withdrawal of the drug or switching to enteric-coated mycophenolate sodium.41, 42, 43 (Table 3).

• 2.Mechanistic/Mammalian target of rapamycin inhibitors (mTOR inhibitors)

Sirolimus (SRL) and Everolimus (EVR) belong to the group of mTOR inhibitors. Sirolimus (SRL) is a macrolide antibiotic that is structurally similar to tacrolimus and binds to the same target (FK-binding protein), but with a different mechanism of action.⁴⁴ EVR is a derivative of SRL with an extra hydroxyethyl group at position 40 and is more lipophilic. They also bind to the FK506 (FKBP12) binding protein inside cells and inhibit the mammalian target of rapamycin complex 1 (mTORC1). They subsequently block signal-3 of T-cell activation, thereby inhibiting IL-2 and IL-15-induced T-cell proliferation. Even though they bind to the same receptor, mTORI and CNI do not compete but act synergistically.⁴⁵ Like the CNIs, sirolimus and everolimus metabolism is occurs the cytochrome P450 system, and therapeutic drug monitoring is recommended.

In a study including 15 patients, Watson et al. in 1999, first reported the effectiveness of sirolimus as an immunosuppressive agent. Some initial studies showed a higher incidence of hepatic artery thrombosis (HAT) and early graft failure in the de novo sirolimus therapy. Subsequently, the US-FDA issued a black box label warning about early post-transplant HAT with SRL use in LT in 2002.⁴⁶ However, several later trials assessed the safety of mTORI introduced 30 days after LT and found no evidence of an elevated risk for HAT.^47,48

Everolimus (EVR) is a derivative of SRL, has a shorter half-life, does not require a loading dose, and the therapeutic window is narrow (3–8 ng/mL). EVR showed good results when combined early with low-dose TAC, reducing rejection rates and improving renal safety. Early conversion to mTORI therapy can improve renal function in patients with CNI-induced nephropathy. Patients receiving mTORI early (30 ± 5 days) or very early (<10 days) post-LT have shown renal function improvement of 8–12 mL/min per 1.73 m² at 12 months after transplantation. But similar improvement is not seen with mTORI in patients with a glomerular filtration rate (GFR) less than 60 mL/min per 1.73 m².^11,14,32

mTORI have antiproliferative effects, and preclinical and clinical studies have shown that they effectively prevent HCC recurrence. They also reduce the risk of posttransplant de novo malignancies. However, recent studies on mTORI and posttransplant recurrence in patients with HCC show conflicting reports. One study on SIR-based immunosuppression in preventing HCC recurrence showed a beneficial effect only in patients meeting Milan criteria. Though current data on the prevention of HCC recurrence is scanty, mTORI may be used in patients with an increased risk for HCC recurrence.^49,50 mTORI may also be beneficial in preventing de novo and recurrent malignancies after LT, and its use is supported by retrospective, cohort, and registry data analysis.^51,52

Complete withdrawal of CNI to introduce mTORI can increase the risk of acute rejections, and a 10–20% risk of acute rejection is seen depending on time from LT. EVR and TAC have no interactions, and the TAC dose is reduced only after the EVR target trough level is reached. EVR can also increase CSA trough levels.53, 54, 55 When required, both of these agents can also be used together so that the minimal possible dose of either agent can be used while minimizing individual drug toxicity. Side effects of mTOR include anemia, thrombocytopenia, leukopenia, dyslipidemia, hypertension, poor wound healing, oral ulcers, interstitial pneumonia, proteinuria, and fluid retention.⁵⁶ (Table-3) Patients treated with mTORI, particularly patients who received LT for NASH, should be counseled on appropriate preventive lifestyle changes. Pleural and pericardial effusions are among the rare complications of sirolimus. Most side effects respond to dose reduction or discontinuation of the drug. The risk of EVR-induced proteinuria (>1 gm/d) is 3 % at three years and usually responds to dose reduction. In cases of severe neutropenia (<1000 mm³), leukopenia (<2000 mm³), or thrombocytopenia (<50,000 mm³), EVR needs dose reduction or withdrawal. mTORIs inhibit the fibroblast growth factor and are also capable of impairing surgical wound healing, which may contribute to more wound complications when introduced very early after solid organ transplantation. Interstitial pneumonitis is dose-dependent and resolves when EVR is withdrawn.⁵⁷ Currently, mTORI is used in LT recipients with kidney dysfunction, HCC, and de novo neoplasms.⁵³ If the use of mTORIs is considered for post-LT patients, it is preferred to be started at least 15-30 days post-transplant to avoid the risk of HAT and also allow enough time for wound healing.^24,58

Corticosteroids

Corticosteroids have been an important part of immunosuppression since the early days of transplantation. They are mainly used in the induction and treatment of rejection episodes. Steroids can also replace CNIs and MMF when these agents need to be held temporarily for different reasons, including drug toxicity and sepsis.⁵⁹ Corticosteroids have multiple targets for inhibition of the immune system. They act as anti-inflammatory and immunomodulatory agents by interacting with antigen-presenting dendritic cells, modulating IL-1 transcription, decreasing the number of circulating CD4+ T cells, inhibiting IL-1-dependent lymphocyte activation, and stabilizing lysosomal membranes.^14,30

Intravenous methylprednisolone in the dose of 500–1000 mg is given in an-hepatic phase, followed by rapid tapering within the initial postoperative days.^31,60 Usually, 100 mg of methylprednisolone is given on the first day with rapid tapering and conversion to oral prednisolone. The current protocol is to taper and stop steroids at 3–6 months in patients without autoimmune conditions or prior rejection episodes.⁶¹ The major concern is their adverse side effects, especially with higher doses or prolonged use. Infections, worsening metabolic syndrome, hypertension, diabetes mellitus, obesity, and hyperlipidemia are the major concerns with the use of high-dose steroids. Prolonged use can also result in osteoporosis, cushingoid features, avascular necrosis of bone, poor wound healing, psychosis, adrenal suppression, and cataracts.⁶² (Table 3).

• B.Antibodies (Biological immunosuppressive)

As T and B lymphocytes express specific antigens on cell surfaces, antibodies to these markers can be used to inhibit or deplete them. These agents can be classified into lymphocyte-depleting agents that deplete T-cells (anti-thymocyte globulin), B-cells (Rituximab), or plasma cells (Bortezomib), and nonlymphocyte-depleting agents (Basiliximab) that inhibit T-cell proliferation (Table 4). Antibodies are not used routinely in liver transplantation but have an important role as steroid-free induction agents and for CNIs sparing immunosuppression, steroid-refractory rejection, ABO-incompatible (ABOi) liver transplantation, and management of antibody-mediated rejection (AMR). The steroid-free regimen can also be used in patients with hepatitis C and nonalcoholic steatohepatitis (NASH). The main limiting factors of the use of these agents are cost and availability (Table 1, Table 4).^63,64

• 1.Polyclonal antibodies

Table 4.

Biological Agents in LT.

Drug	Mechanism of action	Use	Remarks
Muromonab-CD3 (OKT3)	T-cell-depleting monoclonal antibody	Induction of immunosuppression, treatment of steroid resistant rejection	Withdrawn from the market.
Alemtuzumab (campath-1H)	T-cell-depleting monoclonal antibody	Induction of immunosuppression	Variable between centers, a single dose of 30 mg may be used in operating room.
ATG (thymoglobulin, ATGAM)	T-cell-depleting polyclonal antibody	Induction of immunosuppression, treatment of steroid resistant rejection	Variable between centers, for induction 1.5 mg/kg per day iv for 3 d and for treatment of rejection 1.5 mg/kg per day iv for 5–7 d of thymoglobulin may be used. For ATGAM a higher dose of 15 mg/kg per day is usually used.
Daclizumab (Zenapax)	IL-2Ra, monoclonal antibody	Induction of immunosuppression, treatment of steroid-resistant rejection	Withdrawn from the market.
Basiliximab (Simulect)	IL-2Ra, monoclonal antibody	Induction of immunosuppression, treatment of steroid resistant rejection	For induction a 20 mg iv dose is administered within 6 h of reperfusion and another 20 mg on days 4 post Tx.
Belatacept	Anti-CD28 monoclonal antibody	CNI sparing agent	Not recommended in LT

Abbreviations: ATG - Anti-thymocyte globulin; ATGAM - Equine Anti-Thymocyte Globulin; IL-2Ra - Interleukin-2 receptor; CNI - calcineurin inhibitors; LT - liver transplantation.

Adapted From Refs^11,30

Antithymocyte globulin (ATG, thymoglobulin) and antilymphocyte globulin (ALG) are polyclonal antibodies to multiple T-cell antigen, including CD2, CD3, CD4, and CD8.⁶⁵ They are mainly used in the treatment of steroid-resistant acute cellular rejection and are prepared by immunizing animals against mixed populations of thymocytes. They are administered via a central line and can result in profound lymphopenia through complement-mediated cell lysis and the uptake of opsonized cells. Repopulation usually occurs in 3–10 days.^17,66

For ATG induction therapy a daily infusion of 2.5 mg/kg/d of Thymoglobulin for ten days is given. Three-day courses have also shown similar efficacy with fewer adverse effects in LT recipients. Side effects of ATG are infusion reactions, serum sickness, thrombocytopenia, anemia, pruritic skin rashes, and anaphylaxis. Antihistamines and acetaminophen are given before infusion to prevent infusion reactions.⁶⁷

• 2.Alemtuzumab (Campath-1H)

Alemtuzumab (Campath-1H) is a humanized monoclonal, complement-fixing, anti-CD52 antibody. As CD52 is expressed on the surface of T and B lymphocytes, macrophages, monocytes, and eosinophils, it leads to profound lymphocyte depletion. An increased risk for Post-transplant lymphoproliferative disorder (PTLD), opportunistic infections, zoster infection, and cryptococcus infection is reported, along with reports of rapid progression of recurrent HCV.68, 69, 70 Currently, there is a reduced enthusiasm for alemtuzumab due to the low evidence of its safety profile.

• 3.Interleukin-2 receptor antibodies

Basiliximab (Simulect) and daclizumab (Zenapax) are humanized monoclonal antibodies against the IL-2 recepto CD 25. Daclizumab is a fully humanized monoclonal antibody, while basiliximab is a chimeric anti-CD25 monoclonal antibody.⁷¹ They are mainly used in induction therapy as steroid- or CNI-sparing agents. Daclizumab was removed from the market in 2018 due to safety concerns following multiple reports of an increased incidence of inflammatory encephalitis and meningoencephalitis.⁷² Studies have shown that IL-2Ra induction has similar efficacy with fewer side effects than ATG induction. There were fewer acute rejection episodes and better graft survival, with no added risks of PTLD, CMV infections, or HCV recurrence.^73,74

• 4.CD28 Antibodies (Belatacept)

The CD28 receptor present on T-cells is involved in signal-2 T-cell activation and proliferation. Belatacept is a high-affinity fusion protein that binds CD80/86 on antigen-presenting cells (APCs) and is used in renal transplantation. It is given as a monthly infusion and may permit immunosuppression without nephrotoxicity. There have been reports of an increased rate of post-transplant lymphoproliferative disorder in renal transplant recipients, but there is no clear data on its use in LT.^75,76

Treatment of Liver Allograft Rejection

Due to the presence of a large number of resident immune cells, the liver not only provides immune surveillance to external antigens, including pathogenic infections and malignant cells, but also provides a protolerogenic environment. This tolerogenic environment enables the transplanted liver to not only protect itself from the host immune response but also decrease alloreactivity against concurrently transplanted other organs, like the kidney.⁷⁷ Though hepatic allograft rejection is less common than other solid organ transplantations, rejection is still seen frequently and is an important cause of graft failure.

Acute cellular rejection (ACR) presents with a histological triad of portal tract infiltrates, bile duct injury, and venous endotheliitis.⁷⁸ Typically, biopsy-proven acute rejection is treated with pulse intravenous steroid boluses (500–1000 mg of methylprednisolone daily for 3 days). In many cases, mild to moderate rejection can be treated with increasing the levels of CNI alone. Anti-thymocyte globulins (ATGs) are used in steroid nonresponsive rejection.⁷⁹ Prophylaxis against CMV, Pneumocystis pneumonia (PCP), bacterial, and fungal infections is given to patients requiring repeated pulse therapy. As compared to cyclosporine, the use of tacrolimus has been shown to reduce the incidence of both acute cellular rejection and steroid-resistant rejection.³³

Early cellular rejection occurs within 90 days in 10–30% of transplants. Late cellular rejection occurs after 90 days with an incidence of 7.5%–23% and is treated like early cellular rejection. Risks of steroid resistance and progression to chronic rejection become more common in late cellular rejection, resulting in reduced graft survival.⁸⁰ Chronic allograft rejection, which occurs in 1–5% of transplants, is characterized by progressive bile duct loss (defined as ductopenia in atleast 50% of portal tracts) and obliterative arteriopathy with foamy cell infiltration (which may not be picked up by liver biopsy).⁷⁸ Chronic ductopenic rejection is usually not related to acute cellular rejection and can be associated with the pretransplant etiology of liver disease, human leukocyte antigen-matching profiles, and CMV infection. As the pathogenesis of chronic ductopenic rejection is complex, it typically responds to an increase in immunosuppression.⁸¹ Effective treatments for chronic rejection are limited, and patients should be switched to tacrolimus if they are on cyclosporine. Addition of mTORI or corticosteroids is usually not recommended in this condition. In the setting of allograft failure retransplantation is to be considered.⁷⁷

Antibody-mediated rejection (AMR) occurs in <1% of transplants and <5% of sensitized patients. Presence of C4d positivity on staining on histology in the presence of circulating donor-specific antibody (DSA) and exclusion of alternate causes needed for the diagnosis of AMR.⁸² Mild acute AMR usually responds to steroid pulses. Moderate to severe AMR is treated with DSA-depleting therapy like plasmapheresis, intravenous immunoglobulin (IvIg), anti-B cell (rituximab), anti-plasma cell (bortezomib), and anticompliments (eculizumab) agents. Chronic AMR is rarely seen in liver allografts and has no defined treatment strategy.^83,84

Hyperacute rejection is an antibody-mediated graft injury occurring immediately after ABO-incompatible LT and is an extremely aggressive form of rejection that necessitates urgent re-transplantation. Current effective de-sensitization protocols, including pretransplant rituximab and plasmapheresis with or without immunosuppression, have almost overcome this situation.⁸⁵

The Art of Immunosuppression

Current immunosuppression protocols have been established as an art and not just a science. This art is to achieve optimal immunosuppression at the lowest possible dose using a variable combination of agents with stable allograft function while avoiding the risks of rejection and drug-related side effects.^86,87 The choice, dose, and duration of immunosuppressive agents vary with recipient profile, time after transplantation, the initial disease process, the development of toxicity, and graft behavior. Hence, current practice is to develop personalized immunosuppression protocols, considering all the above factors into account (Figure 3).^88,89

Figure 3.

Current protocol of post-liver transplant immunosuppression at our center.

Abbreviations: AIH – Autoimmune Hepatitis, AKI – Acute kidney injury, ALD – Alcoholic liver disease, CLD – Chronic liver disease, CKD – Chronic kidney disease, CNI – Calcineurin inhibitor, CVD – Cardiovascular disease, CMV – Cytomegalovirus, DM – Diabetes Mellitus, HBV – Hepatitis B virus, HTN – Hypertension, HCC – Hepatocellular carcinoma, IS – Immunosuppressant, MMF – Mycophenolate Mofetil, mTORI – Mammalian target of rapamycin inhibitors, NASH – Non -alcoholic steatohepatitis, TAC – tacrolimus; ∗- use carefully in first year post liver transplant; ＃ mTORI - Everolimus (EVL) and Sirolimus (SRL) with target trough level - EVL - 3–8 ng/mL and SRL - 5–10 ng/mL.

Almost all the centers use combinations of drugs from different groups with consideration of the patient profile and duration of the transplant. Though a slight difference in protocol may exist between the centers, the overall crux of therapy remains similar. In our center, we start the therapy within the first 24 h of transplantation, along with intravenous methylprednisolone administered during the an-hepatic phase. Combination therapy of CNIs (preferably tacrolimus-TAC) and antimetabolites (preferably MMF) at initial days only.²⁷ If the patient is suspected to have severe sepsis, major bleeding, and acidosis in the immediate post-transplant period, then only high dose steroid are started until the above conditions are controlled or stabilized.^59,90

Strict monitoring of drug-related side effects, contraindications, and trough levels of CNIs is monitored during the initial posttransplant period. Intravenous steroid is converted to oral prednisolone once enteral feeding starts stably. At discharge, the steroid is usually tapered to 20 mg of prednisolone, with an MMF of 500–1000 mg twice daily, with a trough-level adjusted dose of CNIs. LT recipients with autoimmune conditions like liver disease, inflammatory bowel disease, or who developed repeated episodes of T-cell-mediated rejection remain on a long-term low-maintenance dose of prednisolone (2.5–5 mg/day). In patients with renal dysfunction, basiliximab 20 mg intravenous may be administered on postoperative days 0 and 4.^73,91

CNIs have a narrow therapeutic window, and hence, tacrolimus trough concentrations should be closely monitored. It is usually kept at 6–10 ng/mL in the first month and 4–8 ng/mL within the first year. From the second year onwards, trough levels of 3–5 ng/mL are considered adequate. However, the relationship between the dose and trough level remains highly variable among individuals and even within the same patient over the time.⁹² MMF is given at a dose of 500 mg twice daily and is usually discontinued after 12 months in patients with stable graft function. In patients at risk for kidney injury or hepatocellular carcinoma (HCC) as the etiology for transplant, CNI may be substituted with EVR between postoperative days 15–30.^50,53,93 EVR is started at a dose of 0.25–0.5 mg PO twice daily and subsequently adjusted based on the target trough level. EVR should be avoided in patients with proteinuria, and regular monitoring of proteinuria should be done.^56,94 By the second year, most patients are on monotherapy with TAC or EVR, and the TAC trough level is kept less than 6 ng/mL. Long-term trough levels are unnecessary if the liver biochemical tests remain normal (Figure 3).^8,14

Immunosuppression Practice in Different Scenario

• 1.IS in LDLT versus DDLT

Living donor liver transplantation (LDLT) theoretically may provide an environment favoring tolerance over alloreactivity. This can be due to a less inflammatory surgery with significantly less ischemia in a more controlled setting, as well as biological similarity between donor and recipient in LDLT.⁹⁵ Evidence for LDLT suggests that rates of IS minimization and withdrawal are higher while rejection rates are less common in biologically related donors.^96,97 Furthermore, with less ischemia, improved donor selection, and controlled procedures, LDLT can lead to less human leukocyte antigens (HLA) donor-specific antibody (DSA) formation and consequently a lower rejection rate than deceased donor liver transplantation (DDLT).⁹⁸ Despite all the above facts, the basic immunosuppression strategy remains the same for both LDLT and DDLT.

• 2.IS in Indian versus Western patients

In contrast to west most of the transplant in India are usually perform LDLT.⁹⁹ Though immunosuppression strategy may vary among different Indian centers but the core protocol remains the same as that of western protocol. However, Indian patients are found to require overall lower IS dose than their western counterparts.¹⁰⁰

• 3.IS in patients with sepsis

Post LT patients have increased risk of sepsis especially during early post-transplant period. Use of immunosuppresive agents (ISA) at high dose including steroids is considered important risk factor for development of sepsis. Hence the general practice is to reduce or hold the IS during this condition. However, literatures have shown conflicting results over the use of standard IS during severe sepsis. Post solid organ transplant (SOT) patients with severe sepsis have even shown better inpatient outcome than non-transplant patients admitted with similar severity of sepsis.¹⁰¹ Furthermore, SOT patients who continued with standard IS agents have shown favorable outcomes in comparison to the patient with whom standard IS were discontinued or reduced.¹⁰² However, due to lack of well controlled study over this issue most transplant centers follow the classical practice of IS dose reduction or stoppage.¹⁰³

In our clinical practice, in early ICU sepsis without hemodynamic alteration, we try to reduce the tacrolimus trough level to 3–5 ng/mL and continue same dose of other IS with strict monitoring of laboratory and clinical profile. If patient have progressive renal failure with sepsis, then CNI are completely withdrawn and restarted once they recover clinically from sepsis. If patient have leukopenia (total count <3000 cells/mL) and thrombocytopenia (platelets <40,000 cells/mL) or severe diarrhea anti-metabolite agents are stopped. Once patient develops septic shock, both CNI and anti-metabolites are completely withdrawn and intravenous steroids (for example hydrocortisone 50–100 mg, 8th hourly) given as replacement IS, until inotropic support is tapered off or minimized.

Immunosuppression Minimization and Withdrawal

Though long-term survival is greatly improved after liver transplantation, posttransplant patients still have higher morbidity and mortality than the general population. The chronic and possible lifelong need of immunosuppression plays an important role in the development of these morbidities. Currently, there is growing evidences of immunosuppression minimization and withdrawal in liver transplant recipients after prolonged IS therapy with stable graft function. Although details on this topic are beyond the scope of this article, we will briefly discuss these topics.

Minimization

Minimization in LT is the strategy of using the lowest possible but well-tolerated dose of IS agents while maintaining a stable graft function. The process of IS minimization actually starts in the early posttransplant period, where steroids are progressively reduced along with the introduction of CNIs and antimetabolites. With time, steroids are tapered off (usually within the initial three months) and then antimetabolites (usually within 6–12 months), while maintenance agents are continued. Delayed monotherapy usually includes a CNI, a mTORI, or sometimes an antimetabolite, but the final choice depending on the occurrence of chronic complications of IS like nephrotoxicity or neurotoxicity, dysmetabolism, and de novo or recurrent tumor or allograft disease.^104,105 Generally, doses of CNIs or mTORI are also gradually tapered off with time to the minimum possible level, balancing optimal immunosuppression and the risk of rejection. These adaptations directed toward a single-drug regimen also depend on the experience of the transplant team and the underlying disease of the recipient.

Another important indication of minimizing immunosuppression is with the HCC transplant recipients, where chronic IS is considered one of the risk factors for recurrence. Minimization and even withdrawal of immunosuppression could theoretically reduce the risk of tumor recurrence in this group of recipients.106, 107, 108

Currently, immunosuppression minimization is carried out in an empirical ‘trial and error’ manner. This approach leads to the current strategy, with high rates of rejection seen in a number of clinical trials. This strategy holds a number of demerits, including the risk of developing irreversible graft damage and the development of immune sensitization leading to protracted graft damage.

Immune Tolerance and Withdrawal

Liver being an immunologically privileged organ compared to other transplant organs such as the heart, kidney, or pancreas, the possibility of acceptance of grafted liver without ISA is theoretically possible. The ability of the immune system to respond to pathogens and foreign antigens while maintaining tolerance to its own tissues is called immunological self-tolerance. While transplant tolerance is the acceptance of graft tissue by the body’s immune system without the need for long-term IS while maintaining protective immunity. Tolerance is usually classified as complete immune tolerance (or true tolerance), operational tolerance (OT), and prope tolerance (or near tolerance–a state of stable graft function in the presence of a minimal dose of immunosuppressive agents). Operational tolerance, also called functional tolerance in clinical practice, is the state of long-term functional graft survival with stable graft function and histology in the absence of maintenance IS.^26,109,110 As donor antigens always remain with the recipient, the achievement of complete tolerance is not currently considered.

Up to 20–60% of LT recipients can maintain graft tolerance after ceasing IS; this is known as spontaneous operational immune tolerance.^111,112 Although difficult to predict, some variables that are found to be associated with operational tolerance are: younger age of transplant, older age, prolonged time interval after transplantation, male sex, nonimmune-mediated liver diseases, maternal donor status, and long-term stable graft function. Spontaneous operational tolerance after LT is an ideal outcome considering the fact that it avoids long-term complications associated with chronic immunosuppressive medication use and improves overall quality of life. Thus, achieving operational tolerance is the ultimate goal and the current guiding principle of designing individualized immunosuppression protocols worldwide.^113,114

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 with a highly selected group of patients. On the other hand, some studies have shown that spontaneous operational tolerance is a dynamic and nonpermanent state, and thus, patients may lose tolerance on follow-up. Thus, long-term, regular follow-up, including laboratory monitoring and protocol-based surveillance biopsies, is a must in such cases to rule out subclinical rejection.¹¹⁵

Biomarkers for Tolerance

As normal serum markers of liver injury, including liver biochemical tests, are not considered enough for assuming operational tolerance, there has been ongoing research for reliable biomarkers for OT. The presence of peripheral cell signatures such as a high proportion of FoxP3+ Treg cells, Treg/Th17 subpopulations, Vd2-TCR gdT cells, and a tolerogenic pDC2/pDC1 subset ratio have been identified as potential biomarkers predicting operational tolerance. Other biomarkers studied are the expression of natural killer (NK)-related genes, the expression of genes (in peripheral cell flow cytometry) such as FEM1C, SENP6, miR95, and miR31, and the absence of donor-specific antibodies (DSA) which have also been demonstrated to predict tolerance.^116,117

To date, the use of biomarkers for the guidance of IS withdrawal is still experimental, as most have not been prospectively validated and others have failed to demonstrate external validity.

Therapeutic Immune Tolerance

Immune tolerance induced pharmacologically is the therapeutic operational immune tolerance. Due to immunosuppressive drug nonspecificity, drug toxicity, inconsistent outcomes, and the difficulty of early complete immunosuppression withdrawal, other strategies, including the use of stem cells, regulatory dendritic cells (DCreg), and Treg therapy, have emerged to promote liver allograft tolerance. The pivotal role of many of these cellular subsets in immunomodulation makes them ideal candidates for the use as therapeutic agents.^113,118

T lymphocytes play an important role in the graft immune response for either tolerance or rejection. The balance between cytotoxic T cells and CD4+CD25+FOXP3+ regulatory T cells (Tregs) is the basis of most tolerance protocols. Recipients’ T cells can recognize the donor antigens through both the self and donor antigen-presenting cells (APCs), then undergo expansion and increase the activation of host and donor cells to attack the foreign antigens. Tregs can halt this immune response and induce donor antigen immune tolerance through the inhibition of cytotoxic cell proliferation, the stopping of cytokine production, and the prevention of antibody production.^119,120

Regulatory cells in the response of immune tolerance can be modulated with donor-modified cell therapy. This has been demonstrated in animal models by the ability of donor-treated dendritic cells (DCs) to induce the production of granulocyte–macrophage colony-stimulating factors (GMCSFs) for the proliferation of tolerating cells.^121,122 To date, multiple clinical trials have shown promising results when using Tregs in transplantation, with the subsequent elimination of immune suppression and induction of immune tolerance. A UCSF group (trial #NCT02474199), Kings College group (trial #NCT02166177), and Massachusetts general hospital group (trial #NCT03577431) introduce the use of regulatory T cells (Tregs) for liver transplantation in a clinical setting.^110,119

Artificial Intelligence and Precision Medicine in Liver Transplantation

Precision medicine is an evolving healthcare concept that focuses on tailoring medical decisions, treatments, practices, and products to individual patients based on their genetic, environmental, lifestyle, and other factors. With the advances in computing power, algorithms, and big data, the last decade has witnessed widespread applications of artificial intelligence (AI) in the medical field, including solid organ transplant (SOT). As pre- and post-transplantation patient care usually requires complex decision-making, AI can drive a real paradigm shift in analyzing and synthesizing huge amounts of transplant-related data and transforming it into clinical recommendations, thus tailoring it to personalized therapy.¹²³

The use of AI in SOT, especially renal transplant, has shown promising results, while its application in the field of liver transplantation is still immature.^124,125 In a recent randomized prospective clinical trial, patients with AI-based personalized dosing of tacrolimus after liver transplantation were compared with standard-of-care clinician-guided dosing. Phenotypic personalized medicine (PPM)-guided dosing resulted in a lower fraction of inpatient days where the tacrolimus trough blood level-based group had a large deviation from the target range. PPM patients also had a 33% shorter posttransplant length of stay than patients receiving standard-of-care tacrolimus dosing.¹²⁶

AI is still in its infancy, and, so far, we lack validated algorithms that could accurately drive organ selection, predict potential rejections, or attenuate postoperative complications. Nevertheless, in the last few decades, AI applications have already contributed to a lower incidence of rejection and the fine-tuning of the transplantation and organ preservation processes.¹²⁷

The rate of allograft rejection has been significantly reduced while improving the patient's quality of life over time with immunosuppressive agents and regimens. The combination protocol and personalization of therapy lead to a minimization of both dose and individual drug toxicity. Therefore, the art of immunosuppression is to achieve a balance between effective and optimal immunosuppression in the best possible combination to achieve the desired outcomes while avoiding over-immunosuppression and its long-term consequences. Though ideas of immunosuppression withdrawal with long-term induction of tolerance become an ultimate objective, they are still not practiced currently and may become a viable option in the future. The use of biomarkers of immune tolerance and artificial intelligence will help in the better understanding and management of patients.

Credit authorship contribution statement

Shekhar Poudel: Formal analysis; Visualization; Writing - original draft; Sanjiv Saigal: Conceptualization; Supervision; Writing - review & editing; Subhash Gupta: supervision and review

Conflicts of interest

All authors have none to declare.

Funding

No financial support was sought from any agencies for this study.