Figure 30.1 Intracellular events following interaction of the APC with the naive T cell. The antigenic peptide fragment is loaded on the MHC II complex on the APC. Interaction with the CD3 receptor on the T cell results in signal 1. Interactions between CD28 on the T cell and CD80/86 on the APC result in signal 2, which is a verification step for T cell activation. The whole process then activates intracellular kinases, resulting in activation of calcineurin. Calcineurin activates a transcription factor NFAT, which upregulates expression of IL-2. The IL-2 binds to the CD25 molecule, triggering a cascade of intracellular signalling pathways, resulting in cell proliferation. The various immunosuppressants are shown in the diagram acting on their respective targets. Mab, monoclonal antibody.
30.3.5.4 ROLE OF CALCINEURIN
Calcineurin is a Ca2+-dependent protein phosphatase, which has an important role in the control of intracellular Ca2+ signalling. Both signal 1 and signal 2 are necessary for T cell activation. When an APC interacts with a TCR on a T cell, there is an increase in the intracytoplasmic calcium which activates calcineurin. Calcineurin activates (dephosphorylates) a transcription factor NFAT (nuclear factor of activated T cells), which is then translocated into the nucleus, where it upregulates the expression of IL-2. IL-2 is associated with T cell activation and proliferation, and it is believed that the amount of IL-2 produced by the helper T cells influences the extent of immune response significantly. Calcineurin is the target of calcineurin inhibitor (CNI) drugs such as cyclosporin and tacrolimus [9].
30.3.6 Recognition of graft antigens by the recipient’s T cells
Interaction of donor antigens with the recipient T cells will provoke a fierce rejection response. Three nonmutually exclusive pathways have been described.
30.3.6.1 DIRECT PATHWAY
The donor organ will carry a variable number of passenger dendritic cells with a high density of allo-MHC molecules. These passenger APCs are capable of directly stimulating the recipient’s T cells. In the direct pathway, recipient T cells will recognise MHC molecules on the surface of donor APCs. The degree of MHC polymorphism between individuals will largely affect peptide binding, and consequently the immune response.
Figure 30.2 Cell cycle showing the various phases. MMF, azathioprine and cyclophosphamide inhibit nucleotide synthesis and thereby cell replication. SRL and everolimus inhibit the progression of T cell proliferation from the G1 to the S phase of the cell cycle. S, synthesis phase; M, mitosis phase; G, gap phase.
30.3.6.2 INDIRECT PATHWAY
With time, the donor APCs in the graft will be replaced by recipient APCs, which will then process and present donor antigenic peptides using their own MHC platform.
30.3.6.3 SEMIDIRECT PATHWAY
In this mechanism, it has been proposed that recipient APCs will acquire intact MHC molecules from donor cells and then display this foreign package on their own cell surface for interaction with the host T cell [7,10] (Figure 30.3).
30.3.7 Events that follow naive T cell activation
Newly transplanted organs are subject to severe inflammation due to several nonimmunological factors, such as injury during organ procurement, cold preservation in nonphysiological fluids, surgical trauma and reperfusion injury. This will lead to a hostile cytokine milieu with release of inflammatory mediators. The characteristics of the inflammatory environment in which donor-reactive CD4+ T cells recognise donor antigens determine the immune phenotype of these cells. When CD4+ T cells are activated in the presence of IL-12 (usually produced by activated, mature dendritic cells), they become tissue-destructive, interferon (IFN)-γ–producing T helper (Th1 response). In contrast, CD4+ T cells that are activated in the presence of IL-4 differentiate into Th2 cells, which produce IL-4 and IL-5. In the absence of pro-inflammatory cytokines, transforming growth factor (TGF)-β induces expression of Foxp3 and differentiation of CD4+ T cells into Tregs. In contrast, expression of TGF-β with IL-6 or IL-21 prevents development of the transplant-protective Tregs; instead, the antigen-reactive CD4+ T cells become IL-17-producing T cells (Th17), which are highly cytopathic. The overall outcome is determined by an unpredictable balance between the hostile rejection-promoting helpers (Th1, Th2, Th17 and possibly more) and the tolerogenic Tregs [10].
Figure 30.3 Recognition of graft antigens by the recipient’s T cells. Direct pathway: Dendritic cell (APC) from the donor tissue presents the antigen to the recipient’s T cell (TCR) through its own MHC. Indirect pathway: Dendritic cell from the recipient (host) will process the foreign antigen from the allograft and load it onto its MHC platform for engagement with the TCR on the recipient’s T cell. Semidirect pathway: Here, the recipient’s dendritic cells will acquire intact MHC molecules from the donor cells and use this foreign MHC platform to present the antigen to the recipient T cell.
Several postulated mechanisms of donor cell injury have been described. Cytokine secretion from CD4+ T cells will recruit and activate CD8+ T cells, B cells, NK cells and macrophages, all of which have the potential to mount an immune response. Furthermore, certain cytokines, such as IFN-γ, upregulate expression of MHC on the target donor cell thereby, making it susceptible to the weaponry of CD8+ T cells. The final ‘lethal hit’ for the cytolytic processes is triggering of apoptosis in the target cell [7].
30.4 IMMUNOLOGICAL TOLERANCE IN THE LIVER
The liver tends to be a rather tolerogenic microenvironment for a variety of reasons. The portal venous system drains the gastrointestinal tract and therefore is an inevitable thoroughfare for microbial antigens, microbe-associated molecular patterns (MAMPs), and food-derived antigens. There is a constant traffic of MAMPs, such as lipopolysaccharide (LPS) and other endotoxins. The reactivity of the liver cells towards MAMPs is attenuated by the development of a functional state of refractoriness following repeated exposure and is termed ‘endotoxin tolerance’. The prevalence of MAMPs in the gut and liver is physiological and not usually associated with infection. Therefore, it is important to maintain immune tolerance under these conditions to avoid autoimmunity. Liver has high levels of IL-10 (anti-inflammatory cytokine) and PD-L1 (programmed death-1 ligand-1). Also, the threshold for T cell activation is set rather high, as a result of which a state of immune tolerance prevails and tends to dampen T cell immune response. This is achieved by the cooperation of various tolerogenic liver-resident APC populations and immune regulatory mediators such as IL-10, prostanoids and TGF-β, which are released locally within the liver. These mechanisms are likely to be responsible for the spontaneous acceptance of liver allografts observed in animal models and selected patients. Nevertheless, immunosuppression is a key element of posttransplant care to maintain and enhance survival of allografts [10,11].
30.5 CURRENT STATUS OF IMMUNOSUPPRESSIVE AGENTS IN CHILDREN
Early liver transplantation was complicated by frequent episodes of rejection resulting in graft loss. Posttransplant outcomes have improved significantly with the advent of CNIs, rescue management strategies with more robust immunosuppressive agents and advances in surgical techniques. The wide array of drugs offers the option to use combinations with nonoverlapping toxicity profiles so that doses of individual drugs can be kept at a minimum to avoid toxicity. However, in paediatrics there are only a few clinical studies with robust data to rationalise the use of some useful drugs, leading to frequent off-licence usage in the paediatric age group. Table 30.1 lists the commonly used and newer agents in post–liver transplant immunosuppression. Table 30.2 summarises the mode of action, pharmacokinetics and common side effects of some of the common agents used.
30.5.1 Calcineurin inhibitors
CNIs (cyclosporin* and tacrolimus†) have been used widely and recommended as part of initial immunosuppressive therapy following a liver transplant. The use of cyclosporin (Box 30.2) in liver transplantation has largely been superseded by tacrolimus given its superior efficacy [12]. Tacrolimus is a macrolide immunosuppressant first isolated in 1985 from an actinomycete Streptomyces tsukubaensis, obtained from soil samples from Mount Tsukuba in Japan. It was first used for renal transplantation in Pittsburgh in 1989, and then subsequently was approved for liver transplantation in 1994 in the United States. Like cyclosporin, tacrolimus acts primarily by interfering with T cell activation. Cyclosporin binds to its specific cytoplasmic immunophilin, cyclophilin, and tacrolimus binds to a corresponding protein, FK506 binding protein 12 (FKBP12). These cyclosporin–or tacrolimus–immunophilin complexes inhibit the activity of calcineurin, a calcium-dependent phosphatase. Inhibition of calcineurin impedes calcium-dependent transduction and inactivates transcription factors, particularly NFAT. NFAT is believed to initiate gene transcription for the formation of lymphokines such as interleukin-2 and γ-interferon. The clinical result of this NFAT inhibition is immunosuppression [9,13].
Tacrolimus inhibits lymphocyte activation in vitro 10–100 times more potently than cyclosporin. Its oral bio-availability ranges between 5% and 67%, with the rate and extent of absorption greatest under fasting conditions. Drug concentration monitoring is crucial because tacrolimus has high inter- and intraindividual variability in pharmacokinetics, and a narrow therapeutic index [9].
Tacrolimus has become a common choice for immuno-suppression following liver, kidney and heart transplantation. It is licensed for use in the prophylaxis of transplant rejection in children. Its safety profile and efficacy is well established through robust paediatric studies [14,15]. A meta-analysis of 16 randomised trials comparing cyclosporin with tacrolimus for liver transplantation showed that tacrolimus is superior to cyclosporin in preventing AR [12].
Table 30.2 Pharmacological profile and adverse effects
Drug | Pharmacological action | Pharmacokinetics and dynamics | Adverse effects |
Tacrolimus | Binds to FK506 binding protein 12 and inhibits calcineurin; calcineurin inhibition consequently interrupts T cell activation and proliferation | • Variable oral bioavailability • Peak blood levels after 1–2 h • Half-life of 8–24 h • Highly bound to albumin and a-1 acid glycoprotein • Metabolised by cytochrome p450 isoenzymes | Nephrotoxicity, neurotoxicity, lymphomas and lymphoproliferative disease, cardiomyopathy, anaemia, chronic diarrhoea, onset of diabetes mellitus, electrolyte disturbances |
Sirolimus | Binds to mTOR and inhibits cytokine-driven T cell proliferation | • Hydrophobic drug with low solubility in aqueous solutions • Long half-life • Metabolised by cytochrome p450 system | Hyperlipidaemia, proteinuria, myelosuppression, poor wound healing, pneumonitis, hypersensitivity reactions |
Mycophenolate mofetil | Blocks purine nucleotide synthesis by inhibiting type 2 IMPDH | • Bioavailability is 90%–94% or oral preparations • Has substantial enterohepatic circulation, contributing to its GI toxicity | Gastrointestinal side effects, neutropenia, opportunistic infections |
IL-2 receptor antibodies | Bind to the a chain of the IL-2 receptor, resulting in inhibition of clonal proliferation of the T cells | • Half-life of 7.2 days • Suppressor effects last for 3–4 weeks | Hypersensitivity reactions |
Campath-1H | Monoclonal antibody directed against the CD52 surface antigen, resulting in lymphocyte depletion from the circulation | • Plasma elimination half-life of approximately 12 days | Hypersensitivity reactions |
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