T Cell-Targeted Immunosuppression
The most common form of rejection encountered is T cell-mediated (TMR) (also known as acute cellular rejection (ACR)), occurring in 15–20% of transplants. ACR is characterised histologically by lymphocyte infiltration into the graft (predominantly cytotoxic [CD8] T cells). ACR is orchestrated by CD4 T cells, which are activated by antigen-presenting cells (APCs), such as dendritic cells and B cells, presenting donor antigen to CD4 T cells via MHC class II molecules. For full activation of T cells, APCs also provide a co-stimulatory signal via surface molecules such as CD80/86. Such activated T cells produce large quantities of cytokines, particularly interleukin (IL)-2, which further drive the activation and proliferation of both CD4 and CD8 T cells. Thus, immunosuppressive agents predominantly target T cell activation via four broad mechanisms.
1 Lymphocyte depletion
Lymphocyte depletion has been achieved using biological agents (see Chapter 15), initially polyclonal agents such as ATG and ALG or the T cell-specific anti-CD3 antibody OKT3. Alemtuzumab (CamPath-1H) is a humanised rat IgG1 monoclonal antibody that binds CD52, a glycoprotein present on the surface of lymphocytes, dendritic cells and natural killer (NK) cells. Administration of alemtuzumab leads to rapid and sustained depletion of both T and B lymphocytes. It is principally used at induction and allows a corticosteroid-free maintenance regimen.
2 Disruption of T cell activation by co-stimulatory blockade
When T cells are activated by APCs two signals are required for full activation. First, the T cell receptor must recognise and bind to its specific antigen presented in the context of MHC. The second signal is mediated by the engagement of pairs of co-stimulatory molecules expressed on the surface of T cells and APCs, for example CD28 (T cell) and B7 (also known as CD80/CD86, found on APCs).
CTLA4 is a molecule present on the surface of T cells and is a competitive inhibitor of the CD28:B7 interaction. CTLA4-Ig (abatacept) consists of the extracellular domain of human CTLA4 linked to the Fc portion of IgG1. Abatacept blocks co-stimulation via the CD28:B7 pathway, and in rodent models has prevented rejection of cardiac, renal, pancreatic, hepatic and skin allografts. However, in non-human primate models, abatacept is an ineffective means of prophylaxis against rejection. This led to the modification of abatacept to produce more avid binding to CD86 and thus more potent inhibition of T cell activation. The result was belatacept (LEA29Y), a fusion protein that differed from abatacept by two amino acids. Belatacept is effective in preventing allograft rejection in humans and has been used as a maintenance agent.
3 Cytokine blockade
IL-2 acts as a potent activator and pro-proliferative cytokine for T cells. A number of agents have been developed that inhibit IL-2 synthesis, IL-2 binding to its cell surface receptor, or signal transduction following IL-2 receptor ligation.
Inhibition of cytokine synthesis Antigen presentation to T cells triggers a calcium-dependent intracellular signalling cascade, which results in the activation of the phosphatase calcineurin. Calcineurin dephosphorylates the transcription factor NF-AT, allowing its translocation to the nucleus, where it enhances the transcription of a number of cytokines, including IL-2. Calcineurin inhibitors (CNIs) such as ciclosporin and tacrolimus form complexes with intracellular immunophilins (cyclophilin and FK506- binding protein respectively). These CNI–immunophilin complexes inhibit calcineurin and thus prevent the translocation of NF-AT to the nucleus and inhibit its subsequent actions there. CNIs are useful maintenance immunosuppressants but are nephrotoxic, leading to chronic graft dysfunction.
Corticosteroids, for example prednisolone, have a variety of anti-inflammatory effects including suppression of prostaglandin synthesis, reduction of histamine and bradykinin release, and the inhibition of the production of several pro-inflammatory cytokines. They are widely used and still form a part of many maintenance regimens. In addition, they are also used as first-line treatment of ACR.
Inhibition of IL-2 receptor binding The effects of IL-2 on T cells are dependent on binding to its cell surface receptor. The IL-2 receptor has three subunits, α (CD25), β and γ. During T cell activation the α subunit becomes associated with the other subunits to form a high-affinity receptor. Blockade of the IL-2 receptor by targeting the α-chain profoundly inhibits T cell proliferation. One anti-CD25mAb, basiliximab, is currently in wide clinical use as an induction agent in renal transplantation. Another, daclizumab, has been recently withdrawn from use. Both have proven efficacy in the reduction of the incidence and severity of ACR in renal transplantation.
Inhibition of signal transduction Sirolimus (rapamycin) and everolimus bind to the FK506 binding protein and the resulting complex inhibits an intracellular kinase, the mammalian target of rapamycin complex 1 (mTORC1). mTORC1 is important in a variety of signalling pathways, including that found downstream of the IL-2 receptor. Thus, inhibition of mTORC1 blocks both T cell activation and proliferation by preventing cell-cycle progression. In addition, sirolimus and everolimus also inhibit the VEGF pathway and thus have anti-angiogenic effects, a property that has been used for the treatment of some cancers (e.g. Kaposi’s sarcoma).
4. Inhibition of DNA synthesis
For lymphocytes to proliferate, they must synthesise new DNA prior to division. Thus, agents that inhibit DNA synthesis act as useful immunosuppressants.
Azathioprine (AZA) is a pro-drug which is converted into the purine analogue 6-mercaptopurine (6-MP). It was first used in the early 1960s in renal transplantation and continues to be used in many centres as part of a maintenance regimen. The main disadvantage of azathioprine is that of non-specific bone marrow suppression. The enzyme thiopurine S-methyltransferase (TPMT) deactivates 6-MP, and genetic polymorphisms of TPMT, which are associated with loss of function, can lead to drug toxicity.
Mycophenolate mofetil (MMF) and mycophenolate sodium (MPS) are also pro-drugs, converted in the liver to mycophenolic acid (MPA). MPA is a non-competitive, reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH), the enzyme that controls the rate of synthesis of guanine monophosphate in the de novo pathway of purine synthesis. Most cells can generate guanosine nucleotides by two pathways, the IMPDH pathway and the salvage pathway. Lymphocytes lack this salvage pathway, thus MPA specifically targets lymphocytes while sparing other cells. MPA now forms part of the maintenance immunosuppressive protocol of many transplant centres.