The advances in organ transplantation, so evident in the other sections in this chapter, can be attributed to a great extent to the advances in immunosuppression over the last 40 years. In the 1950s total body irradiation was the only form of immunosuppression available and patients either died of marrow aplasia and overwhelming infection if given sufficient irradiation to prevent rejection of a renal transplant, or rejected the graft if given lower doses of irradiation. Nevertheless, some modest success was achieved in some patients at that time. The introduction of azathioprine in the early 1960s was a major advance in renal transplantation and was quickly applied in renal transplant units throughout the world. As graft survival could now be prolonged in many patients both in the medium and long term, there was a dramatic growth in the numbers of renal transplant units. Steroids were added to azathioprine firstly to treat rejection and then in combination with azathioprine to prevent rejection. Heterologous antilymphocyte globulin, usually made in horses at the time, was introduced to treat steroid resistant rejection in the 1970s, but basically the standard immunosuppressive therapy in all units was azathioprine and steroids for nearly 20 years until cyclosporin became generally available in the early 1980s.

Very quickly cyclosporin-based immunosuppressive protocols became standard therapy and remain so to this day. The introduction of cyclosporin led not only to a marked improvement in renal allograft survival (10–15 per cent), but also to a rapid improvement in liver and cardiac allograft survival. This led to a dramatic increase in the numbers of liver and cardiac transplants throughout the Western world.

During the next 10 years other immunosuppressive drugs will become available and some are already undergoing clinical trials, e.g. FK506 and RS61443. In addition the explosion in the production of monoclonal antibodies recognizing different cell surface markers on lymphocytes promises to add a new dimension to therapy with the hope of producing increased specificity of immunosuppression. Already OKT3, a pan-T-lymphocyte monoclonal antibody, is widely used to treat steroid resistant rejection and is also used as part of induction therapy in many centres. This agent is not a particularly specific antibody in that it recognizes all T cells but monoclonal antibodies against other lymphocyte targets should allow much greater specificity in immunosuppression to be achieved in clinical practice.

One of the major problems in clinical transplantation in the coming years will be the assessment of the large number of potentially valuable new therapies (both drugs and biological reagents) that will become available, remembering that the results of organ transplantation are now relatively good so that only large multicentre trials will be able to establish the true value or otherwise of new therapies. Furthermore, more sophisticated methods of analysis of outcome than mere graft survival will have to be developed.

Azathioprine is a purine analogue, and is essentially an antiproliferative agent, inhibiting both DNA and RNA synthesis by preventing the synthesis of adenylic and guanylic acid from inosinic acid. For 20 years, in association with steroids, azathioprine provided the backbone of immunosuppressive therapy. In the cyclosporin era, azathioprine is still widely used in lower doses with cyclosporin on the assumption that it allows lower doses of cyclosporin to be used, hence decreasing the side-effects of both drugs. The major side-effect of azathioprine is leucopenia, and indeed regular white cell counts are the only method of monitoring the dosage. If used with steroids alone then the usual starting dose is 3.0 mg/kg daily reducing to maintenance levels of 2.0 mg/kg. However, when used as part of a cyclosporin-based protocol it tends to be used in doses of either 100 mg daily or 1.5 mg/kg daily.

Prednisolone or prednisone are used routinely with azathioprine, and also in most cyclosporin-based protocols. Their mechanism of action in suppressing the alloreaction is unclear, but although immunosuppressive to some extent possibly their major action is an anti-inflammatory one. Certainly their concomitant use with azathioprine was essential to produce appropriate immunosuppression, but that is not clearly so in the cyclosporin era. When first introduced soon after azathioprine became available steroids were used in high doses and indeed most of the complications of transplantation in the 1960s and 1970s could be attributed to the use of high dose steroids, e.g. cushingoid changes, avascular necrosis of joints, peptic ulceration, infection, osteoporosis. The demonstration in controlled trials that low dose steroids were equally as effective in preventing rejection as high dose steroids quickly led to the general introduction of low dose steroid protocols, e.g. 20 mg daily during the first 2 to 3 months after transplantation reducing to maintenance levels of 10 mg daily, dramatically reduced the morbidity and mortality of renal transplantation.

High dose steroids are also used to treat rejection and a widely used protocol in this context is either 0.5 or 1.0 g of methylprednisolone given as an intravenous bolus daily for 3 days. This protocol successfully reverses the majority of acute rejection episodes of kidney, heart, and liver, and antilymphocytic globulin and/or OKT3 is reserved for steroid resistant infection.

Cyclosporin is a potent immunosuppressive agent, extracted from two strains of fungi imperfecti. It has a molecular weight of 1200 and comprises 11 amino acids (Fig. 1) 685. Its major mechanism of action is to prevent the product of cytokines such as interleukin-2 (IL-2), which will be discussed in more detail later. When first introduced into clinical practice in renal transplantation and bone marrow transplantation it resulted in superior suppression of rejection and graft-versus-host disease respectively, which was reflected by better graft and patient survival. However side-effects soon became evident, the major ones being nephrotoxicity and hypertension (Table 1) 231. Nephrotoxicity is a major problem of cyclosporin use in all forms of organ transplantation and bone marrow transplantation and although dose related to a great extent there is probably no effective dose of cyclosporin that is not nephrotoxic. The nephrotoxicity is due mainly to vasoconstriction of the afferent arterioles of the glomeruli, leading to a decrease in glomerular filtration rate, and these changes appear to be mediated by inhibition of vasodilator renal prostaglandin metabolites and increased production of thromboxane.

Because of the nephroxtoxicity and other dose related side-effects of the drug a number of cyclosporin-based protocols have evolved in an attempt to reduce the incidence of these side-effects (Table 2) 232.

Although effective it is possible that higher doses of cyclosporin are required than if used with other agents.

This is a commonly used protocol, but is not proven to be better than cyclosporin alone, although it has been suggested that the incidence of nephrotoxicity is lower when steroids are used.

Cyclosporin is used alone or with steroids and then at some given time after transplantation, e.g. 3, 6, or 12 months, cyclosporin is replaced with azathioprine and steroids preferably with some overlap. Undoubtedly renal function improves with conversion but the major drawback of these protocols is the risk of rejection occurring within the 1 or 2 months after conversion. Although these rejection episodes usually respond to steroid therapy, this type of protocol does require close supervision of patients after conversion. Nevertheless the financial aspects of this protocol as well as the improved renal function are attractive. Thus, this Oxford protocol, as it is often known, is widely used in developing countries.

This is a widely used therapy which produces very acceptable patient and graft survival (Fig. 2) 686. It is not a more potent immunosuppressive protocol than other cyclosporin protocols, but it is relatively free of side-effects and easy to use. A typical protocol (as used in Oxford) would comprise cyclosporin 10 mg/kg.day reducing according to whole blood levels, azathioprine 100 mg/day, and prednisolone 20 mg/day reducing to a maintenance level of 10 mg/day, with the further aim of weaning patients off steroids altogether if renal function is stable at 1 year. Indeed our own experience of trials of steroid withdrawal in patients on triple therapy have shown benefits in levels of both hypertension and hypercholesterolaemia.

This is not widely used and as the addition of steroids is required quite often in the early months it has no advantages over triple therapy.

This type of protocol is widely used in North America in all forms of organ transplantation, but its use remains controversial both because improved graft survival has not been clearly established with the use of this type of protocol, and if subsequently steroid resistant rejection requires repeated treatment with either ATG or OKT3 then a substantial risk of developing a fatal acute lymphoproliferative disorder has been introduced. Nevertheless if there is no renal function after transplantation of a cadaveric kidney as a result of acute tubular necrosis, it is an attractive approach to the induction of immunosuppression, for there is reasonably good evidence that the use of cyclosporin in a patient with a non-functioning kidney delays the onset of function and also leads to long-term damage to the kidney.

This regimen is sometimes used in cardiac or liver transplantation. It probably represents an unnecessarily potent immunosuppressive protocol for most patients and is not recommended, except in the highly sensitized patient.

A variety of techniques are commercially available but the most widely used techniques measure trough whole blood levels at either 11 or 24 h depending on whether the patient is taking cyclosporin twice daily or daily (there is no evidence that either dosing protocol is superior to the other). Trough levels are maintained at 200 to 400 &mgr;g/ml in the first 5 weeks and then at 100 to 200 &mgr;g/ml once stable graft function is established. Today most assays use monoclonal antibodies to cyclosporin A, the parent compound, which is essentially responsible for the immunosuppression and side-effects. High trough levels of cyclosporin are likely to be associated with toxicity and low levels with an increased incidence of rejection, but this is not inevitable and the trough levels must be used in association with other clinical, biochemical, and histological parameters.

Another potent immunosuppressive macrolide antibiotic, derived from Streptomyces tsukubaensis, FK506 has an entirely different structure to cyclosporin (Fig. 1) 685 but its mechanism of action is similar in that it prevents the production of cytokines in response to antigen recognition (see later). On a weight for weight basis it is 10- to 100-fold more potent than cyclosporin. Its first clinical use in Pittsburgh was for the salvage of liver transplants undergoing rejection despite the use of high dose steroids and OKT3. In some 70 per cent of instances conversion from cyclosporin to FK506 resulted in improved liver function tests and survival of the liver allografts, an impressive debut. Its use as a primary agent with steroids has also been explored at Pittsburgh in liver, cardiac, and renal transplantation with excellent results in liver (Fig. 3) 687 and cardiac transplantation. In contrast in a prospective controlled trial in renal transplantation, in which FK506 was compared with cyclosporin, no improvement in graft survival was observed and the same degree of nephrotoxicity was noted. Two large prospective controlled trials of FK506 in liver transplantation have been performed in North America and Europe and the results do show that FK506 is of value in liver transplantation. Thus, it would seem that this drug is going to represent a very useful addition to our immunosuppressive armamentarium, especially in liver transplantation. In addition it has allowed a number of successful small bowel transplants in adults and children to be performed in Pittsburgh without a concomitant liver transplant, which does represent a significant advance in this area.

Rapamycin is another macrolide antibiotic, with a very similar molecular structure to FK506 (Fig. 1) 685, but in contrast an entirely different mechanism of action in that it inhibits the proliferation of the activated T cell. It is a potent immunosuppressive agent in experimental models but has not yet been used in clinical trials.

There has been a marked increase in our knowledge of how these agents work, the implications of which are enormous in our understanding both of signal transduction after antigen recognition leading to T-cell activation and proliferation of the activated T cell. All three agents bind to a ubiquitous class of proteins within the cytoplasm known as immunophilins. Cyclosporin binds to cyclophilin while FK506 and rapamycin bind to a different immunophilin, FK binding protein (FKBP). These proteins are isomerases and it was first thought that the immunosuppressive action was due to inhibition of isomerase activity, but as it was realized that very small amounts of cyclosporin or FK506 were immunosuppressive although only binding to a portion of the total amount of isomerase available, other mechanisms were sought. Then another protein was found to be involved, namely calcinurin to which the complex of cyclosporin + cyclophilin and FK506 + FKBP bind, but not the complex rapamycin + FKBP; hence presumably the different effect of rapamycin. The drug–immunophilin complex and calcinurin inhibits the attachment of a protein, NFAT (nucleus factor in activated T cells), to the enhancer region upstream of the gene for IL-2, preventing its transcription and hence the production of IL-2 (Fig. 4) 688. Just how this inhibition of NFAT occurs is not yet understood. It is not known what the complex of rapamycin + FKBP binds to but obviously as this binding prevents proliferation of the activated T cell, i.e. inhibiting the lymphokine-receptor signal, definition will further increase our knowledge of the response to alloantigens by T cells and allow the design of drugs with quite specific actions.

An ester of mycophenolic acid, this is an antiproliferative drug with a quite specific action in the purine pathway, namely the inhibition of the enzyme guanosine monophosphate. Thus it is relatively specific for lymphocytes. In vitro it has proved to be a powerful immunosuppressive agent especially when used together with cyclosporin; in addition it inhibits B-cell activity and hence antibody formation. Early experimental use and clinical trials suggest that this is a potent immunosuppressive agent. It is currently undergoing prospective randomized clinical trials in renal transplantation.

This antiproliferative agent is now seldom used but has been used as a replacement for azathioprine in the presence of hepatotoxicity considered to be due to this agent. It has also been used in India instead of azathioprine because it is cheaper, with success.

Mizoribine, an imidazole nucleoside antibiotic which inhibits RNA and DNA synthesis via the purine biosynthesis pathway, has been used successfully instead of azathioprine in Japan for a number of years.

An antitumour antibiotic, it suppresses macrophage function and inhibits antibody production. It might have a place in sensitized patients but although used for a number of years, especially in Japan, no prospective clinical trials have been performed.

This anticancer agent prevents all proliferation by inhibiting de-novo pyrimidine synthesis. In experimental models of transplantation it appears to be quite a powerful immunosuppressive agent, but as yet has not undergone clinical trials.

Heterologous antisera produced in another species, usually the horse or rabbit, have been used for many years primarily to treat steroid resistant rejection, but more commonly in recent years as inductive therapy to prevent rejection. In general the major action of antilymphocyte antisera is to reduce the T-lymphocyte count, but being heterologous antisera produced by immunization of horses or sheep with human splenocytes or thymocytes, they inevitably have some general antileucocyte action as well as some antiplatelet activity and may produce varying degrees of leucopenia or thrombocytopenia. However, a good biological agent, and several are available, will successfully reverse a steroid resistant rejection in 70 per cent of instances, with relatively few side-effects.

Monoclonal antibodies are produced by immunizing either rats or mice with the particular antigen against which antibodies are required, e.g. T lymphocytes. Spleen cells from the rodent are then fused with a myeloma cell line to produce a hybridoma. The hybridomas are screened to find one producing the desired antibody, which can then be cloned, thus providing a source of only that highly specific antibody (Fig. 5) 689.

Monoclonal antibodies can be produced against a variety of cell surface markers involved the cellular interactions of the immune response (Fig. 6) 690 and hence allow the possibility of more specific approaches to immunosuppression. Unfortunately as the antibody is a rat or mouse immunoglobulin most patients develop anti-idiotypic or anti-isotypic antibodies against mouse globulin which makes them ineffective. However, genetic engineering has allowed chimaeric or humanized antibodies to be made, in which the antigen-binding part of the antibodies (variable part of Fab) is grafted on to the human constant parts of the heavy and light chains. Early experience suggests that immunization is much less likely in this situation.

This represents a second generation of antithymocyte globulins, being a mouse monoclonal antibody directed against the CD3 molecule, which is intimately associated with the T-cell receptor, and hence it is a pan-T-cell antibody. It modulates the CD3 molecule with its T-cell receptor and is extremely effective in reversing a steroid resistant rejection in some 70 to 80 per cent of instances (but not necessarily more effective than a good antithymocyte globulin).

Two major disadvantages, one specific for OKT3 and the other of all monoclonal antibody therapies, are firstly the OKT3 syndrome, associated with the first one or two doses, and secondly the development of antibodies to either the idiotype or the isotype of the mouse globulin. The OKT3 syndrome is characterized by a high fever, dyspnoea, nausea, diarrhoea, and even anaphylactic shock, and is mediated in part by TNF. It can be diminished in intensity by prior administration of 1 g of methylprednisolone. Development of antibodies to OKT3 abrogates the effect of the antibody, and occurs in most patients, preventing its further use.

OKT3 is the only monoclonal antibody to be in widespread use but it is the forerunner of monoclonal antibody therapy in transplantation, where other antibodies are likely to be more specific in their actions.

Antibodies directed against the T-helper cell (CD4+) population, the pivotal cells in graft rejection, are potent in experimental models of transplantation where they may produce tolerance to an organ allograft. Clinical trials are in progress, but it will be surprising if such antibodies do not prove to have a role in transplantation.

The IL-2R is expressed on activated T cells and antibodies against the appropriate epitope of the light chain of the IL-2R block the IL-2 driven proliferation of activated T cells. Such antibodies are extremely immunosuppressive in experimental rodent models, but clinical trials of anti-IL-2R antibodies used as prophylaxis during the first 10 days after transplantation have in general shown a very modest beneficial effect at best.

One of a number of antibodies against adhesion molecules which are involved in the interaction between lymphocytes as well as between T-lymphocytes and endothelium, they have proved to be immunosuppressive in experimental models and are currently undergoing clinical trials in renal transplantation.

This is undoubtedly immunosuppressive in experimental models in the rodent and in primates where tolerance to organ allografts can be achieved. In clinical trials a course of total lymphoid irradiation before renal transplantation has proved to be effective in preventing rejection of renal allografts with the use of minimal immunosuppression after transplantation. Indeed there are several well documented cases of long-term survival in patients receiving no immunosuppressive drugs at all! However, the logistics of using total lymphoid irradiation (up to 21 daily treatments) before transplantation combined with dialysis and the advent of more potent immunosuppression with cyclosporin have led the centres pursuing this therapy to abandon it for the time being.

Apart from the specific side-effects of the drugs or antibodies used in transplantation there are three major side-effects of immunosuppression. These are infection (especially viral infections), cancer, and cardiovascular disease. The increased incidence of cancer is particularly evident in those cancers with a putative viral aetiology—non-Hodgkin's lymphoma, squamous cell carcinoma of the skin, Kaposi's sarcoma, and cervical cancer in women—but all cancers show some increase in incidence. Aggressive atheromatous disease occurs in many transplant patients, and although associated with an increased incidence of hypertension and hyperlipidaemia, it may also be related to the long-term use of immunosuppressive drugs, a possible mechanism being unclear at this time.

Calne RY, et al. Cyclosporin A in patients receiving renal allografts from cadaver donors. Lancet 1978; ii: 1323–7.

Eugui EM, Almquist SJ, Muller CD, Allison AC. Lymphocyte-selective anti-proliferative and immunosuppressive effects of mycophenolic acid in vitro: role of deoxyguanosine nucleotide depletion. Scand J Immunol 1991; 33: 161–73.

Jones RM, Murie JA, Allen RD, Ting A, Morris PJ. Triple therapy in cadaver renal transplantation. Br J Surg 1988; 75: 4–8.

Kahan B. Cyclosporin. N Engl J Med 1989; 321: 1725–38.

Kohler H, Milstein C. Continuous culture of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495–7.

Moller G, ed. Antibodies in disease therapy. Immunol Rev 1992; 129: 5–201.

Morris PJ. Cyclosporin A. Transplantation 1981; 32: 349–54.

Morris PJ. Low dose oral prednisolone in renal transplantation. Lancet 1982; i: 525–7.

Morris PJ. Kidney Transplantation: Principles and Practice, 3rd edn. Philadelphia: WB Saunders, 1988.

Morris PJ. Cyclosporin, FK506 and other drugs in organ transplantation. Curr Opin Immunol 1992; 3: 748–51.

Morris PJ. Kidney Transplantation: Principles and Practice, 4th edn. Philadelphia: WB Saunders, 1994.

Morris RE. Rapamycins: antifungal, antitumor, antiproliferative and immunosuppressive macrolides. Transplantation Rev 1992; 6: 39–87.

Ortho Multicenter Transplant Group. A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplants. N Engl J Med 1985; 313: 337–42.

Salomon DR. Cyclosporin nephrotoxicity and long-term renal transplantation. Transplantation Rev 1992; 6: 10–19.

Shapiro R, et al. Kidney transplantation under FK506 immunosuppression. Transplant. Proc 1991; 23: 920–3.

Sollinger HW, Deierhoi MH, Belzer FO, Diethelm AG, Kaufmann ES. A phase I clinical trial and pilot rescue study. Transplantation 1992; 53: 428–32.

Starzl TE, Todo S, Fung J, Demetris AJ, Venkataramman R, Jain A. FK506 for liver, kidney and pancreas transplantation. Lancet 1989; ii: 1000–4.

Thomson AW. FK506: profile of an important new immunosuppressant. Transplantation Rev 1990; 4: 1–13.

Todo S, et al. Liver, kidney and thoracic organ transplantation under FK506. Ann Surg 1990; 212: 295–305.

Waldmann H. Manipulation of T-cell responses with monoclonal antibodies. Ann Rev Immunol 1989; 7: 407–44.

Winter G, Milstein C. Man-made antibodies. Nature 1991; 349: 293–9.

White DJG, ed. Cyclosporin A. Amsterdam: Elsevier, 1982.

Wood KJ, Pearson TC, Darby C, Morris PJ. CD4: a potential target molecule for immunosuppressive therapy and tolerance induction. Transplantation Rev 1991; 5: 150–64.