Immunosuppression

Clinical immunosuppression has always been a problem of balancing prevention or control of rejection with loss of protection against an array of infectious agents and mutant cells. Immunosuppression is intense immediately after trans plantation and then diminishes during the first year as the allograft loses some of its immunogenicity and the recipient's immune system begins to adapt to the graft in various ways. That is, homeostasis appears but almost never to the point that immunosuppression can be discontinued without risking onset of rejection. Some recipients experience acute rejection despite fairly vigorous immunosup-pression. Most acute rejection occurs within the first few months and nearly all can be reversed by intensifying immunosuppression. Few recipients experience chronic rejection in the absence of a preceding acute rejection episode or non-compliance with long-term maintenance immunosuppression. Thus, transplant centers continually search for regimens that prevent that first acute rejection episode, and they regularly warn recipients about the danger of noncompliance.

Immunosuppressive regimens have evolved steadily from broad attack on nearly all rapidly dividing cells in the body to combinations of drugs and biological agents that interfere specifically with those parts of the immune system most responsible for rejection. Review of the time-line for approval of immunosuppressive agents by the United States Food and Drug Administration during the past 30 years provides a framework for understanding the slow but steady conquest of acute rejection and the application of organ transplantation from the kidney to all vital abdominal and thoracic organs (Table #1). Information taken from the manufacturer's package inset for each of the drugs listed in Table #1 is presented as an appendix to the chapters and essays in this volume.

Clinical regimens for immunosuppression changed with the introduction of each new drug or therapeutic antibody. Typical regimens are shown in Table #2 for the years since the early 1960s. The approximate incidences for acute rejection and graft survival after one year are recorded for each regimen. Prior to 1960 prednisone was used to control various inflammatory processes and as a substitute for hydrocortisone. Mercaptopurine was an antimetabolite used to treat my-elocytic leukemia. The observation in 1959 that mercaptopurine prolonged skin graft survival in rabbits led to its use in human transplant recipients in 1962.

Burroughs Wellcome subsequently introduced azathioprine which was a less toxic modification of mercaptopurine; azathioprine, also known as Imuran, was included in almost all immunosuppressive regimens from 1969 until 1996 when mycophenolate mofetil (CellCept) began to displace it. CellCept, like Imuran, is an inhibitor of cell division and nucleotide metabolism, but unlike Imuran, it affects lymphocytes primarily and largely spares most other rapidly dividing cell populations such as bone marrow and gut epithelium. CellCept inhibits the enzyme inosine monophosphate dehydrogenase, which is crucial to de novo synthesis of guanosine monophosphate. Many rapidly dividing populations of cells have a shunt pathway that bypasses the need for de novo synthesis, but activated T and B lymphocytes have an absolute requirement for de novo synthesis of guanosine monophosphate in order to accomplish clonal expansion. The net effect is immunosuppression with relatively few side-effects. Both Imuran and CellCept can depress bone marrow cell lines and intestinal epithelial cells, but CellCept achieves much more immunosuppression before reaching toxic side-effects such as anemia,

Table 2.3. Immunosuppressive Regimen for Kidney Transplant Recipients, Northwestern Memorial Hospital at Northwestern University

Day Alemtuzumab1

infusion over 2 hours during surgery

Tacrolimus2 MMF

Methyl Prednisone

500 mg at start of surgery

750-1000 mg b 750-1000 mg b 750-1000 mg b 750-1000 mg b

250 mg 125 mg d Discontinue d

1 Alemtuzumab omitted for HLA identical donor recipient pair; 2Maintain trough concentration at 5-10 ng/ml.

leukopenia and diarrhea, and the side-effects are easily controlled by reducing the dose of CellCept.

Equine antithymocyte globulin, introduced as ATGAM by Upjohn in 1972, built on the observation reported in 1960 that rabbit antirat lymphocyte serum prolonged skin graft survival in rats. Antilymphocyte and antithymocyte globulins are extremely potent agents that deplete the host's lymphoid tissue. They are usually given intravenously on a daily basis for as long as 14 days, and are effective as induction treatment to delay (prevent) acute cellular rejection and also to reverse it. Because polyclonal cross species antisera against human lymphocytes/thy-mocytes are easy to prepare in rabbits, goats, and horses, many transplant centers provided their own local product. Their side-effects are limited to fever, leukopenia, thrombocytopenia, and anemia, which respond to prednisone or dose reduction. In 1999 SangStat introduced a polyclonal antithymocyte globulin that had been used effectively in Europe for nearly 20 years; it is prepared in rabbits and marketed as Thymoglobulin.

In 1986 OrthoBiotech (Johnson & Johnson) introduced the first therapeutic monoclonal antibody (orthoclone OKT-3). It was directed specifically against the T lymphocyte CD3 receptor for alloantigen presented by transplanted organs. Like the polyclonal antibodies, it delayed or prevented rejection and also reversed most acute cellular rejection episodes. Because it could be infused into a peripheral vein in less than a minute it was much simpler than ATGAM to administer and quickly became the preferred treatment of prednisone-resistant acute cellular and mild to moderate vascular rejection; it was also less expensive. However, polyclonal antilymphocyte sera are more effective than the monoclonal antibody if the transplant biopsy shows aggressive infiltrates of both T and B lymphocyte lines (i.e., plasma cells). Polyclonal antisera include antibodies against surface antigens shared by both T- and B-lymphocytes regardless of which cell type is used to immunize the animal that produces the antiserum. Until the early 1990s, transplant centers in the Untied States were equally divided between those that used and those that did not use antilymphocyte preparations (ATGAM, OKT-3, or a product prepared locally) as part of initial induction of immunosuppression.

Cyclosporine A (Sandimmune) introduced by Novartis (Sandoz) in 1983 dramatically reduced the incidence of acute-rejection episodes, increased kidney graft survival at one year and permitted rapid development of liver, heart and lung transplantation. Cyclosporine allowed less dependence on prednisone. Cyclosporine is a calcineurin inhibitor that preferentially suppresses activation of T lymphocytes by inhibiting production of the lymphokine interleukin-2. In 1994 Novartis introduced a microemulsion of cyclosporine (marketed as Neoral) which was absorbed more readily in the upper intestine. Side-effects of cyclosporine include fine tremor of the hand, hirsutism, gingival hyperplasia, increased appetite and hyperlipidemia (both cholesterol and triglycerides) which can generally be managed by lowering the dose. But, its main problematic side-effect was constriction of preglomerular arterioles which causes hypertension; if arteriolar constriction is sustained, the arteriole undergoes hyaline degeneration, narrowing, and the ischemic glomerulus becomes sclerotic. Although cyclosporine increased the number of transplants surviving beyond one and two years, nephrotoxicity took its toll not only on transplanted kidneys but also on healthy native kidneys of heart, lung and liver recipients. Despite fewer early rejection episodes in recipients of cadaver kidneys treated with cyclosporine, the fraction of transplanted kidneys that survive beyond five years was not higher than for earlier regimens that did not contain cyclosporine. Cyclosporine nephrotoxicity is such a serious side-effect that 10% of heart allograft recipients lost their native kidneys and were on maintenance dialysis within ten years. Many transplant centers kept maintenance doses and 12-hour blood trough levels lower than the manufacturer's recommendation; they accepted a few more acute rejection episodes rather than risk loss of kidney grafts to drug-induced glomerular ischemia and sclerosis.

Prograf/tacrolimus/FK 506 was introduced by Fujisawa in 1995. Like cyclosporine it is a calcineurin inhibitor, but its side-effects are different and it appears to induce less nephrotoxicity for equivalent immunosuppression. Like cyclosporine it can cause tremor but does not cause hyperlipidemia, hirsutism, gingival hyperplasia or increased appetite. Prograf can induce diabetes but usually not with doses of 3 mg twice daily or less. Prograf increases the bioavailability (AUC-Area Under the Curve) of CellCept when both drugs are taken concurrently.

Monoclonal antibodies against the IL-2a receptor (CD25) were introduced in 1997 by Roche (Zenapax/Daclizumab) and by Novartis in 1998 (Simulect/ Basiliximab). Both are intravenous preparations with no side-effects and both reduced acute rejection episodes significantly during the first six post-transplant months. The Roche preparation is infused just before transplantation and then every other week for four more doses. The Novartis preparation is infused only twice, once just before transplantation and again four days later. Because a course of Simulect requires only two infusions and costs less, it is preferred by many transplant centers. Most transplant centers that used antilymphocyte antibodies (either polyclonal or monoclonal) as part of early post-transplant immunosuppression soon switched to one of the anti IL-2 alpha monoclonals.

Rapamune (rapamycin/sirolimus) is the most recent antirejection drug and was released by the U.S. Food and Drug Administration in 1999. Like cyclosporine and tacrolimus it is a fungal product, but it does not inhibit calcineurin and is not nephrotoxic. It inhibits lymphocyte effects driven by certain cytokines, particularly IL-2. Its major side-effects is thrombocytopenia and hyperlipidemia, which can be controlled by reducing the dose or adding lipid-lowering agents. Rapamycin may also afford protection against chronic rejection by blocking proliferation of vascular endothelial and smooth muscle cells.

From the introduction of mercaptopurine/azathioprine in 1962 until cyclosporine was approved by the FDA 21 years later in 1983, organ transplantation was limited to the kidney. Bone marrow suppression markedly limited the amount of immunosuppression that could be achieved with azathioprine. Relatively high doses of prednisone provided most of the immunosuppressive effect. Even when a fourteen-day course of antilymphocyte serum became the third component of the regimen, steroids still carried most of the load. Bacterial sepsis and slow wound healing were such severe problems that liver, lung and heart transplantations were limited to a few hardy pioneering centers. The main contribution of cyclosporine after 1983 was that it reduced rejection, increased early graft survival, and reduced dependence on steroids; bacterial sepsis decreased; wounds healed more quickly; and successful transplantation of liver, heart and lung was quickly achieved in many centers.

But for kidney transplantation, cyclosporine was a mixed blessing. Despite fewer rejection episodes and increased graft survival beyond a year, the number of grafts surviving beyond the fifth year was scarcely more than before cyclosporine. Many kidney grafts eventually succumbed to ischemia and fibrosis caused by constriction of preglomerular arterioles. Cyclosporine had raised early transplant outcomes to a new plateau where they remained another 12-13 years until the appearance of two new drugs (Prograf and CellCept) and two monoclonal antibodies directed against the IL-2 receptor between 1995 and 1998. The net effect of these four agents has been less nephrotoxicity and much less dependency on pred-nisone. Many believe that Prograf is less nephrotoxic than cyclosporine, and CellCept is not nephrotoxic at all. Optimism has developed in just the last two or three years that some of the most bothersome side-effects of immunosuppression may be on the way out: nephrotoxicity, hyperlipidemia, accelerated vascular disease, osteonecrosis, osteoporosis and other steroid-related problems, and post-transplant diabetes. By 1998 many centers were discontinuing prednisone 6 and 12 months after transplantation. Most kidney recipients tolerated weaning from oral prednisone, but rejection was triggered in 15-20 percent of the weaned recipients. In mid-1998 Northwestern Memorial Hospital's transplant center removed oral prednisone completely from the immunosuppressive regimen used for kidney recipients. The regimen included basiliximab (day 0,3), methylpred-nisolone for 3 days, maintenance tacrolimus and mycophenolate mofetil, but no prednisone. Incidence of first year rejection episodes remained 10-15 percent and graft survival exceeded 95 percent.

Rapamycin/sirolimus (Wyeth-Ayerst Rapamune) was approved in 1999. Sirolimus, cyclosporine microemulsion, azathioprine and steroids limited acute renal allograft rejection to 10 percent with 90 percent first year graft survival. Regimens that combine anti CD-25 induction, early methylprednisolone and tacrolimus with either sirolimus or mycophenolate mofetil, with or without maintenance prednisone limit first year rejection episodes to 10 per cent and permit 95 percent graft survival.

The two most recent agents with immunosuppressive properties, which entered clinical practice in 2001, are monoclonal antibodies against lymphocyte cell surface determinants CD20 and CD52. Rituximab targets CD20, which is found on the surface of both normal B lymphocytes and 90 percent of B cell non-Hodgkin's lymphomas. It produces profound depletion of B lymphocytes in peripheral blood and elsewhere. The primary indication for its use is to treat B cell lymphomas, but it has also been used to reverse antibody mediated rejection in a heart transplant and to decrease production of HLA antibodies in a pre-sensitized potential kidney recipient prior to successful transplantation (14,15). The second antilymphocytic monoclonal is alemtuzumab (Campath), which binds to CD52 found on all B and T lymphocytes, a majority of monocytes, macrophages and NK cells, and a subpopulation of granulocytes. It induces profound depletion of its targeted cells through antibody dependent lysis and is indicated in treatment of non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Alemtuzumab has also been used in the early "induction" phase of several immunosuppressive regimens for kidney transplantation. A single intraoperative infusion of 30 mg induces complete absence of lymphocyte cells from the peripheral blood for 7-10 days with gradual recovery after six months. Alemtuzumab is a murine (rat) anti CD52 developed and humanized at the University of Cambridge in Herman Waldmann's Laboratory (16). Because one or two infusions induce profound lymphopenia for several weeks, Calne treated 31 cadaveric kidney recipients with it in an attempt to induce "prope" (almost) tolerance at Cambridge. Each recipient received a 20 mg intravenous infusion of alemtuzumab on the operative and first postoperative days. Maintenance immunosuppression consisted of a single drug, cyclosporine, in moderate doses. Twenty-nine grafts had good postoperative function beyond the first year. Six rejection episodes during the first year were reversed and one recipient died (17). Knechtle and Kirk report similar experience with alemtuzumab and rapamycin as the only maintenance drug in kidney recipients (18,19). The transplant center at Northwestern Memorial Hospital has treated more than 200 kidney recipients with a prednisone free protocol that begins with a single 30 mg intraoperative infusion of alemtuzumab followed by oral maintenance with tacrolimus 1-2 mg twice daily and mycophenolate mofetil 750-1000 twice daily. Corticosteroids were restricted to the operating room (500 mg) and the first two postoperative days (250 mg, 125 mg). Details of the regimen are shown in table 3. First year patient survival, graft survival and incidence of acute rejection are 99%, 97% and 8%. Mean serum creatinine at one year is 1.3 mg/dl. Incidence of infection of all kinds is less than 5 percent (20,21). The alemtuzumab, tacrolimus based prednisone free protocol has been extended to simultaneous kidney and pancreas recipients at Northwestern with two modifications: corticosteroids are administered for 4-5 days and sirolimus is used in place of mycophenolate mofetil; outcomes are similar to transplantation of the kidney alone (22). As an anti lymphocyte induction immunosuppressive agent, alemtuzumab is at least equivalent in its lymphopenic effect to polyclonal antilymphocyte globulin or muramonab-CD-3 but less expensive, because a single infusion is sufficient. It facilitates prednisone free maintenance but does not eliminate the need for other maintenance drugs in relatively low doses.

Vincenti recently reviewed several new classes of immunosuppressive drugs that are under investigation but not yet approved by the FDA (23). The major targets of new agents are cell-surface molecules important in immune cell interactions (especially the costimulatory pathway), signaling pathways that activate T cells, T cell proliferation and trafficking, and recruitment of immune cells responsible for rejection. The most promising include a humanized OKT-3, humanized anti-CD11a (anti-LFAl), humanized anti B7.1/B7.2, a second generation CTLA4Ig, LEA29y, an anti CD45RB, FK778, (a leflunomide analog), FTY720 and several antagonists to chemokine receptors (CCR1, CXCR3 and CCR5). The FDA is expected to approve modifications or variations on several classes of drugs that are already approved but is unlikely to approve any new class of immunosuppressive drug during the next few years.

To summarize evolution of transplant immunosuppression, seven new classes of drugs have been introduced since 1970, and all of them act primarily or exclusively to deplete lymphocytes or inhibit their function.

Class

Polyclonal antibody

Function

1972,1

Monoclonal antibody

Lymphocyte depletion

Alemtuzumab anti CD52 Rituximab anti CD20

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