Does this patient have too much or too little immunosuppression?
Choosing the proper immunosuppression for a patient involves balancing the risk of immunologic graft injury against the risks of infection and malignancy. This requires an understanding of basic immunology, the mechanism of action and side effects of immunosuppressive medications, and a thorough immunologic risk assessment. Immunological risk is estimated from the patient’s medical history, immunogenetic testing, characteristics of the donor organ (living versus deceased donor), and time elapsed since transplant. Various combinations of immunosuppressants are chosen to target lower and higher risk patients.
Immune system: Concepts and Components
The human immune system is composed of the innate and adaptive components. The innate immune system is composed of macrophages, neutrophils and natural killer (NK) cells as well as nonpolymorphic proteins, such as complement and cytokines, which respond to generic antigens. The cardinal features of the adaptive immune response are specificity to antigenic diversity, memory, and tolerance to self. It is composed of cellular and humoral components (T cells, B cells, antibodies). Though both responses (innate and adaptive) are important in transplantation, the majority of treatment modalities in transplantation are directed against the adaptive immune response.
T cells: T cells are derived in the thymus during embryogenesis and early childhood. CD8 T cells bind to class I MHC molecules and generally mediate cytotoxicity. CD4 T cells bind to class II MHC molecules, secrete cytokines to amplify inflammation, and provide help to induce cytotoxic T cells and antibody-producing B cells.
B cells: B-cell receptors, generally a form of IgM antibody, bind to foreign proteins. Antigen is taken up by receptor-mediated endocytosis, digested, and presented to T cells in the context of class II MHC. CD4 T cells bind B cells, and via transmission of costimulatory signals (CD40 is an important example) induce B-cell differentiation into antibody-secreting plasma cells or into long-living memory B cells.
The immune response induced by a transplanted organ
The host immune system is able to distinguish self from non-self by recognizing foreign HLA molecules (allorecognition) then mounting a highly specific immune response to donor cells in the transplanted organ.
HLA antigens: Grafts transplanted from one member of a species to a different non-identical member of that same species (e.g., one human to another) are termed allografts. In this situation, the two individuals differ at MHC (major histocompatibility complex) loci, also known as human leukocyte antigen [HLA] loci), and/or differ at minor histocompatibility antigens. HLA antigens can be divided into class I and class II. Class I antigens (HLA A, B and C) are present on all nucleated cells, while class II antigens (HLA DR, DQ and DP) are found on antigen presenting cells and can be upregulated on vascular endothelium after ischemia reperfusion injury.
Each person inherits two sets of HLA antigens, one from each parent, which are co-dominantly expressed. The HLA antigens are both polygenic and polymorphic, making it extremely unlikely that two unrelated individuals have the same HLA antigen combination. Even when limited to HLA A, B and DR there are over 100 distinct antigens. While this diversity protects us from pathogens, it also provides a large number of immune targets after organ transplantation. HLA frequencies depend on ethnicity. For example, A2 is found in approximately 50% of all ethnic populations, while HLA-B54 is found almost exclusively in patients of Japanese heritage.
Cellular alloimmunity: Cell-mediated alloimmunity is initiated by antigen-specific T cells that, in concert with other cellular components, result in cytolytic and cytokine-induced damage of a transplanted organ. Initial antigen recognition predominantly occurs in secondary lymphoid organs, where recipient T cells interact with antigens derived from the donor. The primed T cells then migrate back to the graft where they re-encounter antigens and mediate their effector functions.
T cells recognize MHC:peptide complexes through heterodimeric T-cell receptors (TCRs) expressed on their cell surface. In the normal host, T cells are “trained” to recognize foreign peptides expressed in the context of self-MHC molecules and are tolerant to self antigens. T cells also recognize foreign MHC:peptide complexes. This is now known to occur through two distinct but not necessarily mutually exclusive pathways – the direct and the indirect allorecognition pathways. Direct allorecognition of intact surface MHC:peptide molecules expressed on donor cells is a process unique to transplantation.
Direct pathway: Donor dendritic “passenger” cells migrate out of the transplanted graft and into the host’s lymphoid tissue. In the lymphoid tissue T cells recognize the foreign MHC:peptide complex and are activated.
Indirect pathway: Host dendritic cells migrate to the transplanted graft, acquire foreign peptides, and present them to CD4 T cells in host lymphoid tissue. This pathway is thought to be important in late transplant chronic rejection.
T-cell activation: Three signals are required to activate a T cell.
T cell receptor ligation: T cell receptor CD3 complex along with T cell surface molecules CD4 or CD8 interact with its counterpart MHC molecule and antigenic peptide.
Co-stimulation: Positive co-stimulation is required for T cell activation. There are numerous positive and negative co-stimulatory pathways. The best known positive co-stimulatory pathway is the interaction between CD28 on the T cell and B7 found on antigen presenting cells. The combination of T cell receptor ligation and co-stimulation result in T cell activation and production of various cytokines and growth factors including IL-2.
IL-2/ CD25: Activation of the T cell receptor and positive co-stimulation results in the production of the potent T cell growth factor IL-2 and upregulation of the activated IL-2 receptor, CD25. IL-2 works in an autocrine and paracrine manner on the CD25 receptor to induce lymphocyte proliferation.
Rejection – the effector phase of the immune response: The effector phase of the immune response exhibits memory and escalating response to foreign antigen. It requires secondary lymphoid organs (where antigen presentation takes place) as well as both the innate and adaptive immune system.
Innate immune response: Inflammation from ischemia reperfusion injury in the allograft leads to the migration of NK cells, macrophages and neutrophils to the allograft resulting in injury.
Adaptive immune response: CD4 T cells are primed against donor antigens and release cytokines, which direct an immune response in the allograft. This results in CD8 T cells, macrophages and NK cells migrating to the graft and causing cell damage and apoptosis. Cytotoxic CD8 T cells kill their targets in an antigen specific method through direct cell-cell contact in one of two mechanisms. The first is by secreting proteins granzyme and perforin, which result in cell lysis. The second mechanism is by activation of the Fas/ Fas ligand pathway. Fas ligand in the T cell binds to Fas on the graft cell inducing apoptosis. At the same time CD4 T cells activate B cells via the CD40/CD40 ligand pathway, which help B cells transform into antibody producing plasma cells that cause complement mediated damage to the graft cells.
The type and grade of rejection can be defined by the Banff criteria. The Banff criteria are an attempt to standardize rejection criteria in order to facilitate therapeutic studies across various centers. Rejections may be humoral (currently more often called antibody mediated rejection), cellular or a combination of both.
Antibody mediated rejection: Requires the presence of circulating donor specific antibodies (DSA), evidence interaction of DSA with endothelium, usually manifested as C4d (a complement split product) deposits in the peritubular capillaries on biopsy tissue, and histological evidence of tissue injury (for more detailed description, see chapter “Kidney Transplantation- Diagnosis and Management of Early Graft Dysfunction – Intrinsic Causes and Treatments”).
Cellular rejection: Appears as mononuclear infiltration of the tubular basement membrane, i.e. tubulitis, or the vessels, i.e. intimal arteritis (for more detailed description, see chapter “Kidney Transplantation- Diagnosis and Management of Early Graft Dysfunction – Intrinsic Causes and Treatments”).
Induction agents: Used to decrease the risk of rejection early post transplant when the risk is the highest. These agents can be classified as depleting or non-depleting agents.
Polyclonal agents/antithymocyte globulin (ATG): The most commonly used anti-thymocyte globulin is procured from rabbits (Thymoglobulin®). It is used as an induction agent or to treat severe rejection and results in a profound lymphopenia. ATG decreases the risk of acute cellular rejection, however it increases the risk for infections and malignancy. Side effects include infusion reactions (fever, rigors, dyspnea), leukopenia, thrombocytopenia and rarely serum sickness. Methylprednisone, acetaminophen and diphenhydramine should precede the ATG administration to reduce infusion reactions. It is usually dosed at 1.5 mg/kg/day as an IV infusion over 4-6 hours. The dose should be reduced by 50% if the white count decreases to less than 3,000 cells/mm2 or the platelet count decreases to less than 75,000 cells/mm2. ATG should be held if the white count decreases to less than 2,000 cells/mm2 or the platelet count decreases to less than 50,000 cells/mm2, and restarted when the count recovers (usually the following day). The total dose of ATG is usually 1.5 mg/Kg/day for 3-6 days for induction and 1.5 mg/Kg/day for 5-7 days to treat rejection.
Alemtuzumab (Campath 1H®) – Alemtuzumab is an anti-CD52 monoclonal antibody. CD52 is found on T-cells, B cells, monocytes, macrophages and eosinophils. Mechanism of action includes antibody-dependent cellular toxicity (ADCC), complement dependent cytotoxicity (CDC) and apoptosis, all of which result in profound lymphopenia. Its use is associated with an increased frequency of infections. However, recent publication from the INTACT study group revealed similar incidence of infections compared to rabbit-ATG when used in a steroid withdrawal protocol. Infusion reactions include fever, rigors, hypotension and dyspnea, which can be minimized with pre-medication. Alemtuzumab can be given IV or SQ over 2 hours for a dose of 30 mg once or twice.
Rituximab – Rituximab is an anti-CD20 monoclonal antibody approved for use in chronic lymphocytic leukemia, and other diseases. It results in profound depletion of B cells. It has been associated with progressive multifocal leukoencephalopathy (PML) in patients with Lupus and increases the risk for severe infections in transplant recipients when given with ATG. It may be used in patients with pre-transplant donor specific antibodies, in desensitization protocols, to treat post transplant lymphoproliferative disease (PTLD) or as a treatment for antibody mediated rejection. Potential infusion reactions include hypotension, fever, tachycardia and arthralgias. Note that patients with hepatitis B can develop fulminant hepatitis after infusion of Rituximab, and all patients should be screened for hepatitis B before the administration of this medication.
Anti-CD25 antibodies: Basiliximab is a humanized murine antibody with low immunogenicity which causes reversible blockade of CD25. As an induction agent it reduces the risk of rejection, especially in low to moderate risk individuals. It is almost devoid of side effects and is given as a dose of 20 mg on days 0 and 4 post transplantation.
Intravenous Immunoglobulin (IVIg): Pooled human blood product used to “neutralize” pre-existing anti-donor antibodies. IVIg has many immunomodulatory activities including decreasing antibody production, anti-idiotypic antibodies, and neutralizing complement. Side effects include thrombosis and headache. Sucrose-free formulations should preferably be used, as IVIg formulations containing sucrose can cause acute osmotic kidney injury. IVIg is sometimes used as an induction agent for patients with a positive crossmatch and/or those with preformed donor specific antibodies.
Maintenance Immunosuppressive agents
Calcineurin inhibitors (CNIs), cyclosporine and tacrolimus: Cyclosporine binds cyclophilin and tacrolimus binds FK binding protein. The resulting complex binds with calcineurin, which does not permit dephosphorylation of NFAT, thus preventing it from entering the nucleus. This in turn impairs expression of several cytokine genes that promote T-cell activation and proliferation especially IL-2, IL-4, IFN-gamma, TNF-alpha. Therapeutic levels of cyclosporine and tacrolimus reduce calcineurin activity by about 50%. CNIs are not myelosuppressive. Cyclosporine and tacrolimus are both available as generic formulation.
Tacrolimus is available as a once per day medication in two different formulations, Astagraf® and Envarsus®, which are not equivalent. If converting from immediate release to extended release tacrolimus formulations, the extended release daily dose should initially be 70-80% (Envarsus®) and 100% (Astagraf®) of the daily dose of immediate release tacrolimus. Subsequently the dose should be adjusted based on trough levels. The extended release formulations may be associated with less neurotoxicity. They are not commonly used in the US. More evidence of equivalent efficacy and less toxicity is needed before use of these formulations becomes widespread in the US.
Are all formulations of CNI the same?
The different formulations of cyclosporine have different bioavailability and therefore should not be interchanged. Lipid emulsified formulations (Neoral®, Gengraf®) have been developed improving adsorption and leading to more predictable bioavailability, and are generally used.
How are cyclosporine and tacrolimus different?
Tacrolimus is a more potent immunosuppressive and has a slightly different adverse effect profile when compared to cyclosporine (see Table 1).
Adverse effects of tacrolimus versus cyclosporine
Adverse Effect Comments Nephrotoxicity Seen with rental trasnplantation and non-renal solid organ transplant recipients CardiovascularHypertensionHypercholesterolemia Tacrolimus-treated patients may require fewer antihypertensive agents and less lipid lowering agents. Glucose intolerance Tacrolimus is more pancreas islet toxic and results in more post transplant diabetes. NeurotoxicityTremorHeadacheInsomniaParasthesia Seen more often with tacrolimus and generally improves with dose reduction. PRES (posterior reversible encephalopathy sydrome) Clinical/radiographic syndrome of headache, confusion, visual changes, and seizures, with radiographic evidence of posterior cerebral white matter swelling. Reported more often with cyclosporin. CosmeticGingival hypertrophyHirsutismAlopecia Gingival hypertrophy and hirsutism are associated with cyclosporine.Steroid use may exagerate hirsutism and calcium channel blockers can exacerbate gingival hypertrophy.Alopecia may occur with tacrolimus Malignancy Incidence appears to be a function of overall amount and duration of immunosuppression rather than from any specific agent
Nephrotoxicity: Nephrotoxicity can be divided into acute hemodynamic decrease in glomerular filtration rate (GFR) from vasoconstriction on the efferent and afferent glomerular arterioles and chronic renal insufficiency seen with long term use of CNI’s. The acute form of CNI toxicity is related to the CNI level and reversible with dose reduction. Chronic CNI toxicity is manifested by “striped” fibrosis and eccentric hylanosis of arterioles. This is best demonstrated in non-renal solid organ recipients maintained on CNI where the incidence of CKD IV ranges between 7 to 21% at 5 years post transplantation. The exact mechanism of chronic CNI toxicity is incompletely understood but may be related to chronic ischemic nephropathy. Another rare form of CNI nephrotoxicity is thrombotic microangiopathy.
What drug/food interactions do I need to be aware of when using the calcineurin inhibitors?
Many drugs and foods can either increase or decrease the metabolism of calcineurin inhibitors. In addition, diarrhea has been shown to increase the trough values of tacrolimus which is thought to be mediated by loss of P- glycoprotein, an enzyme which metabolizes tacrolimus, on the gut mucosa.
Common interactions that increase the levels of CNI’s (see Table 2) and decrease the level of CNI’s (Table 3).
Interactions that increase CNI level
Non-Dihydropyridine calcium channel blockers Diltiazem Verapamil Antibiotics Erythromycin Clarithromycin Antifungal agents Voriconazole (requires 1/3 dose reduction of CNI) Itraconazole Clotrimazole Cardiovascular drugs Amiodarone Ranolazine Antiviral agents Protease inhibitors (extreme effect sometimes requiring weekly dosing of tacrolimus) Foods Grapefruit and grapefruit juice
Interactions that decrease CNI level
Antibiotics Rifampin Rifabutin (less so than rifampin) Terbinafine IV trimethoprim IV sulfadimidine Imipenim Anticonvulsants Barbituates Phenytoin Carbamazepine Ticlopidine Octreotide Nefazodone Herbal medications St. John’s Wart
Other drug interactions
Combination of CNIs and HMG CoA reductase inhibitors (statins) increase the risk for rhabdomyolysis. Generally only 50% of maximal recommended dose of statin should be used. Simvastatin is contraindicated in patients on cyclosporine.
Sirolomus increases total drug exposure (AUC) of CNI’s without raising trough levels.
Dosing: Both the therapeutic efficacy and toxicity of tacrolimus and cyclosporine vary based on drug exposure. Twelve-hour trough levels correlate with drug exposure and are important monitoring parameters. Two-hour cyclosporine peak levels may correlate better with drug toxicity and exposure (area under the curve (AUC)). Genetic factors play a role in CNI metabolism and there is considerable inter-patient variability of trough levels obtained with a given dose. Children and African Americans are often rapid metabolizers of tacrolimus and may require higher dosing or adjuvant medications to boost the trough level.
Tacrolimus: The initial oral dose of tacrolimus varies from 0.1 to 0.2 mg/kg/day divided in two doses. Trough goal levels depend greatly based on the time from the transplant, whether the patient is on triple immunosuppressive medications or only two medications (steroid-free protocols), history of rejection or infections, HLA match (two haplotype match requires less immunosuppression), and other factors. In general, the first two months tacrolimus trough levels may be around 10-12, and subsequently goal levels can be slowly reduced to 6-8 until 6 months after transplant, and then 5-7 thereafter.
Cyclosporine: The initial oral dose is 6 to 10 mg/kg/day divided into two doses, then maintained based on trough levels.
Trough levels: cyclosporine goal trough levels may vary greatly, as discussed previously for tacrolimus. In general, goal levels of 250 – 300 ng/ml may be maintained for the first 2 months, and subsequently they should be slowly decreased to 150-200 until 6 months after transplant, and then 100 – 150 thereafter.
If the patient is unable to swallow tacrolimus pills, tacrolimus can be given in liquid formulation (which needs to be shaken well before dosing, and may be associated with variable levels), or sublingual. Sublingual administration is associated with greater blood levels, and the dose should be reduced by 50%, and still be given twice per day. Cyclosporine can be administered IV. We try to avoid the IV infusion, due to possible increased toxicity, but if the IV route is necessary, the dose should be given as 1/3rd the total daily oral dose infused over 24 hours.
What if I am unable to obtain the target trough despite very high doses of tacrolimus?
These patients should take tacrolimus on an empty stomach to improve absorption. In addition patients may require additional medications used to boost the tacrolimus level (ex. diltiazem or ketoconazole) or require TID dosing. It is important to realize that TID dosing results in 8 hour troughs which may not correlate as well as 12 hour troughs with drug toxicity.
Mammalian Target of Rapamycin (mTOR) inhibitors
Sirolimus (rapamycin) – mTOR inhibitors bind FK binding protein which then binds to the target of rapamycin (TOR). Inhibiting mTOR prevents signal transduction through several growth factor receptors such as IL-2, thereby inhibiting cell-cycle progression and immune activation. As a class mTOR inhibitors tend to be less immunosuppressive than the CNIs but stronger than other anti-proliferative agents. In addition, many cell lines depend upon the mTOR pathway for proliferation leading to numerous side effects which often limit the use of this drug but have a benefit in certain malignancies such as skin cancer, Kaposi’s sarcoma and renal cell carcinoma.
Side effects related to inhibiting mTOR: apthous ulcers, GI toxicity, mucositis, leukopenia, thrombocytopenia, poor wound healing, lymphoceles. These effects are typically dose dependent.
Other side effects: severe hyperlipidemia (esp. hypertriglyceridemia), non-infectious idiopathic pneumonitis, anemia, thrombocytopenia, myelosuppression.
Nephrotoxicity: Sirolimus has been found to induce or worsen pre-existing proteinuria possibly be affecting podocytes in the glomerulus. It may also lead to hypokalemia and magnesium wasting. In addition, combining sirolimus with CNIs increases total drug exposure, potentiating CNI toxicity. Sirolimus has very rarely been shown to cause thrombotic microangiopathy.
Dosing: Sirolimus has a half-life of 62 hours which allows once daily dosing. It may be started at a loading dose of 2-6 mg, although loading doses of sirolimus may be associated with more side effects, then continued at 1-5 mg/day. Similar to the CNIs, sirolimus trough levels correlate to therapeutic activity and toxicity. Target trough levels vary depending on other agents it is being used with and will be discussed in the immunosuppressive protocols section.
Combined with CNIs: Sirolimus should be administered 4 hours after the morning CNI dose to reduce nephrotoxicity. Interaction is greater with Cyclosporine, compared to tacrolimus. Target troughs may be 4-7 ng/ml with CsA levels of 50 to 100 ng/ml and tacrolimus levels of 3-6 ng/ml.
Combined with MMF: The optimal trough ranges from 5 to 10 ng/ml depending on when it is used. When used in CNI conversion strategies target trough may range from 5 to 8 ng/ml. Optimal timing and dosing will be discussed in the immunosuppressive protocols section.
Everolimus: everolimus has the same mechanism of action as sirolimus, but it has a shorter half life of 23 hrs which requires twice a day dosing. It is approved for treating renal cell carcinoma, and for kidney transplant recipients in the United States when combined with basiliximab induction, cyclosporine and corticosteroids. An ongoing study of maintenance therapy with tacrolimus, everolimus and Prednisone (TRANSFORM STUDY) targets everolimus trough levels of 3-8 ng/mL, in combination with reduced tacrolimus levels. Everolimus side effects are similar to sirolimus.
Dosing: everolimus dosing may start at 1 mg BID, then the dose needs to be adjusted by trough. Target troughs are similar to sirolimus (see immunosuppressive protocols).
Glucocorticoids: Their mechanism of action involves binding to DNA regulatory sequences called glucocorticoid-responsive elements. These elements include sequences in the promoter regions of several cytokine genes and effects are exerted on many cell types. At high doses (pulse steroids) they are directly lympholytic and at low maintenance doses their immunosuppressive mechanism is probably nonspecific and anti-inflammatory.
Side effects: Glucocorticoids have been long known to have many side effects. Immediate effects include psychosis, myopathy and hyperglycemia while side effects related to cumulative and long term dosing include diabetes, weight gain, hyperlipidemia, hypertension, osteopenia, and avascular necrosis among others.
Dosing: Glucocorticoids are given at doses of 250 – 1000 mgs for the first few days post transplant then usually tapered to 5mgs per day by 3 months. Rapid steroid withdrawal dosing involves high doses that are tapered off in 3 to 7 days.
Azathioprine: This pro-drug of 6-mercaptopurine is one of the earliest immunosuppressive agents used in transplantation. It has been largely replaced by mycophenolate mofetil (see below). It works by inhibiting the formation of phosphoribosyl pyrophosphate which is needed in purine synthesis, thereby preventing cell proliferation.
Side effects: The most serious side effect is myelosuppression leading to leukopenia, anemia and thrombocytopenia. It also may lead to cholestatic hepatotoxicity. As azathioprine is degraded by xanthine oxidase it should never be used with an inhibitor of xanthine oxidase – allopuriol or febuxostat (Uloric).
Dosing: The typical dose is 1 – 2 mg/kg per day when combined with a CNI. The dose should be reduced if WBC count is less than 3,000.
Mycophenolate Mofetil (MMF) and enteric-coated Mycophenolate sodium (Myfortic®): MMF is a prodrug of mycophenolic acid (MPA) which inhibits IMPDH – an enzyme needed for de novo purine synthesis. Purine synthesis can occur by two pathways, the de novo and salvage pathway in most cells, except in lymphocytes. It results in impairment of both T and B cell proliferation, inhibits generation of cytotoxic T cells, decreases adhesion molecule function and decreases antibody production. Myfortic® is enteric coated MPA which results in delayed release of MPA at the level of the small bowel in an attempt to decrease gastrointestinal symptoms. It should be avoided in patients with known gastroparesis.
Side effects: The most common side effect of MPA is GI toxicity manifested by nausea, bloating and or diarrhea. Rarely it may lead to mouth and colon ulceration necessitating cessation of the medication. It is also myelosuppressive leading to leukopenia, anemia and thrombocytopenia. The side effect profile of Myfortic® tends to be similar to MMF.
Dosing: 1000 mg of MMF is equivalent to 720 mg of Myfortic®. MPA exposure is reduced with CsA but not with tacrolimus. The dose should be reduced if WBC count drops.
Belatacept: Belatacept is a selective co-stimulation blocker (given IV), which binds surface costimulatory ligands (CD80 and CD86) of antigen-presenting cells. It inhibits T-cell activation, promoting anergy and apoptosis. Studies using it de novo as well as converting from CNI based to Belatacept based immunosuppression have revealed a higher incidence of rejection compared with CNIs when combined with MMF and prednisone; however better GFR. The major concern with Belatacept is an increased risk for post transplant lymphoproliferative disease and the lack of long term data. It is contraindicated in EBV seronegative patients or patients with unknown EBV serostatus as they have a higher risk of PTLD.
When given in combination with Basiliximab induction and maintenance with MMF and steroids, Belatacept is administered IV over 30 minutes and the recommended dosing is 10 mg/kg on the day of transplantation then on day 5, then at the end of weeks 2, 4, 8, and 12. After week 16 it is recommended that the maintenance dose be 5 mg/kg every 4 weeks.
When converting from CNI to Belatacept the studied dose is 5mg/kg IV on day 1, 15, 29, 43, 57 then every 28 days thereafter.
Immunologic risk assessment
Recipient Age: Younger recipients are in general at higher risk for immunologic injury than the elderly.
Recipient Ethnicity: African Americans have been reported to a higher incidence of rejection episodes and overall graft loss despite correction for socio-economic status.
Recipient with HIV infection: Patients with HIV tend to be at a higher risk for rejections and graft loss compared to age matched controls.
Degree of HLA matching: The degree of HLA matching impacts the expected risk of rejection and graft survival in kidney transplantation. Historically the most important HLA antigens have been HLA A, B, and DR. A perfect match using only these antigens would be referred to a 0 antigen mismatch. This is in contrast to a 2 haplo matched transplant where all HLA antigens, including Cw, DQ and DP, are the same (sibling). Greater HLA mismatch increases the risk for graft loss in deceased donor kidney transplantation. Zero antigen mismatch kidney recipients have a graft half life of 16 years whereas 5 to 6 antigen mismatch recipients have a graft half life of about 10 years. In living donor transplantation a 2 haplo matched kidney transplant carries the lowest risk of rejection and best graft survival (half life 26 years) whereas smaller degrees of HLA mismatch do not seem to matter (half life 15-18 years).
Panel reactive antibody (PRA): PRA is an approximation of preformed HLA antibodies that the recipient has against a possible donor pool. Patients with a higher PRA are sensitized and carry a higher risk for chronic rejection and graft loss even in the absence of donor specific antibodies (DSA). This was best seen in the Collaborative study where outcome of 4048 HLA identical transplants were stratified by recipient PRA. Patients with a zero PRA had a 10 year graft survival of 72%, 1-50% PRA of 63% and patients with a PRA of greater than 50% had a graft survival of 56%.
Pre-formed DSA: The different methods to determine DSA will be described below (“What tests to perform” section). In general, the more sensitized the recipient is against donor HLA molecules the higher the risk for rejection and subsequent graft failure.
Deceased versus living donor transplant: Deceased donor organ source carries both a higher risk for rejection and graft loss compared to living donor. This is thought to occur due to ischemia reperfusion injury and detrimental effects of brain death. Deceased donor transplants are more often complicated by delayed graft function (DGF). Patients with DGF are at a higher risk for rejection and graft loss than those with immediate function.
Time on dialysis: Pre-emptive living donor transplants carry the lowest rate of rejection and the best organ survival. Time on dialysis has been shown to have a dose related effect on allograft outcome in deceased donor organ transplantation.
Time elapsed since transplant: The highest risk for rejection is within the first year post transplant. It is believed that there is some degree of graft “accommodation” that occurs as time passes after transplantation therefore reducing the requirement for aggressive immunosuppression.
Most protocols consist of induction therapy and maintenance immunosuppression with a CNI (tacrolimus or cyclosporine), an anti-proliferative agent (MMF or azathioprine) with or without maintenance prednisone. Protocols using sirolimus include combining it with an anti-proliferative agent and prednisone or combining it with a CNI and prednisone. Sirolimus is rarely used as initial maintenance therapy. Finally, Belatacept combined with MMF and prednisone is another option.
What is the most commonly prescribed immunosuppression?
Based on data collected and analyzed up to 2008 by the Scientific Registry for Transplant Recipients (SRTR), 82% of patients received induction therapy. ATG was used most commonly (44%) followed by anti-CD25 (28%) then alemtuzumab in 10% of cases.
The ELITE-Symphony study demonstrated maintenance with low-dose tacrolimus (target trough 3 to 7 ng/ml) to have lower rejection rates and best graft survival as compared to low dose cyclosporine (target trough 50 to 100 ng/ml), standard dose cyclosporine (target trough 150-300 ng/ml first 3 months then 100-200 ng/ml thereafter) and sirolimus (target trough 4 to 8 ng/ml) when accompanied by MMF and prednisone. The results of this study have popularized low dose tacrolimus with MMF and prednisone.
In 2008 the most commonly prescribed immunosuppressive regime at discharge after transplantation was the combination of tacrolimus, MMF/MPA, and steroids (54% of patients) followed by Tacrolimus, MMF/MPA (28%), and cyclosporine, MMF/MPA (4.5%).
Sixty-six percent of patients were discharged on steroids, 88% of patients were discharged on tacrolimus, 7% of patients on cyclosporine, 74% on MMF, 19% on Myfortic, and 4% on sirolimus.
When and what to use as an induction agent?
Induction therapy is given to reduce the risk of acute rejection and to allow drug minimization. Data from the Thymoglobulin® Induction Study Group revealed a lower risk of rejection as compared to anti-CD25 in deceased donor organs at high risk for acute rejection, although at a higher cost of malignancies and infections. Alemtuzumab is also a potent induction agent used in minimization protocols, which has been shown (INTACT study group) to reduce the risk of rejection in low risk patients when compared to basiliximab and had a similar rate of rejection, although more late rejection in high risk patients when compared to Thymoglobulin®.
The choice of the induction agent should be based on patient’s immunologic and infectious risk. Low risk patients may benefit from basiliximab, while higher risk patients may benefit from ATG or alemtuzumab. An alternate approach to the low risk patient is induction with ATG or alemtuzumab and minimize immunosuppression post transplantation. Patients on steroid withdrawal protocols receive an induction agent at the time of transplantation.
What is the optimal dose of CNI?
Target CNI troughs can be adapted from the ELITE Symphony trial when combined with MMF and prednisone (tacrolimus 3-7 ng/ml, cyclosporine 50 to 100 ng/ml) in low risk patients. In the ELITE study the induction consisted of daclizumab, and not ATG, which is more potent and the most frequent induction therapy used in the US. Another confounding factor in this study is the discrepancy between the recommended tacrolimus goal levels (3-7) and the actual tacrolimus levels maintained by the physicians (which were close to 7). In general, the target troughs may be decreased over time after transplant. In patients who receive ATG induction, we suggest tacrolimus troughs of 8-10 ng/ml (triple immunosuppressive regimen with tacrolimus, MMF and Prednisone) or 10-12 ng/ml (regimen with tacrolimus and MMF/steroid free) during the first 2 months after transplant and then decrease the target levels to 6-8 until 6 months after transplant, and 5-7 subsequently. Higher immunologic risk patients (patients with repeat transplants or with DSA) may require higher troughs.
Which CNI should I use?
Trials comparing tacrolimus to cyclosporine have revealed decreased rejection rates in the tacrolimus arm. However, the majority of these trials compared Sandimmune (not a microemulsification of cyclosporine) to tacrolimus. Even better matched studies reveal advantages of tacrolimus in higher risk patients such as those with delayed graft function and African Americans. The enhanced immunosuppression of tacrolimus may be related to higher drug exposure to MMF as compared to cyclosporine. If patients do not tolerate tacrolimus, cyclosporine remains a good alternative.
Which antiproliferative agent should I use?
The most used antiproliferative agent is MMF, which has been shown to decrease the risk of rejection compared to azathioprine, tends to be less myelosuppressive and less hepatotoxic. In patients who cannot tolerate MMF due to GI intolerance, we recommend diving the MMF dose Q6h (for example in patients on MMF 1000 mg twice per day, we divide MMF to 500 mg every 6 hours), and if MMF is still not tolerated we recommend switching to mycophenolate sodium. Azathioprine is an acceptable alternative to MMF/mycophenolate sodium in patients who cannot tolerate them. The use of sirolimus as an antimetabolite is rare, and mostly limited to patients with history of skin cancer.
What is the optimal dose of MMF/mycophenolate sodium?
In most studies patients are started on the equivalent of 1000 mg of MMF twice a day (720 mg mycophenolate sodium is equivalent to 1000 mg of MMF). In patients discharged without steroids, the absorption of MMF is improved. In stable, low to moderate risk patients who received ATG induction and maintenance immunosuppression with tacrolimus, the dose of MMF can be often safely lowered to 500 mg twice per day of MMF at 6 months (if on steroids) or at 1 year post transplant (if off steroids).
What is the best protocol for higher immunologic risk patients?
High risk patients benefit from induction therapy, of which the most potent is rabbit ATG or alemtuzumab. In addition those with donor specific antibodies may require IVIg, rituximab, and occasionally plasma exchange (depending on the result of the cross-match). Currently, the best maintenance immunosuppressive therapy for higher immunologic risk patients includes tacrolimus, MMF/mycophenolate sodium and corticosteroids.
What is the best protocol for low immunologic risk patients?
Kidney transplants from identical twins: These patients do not need induction or long term immunosuppression. Most patients can be given an antimetabolite and prednisone with or without low dose CNI which can be withdrawn within 3 months post transplant.
Two haplo-matched transplants (transplant from siblings who are not monozygotic twin, but who inherited the same HLA from their parents): One approach to immunosuppression includes induction with basiliximab, along with low dose tacrolimus and MMF. Alternative approaches include maintenance immunosuppression with only MMF and prednisone or MMF monotherapy. A prospective study included twenty HLA-identical transplant recipients treated with MMF, tacrolimus and sirolimus which were tapered off by one year post transplant, leaving patients on MMF monotherapy (at a dose of 1000 mg BID). At a median follow up of 633 days there was 100% graft survival and no rejection episodes. This approach has the benefit of avoiding long term CNI toxicity.
Zero antigen mismatch deceased donor transplant: although some transplant centers use induction with basiliximab and maintenance with tacrolimus and MMF, other centers still use ATG induction.
It is important to realize that there is no clear evidence to support steroid withdrawal over standard steroids or basiliximab over antilymphocyte globulin. Deciding on immunosuppression should be based on specific patient’s risk of rejection versus infection and malignancy.
When is rapid steroid withdrawal a good option?
Rapid steroid withdrawal (usually within 4-7 days after transplantation) is a reasonable option in low to moderate risk patients. Steroid withdrawal has shown good 5-year graft survival albeit with a slightly higher rate of acute rejection. The use of ATG and alemtuzumab has resulted in lower rejection rates compared to anti-CD25. Prospective studies show small benefits with rapid steroid withdrawal protocols including a reduction of insulin requiring diabetes, a small decrease in weight gain and reduced incidence of bone disease (combined endpoint of AVN plus fractures) in patients maintained off of steroids.
Is it possible to withdraw steroids after 3 to 6 months post transplant?
Studies of steroid late withdrawal have revealed an elevated risk of rejection and graft failure. This was largely reported in the era of cyclosporine, and data with modern immunosuppression (ATG induction with tacrolimus and MMF) is lacking. In patients with significant steroid toxicity it is reasonable to slowly withdraw steroids when patient receive tacrolimus and MMF, although this can be associated with an increased risk of rejection.
If my patient has a rejection episode should I resume chronic steroids?
It is not strictly necessary to resume steroids after one mild rejection episode, but the benefits and risks of resuming steroids or not should be reviewed carefully and a decision should be taken considering each case individually.
When is mTOR inhibitor a good drug to use?
Studies reveal inconsistent results when using sirolimus as the initial immunosuppressive agent as compared to CNIs. Several single center studies reveal comparable patient and graft survival in patients on sirolimus and MMF compared to CNIs. However, a multicenter trial has been halted due to higher than expected rejection rates in patients receiving sirolimus and MMF. In addition, the randomized multicenter ELITE-Symphony study revealed the worst rejection rate and graft survival in patients treated with low-dose sirolimus (target trough 4 to 8 ng/ml) combined with MMF and corticosteroids compared to CNIs with MMF and prednisone. Finally, SRTR analysis of transplants between 2000 and 2005 reveal the lowest 5 year survival in patients on sirolimus/ MMF compared to CNIs and MMF.
Poor wound healing and lymphoceles also complicate the de novo use of mTOR inhibitors. Furthermore, there is evidence of increased risk of DSA development with mTOR compared to CNI-based immunosuppressive regimen. Due to conflicting data and post operative complications mTOR inhibitors are usually not used as a de novo immunosuppressive agent.
The combination of an mTOR inhibitor and CNI is more immunosuppressive than sirolimus and MMF but infrequently used due to enhanced nephrotoxicity. This combination may be an acceptable treatment option in a high-risk individual that does not tolerate other antimetabolites.
Specific cohorts who benefit from conversion are patients who do not tolerate CNI side effects, have significant CNI toxicity on biopsy or malignancy (Kaposi’s sarcoma or skin cancer). Sirolimus has been associated with less skin cancer compared to other IS medications.
Conversion from CNI to mTOR inhibitor is discussed below.
Is it ok to change to generic formulations of CNI’s and MMF?
Generic medications are the same medication as the brand drug, but may differ in bioavailability (80% and 125% of that of the brand medication). Therefore close monitoring of therapeutic drug levels and renal function should be performed when converting.
When should I convert from a CNI to mTOR inhibitor?
Converting CNI to an mTOR inhibitor has been proposed as a method to reduce long-term nephrotoxicity of CNI’s while avoiding the immediate post op period, where outcomes are inferior with mTOR inhibitors. The large multicenter CONVERT trial found conversion to sirolimus at 6 to 12 months post transplant to be detrimental in patients with proteinuria and/or GFR < 40 ml/min. In addition there was a higher discontinuation rate in the sirolimus group.
Data from two multicenter trials converting CNI to sirolimus and everolimus in low risk patients prior to 6 months post transplant revealed comparable GFR in the mTOR group with a similar rejection rates. Target mTOR troughs in these studies were 5 to 10 ng/ml for sirolimus and 6 to 10 ng/ml for everolimus. Other data showed higher risk of developing DSA in patients on mTOR compared to CNI. As a conclusion, we currently suggest conversion only in patients with skin cancer, or patients unable to tolerate CNIs.
Alternatively to mTOR, patients who do not tolerate CNIs can be converted to Belatacept.
What tests to perform?
The ability to assess the degree of immunosuppression is limited. However, pre-transplant testing can be very informative on post transplant immunologic risk. Immune assays are currently being validated to determine net immunosuppression.
Pre-transplant immune-genetic testing
HLA: HLA typing analysis is currently being performed by DNA based methods, i.e. sequence specific primer PCR (SSP), sequence specific oligonucleotide probes (SSOP). Currently tested class I HLA alleles include A, B and sometimes Cw. Tested class II loci include DR, DQ and sometimes DP. The degree of HLA matching impacts the risk of rejection and graft survival in kidney transplantation.
Detection of HLA antibodies, panel reactive antibody (PRA) and donor specific antibodies (DSA): PRA is the quantification of preformed HLA antibodies the recipient has against a possible donor pool. Donor specific antibodies (DSA) are antibodies against a specific donor HLA antigens. Various methods are available to determine both PRA and DSA.
CDC crossmatch: Donor lymphocytes are mixed with recipient sera, exogenous complement and a vital dye. If greater than 10% of the donor cells are killed then the crossmatch is positive. Positive T-cell crossmatch implies cytotoxic antibody against donor class I antigens while a positive B-cell crossmatch implies cytotoxic antibodies against either class I antigens, class II antigens or surface Fc receptors found on the B cell membrane. Positive T – cell crossmatch is a contraindication to transplantation because of the very high risk for hyperacute rejection. Positive CDC B cell crossmatch is less specific however, does indicate a higher risk of rejection when DSA is confirmed by other methods. CDC crossmatch is repeated after heating serum or adding dithiothreitol (DTT) which removes IgM antibodies. IgM antibodies are usually auto-antibodies and are not detrimental to the transplanted organ. Anti-human globulin (AHG) is now usually being added before complement to increase the sensitivity of the crossmatch.
CDC PRA: Recipient sera is added to a panel of 30 to 60 cells representing most antigens encountered in the general population. Similar to CDC crossmatch, complement and vital dye is added. The amount of dead cells are quantified and PRA is calculated. For example, if 15 of 30 wells contain dead cells the PRA is 50% implying that the patient has antibodies against 50% of the population. Patients with PRA greater than 80% are considered highly sensitized. Sensitized individuals usually experience greater time waiting for a compatible donor and are at higher risk for rejection and graft failure. New organ allocation rules give priority to very highly sensitized patients.
Flow crossmatch: Donor lymphocytes are incubated with patient sera and stained with fluorescence-labeled anti-IgG then run through a flow cytometer. Positive flow crossmatch implies recipient anti-HLA antibodies binding to donor lymphocytes (T or B cells depending on the type of lymphocytes chosen). It is more sensitive than the CDC crossmatch. A positive T flow crossmatch confers a high risk for rejection, while B may less risky, although a positive B flow crossmatch in the presence of known DSA is associated with increased risk of rejection.
Solid phase assays: Either antigen coated trays (ELISA) or antigen coated synthetic microbeads are used to determine specific HLA antibodies. Elisa is about 10% more sensitive in determining HLA antibody than AHG CDC methods and microbead technology is more sensitive than ELISA.
ELISA method: Recipient sera are added to pre-specified antigen coated trays which can be used to determine the presence of specific HLA antibodies.
Synthetic microbeads with recombinant HLA molecules: Recombinant HLA molecules are combined to synthetic beads that display a unique fluorescent pattern (Luminex platform). Depending on the platform, there may be multiple HLA antigens on one bead or a specific HLA antigen on each bead. Recipient sera are added along with fluorescence-labeled anti-IgG then run through either a traditional flow cytometer or on the Luminex platform. The result will be an estimation of HLA antibodies the patient has against the donor pool or, in the case of single antigen beads, specific HLA antibodies in the patients’ serum. Though initially developed as a qualitative test, single antigen bead results are given as a “mean fluorescent intensity (MFI)” which is being used in a semiquantitative manner to determine antibody strength.
Important labs in the post transplant period
CBC with differential: Leukopenia may result from immunosuppressants as well as viral infections (ex. CMV) and malignancy. Commonly attributed medications are anti-thymocyte globulin, alemtuzumab, MMF, azathioprine, pepcid and valgancyclovir. In general CNIs are not myelosuppressive. Total white blood cell count under 3,000 may require further work up or eventually reduction of immunosuppressive medications if safe.
BK virus PCR: BK virus is a polyoma virus, related to JC virus, known to cause nephropathy in kidney transplant patients. It can be measured in the blood by PCR, which correlates with BK nephropathy, or in the urine which is more sensitive but less specific in predicting nephropathy. Once urine BK becomes positive at high levels, BK should be checked in the blood. Most centers routinely check BK in the blood and not in the urine. BK viremia is a disease of over-immunosuppression and is treated mainly by reducing immunosuppression. Studies are under way on possible role of IVIG, pulse steroids and everolimus.
Allograft biopsy: Biopsy is used to detect rejection, polyomavirus nephropathy, recurrent or de novo disease. High risk patients may benefit from protocol biopsies to screen for subclinical rejection or features of chronic rejection. See chapter “Kidney Transplantation- Diagnosis and Management – Intrinsic Causes and Treatments” for more detailed description of pathology findings.
Controversies in diagnostic testing
Monitoring donor specific antibodies (DSA): Donor specific antibodies may be present before transplantation (as in sensitized patients) or may arise de novo in the post transplant period. There are many methods, however, due to limited donor cells, DSA monitoring is often performed by solid phase assay (Luminex). Most studies have shown that the persistence or a rise in pre-existing and de novo DSA are detrimental to allograft survival, as they probably signify low grade chronic antibody mediated rejection. However, it unknown if increased immunosuppression reduces the appearance of HLA antibodies or if increasing immunosuppression after the appearance of de novo HLA antibodies improves graft survival.
Cylex ImmuKnowAssay®: This assay is FDA approved and commercially available. Recipient T cells are stimulated with a T cell mitogen, PHA. The amount of ATP produced from the T cell is quantified and is proportional to T-cell reactivity. Low ATP production has been associated with opportunistic infections, however no prospective data exists on changing immunosuppression based solely on this assay.
Virtual Crossmatch and calculated PRA (cPRA)
By knowing specific HLA antibodies present in a patient’s serum and a donor’s HLA type, we can anticipate positive crossmatches and calculate a PRA value. cPRA is the PRA which is calculated based on unacceptable HLA antigens entered by a center in the UNOS database. These are the HLA antigens against which the recipient has antibodies and, if present in the donor, would very likely cause a positive crossmatch. As a consequence, organs from the donors with known unacceptable HLA antigens will not be offered to this recipient. Based on the unacceptable HLA antigens listed, and based on the frequency of these antigens in the donor pool, a cPRA is calculated for each recipient. For example, if a patient has a strong antibody to HLA A2 we can infer that a crossmatch with a donor expressing HLA A2 would be positive. Since HLA A2 is present in 47% of the donor pool we would be removing 47% of available donors for this patient. Therefore, the cPRA would be 47%. We can then avoid crossmatching the patient with any donors with HLA A2. As a consequence, we basically did a virtual crossmatch. Virtual Crossmatch has been implemented by UNOS as a means for reducing time and resources spent on crossmatching sensitized patients.
How should patients with too much or too little immunosuppression be managed?
Too much immunosuppression: Patients on triple therapy should have prednisone tapered to 5 mg, and may benefit from reducing the dose of the antimetabolite or target CNI trough. Replacing CNI with an mTOR inhibitor may be beneficial in few patients, especially those with history of skin cancer. In general, late steroid withdrawal is not recommended but may be attempted in patients with significant steroid related morbidity.
Too little immunosuppression: Potential approach in increasing immunosuppression include increase target CNI trough, change from cyclosporine to tacrolimus, increase anti-metabolite dose, or resume steroids in patients on steroid free protocols.
What happens to patients with too much or too little immunosuppression?
Unfortunately it is often difficult to ascertain whether an individual patient has the ideal amount of immunosuppression.
Acute rejection episodes: Patients under-immunosuppressed may demonstrate multiple episodes of acute rejection, which is one of the strongest predictors of chronic allograft nephropathy (now known as interstitial fibrosis and tubular atrophy-IFTA). Early low grade rejection episodes which are treated may not affect long term outcome whereas late or aggressive rejection episodes lead to premature allograft failure.
Chronic rejection: Patients under-immunosuppressed may demonstrate pathologic changes consistent with transplant glomerulopathy. Transplant glomerulopathy is often mediated by the humoral immune system but cases without apparent humoral rejection have been described.
De novo HLA antibodies: See controversies in diagnostic testing.
Infections: Over-immunosuppressed patients are often characterized by recurrent infections not explained by anatomic abnormalities. In addition, patients with opportunistic infections > 6 months post transplant suggest over-immunosuppression, for example, late onset PCP pneumonia.
Polyomavirus: BK virus present in the blood and especially on pathologic examination is a marker for over-immunosuppression and should be treated with a reduction in immunosuppression.
How to utilize team care?
Taking care of the transplant patients can be difficult and involves participation of a multidisciplinary team.
The transplant center: Transplant centers include physicians and supportive caregivers who deal with the complexities of transplantation on a daily basis and possess a wealth of information in taking care of transplant recipients. In addition, they may hold valuable information about the patient’s immunologic risk, anatomic details of their transplant, induction therapy and rationale for current immunosuppression.
Transplant pharmacists: Pharmacists play a substantial role in the multidisciplinary team and can provide valuable information in managing the complexities of dosing immunosuppression and the various drug- drug interactions.
The HLA laboratory: The HLA lab is the source of immunologic testing for the transplant center. They hold valuable information of the patients PRA, crossmatch and degree of HLA matching which will impact the decision on how much immunosuppression a patient will require.
Other important team members that are involved in taking care of the transplant patient include transplant nephrologists, transplant surgeons, dieticians, social workers, nurse coordinators, and financial specialists.
Are there clinical practice guidelines to inform decision-making?
KDIGO clinical practice guideline for the care of kidney transplant recipients. Am J Transplant. 9(Suppl 3):S1, 2009
The 2009 Kidney Disease Improving Global Outcomes (KDIGO) clinical practice guidelines on the monitoring, management, and treatment of kidney transplant recipients offer guidelines in immunosuppressive management as well as other transplant related complications.
Guidelines are based on data accumulated until 2009. Our recommendations often, but not always, correspond with these guidelines, and are sometimes the result of the author’s personal experience. They should not be applied indistinctively to specific patients without taking into consideration risks and benefits based on patient’s personal history.
Kidney transplant status – Z94.0
Kidney transplant rejection – T86.11
Other complication of kidney transplant – T86.19
Encounter for aftercare following kidney transplant – Z48.22
Encounter for therapeutic drug level monitoring – Z51.81
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- Does this patient have too much or too little immunosuppression?
- What tests to perform?
- How should patients with too much or too little immunosuppression be managed?
- What happens to patients with too much or too little immunosuppression?
- How to utilize team care?
- Are there clinical practice guidelines to inform decision-making?
- Other considerations