RENAL TRANSPLANTATION IMMUNOLOGY

Following transplantation, the recipient's T cells recognize donor antigens from the graft (the allogeneic antigens, or alloantigens) by two pathways:

(1) Direct pathway: The graft antigens are presented directly to recipient T cells by graft APCs (2) Indirect pathway: the graft antigens are picked up by host APCs, processed (like any other foreign antigen), and presented to host T cells. - Both lead to the activation of CD8+ T cells, which develop into CTLs, and CD4+ T cells, which become cytokine-producing effector cells, mainly TH1 cells. - **The direct pathway may be most important for CTL-mediated acute rejection, and the indirect pathway may play a greater role in chronic rejection**

azathioprine

- Azathioprine contains a nitroimidazoloyl side chain attached to the sulfur of 6-mercaptopurine, which is removed by non-enzymatic reduction by glutathione to yield 6-mercaptopurine. 6-mercaptopurine is converted to thioinosinic acid (TIMP). - The immunosuppressant effects of azathioprine are due to TIMP, which functions as a fraudulent nucleotide. - Azathioprine is toxic to proliferating T and B lymphocytes. - Clinical uses of azathioprine include: Kidney transplantation Autoimmune disorders (e.g. glomerulonephritis, rheumatoid arthritis, hemolytic anemia) Inflammatory Bowel Disease Leukemias - Adverse effects of azathioprine include: Bone marrow suppression (leukopenia, thrombocytopenia, anemia) GI toxicity Hepatotoxicity Pancreatitis The toxic effects of azathioprine may be increased by allopurinol, a xanthine oxidase inhibitor.

Methods of increasing Graft Survival:

- Because HLA molecules are the major targets in transplant rejection, better matching of the donor and the recipient improves graft survival. - HLA matching is more beneficial for living related kidney transplants than for other kinds of transplants, and survival improves with increasing number of loci matched. - However, as drugs for immunosuppression have improved, HLA matching is no longer done for heart, lung, liver, and islet transplantation; in such instances, the recipient often needs a transplant urgently and other considerations, such as anatomic compatibility, are of greater importance. - Immunosuppression of the recipient is a necessity in all organ transplantation, except in the case of identical twins - Suppression of the immune system results in increased susceptibility to opportunistic fungal, viral, and other infections. - Reactivation of latent viruses, such as cytomegalovirus (CMV) and polyoma virus, are frequent complications. - Immunosuppressed patients also are at increased risk for developing virus-induced tumors, such as Epstein-Barr virus (EBV)-induced lymphomas and human papillomavirus (HPV)-induced squamous cell carcinomas.

Acute Cellular Rejection

- CD8+ CTLs may directly destroy graft cells, or CD4+ cells secrete cytokines and induce inflammation, which damages the graft - T cells also may react against graft vessels, leading to vascular damage. - Current immunosuppressive therapy is designed mainly to prevent and reduce acute rejection by blocking the activation of alloreactive T cells. - Acute cellular (T cell-mediated) rejection may produce two different patterns of injury: (1) In the **tubulointerstitial pattern** (sometimes called type I), there is extensive interstitial inflammation and tubular inflammation (tubulitis) associated with focal tubular injury. > As might be expected, the inflammatory infiltrates contain activated CD4+ and CD8+ T lymphocytes. (2) The **vascular pattern** shows inflammation of vessels (type II) and sometimes necrosis of vessel walls (type III). > The affected vessels have swollen endothelial cells, and lymphocytes are seen between the endothelium and the vessel wall, a finding termed **endotheliitis or intimal arteritis** - The recognition of cellular rejection is important because, in the absence of accompanying humoral rejection, most patients respond well to immunosuppressive therapy.

donor-specific tolerance in allograft recipients:

- Costimulatory blockade: It was postulated that recognition of alloantigens in the absence of costimulation would lead to T cell tolerance, and there is some experimental evidence in animals to support this. However, the clinical experience with agents that block costimulation is that they suppress immune responses to the allograft but do not induce long-lived tolerance, and patients have to be maintained on the therapy. - Hematopoietic chimerism: We mentioned earlier that transfusion of donor blood cells into the graft recipient inhibits rejection. If the transfused donor cells or progeny of the cells survive for extended periods in the recipient, the recipient becomes a chimera. Long-term allograft tolerance by hematopoietic chimerism has been achieved in a small number of renal allograft recipients who received a hematopoietic stem cell transplant from the donor at the same time as the organ allograft, but the risks of hematopoietic stem cell transplantation and the availability of appropriate donors may limit the applicability of this approach. - Transfer or induction of Tregs: Attempts to generate donor-specific Tregs in culture and to transfer these into graft recipients are ongoing. There has been some success reported in recipients of hematopoietic stem cell transplants, in whom infusions of Tregs reduce GVHD.

cyclosporine

- Cyclosporine preferentially suppresses cell mediated reactions whereas humoral immunity is affected to a far less extent. - Cyclosporine is used to prevent rejection of kidney, liver, and cardiac allogenic transplants. - Cyclosporine can also be used to treat rheumatoid arthritis and recalcitrant psoriasis. Metabolites of cyclosporine are mainly excreted via the biliary system. - Cyclosporine preferentially binds to **cyclophilin** to form a complex that **inhibits calcineurin** (a phosphatase for transcription factor NFAT that upon dephosphorylation increases the activity of genes coding for IL-2 and related cytokines). - Auto-stimulation by IL-2 is the main stimulus for T cell proliferation. Calcineurin inhibition leads to a decrease in the synthesis of IL-2 by CD4+ Th cells, thereby decreasing T-lymphocyte proliferation. - The major limiting adverse effect of cyclosporine is nephrotoxicity. - The most common cause of a rise in BUN and creatinine levels post-transplantation is from cyclosporine use. Preventative measures can be taken by restricting cyclosporine doses to the lowest effective level. - Other adverse effects of cyclosporine include: Gingival hyperplasia Hirsutism Hypertension Hyperlipidemia Hyperuricemia (gout) Neurotoxicity (tremor) Breast fibroadenomas

Graft-Versus-Host Disease (GVHD)

- GVHD is caused by the reaction of grafted mature T cells in the Hematopoietic Stem Cell Transplantation inoculum with alloantigens of the host . - It occurs when the host is immunocompromised and therefore unable to reject the allogeneic cells in the graft. - In most cases, the reaction is directed against minor histocompatibility antigens of the host because bone marrow transplantation is not usually performed when the donor and recipient have differences in MHC molecules. - GVHD may also develop when solid organs that contain significant numbers of T cells are transplanted, such as the small bowel, lung, or liver. - GVHD is the principal limitation to the success of bone marrow transplantation. - Immediately after HSC transplantation, immunosuppressive agents including the calcineurin inhibitors cyclosporine and tacrolimus, antimetabolites such as methotrexate, and the mTOR inhibitor sirolimus are given for prophylaxis against the development of GVHD. - Despite these aggressive prophylactic strategies, GVHD is the principal cause of mortality among HSC transplant recipients. - GVHD may be classified on the basis of histologic patterns into acute and chronic forms. - Both acute and chronic GVHD are commonly treated with intense immunosuppression, such as high doses of steroids, but many patients do not respond well.

Mechanisms of Graft Rejection

- Graft rejection is classified into: > hyperacute > acute > chronic - on the basis of clinical and pathologic features - Each type of rejection is mediated by a particular kind of immune response.

"one-way" mixed lymphocyte reaction (MLR)

- In the classical form of MLR, peripheral blood lymphocytes from two individuals are mixed together in tissue culture for several days - In the one-way MLR test, donor lymphocytes are inactivated, thereby allowing only the recipient lymphocytes to proliferate in response to foreign histocompatibility antigens

polyomavirus BK virus nephropathy

- Latent polyomavirus BK virus infection is common and commonly reactivated in patients immunosuppressed for kidney transplantation. - Infection of renal tubular epithelial cells causes intranuclear inclusions visible on routine H & E microscopy. - An interstitial inflammatory response is invariably present. - The infection can ruin the transplanted kidney. - Treatment consists of reduced immunosuppression, which may result in transplant rejection.

Antithymocyte globulin

- MOA: Antithymocyte globulin contains cytotoxic antibodies that bind to CD2, CD3, CD4, CD8, CD11a, CD18, CD25, CD44, CD45, and HLA class I and II molecules on the surface of human T lymphocytes. > The antibodies deplete circulating lymphocytes by direct cytotoxicity (both complement and cell mediated) and block lymphocyte function by binding to cell surface molecules involved in the regulation of cell function. - Uses: Antithymocyte globulin is used for induction immunosuppression, although the approved indications are for the treatment and prophylaxis of acute renal transplant rejection in combination with other immunosuppressive agents and for the treatment of aplastic anemia. - A course of antithymocyte-globulin often is given to renal transplant patients with delayed graft function to avoid early treatment with the nephrotoxic calcineurin inhibitors

Muromonab-CD3

- MOA: The antibody muromunab (OKT3) inhibits TCR function via interaction with its CD3 component > CD3 is a component of the TCR complex on the surface of human T lymphocytes > Antibodies directed at the ε chain of CD3 have been used with considerable efficacy in human transplantation. - no longer marketed due to its side effects: It frequently causes cytokine release syndrome and severe pulmonary edema.

mycophenolate mofetil (MMF)

- Mycophenolate mofetil is a reversible inhibitor of inosine monophosphate dehydrogenase in the de novo purine synthesis pathway. It blocks de novo formation of guanosine phosphate (GMP). - Mycophenolate mofetil deprives rapidly proliferating T and B cells of new guanine nucleotides, preventing replication and division. - Absorption of mycophenolate mofetil is decreased if co-administered with bile-acid resins (e.g. cholestyramine) or antacids containing magnesium or aluminum. - Adverse effects of mycophenolate mofetil include: GI symptoms (e.g. nausea, diarrhea) Cytopenias Hypertension Hyperglycemia CMV infection Lymphoma

tacrolimus (FK506)

- Tacrolimus and cyclosporine both inhibit calcineurin, a calcium and calmodulin dependent phosphatase. - In the process of T cell activation, the phosphatase calcineurin dephosphorylates cytoplasmic NFAT, which allows it to be translocated to the nucleus and subsequently upregulate transcription of cytokine genes including IL-2, which further induce T cell activation. - IL-2 is a major stimulus for T-lymphocyte proliferation. - The mechanism of action of tacrolimus is similar to cyclosporine but instead, **tacrolimus binds to FK506 binding protein (FKBP) which inhibits calcineurin.** - Tacrolimus is preferred to cyclosporine, because tacrolimus causes fewer rejection episodes and lower glucocorticoid requirement. - Tacrolimus is used for treatment after lung, liver, and kidney transplants. - Most of the drug and its metabolites are excreted in the feces. - Toxicities of tacrolimus include: Nephrotoxicity Neurotoxicity (tremor, seizures, hallucinations) Hypertension Pleural effusion Hyperglycemia Unlike cyclosporine, tacrolimus does not cause hirsutism or gingival hyperplasia.

Why are immune responses to allografts are stronger than responses to pathogens?

- The frequency of T cells that can recognize the foreign antigens in a graft is much higher than the frequency of T cells specific for any microbe - these strong reactions can destroy grafts rapidly, and their control requires powerful immunosuppressive agents.

Rapamycin (Sirolimus)

- The mechanism of action of sirolimus is: Sirolimus complexes with FK-binding protein 12 (FKBP12) - The sirolimus-FKBP12 complex inhibits mTOR (mammalian target of rapamycin) Inhibition of mTOR prevents the stimulatory action of IL-2 on T and B cells - To compare, sirolimus prevents IL-2 from activating T and B cells, whereas tacrolimus prevents IL-2 transcription (calcineurin inhibitor). - Sirolimus is used in immunosuppression after kidney transplants because it is minimally nephrotoxic. - Sirolimus is also used in combination with cyclosporine and corticosteroids. - Sirolimus-eluting coronary stents are used in balloon angioplasty to prevent smooth muscle proliferation and restenosis of coronary arteries.

Chronic rejection

- an indolent form of graft damage that occurs over months or years, leading to progressive loss of graft function. - Chronic rejection manifests as interstitial fibrosis and gradual narrowing of graft blood vessels (graft arteriosclerosis). - In both lesions, the culprits are believed to be T cells that react against graft alloantigens and secrete cytokines, which stimulate the proliferation and activities of fibroblasts and vascular smooth muscle cells in the graft - Alloantibodies also contribute to chronic rejection. - Although treatments to prevent or curtail acute rejection have steadily improved, leading to longer than 1-year survival of transplants, chronic rejection is refractory to most therapies and is becoming the principal cause of graft failure. - Chronic rejection is dominated by vascular changes, often with intimal thickening and vascular occlusion - Chronically rejecting kidney grafts show glomerulopathy, with duplication of the basement membrane, likely secondary to chronic endothelial injury and peritubular capillaritis with multilayering of peritubular capillary basement membranes. Interstitial fibrosis and tubular atrophy with loss of renal parenchyma may occur secondary to the vascular lesions

Acute Antibody-Mediated (vascular or humoral) rejection

- antibodies bind to vascular endothelium and activate complement via the classical pathway - The resultant inflammation and endothelial damage cause graft failure. - Acute antibody-mediated rejection is manifested mainly by damage to glomeruli and small blood vessels. - Typically, there is inflammation of glomeruli and peritubular capillaries associated with deposition of complement products, which is due to activation of the complement system by the antibody-dependent classical pathway - Small vessels also may show focal thrombosis.

Acute GVHD

- characterized by epithelial cell death in the skin, liver (mainly the biliary epithelium), and gastrointestinal tract. - It is manifested clinically by rash, jaundice, diarrhea, and gastrointestinal hemorrhage. - When the epithelial cell death is extensive, the skin or the lining of the gut may slough off. In this circumstance, acute GVHD may be fatal. - acute GVHD is initiated by mature T cells transferred with HSCs, and elimination of mature donor T cells from the graft can prevent the development of GVHD. - Although GVHD is initiated by grafted T cells recognizing host alloantigens, the effector cells that cause epithelial cell injury are less well defined. - On histologic examination, NK cells are often attached to the dying epithelial cells, suggesting that NK cells are important effector cells of acute GVHD. - CD8 + CTLs and cytokines also appear to be involved in tissue injury in acute GVHD

Chronic GVHD

- characterized by fibrosis and atrophy of one or more of the same organs, without evidence of acute cell death. - Chronic GVHD may also involve the lungs and produce obliteration of small airways, called bronchiolitis obliterans, similar to what is seen in chronic rejection of lung allografts. - When it is severe, chronic GVHD leads to complete dysfunction of the affected organ.

immunosupressives

- cyclosporine - the related FK506 - mofetil mycophenolate (MMF) - rapamycin - azathioprine - corticosteroids - anti-thymocyte globulin - monoclonal antibodies (e.g., monoclonal anti-CD3)

major antigenic differences between a donor and recipient that result in rejection

- differences in HLA alleles - Because HLA genes are highly polymorphic, there are always some differences between individuals (except identical twins)

Prevention of rejection

- immunologic graft rejection is targeted at allogeneic proteins encoded by polymorphic alleles in the recipient not shared by the donor. - Related donors will share more alleles of polymorphic genes, including MHC genes, than unrelated donors, and this will reduce the incidence and severity of rejection episodes. - the major strategy to reduce graft immunogenicity has been to minimize alloantigenic differences between the donor and recipient . > ABO blood typing; > the determination of HLA alleles expressed on donor and recipient cells, called tissue typing; > the detection of preformed antibodies in the recipient that recognize HLA and other antigens representative of the donor population; and > the detection of preformed antibodies in the recipient that bind to antigens of an identified donor's cells, called cross-matching. - Blood typing is uniformly used in renal and cardiac transplantation because kidney and heart grafts will typically not survive if there are ABO incompatibilities between the donor and recipient - greater survival of grafts when donor and recipient have fewer HLA allele mismatches.

Everolimus

- mTOR inhibitor - Everolimus is FDA-approved for treatment of astrocytoma, breast cancer, kidney and liver transplant rejection prophylaxis, pancreatic neuroendocrine tumor, renal angiomyolipoma, and renal cell cancer. - It is chemically closely related to sirolimus but has distinct pharmacokinetics. - The main difference is a shorter t1/2 and thus a shorter time to achieve steady-state concentrations of the drug. - As with sirolimus, the combination of a calcineurin inhibitor and an mTOR inhibitor produces worse renal function at 1 year than does calcineurin inhibitor therapy alone, suggesting a drug interaction between the mTOR inhibitors and the calcineurin inhibitors that reduces rejection but enhances toxicity. - The toxicity of everolimus and the potential for drug interactions seem to be the same as with sirolimus

Acute rejection

- mediated by T cells and antibodies that are activated by alloantigens in the graft. - It occurs within days or weeks after transplantation, and is the principal cause of early graft failure. - It also may appear suddenly months or even years later, after immunosuppression is tapered or terminated. - Based on the role of T cells or antibodies, acute rejection is divided into two types, although in most rejecting grafts, both patterns are present. (1) acute cellular rejection (2) acute antibody-mediated (vascular or humoral) rejection

Hyperacute rejection

- mediated by pre-formed antibodies specific for antigens on graft endothelial cells . - The preformed antibodies may be natural IgM antibodies specific for blood group antigens, or may be antibodies specific for allogeneic MHC molecules that were induced by prior exposure through blood transfusions, pregnancy, or organ transplantation. - Immediately after the graft is implanted and blood flow is restored, the antibodies bind to antigens on the graft endothelium and activate the complement and clotting systems, leading to endothelial injury, thrombus formation, and ischemic necrosis of the graft - Hyperacute rejection is not a common problem, because every donor and recipient are matched for blood type and potential recipients are tested for antibodies against the cells of the prospective donor, a test called a cross-match. - In hyperacute rejection, the affected kidney rapidly becomes cyanotic, mottled, and anuric. - Virtually all arterioles and arteries exhibit acute fibrinoid necrosis of their walls and narrowing or complete occlusion of their lumens by thrombi - Neutrophils rapidly accumulate within arterioles, glomeruli, and peritubular capillaries. - As these changes intensify and become diffuse, the glomerular capillaries also undergo thrombotic occlusion, and eventually the kidney cortex undergoes outright necrosis (infarction). - Affected kidneys are nonfunctional and have to be removed.

Daclizumab & Basiliximab

- monoclonal antibodies that target the CD25 antigen of the IL-2 receptor of T cells. - Uses: Basiliximab (and historically, daclizumab) are used as immunosuppressants for kidney transplants as these agents reduce the incidence and severity of acute rejection in kidney transplantation without increasing the incidence of opportunistic infections; Daclizumab can be used in the treatment of relapsing-remitting multiple sclerosis. - Toxicities: Edema Hypertension Tremor

Cyclophosphamide

- used in leukemia and lymphomas and a variety of other malignancies. - Cyclophosphamide also is FDA-approved for childhood nephrotic syndrome and is used widely off label for treatment of severe systemic lupus erythematosus, MS, and vasculitides such as Wegener granulomatosis. - Cyclophosphamide is the most common alkylating cytotoxic agent used in the treatment of glomerulopathy, and this induces apoptosis or suppression of B and T cell function. - Leukopenia is an important short-term adverse effect. - Infertility in both men and women has been reported with these agents, most commonly following cyclophosphamide, and is likely the most concerning long-term side effect given the often-younger age of patients with glomerular disease. - The other major adverse effect is the risk for malignancy.

Ten things medical students should learn about renal transplantation:

1. Renal transplantation provides a much higher quality of life than dialysis. 2. Transplant rejection can be cellular or humeral (or both simultaneously). 3. Acute cellular rejection is more common in the first few months after transplantation, but often occurs much later (and is still called "acute" then) 4. Primary care physicians in rural areas often inherit the care of transplant patients on new exotic immunosuppressive drugs not in their medical school curriculum years prior 5. Some of the currently most prevalent immunosuppressive drugs are nephrotoxic 6. Opportunistic infection (e.g. polyomavirus nephropathy) can present with clinical manifestations nearly identical to transplant rejection, but require opposite treatment. 7. Hyperacute rejection is vanishingly rare (except in outdated board review materials) 8. Chronic rejection is primarily vascular, increasingly common and minimally reversible. 9. Opportunistic lymphoma driven by Epstein-Barr virus is B-cell, while rejection is T-cell. 10. Humoral rejection can be diagnosed partly by immunostain for complement C4d.

Rejection

a process in which T lymphocytes and antibodies produced against graft antigens react against and destroy the grafts.