Flashcards in Immunology II Deck (60):
An immunodeficiency is an impaired ability to mount effective immune responses to infectious agents.
Impaired immunity may be primary (e.g. primary antibody deficiencies) or secondary (to disease, drugs, infection). The majority are secondary to other condi- tions.
Immunodeficiency results in differing types of infections (bacterial, viral, fungal) depending on the defence mechanisms affected
There are multiple causes of secondary immunodeficiency. It is well recognised that lympho- proliferative disease, including chronic lymphatic leukaemia (CLL) and myeloma, result in impairment of specific adaptive immunity in later stages of disease progression, and increased susceptibility to bacterial infection. Non-neoplastic diseases such as systemic lupus erythematosis (SLE) or rheumatoid arthritis (RA show an inherently increased susceptibility to infections, although the direct cause of the impairment is unknown.
Drug therapy is an important cause of immunosuppression affecting both specific and innate mechanisms. Infections can cause immunosuppression either directly (e.g. HIV-induced T cell destruction) or indirectly (EBV, CMV).
Inflammation can cause transient impairment of immune response – e.g. after surgery, trauma, burns (where there is also loss of serum proteins) – and increased susceptibility to infection, although abnor- malities of functional assays are usually more pro- nounced than clinical problems (depressed CMI skin tests, depressed in-vitro lymphocyte proliferations, alterations in granulocyte and NK functions), reflecting the plasticity of the immune response as a whole. Postoperative infections in patients given periopera- tive blood transfusions appear to be increased due to an ill-defined immunosuppressive effect of some com- ponent of the blood given (RBC or WBC).
Secondary immunodeficiency as a result
The most common way a surgical procedure pre- disposes to infection is by breaching a mucosal barrier. In addition, barriers may be compromised by haemorrhage, gut immobility, ischaemia, burns or mal- nutrition. This would result in increased penetration of pathogens across the mucosa and skin with subsequent defective killing of organisms by phagocytes. The presence of drains or other foreign bodies also provides both routes of entry and niduses of infection.
Surgery results in severe metabolic alterations with an initial hypometabolic phase followed by a hypermetabolic phase. In the first three days after major gut surgery 6–7% of body weight may be lost. There is a potential for infection from endogenous or exogenous sources. A similar impairment of specific and innate defence mechanisms operates in trauma and generalised infla- mmatory responses due to disease or infection, but in elective surgery careful attempts to maintain homeo- stasis during the period of anaesthesia may reduce this impairment.
The precise causes of immunosuppression are unknown but may involve circulating cytokines, loss of blood or plasma (depleting immunoglobulins or complement), hypercatabolic states, or renal and hepatic failure. These effects make it important to perform functional studies on lymphocytes and neutrophils at times when a patient is well. These multiple effects are sometimes referred to as surgical stress.
Cellular effects of surgical stress
Lymphocyte numbers are not consistently altered by surgical stress. CD4 T cell numbers only fall in major trauma (by day 2–4). This may be reflected in the total lymphocyte count. This phenomenon may result from redistribution of cells to peripheral organs or lymph- oid tissue rather than a decline in numbers. CD4/ CD8 ratios are not useful, since they reflect a dynamic ratio of two populations and take no account of abso- lute numbers. NK cell numbers appear stable. B cell numbers may remain stable or transiently decline. Some of the observed changes may be due to the pharma- cological effects of anaesthetic drugs, which can reduce proliferation of B cells. There is no clinically useful correlation between these observations and out- come of surgery.
There is anergy to delayed-type hypersensitivity (DTH) skin tests in postsurgical patients, and impair- ment is more frequent in those with a worse out- come, although it is not clear if this is cause or effect. Patients with burns, viral infections and sarcoidosis all have variable and transient depression of DTH to unrelated antigens, but have other reasons to be sus- ceptible to infections. Likewise, in vitro T cell IL-2 pro- duction and antigen specific proliferation are inversely related to the severity of injury. T cells are activated (increased CD25 (IL-2R) expression), but prolif- eration (specific antigen, allogeneic cells and mitogens) is generally impaired, perhaps due to soluble factors (complement fragments or cytokines), which can sup- press neutrophil chemotaxis and NK cell activity.
Cytokine effects of surgical stress
Failure to produce cytokines such as IL-1 and IL-2 is associated with fatal outcomes. There also appears to be decreased production of IFNγ in trauma, which may impair phagocyte activation and B cell prolifer- ation and increase immunosuppressive PGE2 produc- tion. Many other cytokines are produced, including PAF and TNFα which induces production of IL-1, IL-6 and PGE2.
Complement activation by surgical stress
Both classical and alternative pathways are activated by trauma, the alternative pathway (AP) in burns. This leads to complement consumption in the early stages, with the production of complement fragments which affect phagocyte function. Complement can also be directly activated by drugs, methylmethacrylate resins in orthopaedic surgery and dialysis or cardiopul- monary bypass pump membranes but the effect is often subclinical or results in an adverse reaction rather than immunosuppression.
Antibody production in surgical stress
Any fall in total immunoglobulin levels is due to haemodilution by i.v. fluid replacement or exudative loss of plasma (in severe burns). Defects in specific antibody production to vaccination following major injury have been demonstrated. Thermal injury and trauma reduces vaccine responses to tetanus but not to polysaccharide antigens, suggesting that some of these defects may reflect T cell dysfunction.
Phagocyte dysfunction in surgical stress
A neutrophil leucocytosis is usual and proportional to the degree of inflammation/trauma. This may be due to mobilisation of marginalised neutrophils from pul- monary vasculature or new emigrants from the bone marrow under cytokine control. Neutrophil activation is seen with transiently decreased adhesiveness followed by an increase which parallels changes of adhesion mol- ecule expression on damaged vascular endothelium. This enables homing of neutrophils, activation and extravasation at the site of injury. However, neutrophil chemiluminescence, NBT reduction, and superoxide production are suppressed and antibacterial lysosyme and B12 binding protein are reduced. In vitro chemo- taxis is decreased for up to nine days even after minor trauma, and longer in major trauma. Reduced chemo- taxis correlates with poor outcome in burns patients. Depletion of complement or immunoglobulins due to hypercatabolism, consumption and loss may second- arily impair neutrophil opsonisation and chemotaxis. In severe trauma, acute phase protein production may be depressed.
APC function in surgical stress
Although there is often an initial monocytosis after surgery, with a transient increase in phagocytosis, enzyme content and cytochrome oxidase activity, this is transient and often becomes impaired subsequently. MHC Class II expression may be reduced after surgery or haemorrhage. Impairment of APC function has not been formally demonstrated in humans.
Endothelial effects of surgical stress
Endothelial injury with subsequent coagulation, platelet activation, increased vascular permeability, endothelial cell and platelet production of cytokines or prostaglandins/leukotrienes and upregulation of adhesion molecules are central events in surgical and traumatic injury. Subsequent cytokine-mediated effects on distant organs produce the classical systemic signs of fever (IL-1 and IL-6 are the ‘endogenous pyrogen’ acting on the hypothalamic axis; IL-1 produces leu- kocytosis and activates phagocytes, IL-6 upregulates production of complement and other acute phase pro- teins from mononuclear phagocytes and the liver).
Neuroendocrine effects of surgical stress
The role of the neuroendocrine system is of increasing interest but poorly understood. There are increases in circulating hormones and cytokines, including colony- stimulating factors, corticosteroids and catecholamines, which result in increased neutrophil emigration and production in the marrow as well as pro-inflamma- tory cytokines such as IL-1, IL-2 and IFNγ. Beta- endorphins can increase T cell cytotoxicity in vitro, but the clinical relevance of these changes is unknown.
Immunological impairment after splenectomy I
Severe immunological impairment is caused by splenectomy. Splenic preservation should be attempted whenever possible. Splenectomy removes both sec- ondary lymphoid tissue in the white pulp and a major phagocytic site for the removal of senescent erythro- cytes, opsonised bacteria and intracellular parasites. The spleen is a major site of antibody production, particularily IgM, and is a reservoir of lymphocytes. Splenectomy, therefore, results in a T cell lymphocytosis and an impaired antibody response to the polysaccharide antigens of bacterial capsules.
The result is an increased susceptibility to overwhelming bacterial sep- sis, especially in children. Estimates of risk vary, but the risk is especially high in children less than four-years- old, and in the first few years after splenectomy. It is likely that the underlying disease influences progno- sis, because the risk is greater after splenectomy for pathology, e.g. thalassaemia, than for trauma.
Immunological impairment after splenectomy II
Infections may present insidiously, then rapidly deteriorate. Encapsulated bacteria such as Streptococ- cus pneumoniae, Haemophilus influenzae and Neisseria meningitidis predominate, because antibodies to bacter- ial polysaccharide capsules are important in defence. Mortality from infection is up to 50–70%. Most children receive prophylactic penicillin for five years postsplenectomy, but practice varies in adults, and compliance with long-term therapy may be a prob- lem. Many patients carry prophylactic antibiotics for self-medication, and all need education on the risks and importance of rapid presentation of symptoms to a doctor.
All splenectomised individuals should be immunised with polysaccharide vaccines against Pneumococcus, Neisseria meningitidis (A & C) and Haemophilus influenzae B. These are best given ten days before splenectomy (when a functional spleen is present) or, if pre-immunisation is not possible, two weeks after surgery. They should carry a warning card. If they are traveling abroad they will require additional meningococcal vaccination to cover ACWY strains of Neisseria and appropriate prophylaxis for malaria.
Controlling immunosuppression in the surgical patient
Attempts can be made to reduce immunosuppression after surgery. Homeostasis and pain control reduces any potential neuroendocrine effects. Avoiding ischae- mia improves entry and function of immune effector cells and reduces the likelihood of bacterial infection.
Early wound closure and removal of drains reduces potential portals of bacterial entry. Blood transfusion should be minimised where possible (to reduce possi- bility of blood-borne transmission of infection and the putative immunosuppressive effects of transfusion). It is also helpful, wherever possible, to avoid use of broad spectrum antibiotics which alter normal flora in the gut and increase translocation of pathogens. Nutritional support may be important in some procedures.
Nutritional support in surgery
Nutritional support (calories and protein) to meet the increased metabolic needs following surgery may reduce immunosuppression, particularly in gastro- intestinal procedures. Parenteral nutrition does not appear to have any clinical benefit despite correction of nitrogen balance, perhaps due to mucosal atrophy in the gut, and the invasive procedure itself increases the risk of iatrogenic sepsis. Enteral arginine supplements produce improvements of in vitro tests of lymphocyte function in burns patients which may be of clinical benefit, as may omega-3 fatty acids.
Enteral feeding does not produce the mucosal atrophy associated with parenteral nutrition and thereby may reduce the translocation of pathogens across the gut mucosa and maintain local mucosal IgA secretion. Enteral, but not parenteral, glutamine supplementation may improve mucosal integrity and aid macrophage and lymphocyte function. These interventions have yet to be subjected to double-blind clinical trials of efficacy.
Immunodeficiency in uraemia
Both uraemia and haemodialysis lead to an immuno- compromised state. Infections are a major cause of mortality in renal failure. Vascular access and cuta- neous staphylococcal carriage result in increased risk of infection. Haemodialysis membranes may acti- vate the alternative pathway of complement, leading to C5a generation which affects neutrophil function and causes transient peripheral pooling in the lungs. Metabolic derangement impairs cellular function, and dryness and ulceration of mucosal barriers increases translocation of bacteria. T cell lymphopaenia occurs with impaired proliferation, depressed DTH skin test responses, and some impairment of antibody responses to vaccination.
Imunodeficiency in nephrotic syndrome
Nephrotic patients have a peculiar susceptibility to pneumococcal sepsis. Loss of IgG (180kD) may be relevant in some patients. IgM is generally retained due to its larger size. Complement factor B may also be lost in the urine. Raised complement C3 and C4 levels are usually seen in the nephrotic syndrome due to compensatory hepatic and mononuclear phagocyte production. There is no such feedback regulation of IgG, and low IgG levels persist. There is a demon- strable defect of opsonisation and phagocytosis in vitro, reflecting impairment of antibody, complement and neutrophil function.
Immunodeficiency in connective tissue diseases
Primary immunodeficiencies predispose to auto- immunity, but patients with autoimmune diseases are often immuno-compromised as a consequence of the disease itself, as well as immunosuppressive drug ther-apies. Patients with SLE have acquired abnormalities of complement due to consumption by immune com- plexes and may have dysregulated polyclonal antibody production. Patients with rheumatoid arthritis may have secondary abnormalities of neutrophil function which may predispose to staphylococcal infection, possibly by immune complex formation altering neu- trophil function. Despite these observations, the use of potent immunosuppressive drugs is the major modality of treatment in patients with CTD.
Immunodeficiency in malnutrition
Malnutrition is the most common cause of immu- nodeficiency worldwide, increasing childhood and perinatal mortality from infectious diseases, such as measles. The metabolic demands of established infec- tion (negative nitrogen balance) further compromise the infected host. Impaired DTH, decreased cytokine production, reduced T cell numbers and proliferation to antigen or mitogens is seen. Vaccine responses and total IgG levels are often normal in mild malnutrition, but impaired in severe cases; however, IgA levels often fall. C3 levels fall due to reduced hepatic synthesis and consumption. Neutrophil chemotaxis and opsonisa- tion may be normal but bacterial killing is impaired.
Immunodeficiency as a result of infection
Some impairment of immune responses is common after viral infections where transiently reduced T cell function and DTH anergy are often found (measles, Hep B, EBV, CMV, rubella). The clinical relevance of these functional alterations is not clear, although clearly some viruses gain a survival advantage by sup- pressing host antiviral responses. Herpes viruses (EBV, CMV) appear to directly suppress T cell cytokine pro- duction (IFNγ) and proliferation. Specific antibody production is unimpaired, yet autoantibody produc- tion may be increased.
HIV infection is a special case which causes T cell depletion (and thus causes secondary B cell malfunc- tion) by a combination of direct cytotoxicity and immune-mediated CD8 positive cytotoxic attack on infected T cells and APC. This may eventually lead to clonal exhaustion of T cell precursors (possibly by direct infection of T cell progenitor cells) and eventual loss of antigen specific T cells, leading to total immu- noparesis. Full discussion of the possible pathogenesis of immunodeficiency in HIV infection is beyond the scope of this chapter. Some bacterial infections (TB, leprosy) and fungal infections (Aspergillus) can also cause reduced T cell and macrophage function.
Immunodeficiency as a result of malignancy
An immunocompromised state is often found in dis- seminated lymphoid and non-lymphoid malignancy. Leukaemias and lymphomas cause reduced DTH and mitogen T cell responses, sometimes with impairment of antibody production. CLL can cause hypogamma- globulinaemia and infections, and may require intra- venous immunoglobulin (IVIG) replacement. The host is immunocompromised by radiotherapy, chemo- therapy or splenectomy. Hodgkin’s disease suppresses T cell function and specific antibody responses to carbohydrate antigens by an unknown mechanism, but IgG levels are normal. Myeloma impairs T and B cell function by an unknown mechanism, thus despite normal or elevated IgG levels (which may be predom- inantly monoclonal paraprotein) specific antibody responses to pathogens and vaccines are impaired. Bacterial pneumonia is common.
Premature children have insufficient maternal IgG transfer (predominantly occurs in the last few weeks of pregnancy) and may have transient hypogammaglob- ulinaemia until endogenous production of immuno- globulins restores normal IgG levels at 6–9 months of age. Phagocytosis, T and B cell function, chemo- taxis and complement levels are also impaired in com- parison with normal neonates. IgA production may not reach adult levels until five years of age in many otherwise normal children. Responses to polysacchar- ide antigens are generally poor in normal children before two years of age.
In old age some impairment of immunity is sug- gested by the increased incidence of infections, mono- clonal paraproteins, autoantibodies, DTH anergy, and reduced antibody responses to vaccines and lympho- proliferative disorders. This is reflected in decreased T cell numbers, decreased T cell proliferation and cytokine production. Macrophages are also impaired, with decreased cytokine production or responsiveness. B cell numbers tend to increase with age, while IgE production reduces and many allergies remit.
Immunodeficiency as a result of metabolic disturbances
Diabetes (susceptible to staphylococci and fungi) and cirrhosis (Escherichia coli peritonitis) result in ill- defined defects in cell-mediated and humoral immun- ity. The susceptibility is probably multifactorial and would include tissue ischaemia, increased glucose levels, altered glycosolation of immunoglobulins, cytokines, and other proteins.
This is probably the most common iatrogenic immuno- compromised state. Some drugs have immunosuppres- sive properties which are incidental to their primary
usage (e.g. hydroxychloroquine, dapsone, some antibi- otics and phenytoin).
Some immunodeficient states are inherited, although the expression of the immunodeficiency may in some cases be triggered by environmental triggers at a later stage in life such as EBV in X-linked lymphoprolifera- tive disorder (XLP). These immunodeficiencies may present with unduly prolonged, recurrent or severe infections in childhood or adulthood (Table 6.15). The genetic bases of many are now known (Fig. 6.11). Some of the immunodeficiency states may initially present as surgical complications (e.g. deep seated abscess and inflammatory bowel disease in chronic granulomatous disease; acute abdomen in hereditary angioedema, etc).
Innate immunodeficiency: Phagocyte immunodeficiencies
Defects in neutrophil function include chronic granulo- matous disease (CGD), where there is a genetic abnor- mality in a subunit of the cytochrome b558 enzyme complex (NADPH oxidase). This complex produces oxygen free-radicals in neutrophil and monocyte cytoplasmic phagosomes which kill pathogens in con- junction with hydrogen peroxidase. Some bacteria or fungi produce the enzyme catalase which neutralises hydrogen peroxidase, thus CGD patients get recur- rent, deep-seated and severe infections with catalase positive Staphylococcus, Salmonella and Aspergillus.
CGD is usually X-linked and manifest in males, with female carriers, although autosomal forms exist which may present later in adult life. Patients usually have problems in early childhood and often require surgical drainage of deep abscesses. Prophylactic antibiotics to cover Staphylococcus and Aspergillus are necessary, sometimes augmented with subcutaneous injections of the neutrophil-activating cytokine IFNγ.
Innate immunodeficiency: Leucocyte adhesion deficiency
LAD-1 results from the deficiency of the CD18 chain integrin component of the adhesion molecules LFA-1 (CD18/CD11a), CR3 (CD18/CD11b) and CR4 (CD18/ CD11c). Deficiency results in abnormal neutrophil adhesion and complement receptors, and failure to form pus at the sites of infection because of impaired migration across inflamed endothelium (diapedesis).
Primary antibody deficiency I
Patients with the severe combined T and B cell immunodeficiencies (SCID) will not survive into adulthood without bone marrow transplantation (BMT), which may well restore a functional immune system. SCID will not be mentioned further here. The most common clinical problem resulting from primary antibody deficiency encountered by surgeons is excision of bronchiectatic lung tissue, or ENT sinus drainage procedures for persistent sinus disease. Most primary antibody deficiencies are disorders of B cell develop- ment or function with impaired or absent antibody production. Some have minor abnormalities of T cell function and have an increased incidence of auto- immunity and malignancy. The most common type, common variable immunodeficiency (CVI), has a prevalence of 12–20 per million.
Primary antibody deficiency II
Primary antibody deficiency (PAD) presents with pneumonia, sinus and gastrointestinal infections due to the absence of IgG and IgA. Lack of IgA leads to sus- ceptibility to mucosal pathogens entering by the respira- tory and gastrointestinal tract. Patients get: respiratory infections with encapsulated bacteria (Haemophilus influenzae (usually untypable), Streptococcus pneumo- niae) and mycoplasma; gastrointestinal infections with Giardia, Campylobacter and Salmonella; Mycoplasma arthritis; and rarely Neisseria meningitidis meningitis (more usual in complement deficiency).
Primary antibody deficiency III
Antibody deficient patients usually clear viral infections normally, although they never develop antibody-mediated resistance against re-infection. They usually do not get infections with opportunists or unusual organisms, except in hyper-IgM syndrome (HIGM) due to a T cell defect, where pneumocystis pneumonia and cryptosporidial gastroenteritis occur. X-linked agammaglobulinaemia (XLA) patients occa- sionally get fatal enteroviral meningoencephalitis and myositis. Since XLA is a B cell disorder, this sug- gests that antibodies mediate important enteroviral protection.
Primary antibody deficiency IV
Treatment consists of intravenous immunoglobu- lin (IVIG, predominantly IgG) replacement prepared from a large pool of healthy donors screened for infectious disease. We cannot replace IgA yet, and a major problem with monomeric IgG infusions is poor penetration onto mucosa and the rapid enzymic destruction once there (normal dimeric IgA is specifically secreted and protected by the “secretory component”).
Adjunctive surgery or prophylactic antibiotics may be necessary, especially if end-organ damage such as bronchiectasis or chronic sinusitis becomes established because of delayed diagnosis.
Patients with CVI have an increased risk of malignancy and autoimmunity. Gastric carcinoma, associ- ated with achlorhydria, gastric atrophy and pernicious anaemia, is increased 40-fold, and extranodal B cell lymphomas are increased 100-fold. Occasionally, T cell lymphomas occur. PAD patients may also have auto- immune thrombocytopaenias (which must be mediated by non-antibody mechanisms) and may require splenectomy if unresponsive to treatment.
Primary antibody deficiency V
Diagnosis of autoimmune disease or infections may be difficult because serological tests are useless, since patients do not make antibodies. Vaccinations are unlikely to be useful, and live vaccines are avoided because a concur- rent deficit of cell-mediated immunity in some anti- body deficient patients may lead to fatal dissemination of the vaccine.
PAD patients with low IgG levels (pre- or post- treatment) are susceptible to mycoplasma arthritis. This can result in severe joint destruction and chronic pain requiring operative intervention under cover of tetracyclines.
Sinus disease remains problematic in many patients. Drainage procedures such as Caldwell-Luc procedures were generally unsuccessful in the past. The role of new endoscopic procedures such as FESS (functional endoscopic sinus surgery) remains to be defined.
Regional lymphadenopathy and splenomegaly can occur, particularly in CVI and HIGM. In each case there may be a requirement for excision biopsy of lymph nodes to exclude a lymphoma or other neo- plasm. Approximately one in five CVI patients have a granulomatous variant (GAD), with splenomegaly, reduced CD4 positive T cells, raised CD8 positive T cells, low B cell numbers and sarcoid-like non- caseating granulomata in multiple organs. These may cause diagnostic confusion with mycobacteria (anti- body deficient patients are not unduly susceptible to tuberculosis) and other granulomatous disorders (Whipple’s disease, syphilis, toxoplasmosis). Benign reactive nodular hyperplasia of the gut lymphoid tis- sue is present in many antibody deficient patients, and may mimic other intra-abdominal pathology.
Immunosuppression by disruption of the immune response to a specific antigen is the ultimate goal of immunologists and surgeons and may result from improved understanding of the role of clonal anergy and deletion in the maintainance of self-tolerance and tolerance to foreign antigens (including MHC). Meantime, patients have to live with the inadequacies and potentially fatal side effects of pharmacological immunosuppression. Many of the initial immunosup- pressive drugs were first used in cancer chemotherapy because of their toxicity against proliferating cells. This led to blanket immunosuppression and high incidence of side effects. Immunosuppression can be achieved by targeting various mechanisms:
• depleting lymphocytes
• diverting lymphocyte traffic
• blocking/modifying lymphocyte response
• inhibiting cell proliferation
• inhibiting metabolism
Corticosteroids cross the cell membrane to bind to cytosolic glucocorticoid receptors, which translocate to the nucleus to bind to glucocorticoid responsive elem- ents which activate gene transcription over 6–12h. They also have multiple anti-inflammatory effects on neutrophils, vascular adhesion, cytokine production, wound repair, 5-lipo-oxygenase and cytokine production such as IL-1.
Corticosteroids also affect B cells (reduce antibody secretion, promote apoptosis) and T cells (reduce cytokine secretion and proliferation). Their big disadvantage comes from side effects includ- ing cushingoid features, hypertension, peptic ulcer- ation, poor wound healing, osteoporosis, myopathy, cataracts, stunted growth, acute pancreatitis, avascular necrosis of bone, hypoglycaemia and diabetes, acne, as well as increased susceptibility to infections.
IL-1RA is anti-inflammatory in RA but is not helpful in transplantation.
15-Deoxyspergualin (15DS) binds to heat shock pro- teins (HSP) and interferes with their ability to act in the loading of antigenic peptides onto HLA molecules. 15DS only has a modest effect in transplantation. Cytokines and costimulatory molecule expression are unaffected, but multiple toxicities including leucope- nia limit its utility. 15DS also suppresses B cell prolif- eration, inhibiting antibody formation.
Nucleoside synthesis inhibitors: Azathioprine
Azathioprine is a cytotoxic drug used in transplanta- tion, autoimmune diseases and vasculitis. Azathioprine is a precursor of 6 mercaptopurine which under- goes intracellular conversion to the purine analogue thiosinic acid which inhibits DNA/RNA synthesis. The enzyme thiopurine methyltransferase (TPMT) metabolises azathioprine; the risk of myelosuppres- sion is increased in those with a low activity of the enzyme, particularly in the very few individuals who are homozygous for low TPMT activity. It kills lym- phocytes, phagocytes, megakaryocytes and erythrob- lasts and any other proliferating cell indiscriminately. In transplantation (and autoimmunity), antigen specific, clonally proliferating cells are killed more rapidly than resting cells. The main side effects are thus infections, bone mar- row suppression, hepatotoxicity, hair loss and late malignancy.
Nucleoside synthesis inhibitors: Mycophenolate Mofetil
It is a prodrug that is rapidly hydrolysed to the active drug, mycophenolic acid (MPA) which is a selective, non-competitive and reversible inhibitor of inos- ine monophosphate dehydrogenase (IMPDH) – an important enzyme in the de-novo pathway of guanine nucleotide synthesis. B and T lymphocytes are highly dependent on this pathway for cell proliferation, while other cell types can use salvage pathways. MMF select- ively inhibits lymphocyte proliferation and functions by blocking purine synthesis and is an alternative to azathioprine. Mycophenolic acid also inhibits smooth muscle proliferation and may be useful in preventing chronic vascular rejection.
Cytotoxic therapy: Total body irradiation
Total body irradiation (TBI) was used briefly in human renal transplantation and can induce toler- ance, but has major side effects and may need rescue progenitor cell transplantation. TBI kills lymphocytes indiscriminately in the secondary lymphoid tissues, and the lymphopenia blunts graft rejection responses. Occasionally, Y-mantle irradiation may be used for highly sensitised recipients.
Cytotoxic therapy: Cyclophosphamide
Cyclophosphamide is used in autoimmune diseases, vasculitis and in higher doses in ablation of recipient marrow pre-BMT (conditioning). It is an alkylating agent which chemically modifies the bases of DNA to prevent normal replication by cross-linking. Thus it is both mutagenic and cytotoxic. Cyclophosphamide’s main side effects include mucositis, infertility, infec- tion, bone marrow suppression, hair loss and malig- nancy (bladder and other late tumours). In high doses the drug Mesna is used to neutralise the bladder tox- icity of the acrolein metabolite.
Anti T-cell proliferation/Activation drugs: Cyclosporin
CsA is a lipid-soluble fungal derivative, and was the first T cell specific drug which inhibits cytokine synthesis and clonal proliferation in T cells. It is indis- criminate, because it inhibits all T cells in the early calcium-dependent G0 phase of activation, not just
antigen specific cells. CsA binds to the cell surface receptor cyclophylin, becomes internalised to bind to calcineurin (a calcium/calmodulin-dependent phos- phatase) leading to transcription factor inhibition (preventing NFAT dephosphorylation and nuclear translocation). It thus suppresses cytokine and cytokine receptor production (e.g. IL-2/IL-2R, IL-3, IL-4, IFNγ, TNFα, GM-CSF). Because CsA inhibits T cell proliferation and cytokine production it impairs B cell and macrophage T helper-dependent functions. CsA also inhibits B cells and macrophages directly and acts synergistically with corticosteroids. The main side effects include nephrotoxicity, hepatotoxicity, hypertrichosis, gingival hyperplasia, tremor and infec- tion. There is also an increased incidence of neoplasia, especially lymphomas.
Anti T-cell proliferation/Activation drugs: Tacrolimus
FK506 (tacrolimus) is similar to CsA, acting on the G0 phase of proliferation, but binds to a separate FK506- binding protein (FKBP) which then interacts with cal- cineurin. FK506 has apparent advantages over CsA in liver transplantation but has similar toxicity.
Anti T-cell proliferation/Activation drugs:
Sirolimus (Rapamycin) and Everolimus have a differ- ent mode of action. They have no effect on calcineurin activity. The Rapamycin – FKBP-12 complex blocks a signal transduction pathway triggered by ligation of growth factors and IL-2. It, therefore, allows activation of T lymphocytes by antigen but blocks proliferation by arresting the cells in G1 phase of cell cycle. These cells then die by apoptosis. The Rapamycin: Immu- nophilin complex binds and inhibits the protein kinase named mTOR (mammalian target of Rapamycin). Rapamycin can reverse early allograft rejection.
Monoclonal antibodies: Anti-T cell mAb
Humanised monoclonal antibodies (mAb) promise to be more specific than polyclonal heterologous antisera such as antilymphocyte (ALG) or antithymocyte glob- ulins (ATG) (Table 6.17). New technologies for rapid production of antibody-like molecules such as phage display will probably improve availability. mAb can be divided into those which deplete cell numbers (by lysis or inducing redistribution) and those which block important cell surface costimulatory and adhesion molecules, or a combination of these effects (as with anti-CD3 (e.g. OKT3) or anti-CD4mAb).
Problems include the development of ‘resistance’ due to neutral- ising antibodies against the foreign protein sequences of the species of origin of the mAb. This can be reduced by ‘humanising’ the antibody (i.e. replacing with a human Fc protein sequence but retaining the binding specificity of the original mouse/rabbit mAb). The properties of the final molecule can be adjusted since the Fc portions of each human immunoglobulin isotype have different abilities to activate complement or bind to cellular receptors.
Side effects of mAb include; cytokine release with the first dose (pyrexia, flu-like symptoms, rigors or even hypotension and pulmonary oedema), infections – including opportunistic infections (CMV, fungi), HSV reactivation, and late onset EBV lymphoproliferation or B cell lymphomas (especially with OKT3).
Antibodies can be directly conjugated to toxins for immunotherapy of tumours, but anti-IL-2R-toxin conjugate has been used experimentally for immuno- suppression. In the future, combinations of antibodies are likely to be used.
Cytoreductive antibodies include Campath (CDw52, pan-leucocyte), anti-CD3 (OKT3, anti-T cell murine IgG2a mAb against the CD3ε chain of the TCR complex), anti-CD2 and anti-CD45 (used to deplete passenger APC in experimental grafts).
Anti-costimulatory/adhesion ligand mAb
These antibodies interfere with antigen presentation, T cell proliferation and T/B cooperation at an early stage of T cell activation. They include anti-CD80 (B7.1), anti-CD4, anti-CD25 (IL-2 receptor), anti- LFA-1(CD18/CD11a), anti-ICAM-1(CD54), and anti- CD28. In addition to mAb, chimaeric molecules can be produced using molecular techniques consisting of a receptor or its ligand attached to a human Fc immuno- globulin tail. These agents block the physical inter- action between a receptor/ligand pair. One example is CTLA-4-Ig (which binds to B7.1 and blocks its inter- action with CD28 or CTLA-4 on T cells). Anti-CD4 produces infectious tolerance which can be adoptively transferred by T cells from one animal to another. Anti- CD8 have not been tried, since CD8 cells are not essen- tial for experimental transplant rejection and have had minimal effects in animal models. CTLA4-Ig prolongs allograft survival in animals and can induce tolerance to xenogeneic human pancreatic islet grafts.
Anti-LFA-1/ICAM-1 combination therapies inter- fere with antigen presentation/costimulation and cell adhesion in lymphocytes and phagocytes, and can produce experimental tolerance in primates. ICAM- 1 mAb are being trialled for treatment of rejection and GVHD in humans. Anti-IL-2R mAb are effective in prevention of renal allograft rejection. Anti-IL-4 and
anti-IL-4R may prolong experimental allograft sur- vival, suggesting that other such combinations may be useful. However, prolongation of survival is not the establishment of long-term tolerance, and the redun- dancy of the cytokine network makes single agent therapy unlikely to be sufficient.
Organ transplant recipients
There is a three-fold increase in neoplasia in transplant recipients, usually in young adults about 60 months after transplantation and which is related to the degree of pharmacological immunosuppression, especially with agents such as OKT3. Kaposi’s sarcoma is 500 times more common in renal recipients than age- matched controls. Kaposi’s sarcoma tend to occur ear- lier (mean 23 months), lymphomas later at 37 months, and squamous carcinomas of vulva or perineum after 100 months. Carcinoma of the cervix is increased 14-fold and is human papilloma virus-associated (HPV16 and 18).
Immunosuppressed solid organ recipients also tend to get ultraviolet and virus-induced squamous tumours of skin and lip (human papil- loma virus type 5) which are more aggressive than in immunocompetent hosts, but in contrast do not have an increased incidence of basal cell carcinoma. Non- Hodgkin’s lymphoma (NHL) is increased 28–49 fold, and these are mostly of B cell origin and EBV positive. These statistics demonstrate the importance of a functional immune system in the surveillance of virally- induced tumours. Thus some lymphomas regress on reduction of immuno-suppression (or acyclovir), but as a result the graft may be lost.
Renal transplantation is used for end stage renal failure with 90% loss of nephrons and severe uraemia. glomerulonephritis, pyelonephritis, interstitial neph- ritis, adult polycystic disease and diabetes are the most common causes in all age ranges. Usually an ABO-matched HLA Class I and Class II compatible deceased donor (maintained on life support and clin- ically brain-stem dead) is used, although now 30% of renal transplants are from living donors. HLA- matching is not the only factor important for successful grafting (Tables 6.9 and 6.10, Fig. 6.8); adequate cold preservation, with minimal cold ischaemic time (up to 48 h) and minimal warm ischaemia during reperfusion, is also important. Heart, liver and two kidneys may be retrieved, flushed with cold storage solution and stored on ice. Lymph node and spleen are taken from the donor for tissue typing (because they are rich in lympho- cytes). The kidney is placed in an extraperitoneal position in either the right or left iliac fossa. Immuno- suppression is started 4â€“12h before transplantation. The most appropriate immunosuppressive regimen will vary depending on the risk factors present includ- ing cold ischemia time, age, recipient sensitization level, prior transplant and others. One-year survival is almost 90% (0â€“3/6 HLA mismatch). The five-year kidney graft survival is dependent on number of HLA mismatches and is around 68% for 0/6 match and is 55% for 6/6 mismatch.
Bone marrow transplantation
The ideal is to obtain complete HLA-A, -B, -DR, -DQ matching by serological and PCR techniques for the best possible match using first degree relatives or volun- teer donor panels. There is a much higher risk of graft- versus-host disease than in solid organ grafts, because of the transplantation of immunocompetent donor T cells. Best results are seen with haplotype-matched first degree relatives. Unrelated donors, who inevitably include other antigen mismatches even if HLA matchappears good, have a worse prognosis. Therefore, if the donor is unrelated, only a single minor mismatch is allowed, whereas if the donor is related a single anti- gen major mismatch may be accepted.
Cardiac transplantation for end-stage cardiac disease began in 1967 and now has a 75% five-year survival. HLA matching is usually impractical due to limited organ preservation times (4â€“6 h) and low organ avail- ablility, but a known positive anti-HLA antibody cross-match contraindicates transplantation.HLA-DR matching may reduce early cardiac rejec- tion, but it is uncertain whether this improves sur- vival or reduces the accelerated atherosclerosis seen in 40% of recipients at five years (also seen in chronic rejection of kidneys and liver). It is hypothesised that accelerated atherosclerosis may be immune-mediated and secondary to endothelial damage, since it is worse in MHC Class I mismatches and allosensitised individuals. Antiplatelet drugs may slow progression, but retransplantation is the main therapeutic option. Immunosuppressive requirements are stricter than for renal transplants, with CsA, steroids, azathioprine, and ATG (antithymocyte globulin) or Campath (a lytic monoclonal antibody directed against surface CD52 on all leucocytes) being used. One or more rejection episodes occur in 85% of recipients. Weekly endomyocardial biopsies may be performed to moni- tor rejection. Focal and perivascular interstitial lymphocyte infiltrates (neutrophils if severe) are seen in rejection, similar to renal rejection. Rejection may be aborted with steroids, ATG or OKT3. Infections are the most common cause of death within three months of transplantation.
Common indications for lung tranplantation include COPD, interstitial lung disease, cystic fibrosis, and alph-1 antitrypsin deficiency. Matching criteria are the same as for heart-lung transplantation, with a three- year survival of 60% for COPD and cystic fibrosis. No hyperacute rejection has been documented, but there is no time to cross-match anyway due to time con- straints on organ preservation (6h). Acute cellular rejection produces perivascular lymphocytic infiltrates and bronchiolitis. Chronic rejection produces bronchi- olitis obliterans.
This is used for end-stage liver failure. The most common indication in children is biliary atresia and in adults primary biliary cirrhosis. Cross-matching is not routinely done for anti-HLA antibodies although a positive cross-match for anti-HLA Class I anti- bodies may predispose to chronic rejection. The degree of HLA-A, -B, -DR, -DQ mismatch may determine the need for immunosuppression retrospectively. Re-infection of the graft is a particular problem with liver transplantation for end-stage hepatitis B or C infec- tion in the immunosuppressed recipient. Autoimmune diseases such as primary biliary cirrhosis (PBC) or autoimmune chronic active hepatitis (AICAH) rarely recur in the graft. CsA, corticosteroids, azathioprine, tacrolimus and OKT3 are used for immunosuppression and treatment of acute rejection. Chronic rejection produces intraluminal biliary fibrosis analogous to the vasculopathy in other types of organ transplantation.
Small bowel transplantation
This is a potential solution for intestinal failure. The small bowel contains large amount of lymphoid tissue in Peyerâ€™s patches and mesenteric lymph nodes, thus rejection and graft-versus-host disease are a greater problem than with other organs, and the bowel is very intolerant of ischaemia. In addition, the infection risk is high because the bacteria in the gut translocate eas- ily across damaged mucosa, causing sepsis. A com- bined small bowel and liver graft may be performed. Graft survival is improved by CsA, prednisolone, azathioprine, OKT3, and tacrolimus. Prostaglandin (PGE1) infusion may be beneficial. Graft survival of up to 75% has been reported at one year. Cadaveric donors with minimal graft cold ischaemia (6 h) are used. Acute and chronic rejection occurs.
This is usually carried out for juvenile-onset diabetics who have concominant renal failure and require kidney transplantation in addition. The aim is to prevent the development of other microangiopathic complications.Whole organ pancreatic transplantationThe kidney and pancreas from the same donor are usually transplanted simultaneously, one organ into the right iliac fossa, the other into the left iliac fossa. The use of pancreatic transplantation alone to prevent the complications of diabetes is increasing.
Pancreatic islet transplantation
In 2000, Dr. James Shapiro and colleagues published a report describing seven consecutive patients who achieved euglycemia following islet transplantation using a steroid-free protocol and large numbers of donor islets, since referred to as the Edmonton proto- col. This protocol has been adapted by islet transplant centers around the world and has greatly increased islet transplant success. For an average-size person (70 kg), a typical transplant requires about one million islets, extracted from two donor pancreases. Healthy islets are isolated from a donor pancreas, purified, and then infused through a small tube into the portal vein of the liver. ABO and anti-HLA Class I cross-matching are essential for this tissue. No other matching is prac- ticable for vascularised gland or isolated islet cells. The latter approach excludes passenger leucocytes and reduces rejection potential. The islets themselves do not express costimulator molecules and thus do not invoke rejection (and may even tolerise the host). Loss of APC is accelerated experimentally by in vitro culture in 95% oxygen or UV irradiation before trans- plantation. While significant progress has been made in the islet transplantation field, it still remains an experimental therapy.
No matching is required, because this non-vascular graft is a relatively immunoprivileged site. The cornea can be stored for 28 days. HLA-A and -B matching is used only for high risk grafts with previous rejection or
Heart lung transplantation
The main advantage of this approach (for lung dis- ease, cystic fibrosis and pulmonary hypertension) over lung transplantation is not immunological but preser- vation of tracheal blood supply. Two-year survival for heart-lung transplants approaches 50%. HLA match- ing is impracticable due to the cold ischaemia time limit of 6h. HLA-A, -B, -DR, -DQ matching may determine the level of immunosuppression required subsequently. Immunosuppressive regimes are similar to those of heart transplantation. The lungs are very vascular and susceptible to immunological attack, showing the first signs of rejection. Monitoring of graft function (FEV1, PO2, CXR), bronchiolar lavage and transbronchial biopsy for the interstitial perivas- cular mononuclear infiltrates of rejection are used. Obliterative bronchiolitis occurs in 50% of recipients at 8â€“12 months as result of chronic rejection with intimal vasculopathy. Obliterative bronchiolitis is treated with steroids and ATG, but the prognosis is poor.
Immunosuppression in transplantation
In the absence of mechanisms for producing donor- specific tolerance, we are left to fall back on general impairment of the host immune responses in order to prevent immune-mediated graft rejection. These drugs, however, predispose the patient to infections (Table 6.11) and neoplasia. Post-transplantation EBV positive lymphoproliferative disorders are increased with prolonged immunosuppression (particularly with ATG, ALG, OKT3, CsA) and in those with primary EBV infections post-transplantation.The mode of action of immunosuppressive drugs is described later. Azathioprine was first used in renal transplantation in the 1960s. Corticosteroids are still used for their multiple anti-inflammatory and immunomodulatory effects. CsA and, more recently, FK506 (tacrolimus) have markedly improved the outlook in clinical solid organ transplantation. Antilymphocyte immunoglobulins (ALG, ATG) and anti-T cell mono- clonal antibodies (ATG, Campath, OKT3) are effec- tive in T cell depleting bone marrow and treating cellular rejection. Newer drugs such as mycophenolic acid, rapamycin, Brequinar, 15-deoxyspergualin, and antibodies to costimulatory or adhesion molecules on T cell and APC surfaces are promising new alterna- tives, the latter holding out the possibility of specific tolerance induction.
Graft versus host disease
The transfer of immunologically competent T cells (and their precursors) may result in an attack on the host by donor lymphocytes. These cells clonally pro- liferate in the new host. This is a major problem in bone marrow transplantation but is also occasionally seen in solid organ transplantation (particularly small bowel) depending on the number of lymphoid cells in the graft. Acute graft-versus-host-disease (GVHD) (onset 100 days post-transplant) may resolve with treatment. Chronic GVHD (100 days) is an aggressive disease with autoimmune-like features and multi- ple organ involvement with fibrosis. Preventative drug strategies, including methotrexate and CsA, are manda- tory for allogeneic bone marrow transplantation, and some estimation of risk can be made from specialised quantitation of precursors of cytotoxic recipient-reac- tive T cells in the donor (CTLPp). The mechanisms regulating the balance between long-term chimaerism (where donor lymphoid cells persist in the new host without damage), and GVHD are unknown.
Tumour immunobiology I
It has been assumed for many years that the immune system is important in the suppression of neoplastic growth. Immunodeficient or immunosuppressed individ- uals (particularly those with T cell dysfunction or renal recipients treated with OKT3) have a clearly increased incidence of certain tumours (viral(EBV)-induced B cell lymphomas and non-viral lymphoid tumours). Some primary antibody-deficient patients (CVID) also have an increased incidence of neoplasia (particularly stomach and B cell non-Hodgkinâ€™s lymphoma (NHL)). HIV infection has increased knowledge of the role of intact immunity in tumour suppression and refocused attention in the potential role of oncogenic viruses such as HPV 16/18 (cervical cancer), EBV (Burkittâ€™s lymphoma), HTLV-1 (T cell leukaemia) and HSV-8 (Kaposiâ€™s sarcoma) in producing tumours in humans.