Syb's Immunology Year 2

Sybghat Rahim

Year 2 Immunology Hypersensitivity and Allergy

by Professor Sebastian Johnston

About 50% of us have allergic sensitisation (atopy), and 30% of us have allergic disease symptoms (allergy).

We all make ‘appropriate’ immune responses to things that may harm us, such as viruses, bacteria, fungi and parasites. These immune responses are necessary to eliminate the pathogens and prevent or limit disease.

There may be concomitant tissue damage as a side effect, but as long as the pathogen is eliminated quickly there is minimal damage and it is repaired easily. The immune responses involve antigen recognition by antibodies and cells of the immune system.

Hypersensitivity reactions occur when immune responses are mounted against harmless foreign antigens (allergy, contact hypersensitivity), or autoantigens (autoimmune disease), alloantigens (serum sickness, transfusion reactions, graft rejection), and infectious agents (e.g. TB or Leprosy that are not cleared and lead to chronic immune mediated damage).

A common feature in all of these hypersensitivity reactions is inflammation. This is a consequence of the action of immune molecules (antibodies, complement, cytokines, etc) and immune cells on sites of injury and/or infection. There is local vasodilation and increased blood flow to allow increased access of cells to the affected area, and there is also increased vascular permeability. This presents as redness and heat.

Inflammatory mediators, cytokines and cells cause swelling and tissue damage may cause pain.

Increased vascular permeability is caused by C3a, C5a, histamine and leukotrienes. Cytokines such as IL-1, IL-6, IL-8, IL-2 and TNF are chemokines. The inflammatory cells are trafficked by chemotaxis. Neutrophils, macrophages, lymphocytes and mast cells are activated.

Hypersensitivity Classification by Gell & Coombs Type I: Immediate Hypersensitivity, IgE mediated.

Type II: Antibody-dependent Cytotoxicity Type III: Immune Complex-Mediated Type IV: Delayed Cell Mediated, T cell mediated.

Type I is immediate hypersensitivity .This includes anaphylaxis, asthma, rhinitis (seasonal or perennial) and food allergies. On a primary antigen exposure, IgE antibodies are produced and they bind to mast cells and basophils. You are now sensitised to the antigen. On the secondary antigen exposure there are even more IgE antibodies produced and the antigens cross-bridge IgE molecules on the mast cells and basophils to cause degranulation. This releases all the inflammatory mediators and there can be a systemic reaction.

Type II is antibody dependent hypersensitivity. Clinical presentation of this type of hypersensitivity depends on the target tissue involved. Organ-specific autoimmune diseases are type II reactions, these include myasthenia gravis (ACh receptor antibodies in skeletal muscle = muscular weakness and tiring), glomerulonephritis (anti-glomerular basement membrane antibodies = renal failure), and pemphigus vulgaris (antibodies to epithelial cell cement = blistering skin eruption). There are also autoimmune cytopenias (blood cell destruction), including haemolytic anaemia, thrombocytopenia, and neutropenia. Haemolytic disease of the newborn (rhesus positive child born to rhesus negative mother = antibodies) is another example of a type II reaction.

Type II reactions can be caused by drug allergies, hyper-acute graft rejection, transfusion reactions, pernicious anaemia (intrinsic factor blocking antibodies), and by idiopathic urticaria (antibodies against IgE receptor).

In a type II reaction there can be antibody interaction with the cell surface antigen. Complement activation causes cell lysis and mast cell activation, and inflammation attracts cytotoxic cells such as neutrophils, eosinophils, monocytes and killer cells. Tissue damage results in altered function.

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Sybghat Rahim To diagnose type II hypersensitivities, there are tests that can be done for specific autoantibodies if you have the antigen. There are also immunofluorescence tests where you take the tissue on a slide, add the patient’s serum, and if there are antibodies in the serum it will bind to the tissue and this will be detected by a fluorescent label. Pemphigus can be seen by immunofluorescence. There is also the ELISA test for identified antigens.

Treatments generally involve trying to suppress production of the antibodies. Immunosuppressants, steroids and cyclophosphamide are all used for this. You can also use plasma exchange, where you remove proteins from the patient’s plasma and replace them. A splenectomy could also help, or giving intravenous globulins is also used.

Type III reactions are immune complex mediated hypersensitivity reactions. This is where there is formation of antigen-antibody complexes and deposition of complexes in a tissue. If this builds up and cannot be cleared by the immune system, this activates complement and consequent cell recruitment and consequent inflammation, and it also activates other cascades e.g. clotting. The resultant tissue damage (vasculitis) is seen in diseases like systemic lupus erythematosus (SLE) and vasculititides. The most common places where small vessel vasculitis occurs are in the kidneys, skin, joints and in the lungs.

Type IV reactions are delayed hypersensitivity responses. This occurs in a number of different conditions, like chronic graft rejection (receive tissue graft, body rejects), graft vs host disease (bone marrow graft, bone marrow rejects body), Coeliac disease (small intestine, chronic inflammation due to T cell delayed response to proteins in wheat), contact hypersensitivity (most commonly to nickel in jewellery), tuberculosis & leprosy (infectious agents not cleared = chronic exposure to antigens = delayed response), asthma, rhinitis and eczema (although these three are different because they are Th2 mediated responses with IL-4, IL-5 and IL-13, whereas the others involve Th1 reactions with IFN-γ, so they are slightly different in terms of polarisation).

Type IV delayed hypersensitivity responses are of three varieties in terms of provoking inflammatory responses - Th1, cytotoxic or Th2.

Mechanisms involve transient or persistent exposure to antigens, activation of T cells (which leads to activation of macrophages and cytotoxic lymphocytes), IL-2 production and further proliferation of T cells.

FGF release means fibroblasts also proliferate, causing fibrosis, and macrophages produce TNF, which is the cause of much tissue damage.

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Sybghat Rahim Allergy Common allergens include cats, house dust mites, various pollens and fungal spores. Allergy is very common, and the prevalence of atopy is 50% in young adults in the UK. Severity varies from mild occasional symptoms to severe chronic asthma and life threatening anaphylaxis. Risk factors are both genetic and environmental.

About 80% of atopic people have a family history. It is polygenic, and there are a lot of studies trying to find asthma genes. Over 70 different genes have been linked to asthma and atopy. The most important genes seem to be of the IL-4 gene cluster (IL-4, IL-5 and IL-13 on chromosome 5), which are linked to raised IgE, asthma and atopy. Genes on chromosome 11q (IgE receptor) are also linked to atopy and asthma.

Environmental influences then determine whether you get allergic disease or not. Age is very important, asthma and allergies are increased in children, peaking in teens, and then reducing in adulthood. Gender is another factor, as asthma is commoner in males in childhood and in females in adulthood (hormones therefore may have some influence). Family size is also important, as if you are an only child you are twice as likely to have allergies as a child born into a family of 6, but in that larger family birth order is also important (first born = twice as likely as last born). Exposure to infection is also very important, as early life infections particularly by parasites protect against developing allergies. Animal exposure can achieve the same effect, being exposed to animal microbes. Diet is another important environmental factor - breast feeding, antioxidants and fatty acids are all protective against allergy.

Allergies like hay fever, eczema and asthma have been increasing in the UK over the past 50 years (roughly tripling or more). This is most likely due to changes in our environment, particularly the fact that there is less exposure to infectious disease than before.

Anaphylaxis, urticaria and angioedema are all type I hypersensitivity reactions (IgE mediated). Chronic urticaria can be type II hypersensitivity (IgG binding to IgE receptors, without an allergen being involved). Asthma, rhinitis and eczema are mixed pictures, with some IgE mediated and some type IV (chronic) causes of inflammation.

The expression of allergic disease requires sensitisation to allergens (primary response, usually occurs early in life), and then a subsequent exposure to that allergen again causing a memory response with increased IgE production and T cell activation.

The picture on the right shows e.g. a bronchus. The allergens inhaled are picked up by dendritic cells which reside in the epithelium. They process the antigen, chop it up into small peptides, and present those small peptides to CD4 T cells. The T cells then become specific for that allergen. The naĂŻve T cells can then proliferate in three pathways to Th2 cells, which are involved in allergy (IL-4, IL-5 and IL-13 production). B cells switch from IgM production to IgE production. They are now antigen specific too. They differentiate into plasma cells which secrete IgE specific to the peptide from the allergen inhaled.

On the other hand, if it were an infectious agent being presented instead of an allergen, it would cause Th1 production. T regulatory cells are where inflammation and immune responses are suppressed by a cytokine called IL-10. This is an important way of preventing allergic disease. Th1 and Th2 counter-regulate each other (IFN-Îł suppresses Th2 production, and IL-4 and IL-13 can suppress Th1 responses).

Any time in life after the sensitisation, a subsequent exposure follows the same process. The allergen is processed by dendritic cells and presented to the memory T cells, which then proliferate very quickly to cause an amplified immune response to the allergen. More IL-4 and IL-13 are produced, more IgE is produced, more eosinophils are activated (IL-5), and the IgE binds to mast cells which activates them. Inflammatory mediators are released, which causes the acute or chronic hypersensitivity response.

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Sybghat Rahim Eosinophils make up 2 to 5% of blood leukocytes. They are present in blood, but most reside in tissues. They are recruited during allergic inflammation in response to IL-5. They also switch on increased production of eosinophils in the bone marrow. Eosinophils are easily identifiable - they have a polymorphic bilobed nucleus, and contain large granules (toxic proteins) which lead to tissue damage.

Mast cells are tissue resident cells, and have IgE receptors on their cell surface. When their IgE recognises the antigen they are specific for, this leads to cross-linking, which causes mediator release. Pre-formed mediators include histamine, pro-inflammatory cytokines, and toxic proteins. Newly synthesised mediators include leukotrienes and prostaglandins which cause acute inflammation.

Neutrophils are important cells in virus induced asthma, severe asthma and atopic eczema. Neutrophils form 55 to 70% of blood leukocytes. They are identifiable as their nucleus contains several lobes. Their granules contain digestive enzymes, and also synthesise oxidant radicals, cytokines and leukotrienes.

Asthma Acute inflammation of the airways is mainly caused by mast cell activation/degranulation. The important prestored mediator is histamine. The important newly synthesised mediators include prostaglandins and leukotrienes. Release of these mediators causes acute airway narrowing, vascular leakage (oedema), induction of mucus secretion and contraction of smooth muscle (further narrowing).

Chronic inflammation of the airways is a mixture of a type I response and a delayed response. It is caused by infiltration by Th2 lymphocytes and eosinophils. There is smooth muscle hypertrophy, mucus plugging, epithelial shedding and sub-epithelial fibrosis.

Asthma is reversible generalised airway obstruction leading to chronic episodic wheeze as the main symptom.

Bronchial hyper-responsiveness is another feature (bronchial irritability). Cough is a prominent feature, as well as increased mucus production, breathlessness and chest tightness. Asthma does respond to treatment, which is good. There is spontaneous variation, but can be diagnosed by a reduced and variable peak flow (PEF).

Allergic Rhinitis This is the most common allergic disease. Hay fever is the best known form, which is seasonal with grass and tree pollens. There are also perineal allergens like house dust mites and animals. Symptoms include sneezing, rhinorrhoea (runny nose), itchy nose and eyes, nasal blockage, sinusitis, loss of smell/taste.

Allergic Eczema This is a chronic itchy skin rash due to hypersensitivity, most commonly to house dust mites. Combined with dry skin this can cause cracking of the skin and better penetration of antigens into the skin = immune responses. The flexures of the arms and legs are common places in childhood. Eczema can be complicated by bacterial and (rarely) viral infections (herpes simplex). 50% clears by 7 years, and 90% clears by adulthood.

Food allergy This is very common in infancy. The most common allergens for infants are egg and cow’s milk, but as children get older they usually lose this hypersensitivity. In children and adults, food allergies can also develop, most commonly to peanuts, shell fish, nuts, fruits, cereals, soya, etc. Food allergies can be mild (itchy lips, mouth, angioedema, urticaria), or can be quite severe (nausea, abdominal pain, diarrhoea, anaphylaxis).

Anaphylactic shock This is a severe generalised allergic reaction. It is uncommon, but is potentially fatal, and there are around 200 deaths per year in the UK from anaphylaxis. It is the generalised degranulation of IgE sensitised mast cells.

Cardiovascular effects are vasodilation and cardiovascular collapse. Respiratory effects are bronchospasm and laryngeal oedema. Skin effects are vasodilation, erythema, urticaria and angioedema. GI effects are vomiting and diarrhoea.

Symptoms include itchiness around mouth, pharynx and lips, swelling of the lips, throat and other parts of the body, wheeze, chest tightness, dyspnoea, faintness, diarrhoea and vomiting, collapse, and death if severe and untreated.

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Sybghat Rahim Investigation and diagnosis The most important thing is taking a careful history. Further to this, if the history does not reveal all, skin prick testing can be done. There is also RAST (testing blood specific IgE). It is also a good investigation to measure total serum IgE. Treatment Emergency treatment is an EpiPen (prefilled with adrenaline) and an anaphylaxis kit (adrenaline, antihistamine, steroid). If the patient has time to get to A&E of course they should seek medical aid. Prevention is the best treatment, so patients should avoid any known allergens, and always carry a kit and EpiPen just in case. Patients should inform immediate family and caregivers, and always wear a MedicAlert bracelet.

Allergic rhinitis can be treated with anti-histamines (sneezing, itching, rhinorrhoea), nasal steroids (nasal blockage), and cromoglycate (children, eyes).

Eczema is treated with emollient creams and topical steroid creams.

Asthma treatment involves four steps. First, use β2 agonist drugs as required by inhalation to cause bronchodilation (Salbutamol). The next step is low dose inhaled steroids like Beclomethasone or Budesonide to suppress the Th2 immune responses driving the asthma pathogenesis. Fluticasone is a more recent inhaled steroid, which is twice as active so you need half the dose. If even this isn’t working, the next step is to add a long acting β2 agonist and a leukotrienes antagonist. You could also use high dose inhaled steroids via a spacer. The final step if none of this is working is to add courses of oral steroids like Prednisolone, at 30mg daily for one or two weeks.

Immunotherapy is quite effective for venom allergies such as bee or wasp stings. This is treatment against a single antigen, and the antigen used is purified. Immunotherapy is also effective with pollen induced allergies by sublingual immunotherapy (SLIT). Immunotherapy is likely to become more used as the years go by.

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Sybghat Rahim

Tolerance and Autoimmunity

by Dr Keith Gould

Autoimmunity = adaptive immune responses with specificity for “self antigens” (autoantigens). Adaptive immune reactions against self use the same mechanisms as immune reactions against pathogens and environmental antigens. Autoimmune diseases involve breaking T-cell tolerance. Because self tissue is always present, autoimmune diseases are chronic conditions. Effector mechanisms resemble those of hypersensitivity reactions (types II, III and IV).

Over 70 chronic disorders have been identified which relate to aberrant immune responses causing the body to attack its own tissues. About 5% of individuals in “developed” countries are affected by autoimmune disease. Nearly 80% of affected individuals are female, although there are some diseases like type I diabetes where actually males are more affected than females. The incidence of autoimmune disease (and hypersensitivity) is increasing. There is probably a link between autoimmunity and hypersensitivity.

About 1 in 30 people in the USA have an autoimmune disease. Among the most common include rheumatoid arthritis, which affects 1 in 100 people. There are 2,100,000 cases of rheumatoid arthritis is the USA. Another common autoimmune disease is type I diabetes, which affects 1 in 800 people. Type I diabetes often starts in teenagers. Multiple sclerosis affects 1 in 700 people, and causes 25,000 hospitalisations per year. In the USA there are also 240,000 cases of systemic Lupus Erythematosus (SLE). Another common autoimmune disease is autoimmune thyroid disease, which includes Hashimoto’s and Graves’ disease. There are 5 cases per 1000 women, and 0.8 cases per 1000 men.

Autoimmune disease generally affects more females. Oestrogen is implicated in this, but the mechanisms are not completely understood. In SLE, in pregnancy where the levels of oestrogen increase, the symptoms get worse. But there are other diseases like MS where symptoms get better. So the reason why females are more affected than males is not completely understood.

Autoimmune diseases are classically classified by the organs affected, the involvement of specific autoantigens, and the types of immune responses. Autoimmune diseases affect a wide range of organs and tissues. The thyroid is affected by Graves’ and Hashimoto’s disease, the pancreas is affected by type I diabetes, the kidney by Goodpasture’s syndrome, the stomach by pernicious anaemia, the liver by primary biliary cirrhosis, muscles by myasthenia gravis, blood vessels by vasculitis, joints by rheumatoid arthritis, and multiple targets by SLE. Autoantigens have been identified in various autoimmune diseases which play a direct role in the immunopathogenesis. Early experiments showed that autoantibodies against red blood cells were responsible for autoimmune haemolytic anaemia in humans. The antibodies would bind and activate the complement system via the classical pathway, and this can lead to direct lysis or recruitment of effectors like neutrophils and the subsequent release of pro-inflammatory mediators etc... The result is the clearance or complementmediated lysis of autologous erythrocytes. There is a direct link between autoantibodies and disease.

Immune reactions are known to play a direct role in the pathology of human autoimmune disease. For example, an antibody response to cellular or extracellular matrix antigen (type II), an immune complex formed by antibody against soluble antigen (type II), or a T cell mediated disease (delayed type hypersensitivity reaction, type IV).

In Goodpasture’s syndrome, the autoantibody IgG is produced, and this reacts with the collagen in the basement membrane. This is particularly important in the kidneys, as you end up with glomerulonephritis.

Once the antibody is bound, you get a massive influx of neutrophils, which damages the tissue and causes inflammation. This impairs kidney function.

Graves’ disease is where the specific IgG antibodies bind to the surface of thyroid cells and this stimulates the cells to keep producing more thyroid hormones. The feedback loop doesn’t work properly, because although the pituitary is inhibited, the antibodies have bound to the thyroid receptors = hyperthyroidism.

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Sybghat Rahim

Systemic Lupus Erythematosus is a classic type III hypersensitivity response - immune complex disease. There are a lot of IgG autoantibodies produced against normal nuclear components of cells including histones, DNA and ribonucleoproteins. Characteristically, there are antibodies that can bind to double stranded DNA. Because all cells have DNA, potentially all cells are target antigens! Dying cells release DNA in the circulation (for example), antibodies recognise this, immune complexes form, and if there are too many, the immune system can’t clear them properly they will be deposited in blood vessels, and in particular cause problems in the glomeruli of the kidneys.

Type IV are T cell mediated diseases. In type I diabetes the autoantigens are pancreatic β cell antigens, and there is β cell destruction. In rheumatoid arthritis the autoantigen is an unknown synovial joint antigen, and there is joint inflammation and destruction. In multiple sclerosis, the autoantigen is myelin basic protein (proteolipid protein), and there is brain degeneration (demyelination) and weakness/paralysis. It is clear in all these diseases that CD8 cytotoxic and CD4 helper T cells are involved.

T cells have T cell receptors, which only recognise antigens presented by MHC molecules. MHC Class I presents to CD8, and MHC II presents to CD4. If there is recognition, this can lead to a response. If it is a naive T cell, it needs co-stimulation to illicit a response.

T cells are important because human MHC (HLA) is the most important genetic factor affecting susceptibility to autoimmune disease. Different HLA molecules are associated with different diseases. For example, HLA-B27 is associated with the rheumatic disease Ankylosing spondylitis. The relative risk factor is quite high, but most people with HLA-B27 won’t get the disease. It is still unclear what the actual HLA-B27 mechanism is.

Mechanisms in autoimmunity are the same as in normal responses against foreign antigens. Immune responses to autoantigens (self) have a direct role in the pathology of autoimmune diseases. Both B cells (antibody) and T cells can be involved. HLA associations strongly imply a role for T cells in initiating autoimmune disease.

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Sybghat Rahim Why are autoimmune diseases not even more common if most people have lymphocytes capable of recognising self?

There are mechanisms which normally prevent our immune system from attacking our own tissues. Evidence for tolerance against self includes freemartin cattle. They have fused placentas and exchange cells and antigens in utero. Non-identical twins have different sets of blood group antigens. As these twins are nonidentical, as adult cattle they would normally be expected to react to each other’s cells and tissues. However, adult cattle tolerate blood transfusions from a non-identical twin. They also accept skin grafts from each other.

This shows that if you have exposure to an antigen at an early stage in life, you can become tolerant to them and won’t form an immune response against them.

Medawar et al also showed that if you had two different strains of mice, you could take spleen cells from one type and transfer them to another at neonatal stage, and at adult age they would accept skin grafts. However, if you transferred spleen cells at adult age, and then tried a skin graft, it was rejected. Tolerance has antigen specificity, and this was shown by using a further different strain of mouse, whereby the skin graft was rejected by the same mouse that would have accepted the other graft.

Tolerance is defined as the acquired inability to respond to an antigenic stimulus. The 3 A’s are: Acquired: involves cells of the acquired immune system and is “learned”.

Antigen specific Active process in neonates, the effects of which are maintained throughout life.

There are several mechanisms that are involved in the generation and maintenance of the tolerant state: central tolerance and peripheral tolerance (anergy, immune privilege/ignorance, and regulation). Failure in one or more of these mechanisms may result in autoimmune disease.

Central Tolerance All lymphocytes come from stem cells in bone marrow. B cells develop in bone marrow, and T cell stem cells leave the bone marrow and mature in the thymus. In both types of lymphocyte there is rearrangement of the gene segments that form the antigen receptors and then there is selection of the cells to try to eliminate self-reactive cells.

For T cells, in the thymus they interact with MHC molecules presented on thymic epithelial cells and dendritic cells (professional antigen presenting cells).

Cells that have a T cell receptor that can’t see MHC are useless and die of apoptosis. Useful T cells can see MHC plus the peptide relatively weakly, and these are positively selected. The ones that are dangerous and bind very strongly are negatively selected and are eliminated. Only 5% of thymocytes survive selection.

For B cells, maturation takes place in the bone marrow. If the developing B cell has an antigen receptor that is cross-linked by something it is exposed to (antigen), then the cell is deleted by apoptosis. If there is no crosslinking during its development, the cell is self-tolerant and able to respond to foreign pathogens. It will survive and be useful in immune responses.

These processes aren’t perfect, and don’t work for eliminating all self-reactive lymphocytes. Central tolerance does matter, because there are rare autoimmune diseases like APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy) caused by a single gene defect. APECED is a rare autoimmune disease which affects the endocrine glands (thyroid, kidneys, chronic mucocutaneous candidiasis, gonadal failure, diabetes mellitus, and pernicious anaemia).

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Sybghat Rahim APECED is caused by mutations in the transcription factor AIRE gene. AIRE is important for the expression of “tissue-specific” genes in the thymus, and is involved in the negative selection of self-reactive T-cells in the thymus. So in APECED there is the persistence of autoreactive cells, which leads to autoimmune disease.

Most autoimmune diseases, however, are associated with multiple polymorphic defects and genetic traits. A good example is SLE. In SLE there are genes affecting multiple biological pathways that may lead to a failure of tolerance: Induction of tolerance (B lymphocyte activation: CD22, SHP-1): autoantibody production Apoptosis (Fas, Fas-ligand): failure in cell death Clearance of antigen (complement proteins C1q, C1r and C1s): abundance/persistence of autoantigen T-cell selection occurs in the thymus, and is dependent on MHC-peptide-T-cell receptor interactions. Most cells die by neglect if they have no or very weak recognition. Negative selection occurs for cells with high affinity Tcell receptors, which die by apoptosis. Surviving cells are MHC-restricted, with low/intermediate affinity for self-peptide. B-cell selection occurs in bone marrow, and cross-linking of surface immunoglobulin by polyvalent antigens expressed on bone marrow stromal cells facilitates deletion. Failure in central tolerance can lead to autoimmunity. In most diseases, a complex interaction of multiple factors is usually involved.

Peripheral Tolerance Some antigens may not be expressed in the thymus or bone marrow, and may be expressed only after the immune system has matured. So other mechanisms are required to prevent mature lymphocytes becoming auto-reactive and causing disease. The three main mechanisms are anergy, ignorance of the antigen, and suppression by regulatory T cells.

Anergy happens when there is an MHC-T-cell receptor interaction in the absence of any co-stimulation. Naïve T cells require co-stimulation for full activation: CD80, CD86 and CD40 are examples of co-stimulatory molecules expressed on APC. These are absent on most cells of the body. Without co-stimulation then cell proliferation and/or factor production does not proceed.

Subsequent stimulation, even in the presence of costimulatory molecules leads to a refractory state termed anergy. B cells anergy is induced by high concentrations of soluble antigen.

Immunological ignorance is where the antigen concentration is too low peripherally, or is zero. It occurs when the relevant antigen presenting molecule is absent: most cells in the periphery are MHC class II negative. It occurs at immunologically privileged sites where immune cells cannot normally penetrate: for example in the eye, central and peripheral nervous system and testes. In this case, cells have never been tolerised against the auto-antigens. An interesting example of disease caused by failure of this immunological ignorance is sympathetic ophthalmia. Trauma to one eye results in the release of sequestered intraocular protein antigens.

Released intraocular antigens are carried to lymph nodes and activates T cells. Effector T cells return via the bloodstream and attack the antigen in both eyes.

Suppression by T-reg cells is the third mechanism of peripheral tolerance. Auto-reactive T-cells may be present but do not respond to autoantigen. They are controlled by other cells types. Regulatory T cells are a special population of cells (CD4+, CD25+, CTLA-4+, and FOXP3+). CD25 is the IL-2 receptor. CTLA-4 binds to B7 and sends a negative signal. FOXP3 is a transcription factor required for regulatory T cell development.

IPEX (Immune dysregulation, Polyendocrinopathy, Enteropathy and X-linked inheritance syndrome) is a severe disorder caused by failure in the regulation of peripheral tolerance. It is a fatal recessive disorder presenting early in childhood. There is a disorder in the maturation in the FOXP3 gene which encodes a transcription factor critical for the development of regulatory T-cells. Symptoms include early onset insulin dependent DM, severe enteropathy, eczema, variable autoimmune phenomena, and severe infections. There is an accumulation of autoreactive T cells because there aren’t enough T-reg cells. This is an example of why T-reg cells are important in peripheral tolerance.

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Sybghat Rahim Does infection ‘break’ peripheral tolerance? Various autoimmune diseases have been associated with different infections, particularly viral infections. But it has been difficult to say that any infection can trigger a particular auto-immune disease. It seems more likely that certain types of infection may be associated with certain autoimmune diseases.

Infection can affect tolerance in a number of ways: Molecular mimicry of self molecules Induce changes in the expression and recognition of self proteins Induction of co-stimulatory molecules or inappropriate MHC class I expression: pro-inflammatory environment Failure in regulation: effects on regulatory T-cells Immune deviation: shift in type of immune response e.g. Th1-Th2 Tissue damage at immunologically privileged sites Induction and maintenance of peripheral tolerance will depend on: site of antigen expression (MHC expression, immune privilege), timing of antigen expression, amount of antigen expression, co-stimulation, T cell help for B cell responses, and regulation. Infections may help break tolerance by a variety of mechanisms.

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Sybghat Rahim

Transplantation

by Dr Candice Roufosse

Organs are transplanted when they are failing or have failed, or for reconstruction. Organ transplants can be life-saving (e.g. in heart failure) or life-changing (e.g. kidney transplant). Kidney transplants are an alternative to dialysis. Once you are past the risks of death associated with the surgery, the risk of death is significantly decreased after a kidney transplant. Patients with a transplanted kidney generally have a better quality of life than patients on dialysis.

Autografts are transplants within the same individual. Isografts are between genetically identical individuals of the same species, i.e. twins. Allografts are between different individuals of the same species. Xenografts are between individuals of different species. The animal most commonly used in the pig, because of the size of their organs and immunological similarities. Prosthetic grafts are plastic or metal, and a good example is hip replacement.

Examples of autografts include coronary artery surgery, in which parts of the left internal thoracic artery, radial artery, or saphenous vein can be grafted to coronary arteries. Another example is reconstructive surgery, with skin grafts, jaw from fibula, hair, ACL reconstruction etc...

Ongoing research in autografts involves stem cells from bone marrow. Marrow cells are aspirated and the stem cells are purified. Malignant or deficient bone marrow cells are eradicated, and the purified stem cells are re-colonised. There is the possibility of using pluripotent stem cells to make other tissues, but this is very much an area of research in its early stages.

The most common Xenografts are using the heart valves of pigs, and also sometimes skin transplantation can be done in this way.

Allografts are done with solid organs (e.g. kidney, liver, heart, lung, pancreas). Small bowel allografts are a relatively new area in development. There is also transplantation of bone marrow and pancreas islets. Blood transfusions are also allografts (temporary), and skin can be used in the same way for burns. Corneal transplantations are also done, and also a variety of different tissues such as bone, cartilage, tendons and nerves. In recent times there have been composite transplants of the face and hands.

There are two types of allografts – those from deceased donors and those from living donors. Deceased donors can give solid organs like the kidneys, heart, pancreas, lungs and liver, as well as corneas, heart valves, bone, skin and composites. Living donors can give a limited number of things, but most commonly bone marrow, a kidney, or parts of the liver.

Deceased Donors There are two types of deceased donors. The main source of donated organs is from brain dead, heart beating donors (DBD donor after brain death). Often these may be patients who have been in a road traffic accident and suffered massive cerebral haemorrhage. After confirming brain death, you can harvest the organs and cool them to minimise ischaemic damage. Due to shortage of these sorts of donations, there are also non-heart beating donors (DCD donor after cardiac death). The heart stops beating before the organ harvesting.

Unfortunately there is a longer period of warm ischaemia time, and many organs are more likely to fail after being transplanted. It is usually suitable for kidney transplants.

DBD donors have irremediable structural brain damage of a known cause. They are in an apnoeic coma not due to depressant drugs, metabolic or endocrine disturbance, hypothermia or neuromuscular blockers. It is important to demonstrate a lack of brain stem function - pupils both fixed to light, corneal reflex absent, no eye movements with cold caloric test, no cranial nerve motor responses, no gag reflex, no respiratory movement on disconnection (with PaCO 2 > 50mmHg). Consent is essential.

It is important to exclude viral infection (HIV, HBV, HCV), malignancy, drug abuse, overdose or poison, and disease of the transplanted organ. Removed organs are rapidly cooled and perfused. The absolute maximum cold ischaemia time for the kidney is 60 hours (ideally should be less than 24 hours). This is much shorter for other organs, except for the cornea (96 hours) which can last longer with cryopreservation.

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Sybghat Rahim Live Donors Donors may be related genetically or unrelated (but these are often emotionally related, e.g. spouse). The number of living donors has increased 3 fold over the past 10 years.

Why Do Organs Fail?

Cornea - degenerative disease, infections, trauma.

Skin - burns, trauma, infections.

Kidney - diabetes, hypertension, glomerulonephritis, hereditary conditions.

Liver - cirrhosis (viral hepatitis, alcohol, auto-immune, hereditary conditions), acute liver failure (paracetamol).

Heart - coronary artery or valve disease, cardiomyopathy (viral, alcohol), congenital defects.

Lungs - COPD/emphysema (smoking, environmental), interstitial fibrosis/interstitial lung disease (idiopathic, autoimmune, environmental), cystic fibrosis (hereditary), pulmonary hypertension.

Pancreas - type I diabetes.

Bone Marrow - tumours, hereditary diseases.

Small Bowel - mainly children, hereditary conditions or related to prematurity (in adults = Crohn’s, vascular disease).

The first initial attempts at kidney transplants were in the 1950s, and the first attempts at heart, liver, lung, pancreas and small bowel transplants were in the 1960s. By the 1980s there was a much better understanding of immunology, immunosuppressive drugs and operative procedures.

Organ transplantation requires a multidisciplinary approach. People involved include the organ donor and family, the organ recipient and family, the transplant co-ordinator, physicians, surgeons, nurses, radiologists and pathologists.

There are multiple challenges to successful transplantation, and these are a mix of clinical, surgical, scientific, ethical and psychological, legal and organisational.

Who Needs a Transplant?

First of all, access to the waiting list requires selection. Then access to an organ requires allocation. People receive transplants if they will be life saving or life enhancing. In the case of life saving transplants, other life supportive methods will have probably not been fully developed (LVAD = heart transplant, liver transplant) or will have reached the end of their possible use (total parenteral nutrition with venous access problems = small bowel transplant). Life-enhancing transplants will be done if other life-supportive methods are less good (dialysis = kidney transplant, insulin injections = pancreas transplant).

Factors affecting access to the waiting list include if the patient is too ill, if the patient does not want a transplant, surgical/technical problems (obesity, atherosclerosis etc), or if it is too early for the patient to be placed on the waiting list.

Organ allocation and distribution will depend on ethical determinants and biological determinants. Ethical determinants are about what is ‘fair’, for example if it is a super-urgent transplant (imminent death e.g. liver or heart), the time spent on the waiting list, etc. Biological determinants include cold ischaemia time (geography), which is particularly important for the heart, organ size (lungs), and histocompatability (kidney).

NHS Blood and Transplant (NHSBT) provide a reliable, efficient supply of blood, organs and associated services to the NHS. They also monitor allocation. Rules for organ allocation are established by the medical community / health professionals / advisory groups / DH. The Organ Donation Taskforce was made in January 2008 when it was seen that donations were very low. The aim was to increase the number of organ donors by 50% by 2013, so organ donation would become usual.

Areas of work included addressing legal and ethical concerns, public promotion, training for NHS staff, reimbursing Trusts for donation activity, having a Donor Transplant Co-ordinator, etc.

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Sybghat Rahim The Donor Transplant Co-ordinator is a role and responsibility under review. The role is usually for registered nurses with experience in critical care, but the employment will probably shift from transplant centres to NHSBT. It is mainly for potential donors for A&E and ICU, and they also carry out family interviews and are part of bereavement services.

Other strategies to increase transplantation activity include finding marginal donors (elderly or sick), carrying out more high risk transplantations (across tissue compatibility barriers), paired-exchange (live donation): donor swaps for better tissue matching, xenotransplatation, and stem cell research.

Immunology The immune system recognises someone else’s organ as foreign. The most relevant protein variations in clinical transplantation are of course the ABO blood group and the HLA coded on chromosome 6 by MHC. A and B proteins are found on red blood cells but also on the endothelial lining of blood vessels in a transplanted organ. There are naturally occurring anti-B antibodies in A patients and vice versa. Blood group O patients have both anti-A and anti-B. Here is the scenario for a mismatch. If a patient has blood group A, then their red cells will express A, and their serum will contain naturally occurring anti-B antibodies. A heart transplant from a blood group B donor means the cells will express B proteins. The circulating, pre-formed, recipient anti-B antibodies bind to the B blood group antigens on the donor endothelium. This activates complement, which leads to complement mediated lysis, opsonisation and increased permeability. Other cells like phagocytes are rapidly recruited. There is also disruption of the endothelium, with platelets activated, inflammation and thrombosis. This causes hyperacute rejection.

However, in recent years, it has become possible to remove the antibodies in the organ recipient with good outcomes: ABO-incompatible transplantation.

HLA (human leukocyte antigen) was first discovered in the 1940s after the first failed attempts at human transplantation - graft rejection. They are cell surface proteins with a highly variable portion. The variability of HLA molecules is important in defence against infections and neoplasia. Their primary function is to protect the organism by recognising foreign proteins and presenting them to immune cells (T cells). This is in the context of HLA molecules recognised by the immune cells as “self”.

HLA Class I (A, B, C) is expressed on all cells. Class II (DR, DQ, DP) is expressed on immune cells, but can also be up-regulated on other cells. HLA is highly polymorphic, and there are lots of alleles for each locus.

Each child will at least have 1 haplotype matching with a parent. Amongst siblings, 25% have 2 haplotype matches, 50% have 1 haplotype match, and 25% have 0 haplotype match.

In the case of a mismatch, the recipient’s immune system mounts a reaction against the donor’s HLA, as if it were an infection or cancer. This results in “rejection” - destruction of the graft by cellular and antibody-mediated immune processes, eventually resulting in graft failure.

Minimising HLA differences between donor and recipient improves transplant outcome.

HLA matching in organ transplantation is important in kidneys and bone marrow, controversial in the liver, and not so important in the heart and lungs.

Rejection is the most common cause of graft failure. It can be mediated by T cells (T cell mediated rejection) or

by B cells (antibody mediated rejection). The gold standard for diagnosis is a histological examination of a biopsy. Treatments include suppressing the immune system with drugs.

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Sybghat Rahim In acute T cell mediated rejection, there is recognition of the donor HLA antigens by CD4 T cells. These CD4 T cells are activated, and they produce cytokines that help CD8 cells, help B cells and cause recruitment and activation of macrophages and neutrophils. This is a type IV hypersensitivity response.

In antibody-mediated rejection, antibodies are made against the graft HLA and AB antigen. Antibodies can be present before transplantation (recipient has seen the antigen before) or arise after transplantation. The antibodies activate complement and macrophages. Activated complement = complement mediated lysis, opsonisation and increased permeability and phagocyte recruitment. Disruption of endothelium = platelets activated, inflammation and thrombosis.

Rejection is diagnosed in the kidney, liver and pancreas when graft dysfunction is detected by regular blood tests (creatinine, liver function, amylase) ďƒ graft biopsy and histological interpretation. For the heart there is no good test for dysfunction, just regular biopsies.

To prevent rejection, HLA compatibility should be maximised and the immune reaction of the recipient should be suppressed by immunosuppressive drugs. Treatment of rejection is just increased immunosuppression.

Immunosuppressive drugs target T cell activation and proliferation, B cell activation and proliferation, and may be life long.

T cell:

B cell:

Modern transplant immunosuppression involves: An induction agent to deplete immune cells before transplantation (e.g. anti-CD52 Campath) A baseline immunosuppressant, which is variable. Signal transduction blockade is usually done by a CNI inhibitor like Tacrolimus or Cyclosporin, or sometimes an mTOR inhibitor like Rapamycin. Antiproliferative agents include MMF or Azathioprine, and corticosteroids may also be used.

Treatment of episodes of acute rejection are both cellular (steroids, anti T cell agents) and antibody mediated (IVIG, plasma exchange, anti-C5).

Post Transplantation Risks Immunosuppression means there are increased risks for conventional infections (bacterial, viral, fungal).

Opportunistic infections are normally harmless infectious agents which give severe infections because of the immune compromise in these patients (e.g. Cytomegalovirus, BK virus, Pneumocytis carinii).

There may also be malignancies like skin cancer, post transplant lymphoproliferative disorder (EBV driven) and others.

Very shortly after the transplantation the risks are technical complications, delayed graft function, acute rejection, infections (bacterial/fungal), and drug toxicity. Later on the risks are infection (viral, fungal, bacterial, parasitic, toxoplasma), drug toxicity causing hypertension, hyperlipidaemia and CVD, and also acute rejection.

Later still the risks are infection, chronic rejection, cancer and CVS.

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