blood-immune-lecture-notes.pdf

16 - Blood composition and function Circulation of blood What are the 2 purposes of blood Even and efficient flow through the small capillaries. pressure? Low enough pressure to prevent capillary leakage, but high enough to avoid coagulation. How is blood circulate...

16 - Blood composition and function Circulation of blood What are the 2 purposes of blood Even and efficient flow through the small capillaries. pressure? Low enough pressure to prevent capillary leakage, but high enough to avoid coagulation. How is blood circulated? Describe The heart pumps blood to the lungs. The pulmonary artery extends from the right ventricle to the using the terms: heart, lungs, lungs where unoxygenated blood is bathed in the oxygen that you breath in through the lungs and is pulmonary artery, right ventricle, drawn back to the left atrium by the pulmonary vein. left ventricle, atrium, pulmonary The left ventricle then pumps blood out through the aorta and through the arterial system to the vein. tissues and organs. Is the pressure of arterial blood The pressure of arterial blood is quite high. high or low? Describe why. This high pressure is maintained by elastic vessel walls that contain an abundance of smooth muscle. Arteries are muscular capillaries. When the left atrium pumps, the walls expand, carrying the pressure from the heart. Is the pressure of venous blood Venous pressure is lower because veins are not elastic. high or low? Describe why. What is systolic pressure? The blood pressure of the blood in the arteries when the heart pumps. What is a normal blood pressure? 120/80. This means 120 mm of mercury. At this pressure, the blood is at full compression, which is when the left ventricle is squeezed at its tightest and the arteries are expanded at their greatest. What is diastolic pressure? Diastolic pressure is when the heart is at complete rest. What does a high or low BP A low BP means that you don’t have enough blood pumping in your veins. mean? A high BP means that your arteries are not expanding or contracting normally. What is the purpose of the venous The venous system is there to provide back flow because there is no blood pressure in the venous system? system. The blood is draining back to the right atrium, that is not under as great pressure as the arterial system. This ensures that the blood is always flowing in one direction. What is the significance of blood Blood volume must be maintained to retain BP. volume to BP? What is hypertension? Hypertension is caused by a narrowing or hardening of the arteries, reducing flow and resulting in unwanted coagulation. Components of blood What are myeloid cells? Cells that engage in some form of innate immunity. What are lymphoid cells? Cells that engage in adaptive immune response. What proteins are present in the - Albumin: most abundant protein in blood. Provides osmotic pressure, maintains blood? hypertonicity and osmotic pressure. - Haemoglobin: component of RBC that carries oxygen. - Fibrinogen: constitutes 7% of blood and forms blood clots. - Immunoglobulins What lipids are found in the blood? Lipids in blood are bound in lipoproteins: HDL, LDL, VLDL. LDL (low density lipoprotein) is bad whereas HDL and VLDL are good. Other components? - Electrolytes: salts and minerals (HCO3-, Na+, Cl-, Ca2+, Mg2+, K+, creatine and creatinine) - Vitamins - Hormones - Glucose What is the relevance of pH in A pH variance of more than 0.2 results in severe stress - acidosis or alkalosis. Blood is tightly blood? maintained at a pH of 7.4. What regulates blood pH? Blood is effectively buffered by albumin, phosphate, bicarbonate, creatinine and other compounds. Centrifugation of blood What is centrifugation? Centrifugation is one of the simplest ways of separating blood. What does blood separate into - Packed RBC (approx 40-45%) during centrifugation with an - Buffy coat (approx 10%) containing white cells such as lymphocytes, myeloid cells, anticoagulant? leukocytes, etc. - Plasma (approx. 50%) which is blood that still has fibrinogen and hasn’t been clotted. Plasma contains dissolved substances and formed elements (cells and cell fragments). Plasma is 91.5% water and 8.5% solutes. What is serum? Serum is blood without fibrinogen - it is essentially plasma without fibrinogen. It is the liquid that remains after coagulation/clotting. Blood separation by electrophoresis What is serum electrophoresis? A common means of separating blood serum proteins using an electric field. Electrophoresis separates serum into 5 major protein fractions - albumin, ɑ1, ɑ2, β and γ fractions. What are the five major proteins in - Albumin - constitutes 50% of total blood protein. Maintains colloidal osmotic pressure. Binds blood? What are their roles? and transports many small molecules and hormones. Can be seen as a protein sponge that absorbs a lot of the fluid in your blood. Allows that fluid to be balanced, preventing fluid from leaking from the capillaries. A lot of pharmaceuticals take into account how well a drug binds onto albumin because albumin makes up a high concentration of blood protein. - Fibrinogen - is the second most abundant protein. Activated through the coagulation cascade to form cross-linked fibrin wherein fibrinogen is cleaved by the enzyme thrombin to form cross-linked fibrin that forms blood clots. - Immunoglobulins (Ig) - aka antibodies. Diverse repertoire of antigen binding proteins that are produced by B lymphocytes. Ig are found in the γ fraction. - Complement (C’) - 9 proteins that “coat” bacteria, targeting them for phagocytosis. C3 is a major complement. Main role is to tag foreign objects. The complement proteins will coat the pathogens in a process called opsonisation. This then signals white cells to migrate to the site of infection, to digest or phagocytose the pathogen. - Neutrophils are the first cells to migrate to a site of infection. They are driven to migrate from the capillaries to the site of infection by the activation of complement. - Coagulation factors - 13 proteins that initiate the cleavage of fibrinogen to fibrin to form the clot. Thrombin is the central enzyme that cleaves fibrinogen. - Ca2+ is essential to coagulation. - Haemophilia results from a missing component, therefore blood does not clot properly. Types and functions of blood cells What are erythrocytes? - RBCs that carry out oxygen transport/ gas exchange. ~5-6 million. - Don’t have a nucleus - are anucleated. - Major protein is haemoglobin. What are leukocytes? - White blood cells that make up immune defence. ~10,000/ml. - Most abundant leukocyte is neutrophil. Neutrophils migrate very quickly from the capillary tissue to the site of infection and begin to engulf the infection. What are platelets? - Coagulation and tissue repair. ~400,000/ml. - Important in releasing a number of factors that regulate the homeostatic mechanism of tissue repair. Without platelets, tissue repair would be much slower. - Prevent blood loss at sites of vascular injury, promote tissue repair and play a role in angiogenesis (blood vessel formation), inflammation and the immune response. What are myeloid cells? Myeloid cells provide you with innate immunity and phagocytosis as a key mechanism. These cells have a range of receptors that bind immune complexes. Microbes are rapidly phagocytosed and killed once they have been opsonised by complement proteins. What types of myeloid cells are - Neutrophils - approx 60-70% of all WBCs. Main function is phagocytosis which is achieved there? What are their functions? by destruction of bacteria with lysosomes, defensins and strong oxidants such as superoxide anion, hydrogen peroxide and hypochlorite anion. - Monocytes (become macrophage) - approx 3-8% of all WBCs. Main function is phagocytosis (after transforming into fixed or wandering macrophages).  - Basophils - 0.5-1% of all WBCs. Liberates heparin, histamine and serotonin in allergic reactions that intensify overall inflammatory response. - Eosinophils - 2-4% of all WBCs. Combats effects of histamine in allergic reactions, phagocytizes antigen-antibody complexes and destroys certain parasitic worms. What are the types of lymphoid - B lymphocytes - B cells develop into plasma cells which secrete antibodies. Part of the cells? What are their functions? humoural (because of antibody) adaptive immunity. - T lymphocytes - T cells attack invading viruses, cancer cells and transplanted tissue cells. Part of the cellular adaptive immunity. - Natural Killer cells - attack a variety of infectious microbes and certain spontaneously arising tumour cells. Name examples of granular - Granular leukocytes - neutrophils, eosinophils, basophils leukocytes and agranular - Agranular leukocytes - lymphocytes, monocytes leukocytes. Haematopoiesis What is HSC? HSC is a human stem cell from which all blood cells come from. HSC is found mainly in the bone marrow. HSC is characterised by a CD34 cell surface marker antigen used to isolate these cells. HSC is relatively rare (about 1 in 10,000 white cells are CD34+) and are more abundant in core blood (placental blood). What is the purpose of storing Multipotential hematopoietic stem cells are relatively rare, but can be saved and stored in liquid HSC? nitrogen in case the baby develops leukemia later in life. The leukemia can be treated using radioablation and chemicals, and then the core blood is transplanted back into the patient and thus hematopoietic stem cells can repopulate the bone marrow and regenerate a normal blood system. What is CD34+? CD34+ is a surface antigen marker on HSCs. An anti-CD34 antibody can select and concentrate HSCs from blood prior to a bone marrow transplant. How do we treat leukemia in adult We use a technique that isolates HSC using a monoclonal antibody (with a fluorescent tag or patients? magnetic bead which bind with the CD34+ antigen) which recognises the CD34+ antigen. All cells with a CD34+ antigen will bind to the magnet, allowing the other cells to be washed away. When you have a relatively pure sample of stem cells, you treat the patient with radiation, which destroys cells, and then transplant back the isolated CD34+ cells. What cells does CD34+ HSC give Myeloid or lymphoid progenitors. rise to? What are myeloid progenitors? Can give rise to erythrocytes, a platelet producing megakaryocyte, mast cells or myeloblasts. Myeloblasts further differentiate into either neutrophils, basophils, eosinophils or monocytes. What are lymphoid progenitors? Differentiate into B or T lymphocytes. Immature T lymphocyte differentiates into CD4 or CD8. What are the 3 factors that drive - GM-CSF - granulocyte macrophage colony-stimulating factor. Produced by macrophages, T haematopoiesis? What are their cells, endothelial cells and fibroblasts. Stimulates the production of neutrophils, eosinophils, functions? basophils and monocytes. Is a major driver of the myeloid lineage. Stimulates the production of granulocytes by binding to myeloid progenitor cells. - EPO - erythropoietin. Drives the production of erythrocytes. Produced mainly by the kidney during adulthood and liver in prenatal. - G-CSF - granulocyte colony stimulating factor. Produced by many different cells. Stimulates the production of granulocytes but also acts to mature neutrophils. GCSF and GMCSF are administered to re-populate white cells in leukemia patients following radio ablation. Hemoglobin and oxygen transport What is hemoglobin? Haemoglobin is a major protein in RBCs and constitutes 96% of the RBC’s dry weight. How does gas exchange occur in Each haemoglobin molecule contains 4 lobes, each containing a haem. Each haem molecule the hemoglobin of RBCs? contains 1 iron atom in the ferrous form (Fe2+) which allows the oxygen to bind and dissociate. Under the pressure of oxygen in the blood (~100 mm Hg) oxygen binds freely to the ferrous iron. As the pressure of oxygen drops in the tissue, oxygen dissociates and is displaced by carbon dioxide. Can other molecules bind even Carbon monoxide can bind a little tighter, therefore displaces oxygen. tighter than oxygen to Cyanide binds even tighter than carbon monoxide and oxygen because it binds very tightly to iron. hemoglobin? What is the colour of blood when - O2 - bright red, oxyhaemaglobin, normal oxygenated blood the haemoglobin is binded to: - CO2 - dark red, carbamihaemaglobin, venous blood oxygen, carbon dioxide, carbon - CO - cherry red, carboxyhaemaglonin, carbon monoxide poisoning monoxide and cyanide? - CN - pink, cyanohaemaglobin, cyanide poisoning Complement cascade What is a complement? Complement is a set of 9 major plasma proteins that opsonise and phagocytose foreign organisms. What are the 3 pathways that Classical (antibody) activation, lectin activation and alternative activation. activate complement? Explain classical pathway. Classical activation is initiated by antibodies (IgM or IgG) binding to the surface of a microbe. C1, C2, C4 and C3 condense on the antibody to form a bound C3 convertase on the microbe surface. Explain alternative pathway. Complement C3 is activated by just being close to the surface of the microbe. This activates another type of C3 convertase. Explain lectin pathway. Lectins are carbohydrate binding proteins in blood that bind to unusual carbohydrates found only on microbes. Complement condenses on these bound lectins. How does the complement Complement C1 recognises antibodies bound to the surface of the bacteria. C1 creates a proteolytic cascade occur? enzyme that cleaves the complements C4 and C2 which then cleaves complement C3. This results in the release of anaphylatoxins (C5a, C4a, C3a). Macrophages sense the anaphylatoxins being released from the bacteria. The macrophage has a receptor, mainly the C5a receptor, which tells the macrophage to move to the direction of the bacteria because that’s where it’s sensing that concentration of C5a. The C3 and C5 proteins bind irreversibly to the surface as it is a covalent interaction, forming convertase complexes. This means that once bacteria is coated, it is coated forever. This process is known as opsonisation. The later stage of complement goes on to form lytic pores/MAC complex, which then forms a pore which some types of bacteria can insert and are automatically killed. What is opsonisation? Deposition of complement on microbes, which is essential for phagocytosis. Deposited complexes are called convertases which activate more complement that then deposits even more to the surface. What is the most abundant C3 complement component in serum? What is end stage complement? The surface bound convertases activate complement C5-9. This forms a pore that inserts into some bacterial membranes to cause lysis. This pore is called the MAC or membrane attack complex. What are anaphylatoxins? Small polypeptides generated by cleavage of larger complement proteins are powerful chemoattractants that recruit and activate phagocytes to the site of infection. What are phagocytic cells relevant Neutrophils and macrophages have complement receptors that bind complement and initiate to complement? phagocytosis. The coagulation cascade What are the 3 pathways in the Intrinsic pathway, common pathway and extrinsic pathway. coagulation cascade? Describe the intrinsic pathway. In the intrinsic pathway, factors XII, XI, IX and VIII (12, 11, 9 and 8) lead to the cleavage of factor X (10) that converts prothrombin to thrombin. This is caused by contact with surfaces. Describe the extrinsic pathway. In the extrinsic pathway, factors VII (7) and tissue factors (TF) combine to activate factor X. This is caused by tissue damage. What does Factor X do? Factor X activates thrombin by cleaving prothrombin. What is thrombin? Thrombin is an enzyme that cleaves fibrinogen to fibrin, which cross links to form a blood clot. What is plasminogen? Plasminogen is a protease that is activated by tissue plasminogen activator (TPA) or streptokinase. Plasmin cleaves the fibrin clot, resulting in thrombolysis. People who have had a thrombosis are given molecules (TPA or streptokinase) that activate plasminogen so that the clot is released, thus saving the patient from severe tissue damage. List examples of anticoagulants. Heparin and warfarin are anticoagulants that mostly block thrombin. Coagulation is what type of Coagulation is a proteolytic activation cascade. activation cascade? What is the relevance of calcium Calcium is essential at a number of steps. Without calcium, blood will not clot. to the coagulation cascade? 17 - Infection and innate immunity Basic concepts of innate immunity Describe the difference between Soluble factors are part of your humoral system and include things like antimicrobial peptides that cellular and humoral immunity. directly kill bacteria and antibodies which are produced by B lymphocytes. Cellular immunity consists of either myeloid cells (innate) or lymphoid cells (adaptive). List examples of humoral innate - Complement. immunity. - Lectin binding proteins which activate complement. This recognises unique carbohydrates found on the surface of bacteria. I.e. mannose binding lectin which is found on primitive organisms/prokaryotes. In higher organisms, manose does not terminate glycoproteins, usually it is sialic acid. - Antimicrobial peptides in the gut, saliva and other parts of the body that come into contact with the environment, that bind to the surface of bacteria and cause them to lyse. Describe the difference between Your innate immune response does not change or strengthen over time whereas your adaptive innate immunity vs adaptive immune response strengthens or adapts the longer you’re exposed to the antigen. Innate immunity is immunity. immediate and is your first line of defence against an infection. Innate immunity is more primordial than adaptive immunity. For example, bacteria can defend against bacteriophage infection by recognising and cleaving viral DNA that has been inserted. CRISPR/Cas9 gene editing technology has been developed from one of these innate bacterial anti-viral systems. What is CRISPR Cas9? A gene-editing set of genes that are part of the innate immune response of an organism’s strep pyogenes. Strep pyogenes use CRISPR Cas9 to defend itself against bacteriophage infections. CRISPR Cas9 is simply a mechanism which designs these gene editing enzymes that edit and cleave any sequence that represents a bacteriophage sequence. What are the 3 main processes - Complement - opsonisation of microbes by blood proteins and the production of that regulate innate immunity in anaphylatoxins that attract and activate phagocytes. mammals? - Phagocytosis - engulfment of the microbe by phagocytes (neutrophils and macrophages) that destroy the organism. - Pattern recognition receptors (PRR) - receptors found on many myeloid cells that recognise complex microbial molecular patterns. Types of different pathogens What are viruses? Intracellular pathogens that use host cell machinery for replication. Need a means of detecting infected cells and destroying them while leaving normal host cells alone. What are bacteria, yeast and These are predominantly extracellular pathogens that are engulfed and destroyed by phagocytic cells. fungi? Most bacteria are distinguished by the gram stain. Gram positive bacteria have a thick cell wall and are resistant to direct complement MAC lysis. Gram negative bacteria have a thinner peptidoglycan layer and an outer membrane and are often more sensitive to complement MAC lysis. List examples of gram positive and Gram positive bacteria can be S. Aureus, S. pyogenes. These significantly light up with the gram gram negative bacteria. stain due to the thick peptidoglycan wall. Gram negative bacteria can be E coli, H. influenza. These do not light up the gram stain. How do antibiotics affect bacteria? Antibiotics such as penicillin disrupt the synthesis of a cell wall, thus preventing bacteria from being able to synthesis a cell wall therefore cannot reproduce effectively. What are protozoa and other These are complex organisms that are often multicellular and highly developed (helminths - worms). parasites? Can live inside (malaria - plasmodium falciparum lives inside the red blood cells) or outside cells. These are too big to be engulfed by macrophages so basophils, eosinophils and mast cells secrete inflammatory mediators and cytotoxic chemicals that kill. Neutrophil extravasation and chemotaxis What is neutrophil extravasation? The ability of neutrophils to identify the site of infection by recognising the endothelial cells on the inner wall of the capillary that is closest to the infection. Describe the importance of Neutrophil extravasation is a unique mechanism. This is important because microorganisms such as neutrophil extravasation. staph aureus produce virulence factors that target almost every step in the pathway of neutrophils finding their way through the blood to the site of infection. What processes are linked? Opsonisation, chemotaxis and phagocytosis. What are the 5 steps to neutrophil 1) Activation - chemokines from tissue injury or inflammation activate the local endothelial cells extravasation? lining an adjacent capillary wall. 2) Tethering - neutrophil slows down and tethers to the inside capillary wall. Mediated by selectins upregulated on endothelial cells and sialyl Lewis X (sLeX), a carbohydrate antigen on neutrophils. 3) Adhesion - strong binding between neutrophil integrins and ICAM-1 on the endothelium. Neutrophil immobilises and flattens. 4) Diapedesis - neutrophil squeezes between endothelial cells into the interstitial space. 5) Chemotaxis - neutrophil migrates along a chemical gradient to the site of infection. - When neutrophils migrate up the chemokine gradient, they polymerise actin filaments at their leading edge and depolymerise these filaments at their trailing edge. What is the relevance of Neutrophils and macrophages engulf opsonised bacteria but ignore non-opsonised cells. Neutrophils complement to neutrophils? have receptors that bind to deposited complement proteins, mainly C3b on the surface. What does opsonisation do in The deposited complement proteins (complex convertases) on the surface of bacteria release relevance to neutrophil chemoattractants such as C5a which are sensed by neutrophils. extravasation? What are complement receptors? Complement receptors are myeloid cell receptors that bind activated complement components deposited on bacteria. Cross linking of the surface CRs initiates phagocytosis. CR1 is the most important and it is the one that recognises/binds to the C3b component that is part of the complement convertase, covering the bacteria. What are the 5 steps to 1) Ingestion - the bacterium is captured by receptors, membrane invaginates into a phagocytosis? phagosome. 2) Fusion - the phagosome and lysosome fuse to form a phagolysosome. 3) Acidification - the phagolysosome acidifies with H+ pumped in. 4) Digestion - acidification activates protease and stimulates the production of superoxides such as H2O2 (peroxide) and HOCl (hypochlorous acid) which kill bacteria. 5) Exocytosis - expulsion of the digested microbe. How does FcR (antibody) There is a classical complement that requires antibodies to activate it. In this case, FcR are the mediated phagocytosis work? receptors that recognise antibodies that have coated the surface of the bacteria. 1) Antibody (IgM and IgG) bind to bacterial antigens, causing the bacteria to be coated by antibodies. 2) Exposes the antibody Fc region 3) Neutrophil FcR binds multivalent Fc 4) Activates phagocytosis 5) Membrane invaginates forming a phagosome 6) Phagosome fuses with lysosome to form a phagolysosome 7) Phagolysosome acidifies and superoxides kill bacteria. PRRs and PAMPs What are PRRs? Pattern recognition receptors (PRR) bind complex molecules that are unique to microbes. PRRs are essentially the power switch that tells the adaptive immune response to then start making the antibodies that you want. The best known PRRs are Toll-Like receptors (TLRs). TLRs are leucine rich repeat (LRR) receptors that look like a slinky. What are PAMPs? Molecules unique to microbes. PAMPs are recognised by PRRs. Aka pathogen associated molecular patterns. List examples of PAMPs and the - Pam3CSK4, PGN and Zymosan on gram positive bacteria (e.g. S. aureus) are recognised PRR that recognises them. by TLR1, 2 and 6 - LPS and lipid A on gram negative bacteria (e.g. E. coli) are recognised by TLR4 - Flagellin on bacteria and flagellum are recognised by TLR5  - dsRNA on virus are recognised by TLR3 - ssRNA on virus are recognised by TLR7 and 8 - CpG DNA on bacteria and DNA are recognised by TLR9 What is LPS? Lipopolysaccharide (LPS) is a component of gram negative bacteria. LPS is a highly complex glycolipid molecule that stimulates TLR4 cross-linking to signal a powerful inflammatory response. What is a septic shock? LPS is a powerful pyrogen. Tiny amounts in the bloodstream can cause fever, rigors and hypotension and can be fatal if not controlled. This condition is known as septic shock and is often a fatal consequence of an uncontrolled gram negative infection in blood. https://quizlet.com/515989372/blood-and-immune-finer-details-flash-cards/?new 18 - antibodies and gene rearrangement Origins of adaptive immunity How did adaptive immunity Adaptive immunity is only found in higher vertebrates. An event occurred ~500 mya in jawless fish originate? which allows a region of the genome to rearrange. The immunoglobulin (Ig) and T cell receptor (TcR) gene regions or loci use the same mechanism of rearrangement as transposons (jumping genes) that are found in primitive organisms. It is most likely that an ancient transposon inserted itself into the DNA of a germ cell in jawless fish, within a primordial receptor gene and then the transposase (the enzyme that cuts and shifts the transposon) moved to another part of the genome leaving the recognition sequences (RS) behind. The transposase could then operate (rearrange) bits of the genome with these recognition sequences attached. The ancient transposase is still present in our genome and are called RAG1 and RAG2 (recombination activation genes). The RS are located at the ends of all the Ig and TcR gene segments. What are the properties of Adaptive immunity has memory and changes with both time and persistence of antigen. A secondary adaptive immune response? response is stronger and more rapid than the naive response. This is the basis for vaccination. Describe the primary and The 1st immunisation with antigen results in a rise in antigen specific low affinity IgM in blood peaking secondary response that occurs at about 2 weeks post immunisation but then diminishes rapidly. Note that IgM increases first and with vaccinations. then IgG increases. The 2nd and 3rd immunisations (boosters) generate a rapid and intense burst of antigen specific high affinity IgG that lasts a month - sometimes years. These are both B cell responses. What makes the best vaccines? The best vaccines are inactive variants of bacteria toxins that rapidly produce high affinity neutralising IgG that binds to the toxin before it binds to their target receptor. I.e. Tetanus Toxoid as an antigen in the tetanus vaccine. What are transposons? Bits of DNA that have the ability to move themselves from one location to another location. Transposons have recognition DNA sequences at its ends. What is transposase? Enzyme that does the cutting, insertion and reinsertion of transposons. Transposases works on the recognition sequences on the transposons to chop it up and move it elsewhere. Lymphatic system What is the lymphatic system? A circulatory system for immune cells that connects hundreds of small lymph nodes, draining sites that interface with the environment (throat and gut). The lymphatic system carries clear fluid called lymph that drains tissues from the interstitial space between cells. The bone marrow and thymus are the primary lymphoid organs while lymph nodes and spleen are secondary tissue. Lymph nodes and spleen are filled with lymphocytes. This is where b cells from follicles that contain germinal centres encounter antigens and undergo affinity maturation. What are B cells? B lymphocytes originate from the bone marrow and then mature in secondary lymphatic organs (such as the spleen and lymph nodes). B lymphocytes produce antibodies and form the humoral (soluble) arm of the adaptive immune response. What is the B cell receptor? The B cell antigen receptor is a membrane-bound IgM molecule that is associated with intracellular molecules that transmit an activation signal through phosphorylation. What are T cells? T lymphocytes begin as immature lymphocytes in the thymus and then later mature into functional T cells and provide cellular adaptive immunity. What is the T cell receptor? The antigen receptor on T lymphocytes is called the T cell receptor (TcR). It is an immunoglobulin-like surface molecule coded for by a separate gene locus. The TcR is associated with a number of surface molecules and two important ones are CD4 and CD8 which distinguish two functionally different types of T lymphocytes. Antibody protein structure and function Describe the structure of the An Ig domain consists of two anti-parallel β-sheets made up of seven (constant) or nine (variable) immunoglobulin fold. β-strands. The two β-sheets lie parallel but at 30° twist to each other forming a very soluble β-barrel structure. A single disulphide bond between the sheets further stabilises this structure. The strands are connected by loops. The loops are not constrained in the structure thus allowing extreme amino acid diversity without compromising the overall stability. These loops form the antigen binding site. Describe the basic structure of an Antibodies consist of 2 heavy chains (50-75kD each) and 2 light chains (25kD). IgG antibody. The sequence of the protein chains is L-s-s-H-s-s-H-s-s-L. The H chains are joined by disulphide bonds and the L chains are joined to the H chains by disulphide bonds. Secretory IgA and IgM also exist in dimer and pentameric forms respectively. The total molecular weight of an IgG molecule is 150kG (always a multiple of 25kD). The Fab domain carries the antigen binding region whereas the Fc domain carries the effector function. Fc is bound by things like phagocyte Fc receptors or complement. Once the antibody is bound to the surface of the bacteria, the Fc structure changes slightly which allows complement to bind or phagocytes to recognise the antibody bound to the surface and so initiate phagocytosis. Five classes of immunoglobulins and their functions How are the classes of Ig By the heavy chain gene used. determined? What is IgM? IgM is the default antibody made by all immature B cells. It comes in a membrane bound (monomer) and soluble (pentamer) form. The membrane form is the B cell antigen receptor (BCR). The soluble form has 10 antigen binding sites, therefore avidity is very high. An IgM molecule binds by changing its structure to a crab like structure on the surface of the bacteria, which then exposes the effector region, which is then bound rapidly by the initiators of complement or phagocyte receptors. IgM is designed to be low affinity, but high avidity. What is IgG? When a B cell encounters an antigen and activates, it switches to use the ɣ gene to produce an IgG molecule. IgG and IgM are good at activating complement as they both bind with avidity. IgG can transfer through the placenta. What is IgE? IgE is the rarest class of Ig. IgE is strongly associated with allergies because it has the ability to bind to large molecules like pollen grain. There is also a receptor on mast cells that are present in mucosa sites which bind strongly to IgE, causing the mast cell to degranulate and release histamine, thus causing an atopic allergy. What is IgA? IgA is found primarily in secretory sites; tears, saliva, breast milk, etc. IgA is long lasting in the gut. Babies get their IgA from their mother, hence why mothers must breastfeed their babies. What is IgD? IgM and IgD are the only ones where there is actually a membrane bound form. Affinity, avidity and complimentarity Describe affinity. The quantitative term for the forces between two attractive components. Describe avidity. When two or more binding sites contribute - like velcro. Describe complementarity. An antibody can form complementarity to virtually anything because the potential amino acid diversity at the antigen binding site is vast. This means that antibodies can be developed to virtually any surface that you want. We can make antibodies just the way we want them. Antibodies ending with -mib are all modified humanised manufactures antibodies. Antibody binding site How is the antigen binding site of It is formed from the 6 protein loop regions that connect the β-strands in the Ig variable domain (the Ig and TcR formed? first N terminal domain in H chains and L chains). There are 3 H chains and 3 L chain loops that form a “flat” surface of about 800Å2. The loop regions are called complementarity determining regions (CDR) and contain massive amino acid diversity caused through the rearrangement and imprecise joining of germline gene segments in the Ig and TcR locus. Where is amino acid variation In 3 discrete regions called CDR which are the 3 loops that connect the strands in the 1st domains found? of the H and L chains. There is CDR1, CDR2, CDR3 on each of the chains. Located specifically on the heavy and light chains on each side. Hence there are 6 on each side. Total is 12 for both. How can so many different These genes are able to undergo somatic hypermutation so that once they’ve rearranged, they antibody molecules be produced continue to chain their base pairings through random mutations. Some of those will occur in the from so few genes? antigen binding site and an even smaller number of those will produce a result where the antigen affinity increases. That antibody molecule is selected in successive rounds of affinity maturation selection. This all occurs in germinal1 centres in the lymph nodes. Genetic recombination What regions are the germline Ig Variable, Diversity, Joining and Constant regions. and TcR gene loci segmented Within these regions are many segments. There are hundreds of V segments in the human H chain into? locus. The L chain locus has no D segments. What is responsible for the RAG1 and RAG2 (recombination activation gene) are responsible for rearrangement and are only rearrangement? active in B and T lymphocytes. How does rearrangement occur? An immature B lymphocyte in the bone marrow first rearranges an H chain D segment to join to a J segment then a V segment joins to the D segment. This forms a pre-RNA which is then spliced to a C region segment. Because the joining is very imprecise, this leads to a huge diversity in the finally joined DNA sequence (and thus amino acids). What does the VDJ join region CDR3 code for? Does the L chain rearrange? The L chain also rearranges, but there are no D segments so V joins to J. Again, imprecise joining results in massive diversity in amino acids of the L chain. Where is the CDR3 region? The CDR3 regions are in the middle of the antigen binding surface so most of the diversity that occurs in amino acid sequences occur in this middle section. Clonal selection, affinity maturation and vaccination What does the clonal selection That for every single B cell, there is one antigen specificity. theory claim? What does clonal selection result Clonal selection results in high affinity, reactive memory B cells that sit in your lymphoid tissue. If in? an individual is faced by the same pathogen in the future, these memory B cells rapidly produce plasma cells that make high affinity soluble IgG. What is the fundamental Affinity maturation. mechanism behind vaccination? Affinity maturation drives weak naive B cells to undergo rearrangement and extensive hypermutation to eventually secrete a high affinity IgG. Where does clonal selection In the lymph node follicles: cervical lymph nodes, thoracic duct, lymphatics of the mammary gland, occur? thymus, axillary lymph nodes, cisterna chyli, spleen, lumbar lymph nodes, lymphatics of the upper limb, pelvic lymph nodes, inguinal lymph nodes and lymphatics of the lower limb. These are concentrated around areas exposed to the environment/ regions which are likely to drain from external sites. How does affinity maturation work A massive repertoire of naive B cells is generated stochastically before birth, each with a unique B cell in B cells? receptor. B cells encounter antigens in the lymph nodes. An antigen expands a small number of B cell clones with receptors that weakly bind the antigen. Somatic hypermutation of the Ig gene results in some clones with a higher antigen receptor affinity. After successive rounds of this, the mature B cell becomes a plasma cell secreting soluble IgG. Some B cells reside in lymph nodes as long term memory cells. Describe Tetanus case study. Clostridum tetani is a common soil bacterium that produces Tetanus Toxin (TT). TT is a neurotoxin that binds to the presynaptic membrane of the neuromuscular junction and causes spastic paralysis. The patient’s jaw freezes and other muscles spasm. 19 - Cellular immunity and histocompatibility Thymus and T lymphocytes What is the thymus a site for? The thymus is where immature lymphocytes from the bone marrow migrate to and mature. The thymus educates T cells to become either CD4 or CD8 populations of mature lymphocytes. Where is the thymus? Thymus is a gland that sits above the heart. When is the thymus largest? At birth and reduces with age. How do T cells mature? T lymphocytes express both the CD4 and CD8 surface antigens (or coreceptors) at this stage. This is the only time that a T cell will express both of these receptors together. These are called double positive immature lymphocytes. T lymphocytes encounter MHC class I and class II molecules for the first time expressed on thymic epithelium and respond accordingly or die by neglect. If they respond to MHC class II, they become CD4 helper T cells. If they respond to MHC class 1, they become CD8 cytotoxic T cells (CTL). T cells mature into either CD4 or CD8 cells prior to entering the bloodstream. How does negative selection work Only a small percentage of T cells survive the thymus as mature T cells. Most die because they in the thymus? haven’t recognised the right antigen to allow them to activate and undergo mitosis. This is negative selection. There is another group of T cells that have recognised the right antigen but have responded too strongly and they are actively killed by apoptosis. This is positive selection. What are the 2 major functional CD4 (80% of blood T cells) are helper T cells and respond by secreting cytokines. subsets of T lymphocytes? CD8 (20% of blood T cells) are cytotoxic T cells that kill other infected cells. What are the 4 major types of - Treg - important role in regulating the immune response/ suppresses the immune helper T cells? response - Th1 - tends to drive a cellular response, so if the pathogen that you’re infected with demands a cell immune response, then Th1 cells predominate and drive that response - Th2 - tends to drive infections demanding more of an antibody or humoral response and also tend to regulate and moderate B cell response making B cells produce more antibody - Th17 - important in the control of inflammatory response What is CD? CD = cluster differentiation, which is a way of naming cell surface markers. T cell receptor and MHC restriction What is a TcR? The T cell receptor (TcR) is an Ig like molecule on the surface of all T lymphocytes. The TcR gene locus undergoes rearrangement just like in B cells. There are 2 gene loci (and alpha and a beta chain). What does TcR recognise? Unlike antibodies, TcR only recognises one antigen which are proteins of the major histocompatibility complex (MHC) because the T cells were “selected” during thymic development for their ability to respond to self MHC molecules. Unlike Ig, the TcR does not undergo affinity maturation because you don’t want the affinity of TcR to increase to a self-antigen (MHC). How does MHC work? T cells recognise foreign antigens from both virus and bacteria but only when there are MHC molecules present on the surface of the cells. This is how T cells detect host cells that have become virally infected. They detect small fragments of the virus picked inside the cell which are then expressed in the binding groove of MHC molecules on the cell surface. MHS is unique to every individual except for twins. What does MHC restriction imply? MHC restriction implies that T cells see two antigens at the same time. T cells see foreign peptide antigen (non-self) embedded in MHC and MHC molecules (self). How was MHC restriction MHC restriction was found in an experiment using cytotoxic T cells from congenic mice. The congenic discovered? mice were genetically identical except for the locus that controls tissue rejection. Cytotoxic T cells only killed virally infected cells from their own strain. This meant that the CTL were restricted by MHC and explained for the first time how viral immunity depends on MHC to “present” antigens. It also explained why transplanted tissue is rejected. What is the molecular mechanism The antigen binding surface of the TcR (top molecule) binds to the top of MHC which represents behind MHC restriction? the “peptide groove” containing the foreign peptide antigen. Thus the TcR has affinity towards the combination of MHC. What are the 2 classes of MHC? MHC has 2 forms; class I and class II. Class I picks up peptide antigens from inside the cell (intracellular) and presents them to CD8 cytotoxic T cells. CD8 cells can effectively kill the presenting cell. Class II molecules pick up digested antigens from the phagolysosome (extracellular pathogens) and present them to CD4 helper T cells. What are human MHC molecules HLA or human leukocyte antigen. There are 6 different molecules expressed on human cells. Class called? I molecules are A, B and C. Class II molecules are DR, DP and DQ. The genes for MHC are codominant. What are mouse MHC molecules H-2 or 2nd histocompatibility locus called? The role of CD4 and CD8 molecules Summarise MHC class I. Peptide source: intracellular, pathogen: virus, responding T cell: CD8, effector function: cytotoxic Summarise MHC class II. Peptide source: extracellular, pathogen: bacteria, responding T cell: CD4, effector function: help Describe HIV and AIDS case The HIV virus uses the CD4 antigen as its receptor to enter and replicate in helper T cells. AIDS is a study. condition resulting from long term depletion of the CD4 T cell population. A person with AIDS slowly loses their ability to respond to simple infections. A person with AIDS will have many hundreds of viral mutants making it impossible to define a vaccine that will cover all those variants. How do CD4 and CD8 activate CD4 and CD8 have intracellular tyrosine kinases associated with their cytoplasmic tails that immunity? initiate T cell signalling through phosphorylation. They are crucial to immune activation. MHC polymorphism Why is MHC so polymorphic? There are 3 class I and 3 class II molecules expressed and both maternal and paternal genes are codominantly expressed which means each person has a total of 12 separate polymorphic MHC molecules all presenting antigens to the host T lymphocytes. This means that except for identical twins, no two people share exactly the same MHC profile. What is a consequence of MHC Transplant rejection. People who receive a transplant must take an immunosuppressive drug polymorphism? (cyclosporine) for the rest of their lives to suppress the T cell response. In addition, MHC polymorphisms are strongly linked to many autoimmune diseases. Describe the link between MHC The MHC locus is the only polymorphic part of your entire genome, therefore it is strongly associated polymorphism and autoimmune with many autoimmune diseases. Diseases associated with MHC polymorphism include: Addison’s disease. disease, type 1 diabetes, rheumatoid arthritis, multiple sclerosis and ankylosing spondylitis. If an individual has one of these conditions, it is highly likely that they will carry one or more of the MHC haplotypes. Review What regulates cellular immunity? Cellular immunity is regulated by MHC, a set of highly polymorphic genes coding for a group of membrane molecules called HLA in humans. What is TcR? T cell receptor is an Ig like membrane molecule. Its gene locus is segmented and undergoes rearrangement. What did graft rejection imply? The experiment using congenic strains of mice showed that viral immunity requires both self MHC and the foreign antigen - MHC restriction. Why is tissue transplant so Because of MHC polymorphism. difficult? What are CD4 and CD8? Accessory molecules associated with the T cell receptor responsible for intracellular signalling through associated tyrosine kinases. 20 - Allergy, hypersensitivity and review Allergy, anaphylaxis What is an allergy? The most common form of immune disorder. Affects 30% of people. What is hives caused by? Hives/urticaria is caused by the release of histamine into the tissue from mast cells in the skin. What is oedema caused by? Oedema/swelling is caused by leakage of fluid into the spaces between cells. What is anaphylaxis? Anaphylaxis and anaphylactic shock are serious consequences of allergic reactions. This is when oedema and swelling occurs at multiple anatomical sites that are distant from the original site of allergen challenge. Most commonly, the lips, eyes and throat swell, but this can also extend to airways and the gut. Treatment is the immediate injection of epinephrine (addrenaline). 4 classifications of hypersensitivity What are the 4 types of allergies? - Type I - atopic allergy (IgE mediated), immediate - Type II - complement mediated, medium - Type III - serum sickness (immune complexes), medium - Type IV - delayed type (DTH), slow response IgE and the mechanism of type I allergy Describe the mechanism behind Mast cells in the skin possess a receptor for IgE called the FcƐR (Fc epsilon receptor). FcƐR has an type I allergy. exceptionally high affinity toward IgE that are bound to its antigen (i.e. pollen). When first exposed to an allergen, the body mounts an inappropriate B cell response that produces IgE. When next challenged, the allergen cross-links pre-sensitised IgE coated mast cells which degranulate, releasing histamine and other compounds. The chemicals released are highly toxic and cause a local inflammatory response, vascular leakage, smooth muscle/ blood vessel constriction, mucous glands to produce more mucus causing oedema, sensory nerve ending stimulation which results in pain and recruitment of other innate cells such as eosinophils and basophils which also release granules. What is histamine? Histamine is a compound which has different effects on many different cell types, but it is the main cause of tissue oedema. Antihistamines block the action of histamine with its receptor. What are mast cells? Mast cells are innate cells of myeloid lineage that reside in the skin. Mast cells provide protection against complex organisms. These are organisms that can’t be engulfed by phagocytosis. How does an IgM producing B cell An allergen triggers the B cell receptor, causing the B cell to recruit a helper T cell, which causes an switch to producing IgE? interaction between MHC class II and the TcR. The helper T cell is then producing cytokine to help to turn the IgM producing B cell into an IgE producing B cell instead of an IgG producing B cell. One of the main cytokines that regulates that is called Interleukin-4 (IL4). The T cells are either Th1 or Th2 and these are the ones that drive this inappropriate response which makes B cells switch to making IgE instead of IgG. The net effect is that we end up with memory cells that are resident in our lymph nodes which are IgE producing instead of IgG producing. These generate more plasma cells which produce more IgE. Type II hypersensitivity Describe the rhesus (acute In a condition called RhD, there is an antibody present in a newborn baby that reacts to a protein on haemolytic anaemia) case study. the surface of their RBCs. This induces a response by attracting neutrophils so complement is deposited and the net effect is that the neutrophil becomes frustrated and tries to digest the membrane of the RBCs. This results in the lysis of RBCs, causing haemolytic anaemia. This condition is dependent on anti RhD, which is an antibody that the mother has developed abnormally, against the blood of the foetus while it’s resident in the womb. What is rhesus caused by? A blood group antigen RhD on the surface of RBCs. Maternal antibodies developed to fetal RhD cause lysis of the newborn’s RBCs. Rhesus factor is a gene which dictates whether an individual has  the condition. What is required to have a rhesus The mother must be rhesus negative (her RBCs don’t have the rhesus antigen) and have a child with baby? someone who is rhesus positive. Because the genes are dominant, the baby will always be rhesus positive. Sometimes, not always, RBCs can pass across the placenta and leak into the mother’s blood, so the mother will develop a very strong antibody against the rhesus factor in the foetal RBCs. This won’t be bad for the first born baby, but unfortunately the mother will continue to have those B cells producing anti-RhD in her blood. So in the next baby that is born, anti-RhD IgG will pass across the placenta and into the baby. When the baby is born, it has haemolytic anaemia because that antibody induces type II hypersensitivity. What is the treatment for rhesus? If the mum is rhesus -ve and the dad is rhesus +ve, the mum will receive an antibody that is anti-rhesus which effectively kills any RBCs that might have gotten into her blood when she’s carrying the newborn. Treatment of allergies How is desensitisation used as a A skin scratch test identifies the allergen(s). If an allergen is identified, the next step is to inject very treatment? small amounts of those antigens into the patient over a long period of time. This slowly increases the concentrations of the allergens. The idea is to slowly drive the B cells to produce IgG. Hopefully, what will happen is that when you are challenged with the allergen, the IgG binds to the allergen before the IgE. How are monoclonal antibodies A mouse is injected with an antigen. After a 2-3 week wait, the mouse has had a strong IgG response. produced? Splenocytes containing the B lymphocytes are mixed with mouse myeloma (a malignant B cell line) creating a hybridoma. The hybridoma, more often than not, still produces antibodies that the B cell is producing. The hybridoma is isolated and left to replicate. All of the clones will be producing a monoclonal antibody. What are the pros and cons of Pros monoclonal antibodies? - Highly specific for the intended target, so no “off target” effects - Can be tailor-made with just the right affinity - Humanised so they stay in the bloodstream for months - No adverse reactions or toxicity to the antibody - Can be modified to be “bi-specific” for even greater potency Cons - Expensive to develop and make commercially - Side effects of their function can be serious Summary How are the 4 classifications of By how long they take to appear. hypersensitivity defined? Summarise Type I It is immediate and is mediated by IgE and mast cells with a high affinity Fc receptor. hypersensitivity. Summarise Type II Type II hypersensitivity is slower and involves antibody, complement and neutrophils. hypersensitivity. Why are allergies and Th1 and Th2 paradigm. autoimmune diseases more prevalent in industrialised countries? What does the hygiene hypothesis Children are not challenged appropriately with old germs and develop an imbalance Th2 mediated argue? anybody response with fewer regulatory T cells. What are monoclonal antibodies? Single specificity antibodies that are now used as powerful drugs to treat a range of conditions.