Immunology Notes PDF

**Immunology** Who discovered phagocytosis? Elei Metchnikoff 1883-1905 - Immunity via macrophages Who discovered antibody activity? Emil Von Behring and Kitasato Shibasaburo What are the main differences between the innate and adaptive immune systems? +-----------------------------------+--...

**Immunology** Who discovered phagocytosis? Elei Metchnikoff 1883-1905 - Immunity via macrophages Who discovered antibody activity? Emil Von Behring and Kitasato Shibasaburo What are the main differences between the innate and adaptive immune systems? +-----------------------------------+-----------------------------------+ | Innate | Adaptive | +===================================+===================================+ | - Rapid | - Slower than innate | | | | | - Generalised | - Specialised | | | | | - Non-specific | - Has memory | | | | | - Doesn't involve antibodies | - Production of antibodies (B | | | cells and T cells) | | - No memory | | +-----------------------------------+-----------------------------------+ What are the first and second lines of defence in the innate immune system? +-----------------------------------+-----------------------------------+ | First line | Second line | +===================================+===================================+ | Mechanical barriers | Serum -- complement | | | | | - Skin/mucous membranes | - Proteins adhere, initiate | | | inflammation, kill cells | | Normal flora | | | | WBC | | - Competition | | | | - Phagocytic -- n/m | | Secretions | | | | - Inflammatory chemicals -- e/b | | - Mechanical removal and | | | germicidal action | - Natural killer cells | +-----------------------------------+-----------------------------------+ How does inflammation come about and what are it's effects? - Response to injury -- trauma, chemical, infection, heat - Release of mast cells and resident cells - Effects - Prevents spread - Removal of debris/infectious agent - Repair n - Cardinal signs- redness, heat, swelling, pain, limitation of joint movement Describe humoral and cell mediated immune systems. Humoral Cell mediated ------------------------------------- ------------------------------------------------- Production of antibodies by B cells Killing of infected or damaged cells by T cells Describe the primary and secondary immune responses, and how they come about. Primary -- initial exposure slower and smaller response Secondary -- B cells have memory of initial exposure and on second exposure, the response is faster and much larger What are antigens? Substances that induce acquired immune response What are antibodies? Bind to specific epitopes What is an "epitope"? Part of an antigen in which antibody binds to List the granulocytes and describe their functions. **Neutrophil** -- exit blood to engage foreign molecules, phagocytic, produce peroxide and superoxide radicals that are toxic to microorganisms **Eosinophil** -- release enzymes to digest parasitic worms, allergies and asthma, modulating IR **Basophil** -- contain histamine which acts as a vasodilator to attract WBCs to infected area What are the functions of T and B cells? T cells -- activate macrophages, destroy viral infected particles, initiate adaptive IR B cells -- become plasma cells which produce antibodies What do NK cells do? What arm of the immune system do they belong? Innate killing of virus infected and cancer cells Where are Mast cells found and what is their function? What cell type in blood is similar? Similar to basophils but found in tissues rather than blood. Contain histamine which acts as a vasodilator to attract WBCs to infected area List the types of macrophages. Osteoclasts -- bone Kupffer cells -- liver Alveolar -- lung Spleenic -- white pulp in spleen Peritoneal -- peritoneal fluid Microglial -- CNS Describe the haemopoiesis of Macrophages and Dendritic cells. Starts with hematopoietic stem cell in bone marrow which differentiates into a myeloid precursor. The myeloid precursor then differentiates into a monocyte in the blood. Monocyte then differentiates into macrophages and dendritic cells in the tissues. What is the function of Dendritic cells? Phagocytosis of pathogens and migration to lymph node where they mature and present antigen to T cells and therefore activate adaptive IR Describe the basic way the immune system would deal with bacteria and with viruses Upon exposure to bacteria, B cells produce antibodies which bind to epitope of bacteria and destroy bacteria. Memory cells are produced so that on second exposure, response is quicker and larger. Upon exposure to viruses antibodies are produced and bind to virus infected cells. T lymphocytes then phagocytose infected cells **Innate and adaptive immunity** List and describe the features of the innate immune system. First natural defence Prevent pathogens entering body Doesn't differentiate between one pathogen or another Mechanical barriers - Skin - Mucous lining -- traps pathogen Chemical - Lysozyme eyes Normal flora -- compete with pathogens Inflammation -- mast cells: release histamine which causes inflammation Phagocytes -- neutrophils (kill infected cells and die = pus) and macrophages (consume pathogens) NKC -- detect when MHC isn't being produced and kill cells that don't Dendritic cells -- link between innate and adaptive ( eat pathogen and carry info to adaptive immune cells) pass on info to T cells Quicker and non-specific List and describe the features of the adaptive immune system. Differentiate between different pathogens T cells intitial and B cells activated when hasn't caused an infection yet Helper T form memory T cells Cytotoxic T cells kill infected cells B cells produce antibodies which attach to antigens -- tag and signal macrophages, produce B memory cells How is the innate system stimulated and why does the innate system lack specificity? Pathogen-associated molecular patterns - Different structures on pathogens that are recognised by cells of innate IS Damage-associated molecular patterns - Molecules released by damaged cells - Endogenous List the cardinal signs of inflammation. Redness Pain Swelling Heat Loss of function In an inflammatory response what do cytokines, chemokines do? Cytokines -- proteins released by cells that affect other cells Chemokines -- proteins released by ells that attract other cells to area How is the adaptive system stimulated and what makes it so specific? If Toll-like receptor 3 (TLR3) is stimulated what is the outcome? What stimulates TLR3? Which TLRs are stimulated by bacterial proteins? TLR1, 2, 5, 6 Where are the NOD-like receptors located? Cytosol Describe the two mechanisms by which NK cells are activated. Exposed Fc receptors Absence of surface MHC class I molecules What do Dendritic cells and macrophages do? Are they part of the innate or adaptive immune systems? Antigen presenting cells that move around and detect foreign antigens. Deliver message to adaptive to innate. What are the down stream effects of an antibody binding to an antigen? Antibody Dependent Cell Mediated Cytotoxicity -- NK cells can attach to antibodies on surface of cells List the classes of antibodies and there function. IgG IgA IgM IgE IgD What are primary and secondary immune responses? How do cytotoxic T cells (Tc) recognise cells for destruction? Infected cells express antigen on surface by MHC. T cell receptor binds and destroys cells expressing antigen with MHC 1. Kills by release of toxic molecules. Describe the immune steps that happen when a person is infected with agent. **Immunogens and antigens** What is an antigen? Which part of an antigen interacts with the immune system? Any molecule or agent that is able to specifically interact with a component of the immune system What is the difference between a hapten and an immunogen? Hapten -- Cant stimulate an immune response by itself Immunogens -- can stimulate an IR Penicillin is an example of a drug that is classified as a hapten. What must happen inside the body for this hapten to stimulate an IR? Must attach to hapten carrier molecules so that they become immunogenic Name the four major classes of antigens? Which two are considered poorly immunogenic by themselves? Polysaccharides Proteins Lipids -- poorly immunogenic on own Nucleic acids -- poorly immunogenic on own List the 4 principal requirements for immunogenicity and explain each with a sentence? 1. Foreignness -- more foreign = more immunogenicity 2. Molecular size -- larger molecule = higher immunogenicity 3. Chemical complexity -- increased structural complexity = higher immunogenicity 4. Susceptibility to antigen processing and presentation (degradability) What is an epitope? Part of antigen that interacts with the immune system List three components of the immune system that can recognise epitopes? Antibodies, B and T cells What kind of antigens are recognised by T cells compared with B cells? (native vs. processed, types of epitopes, classes of antigen recognised) B cells: native: accessible, sequential or non sequential: protein, polysaccharide, lipid T cells: processed: linear: peptide What is the difference between a sequential epitope and a conformational epitope? Sequential binds to the sequence of AA but conformational binds depending on the 3D structure What is an adjuvant? Substance that when mixed with antigen, enhances immune response Explain what a neo-antigenic determinant is? An epitope on a neoantigen. Neoantigen is an antigenic substance produced by a tumour cell Name one advantage of cross reactivity? Could you explain this in a paragraph? Enables broader immunity against pathogens Name one disadvantage of cross reactivity? Could you explain this in a paragraph? Antibody response to self (autoimmune) **Antibody Structure** Excluding disulphide bonds, what are the four primary components of an immunoglobulin molecule? Light chain Heavy chain Constant regions Variable regions Which part of the immunoglobulin molecule is responsible for antigen recognition? Variable region Name two cell types that express immunoglobulin on their surface? Memory cells, NK cells Name the five classes of human immunoglobulin? Draw a basic schematic diagram of each. IgG IgA IgM IgD IgB Which cell type is responsible for actively secreting immunoglobulin? Which of the human immunoglobulin isotypes is not actively produced by this cell? Plasma cells, IgD What must happen for a memory B cell to transform into a plasma cell and begin producing antibody? Re-infection and exposure to antigen Which isotype of antibody will be produced if there is no T cell help? IgM Which two human immunoglobulins have an extra fold domain in their heavy chains? IgM and IgG  Which part of the immunoglobulin molecule is responsible for biological activity (once antigen has been bound to the Aby molecule)? Fc region Using diagrams, explain the fragments that are produced when an immunoglobulin molecule is treated with a. Pepsin: One Fab fragment b. Papain: 2 fab fragments and fc portion c. Mercaptoethanol: 2 separate light chains and 2 separate heavy chains  Which human immunoglobulin is the most prevalent in serum and which can cross the placenta? IgG Which human immunoglobulin can best activate complement? IgM Which human immunoglobulin (only one) can bind to the Fc receptors on PMN, Mϕ, NK cells? IgG What is the main structural difference between the human subclasses of IgG? Variation in number of disulphide bonds During a humoral immune response, which immunoglobulin isotype is produced first? Which is produced second? IgM and IgG What is the difference between antibody affinity, avidity and valency? Affinity = strength of binding between one antibody binding site and one epitope Avidity = overall strength of whole ab and epitope Valency = number of contact points Which class of antibody would be expected to have the strongest avidity? Why? IgM Which human antibody isotype is most prevalent in the body? IgA **Antibody Function** What does the term 'antibody specificity' mean? One ab only recognises a specific antigen Which pathway of complement may be activated by antibody binding to antigen? Classical What is the end result of the activation of this pathway of complement? Cell lysis With traditional/typical ADCC, which immune cell is activated, which isotype of immunoglobulin is involved, and which cells are targeted by this immune response? What about with the other form of potential ADCC? NK cells, IgG, tumour cells and virally infected cells IgE and parasites Only one class of maternal immunoglobulin is transferred placentally. Which one, and what biological purpose does this passive transfer have? IgG -- transfer of immunoglobulins from mother to fetus Use a schematic diagram to explain how antibody molecules can neutralise bacterial toxins/venom? Label ALL interacting particles and components  IgG, IgA and IgM can all bind to viral particles. How can this attachment potentially inhibit viral infection? What conditions must exist for this process to be successful? If sufficient amount of antibodies, they block all available receptors and prevent viral entry into cell. Can also block adhesion of virus to cell membrane Describe the process of antibody opsonisation of an immune target. Which isotype of immunoglobulin is involved? Ab binds to epitope on antigen on target cell and coats particle, marking it for destruction. Why is IgM the isotype of immunoglobulin that has the best agglutinating ability? Why is IgA the next best? Has a larger conformation and IgA has second largest conformation Briefly describe the events that occur during the primary and secondary exposure of the immune system to an allergen and explain the role of IgE in this process? Sensitisation 1. Allergen invades body 2. Plasma cells produce IgE 3. IgE binds to mast cells and basophils Secondary 1. More allergen invades 2. Antigen combines with IgE attached to mast cells and basophils which trigger degranulation and release of histamine 3. Histamine = BV dilation and which causes leaking and edema. Mucus is secreted and causes smooth muscles to contract Why can the release of histamine cause asthma in susceptible people? i.e., what biological effect does it have? The body has an allergic recation to an allergen and mast cells/basophils release histamines. Histamines cause the smooth muscle walls of the trachea to close and cause difficulty breathing What determines whether a plasma cell secretes IgM or one of the other isotypes of immunoglobulin? Recognition of specific antigens. Antibody specificity If an antisera against the venom of the blue ring octopus was made in a rabbit, what would the correct name of the antibodies in that formulation be described as? Rabbit anti blue ring octopus antiserum In haemolytic disease of the newborn, which class of immunoglobulin is involved in the manifestation of the disease? Are these immunoglobulins manufactured in the mother or the infant? What antigen do the immunoglobulin molecules target? What causes the production of these antibodies? IgG anti D antibodies from mother bind to D antigen in foetus **Basic Antibody Based Immunoassays** What is an epitope? Part of an antigen that antibodies bind to If you injected a goat with human IgM what would you call the resulting antibody made from the goat? Goat anti-human IgM What is crosslinking? When antibodies conjugate together to form larger molecules and cause agglutination What is the difference between agglutination and precipitation in relation to antigen-antibody reactions? Agglutination is when antigens and their respective antibodies clump together. Precipitation is when antibodies bind to their soluble antigens Why would IgM be the best antibody isotype to crosslink? Has a larger conformation Explain the difference between haemagglutination assays and haemagglutination inhibition assays Haemagglutination - Inhibition -- the serum containing suspected antibody is treated with antigen to remove it prior to agglutination. If the antibody is present, agglutination is inhibited How are latex particle agglutination assays used to detect antigens and how are they used to detect antibodies? Latex particles are coated in a virus antibody. The antigen then binds forming aggregates What is the zone of equivalence? When there are optimal amounts of antibody and antigen Explain how single radial immunodiffusion is used to determine concentration of protein. Gel is made with antibodies in agar. A hole is made in the gel and antigen is added. Antigen diffuses through gel and contacts antibody. Zone of precipitation is formed and measured to determine the concentration of antigen Explain the basic principle behind an Ouchterlony gel. Two adjacent wells in gel. One well contains antigen the other contains antibody. They both diffuse through gel forming a ab/ag complex and line of precipitation forms. What precipitation reactions would you see for an identical, non-identical and partially identical antigens in an Ouchterlony gel? Describe an immunofixation electrophoresis method and what it is used for. Used to detect paraprotein in serum (multiple myeloma) Serum is electrophoresed in agarose gel and then overlayed with specific antisera. Ag/ab complex precipitates in gel Explain the nephelometry method of determining protein concentrations Antigen mixed with antibody in tube causes turbidity as aggregate forms. Measured by passing light through tube and detecting scattering of light. What is a "conjugate"? Labelled antibody **Advanced Antibody Based Assays** What does ELISA stand for? Enzyme linked ImmunoSorbent assay Describe the different types of ELISA. Direct -- antigen immobilised on surface of plate and detected with antibody specific to antigen Indirect -- primary ab specific for antigen binds and then a labelled secondary antibody against host binds to primary Sandwich -- two antibodies specific to different epitopes of antigen. One of the antibodies are used to coat surface of multi-well plate where it serves as a capture antibody to facilitate immobilisation of antigen. Other antibody is conjugated and facilitates the detection of antigen Competitive -- detects signal interference. Sample antigen competes with reference antigen. More antigen = less interference signal  How would you use an ELISA to measure the concentration of a particular serum protein? Describe a competitive RIA. What is the difference between Direct and Indirect immunofluorescence? Direct uses dye-conjugated antibody to stain target protein Indirect involves first binding primary antibody to target then detecting primary antibody using conjugated secondary antibody Compare and contrast Immunohistochemistry to Immunofluorescence and ELISA methods. Similar to ELISA but on tissue Similar to immunofluorescence except no fluorescent dye Uses enzyme labelled antibody A diagram of a person\'s reaction Description automatically generated What is "antigen retrieval" and in what circumstances is it used? Used to unmask epitopes from frozen sections used in IF and IHC What parameters are measured by Flowcytometry and what information is obtained from each parameter? Measures forward scatter (size), side scatter (granularity), fluorescence (surface marker) What cell types have CD3, CD4, CD8 and CD19 expressed on their surface? 3/4 T cells 8/19 B cells Describe the SDS-PAGE and Western Blot methods. SDS-PAGE -- Charged molecule migrates to opposite electrode. Separates based on size. Smaller molecules move through the gel faster. Westernblot -- Electrophoresis in SDS-PAGE and then electric current is used to force protein from gel to nitrocellulose. Then stained with appropriate labelled antibody Why is the advantage of doing a Western Blot to detect a serum protein? Places specific protein on matrix Highly specific protein ID EXAM **Infectious diseases** What enzyme and what cells type is involved in X-linked agammaglobulinaemia? Decreased number of B cells = Decreased Ig Mutation in B cell tyrosine kinase gene on X Describe DiGeorge syndrome Failure to form T cells due to hypoplasia of thymus Symptoms: cleft palate, facial cleft, low set ears, absence of parathyroid gland, heart malformed Vulnerable to virus and intracellular bacterial infection After age 5 most T cells increase What is SCID? Severe combined immunodeficiency Failure to develop B and T cells Thymus and lymphoid tissue decreased Adenosine deaminase deficiency = accumulation of toxic waste in lymphocytes HIV destroys what cell type and what is the effect? Thelp cells (CD4) destroyed Severe immunosuppression Describe what happens to CD4 T cells, antibody levels and virus levels in the course of AIDS. CD4 T cells decline as they are destroyed by HIV Antibody levels high in beginning but decreases after CD4 cell count decreases and ab can't be made efficiently Virus levels peak then low during latent phase as immune system is suppressed. High again after List the four types of hypersensitivity Type I -- immediate hypersensitivity -- th2 response leading to excessive IgE to ag Type II -- circulating Ab bind to Ag on cell surface to tissue inappropriately -- complement activation Type III -- Soluble ag reacts with ab and complexes deposit Type IV -- delayed type hypersensitivity What antibody type and cells types are involved in an allergic reaction? Th2, IgE, mast cells, eosinophils List the factors that may result in autoimmune disease Sequestered antigen Cross reactivity with microbial agent Polyclonal activation Non-infectious triggers Why do most people not make an immune response to self? Self tolerance Describe the mechanisms of self tolerance. Anergy -- non-responsiveness of cells upon contact with ag Receptor editing -- genetic rearrangement of variable region of BCR and TCR -- no longer specific for ag Clonal deletion -- autoreactive T and B cells eliminated by apoptosis during development Clonal ignorance -- cells that remain inactivated due to low affinity with self antigen T cell suppressors -- Treg cells regulate/suppress T cells non-specifically Describe the immunopathology of Myasthenia Gravis. Ab directed to acetylcholine receptor at neuromuscular junction Blocks binding of acetylcholine Weakness and fatigue of voluntary muscles Why do some people produce anti-nuclear antibodies (ANA)? 1. Genetics 2. Environmental -- infections, UV, medications 3. Immune system dysregulation -- imbalance in IS = activation of autoreactive B cells 4. Loss of self-tolerance 5. Chronic inflammation Describe the immunopathologies of SLE and RA. SLE Mediated by autoab with type I and II hypersensitivity Tissue damage due to deposition of dsDNA ab complexes RA Unknown trigger Autoreactive CD4 t-lymphocytes activate macrophages Production of pro-inflammatory cytokines Cytokines induce production of metalloprteinases and RANK ligand by surrounding fibroblasts What clinical findings and lab tests are used to differentiate SLE and RA? SLE usually younger women RA have joint erosion and deformity -- women over 40 Blood tests -- RF in 70% of RA and less than 30% SLE ACPA in RA Describe the staining patterns of ANA and list the diseases associated with each pattern Homogeneous -- even staining nucleus, dense staining mitotic body Speckled -- uneven stained nucleus, unstained mitotic body Centromere -- fine speckled staining in nucleus, lining staining in mitotic body Nucleolar -- 2-5 dense staining granules in nucleus What are the two ANCA patterns and what antigens are involved in each? Cytoplasmic granular pattern -- proteinase 3 Perinuclear pattern -- myeloperoxidase What is a cryoglobulin? What types of cryoglobulins are there? Immune complexes that can deposit in extremities and form purpura 1. Monoclonal ig 2. Rheumatoid Factor (monoclonal IgM) **Complement** Name the three different complement pathways, and describe how each is activated Classical pathway Activation - ab-ag complex Key component - C1q binds to Fc regions on IgM or IgG bound to ag C1s becomes enzymatically active (C1s esterase) -- cleaves C4 into C4a and C4b C4b binds to C2 which is cleaved into C2b and C2a and associates to form C4b2a MC-Lectin pathway Activation -- MBL bind to surface of pathogens Key component -- MBL + MASP1 and 2 which cleave C4 and C2 = C3 convertase Alternative pathway Activation -- spontaneously at low levels of C3 -- enhanced when C3b binds to foreign surfaces Key component -- C3bBb on pathogen surface Properdin stabilises -- rapid response What role does properdin play in the complement cascade? Stabilises C3bBb All three pathways converge with the formation of C3 convertase How are the components of the classical pathway named? C followed by number of order of discovery List 3 functions of complement 1. Opsonisation 2. Inflammation 3. Lysis What is the MAC? Describe how it is formed Membrane attack complex C6 first binds C5b on cell surface, followed by C7 and C8 -this complex penetrates into the cell membrane. C8 is first component to penetrate the membrane. C5b-8 complex acts as receptor for C9 -many C9 molecules interact with the complex, which results in polymerization of 10 to 16 x C9 molecules into a pore What is the C1 complex? - Recognises and binds to pathogens - Triggers cascade - C1q binds to Ab-Ag or Ag - C1r activated which activates C1s - C1s cleaves C4 and C2 = C3 convertase What happens when C1s binds to the antibody-antigen complex? It becomes enzymatically active and becomes C1s esterase What are opsonins? Molecule that enhances phagocytosis of particles by macrophages and neutrophils Draw an overview of the complement pathways -- include the name of each pathway, how it is activated and list the complement components involved in each. Show where they converge and include the terminal pathway **Purification** Describe the layers seen after blood has been centrifuged. What is a buffy coat? RBC layer, WBC layer and plasma layer WBC layer is buffy coat How do density gradient centrifugation improve separation of WBCs? - Improved purity of WBC - Enhanced recovery and yield - Specificity for different WBC subtypes  Describe the layers and what cells are found in each layer after centrifugation using Ficoll Hypaque? Mononuclear cells layered on top of Ficoll Hypaque RBC and granulocytes layered under Ficoll Hypaque Platelets and plasma layered over mononuclear cell layer Describe the magnetic bead separation technique Small magnetic beads coated with monoclonal Ab Added to blood and incubated -- specific cell type binds to beads forming rosettes Magnet placed near tube and cell rosettes migrate to magnet What information does forward and side scatter give in flow cytometry? Forward scatter -- size Side scatter -- granularity Describe the components of a flow cytometer Fluidics -- cell suspension in single file Optics -- generates and collects light Electronics -- process optic signals How does cell sorting on a flow cytometer separate cells? Cells labelled with antibodies with fluorescent tags and directed to specific surface markers Cells are then measured by laser Cells become charged and can be separated by electric field List the steps in the serology based HLA typing technique Serum containing known specific anti-HLA antibody in tray wells Lymphocytes added to serum -- read with specific ab if express specific HLA type Rabbit complement added -- lymphocytes with ag-ab complex killed Detect killing by addition of eosin dye, ethridium bromide -- killed cells larger and stained Describe the neutrophil functional assays Phagocytosis -- measures uptake of bacteria or latex particles by counting or label Intracellular killing -- use staph aureus -- test for viability Directional migration -- movement through filters or gradients Measure up regulation of surface markers using monoclonal ab Describe the cytotoxic T cell functional assay Measure of how well cytotoxic T cells can kill target cells Target cell labelled with radio isotype Mixed with lymphocytes and incubated Release of ^51^Cr surrounding media is a measure of T cells ability to kill target **B and T lymphocytes** Where do T cells come from and where do they mature? Mature in thymus and primed by ag in spleen and mucosal and cutaneous lymphoid tissue Where do B cells come from and where do they mature? Mature in bone marrow and primed by ag in spleen, lymph nodes and mucosal lymphoid tissue Where are dendritic cells located in the body? Spleen Which type of dendritic cell is a specific B cell presenter? Follicular dendritic cell Which type of APC presents to T cells? Dendritic cells What type of antigens do B cells interact with? What type of antigens do T cells interact with? Do both react to native antigen? B cells -- Ag directly (native) T cells -- processed ag that are presented by APCs, presented by MHC What is the biological role of the CD4 and CD8 antigens on T cells? CD4 -- T helper cells -- activate macrophages and stimulate killing of phagocytosed antigens CD8 -- cytotoxic T cells -- bind to and kill infected target cells Which class of MHC receptor do CD4+ and CD8+ T cells bind to? Which cells express class I and II? CD4+ binds to MHC class II -- APCs CD8+ binds to MHC class I -- on all nucleated cells List the three types of T helper cell (other than CD4+ T reg cells) that can be produced following antigen presentation by an APC to a naïve T helper cell? Th1, Th2, Th17 List the key effector function of Th1 cells with respect to humoral immunity? Enhances macrophage and APC killing of ingested microbes B cells stimulated to produce IgG1 and 3 Activation of cytotoxic T cells List the key effector function of Th2 cells with respect to humoral immunity? Alternative macrophage activation Intestinal mucous secretion and peristalsis Isotype switching of B cells to produce IgG4, IgE, IgA Do B cells present antigen to Th1/Th2 cells or do these T helper lymphocytes present antigen to the B cells? T helper lymphocytes present antigen to B cells What is the main function of activated CD8+ lymphocytes (irrespective of T helper involvement in their activation)? Killing of infected cells What is T dependent humoral immunity? Explain what the benefit of this process is over T independent stimulation of B cells by antigen? The presentation of protein/peptide antigen by B cells primed to T helper cells in the spleen and/or other lymphoid tissue What process/cellular interactions are required to produce long lived plasma cells and memory B cells? T dependent humoral immunity Outline the steps/processes involved in the adaptive immune response to an extracellular infection? 1. Recognition of pathogen -- recognises PAMPs 2. Antigen processing and presentation -- APCs anf MHC class 2 presentation 3. Activation of CD4+ - DC present to CD4. Differentiate into Th1, 2, 17 4. Activation of B cells = Ab production 5. Neutralisation, opsonisation, complement activation 6. Activation of macrophages by Th1 cells 7. Pathogen clearance and immune clearance 8. Formation of memory **Cytokines** Briefly describe the key properties shared by most cytokines - Low molecular weight proteins - Synthesized in active and inactive forms - Rapid secretion - Secretion is brief and self-limiting - Active at very low concentrations Interferons were the first cytokines to be identified True Cytokine functions can be described as redundant because several cytokines perform the same function. True Name two pro-inflammatory cytokines TNF, IL1, IL6 Differentiation of CD4+ Th0 T cells into the Th1 T cell subset depends on: 1.Interleukin (IL)-4. 2.IL-6. 3.Interferon-gamma (IFNg). 4.IL-2. 5.Transforming growth factor-beta (TGFb). **B and T cell activation** Where in the body is a T Cell is first activated (primed)? Lymph node T cells are directly activated by antigen in its native form, i.e. by exposure to the intact (or complete) protein molecule. True B cells are directly activated by antigen in its native form, i.e. by exposure to the intact protein molecule. False Antigen presenting cells must be activated before they can activate a T cell. True/false True Both T cells and B cells require two signals to be fully activated. Briefly describe them. T cells 1. Antigen in MHC molecules binding to TCR 2. Activated APC that express co-stimulatory molecules B cells 1. Epitope binding to BCR 2. Cytokines secreted by activated T cells Briefly describe how B cells get help from T cells. 1. B cell ag recognition 2. Ag processing and presentation 3. Interaction with CD4 4. Co stimulatory signals 5. Cytokine secretion 6. B cell activation and secretion Major Histocompatibility (MHC) Class I molecules on antigen presenting cells: A.Present extracellular antigens to CD8+ T cells only. B.Present intracellular antigens to CD4+ T cells only. C.Present intracellular antigens to CD8+ T cells only. D.Present intracellular and extracellular antigens to B cells. E.Present intracellular and extracellular antigens to CD4+ and CD8+ T cells. **Hypersensitivity** List and describe the four hypersensitivity types Type I (immediate hypersensitivity) -- Mediated by IgE -- within 30 minutes Type II (Cytotoxic) -- mediated by IgG or IgM binding to antigens on cell surfaces -- activating complement cascade -- cell destruction Type III -- mediated when immune complexes activate complement; granulocytes are attracted to site of activation and damage is caused by the release of lytic enzymes (reaction within hours) Type IV (delayed type hypersensitivity) -- involves cytotoxic T cells and Th1 cells; mediated by Th1 which release cytokines -- accumulation and activation of macrophages and cytotoxic T cells -- local damage (days to week) What is Atopy? Name a condition that is atopic IgE mediated hypersensitivity -- tendency to develop allergic diseases Eczema, arthritis, asthma What are the steps in an allergic reaction? 1. Sensitisation -- IgE produced and minds to mast cells 2. Activation -- re exposure to antigen triggers mast cells to release contents of granules 3. Effector step -- complex response results from effects of inflammatory mediators released by mast cells In what circumstances is IgE production favoured? Parasitic infections and allergic responses Name two mediators in the effector step of an allergic reaction Histamine Cytokines and chemokines Name two possible clinical consequences of an allergic reaction Allergic rhinitis Food allergies In a type 2 reaction how is the target cell damaged or destroyed? Opsonisation and phagocytosis by macrophages or neutrophils expressing receptors for C3b Lysis by MAC How is damaged caused in a type 3 reaction? C3a and C5a increase vascular permeability and neutrophil accumulation = release of lytic enzymes C3a and C5a induce mast cells to release mediators = more toxic mediators Type 4 hypersensitivity is also known as? Delayed type hypersensitivity Explain how contact hypersensitivity occurs Contact sensitising agent penetrates skin and binds to self-proteins which are taken up by Langerhans cells Langerhans cells present self-peptides haptenated with contact sensitising agent to th1 cells, which secrete IFNy and other cytokines Activated keratinocytes secrete cytokines such as IL1 and TNFa and chemokines Products of keratinocytes and th1 cells activate macrophages to secrete mediators of inflammation Give one clinical example of each of the hypersensitivity types and describe example. Type I -- food allergies -- mast cell mediators lead to localised smooth muscle contraction and vasodilation = vomiting and diarrhea Type II -- Transfusion reactions -- transfusion of incompatible blood; if wrong type of blood = Transfused RBC bind IgM in serum -- activate complement and transfused cells destroyed Type III -- Farmers lung -- large amounts of IgG to spores of moulds that grow on rotting hay. Re-exposure = insoluble immune complexes, vessel wall damage, haemorrhage and necrosis Type IV -- Poison ivy dermatitis -- penetrates skin and forms hapten carrier complex. Initial -- allergen presented by Langerhans cells = expansion of th1 clones recognising presented allergen. Re-exposure -- intraepithelial blister **Tumour immunology** List 3 key concepts in tumour immunology 1. Tumours express Ag that are recognised as foreign by host IS 2. IR frequently fail to prevent growth of tumours 3. IS can be activated by external stimuli to effectively kill tumour cells and eradicate tumours Describe the three phases related to the tumour growth and the immune system Immune surveillance -- recognition and elimination of abnormal cells Immune editing -- elimination -- IS targets and kills some tumour cells. Equil -- mutation to evade IS Tumor escape -- additional mutations to enable escape from IS -- Tumor growth What is cancer immunoediting and in what phase does it occur? Where immune system suppresses growth of tumour cells and promotes them towards malignancy Which lymphocyte subsets are involved in direct killing of tumour cells and describe the mechanisms by which they destroy the target tumour cell Cytotoxic T -- Perform (pores) and Granzymes (induce apoptosis) Natural killer -- Perforin and granzymes. Cytokine release -- TNF and IFNy How do tumour antigens come about? List two types of tumour antigen. 1. Mutations -- oncogenes or tumour suppressor genes 2. Overexpression -- proteins overexpressed in tumours 3. Viral ag -- in cancers ass. With viral infections 4. Post translational mod -- glycosylation can create neoantigens How do cancers evade immune surveillance? 1. Immune checkpoint activation -- checkpoint proteins "brake" IR preventing T cells from attacking 2. Immunosuppressive factors -- directly inhibit immune cells (IL-10 TGFb) 3. Ag loss or mod -- down regulation or mutate expression of Ag -- reduced visibility to immune cells Macrophages can inhibit or promote tumour growth. Which phenotypes are involved and how do they affect the tumour? M1 -- Kill tumour cells -- production of NO M2 -- tumour progression -- secrete VEGF, transforming TGF-B and other soluble factors that promote tumour angiogenesis Why do cancers with high mutation loads more likely to elicit an immune response? Higher mutation = neoantigens (foreign) -- more likely to recognise. Wider variety of Ag List and describe two immunotherapies currently being tested. CAR-T cell -- modifying own T cells to recognise and attack cancer cells -- genetically engineered to express chimeric antigen receptor that targets specific ag Checkpoint inhibition -- monoclonal ab that block proteins that inhibit T cell activation and proliferation Explain adoptive therapy in relation to treatment of cancer. 1. Lymphocytes of a patient may be expanded by culture in IL-2 and infused back 2. Lymphocytes may be transferred by CAR genes Can lead to tumour regression Promote robust T cell activation **MHC and HLA** Where are the genes of the MHC located? Chromosome 6 What is the function of MHC molecules? Breakdown proteins of invaders and together move to surface to present antigen to T cells What different forms of antigen do B and T cells recognize? B cells -- recognise antigen in soluble form (proteins, nucleic acids, polysaccharides, some lipids, chemicals) T cells -- Non soluble antigens (fragments of protein) What size peptides do MHC class I and class II molecules bind? MHC class 1 -- 8-15 MHC class II -- 11-30 Which domains of MHC class I molecules most polymorphic and which are most conserved? A1 and a2 To what T cell types do MHC class I and class II present peptides and what specific T cell molecule is expressed for each type? Class I -- present to cytotoxic T cells Class II -- present to T helper cells What cell types are MHC class II molecules expressed? Expressed on professional APC How are MHC molecules able to bind a large variety of different peptides? Binding site is flexible at early intracellular stage -- Binding site folds around peptide Describe how exogenous and endogenous antigens are processed. Exogenous - Ag taken into cell by phagocytosis - Ag internalised and contained in intracellular vesicle - Fuses with endosome or lysosome - Ag broken down due to low pH and enzyme activity - MHC class II formed in rough ER in ass. With CD74 -- moves to GA then to acidic vesicle - Degradation of CD74 removes most but leaves part in binding groove - Fusion with endosome/lysosome - HLA-DM facilitates exchange of clip for Ag - MHC class II/peptide complex moves to cell surface for presentation Endogenous - Proteasome cleaves protein - Cytosolic enzymes cleave further - Transported to ER by TAP-1 and TAP-1 Why are there no T cell responses to self proteins when most of the MHC molecules on the surface of cells are bound to self proteins? T cells reactive to self antigens removed during differentiation in thymus Pathogens produce second signal which isn't produced by self proteins **Monoclonal Antibodies** Describe the structure of an antibody Heavy and light chains Constant and variable regions What is "clonal selection"? Clonal proliferation of B cells Those cells triggered by contact with Ag are able to undergo successive waves of proliferation = large \# of genetically identical plasma cells What are "Specificity" and "Cross-reactivity" in relation to antibodies? Ability of Ab to distinguish between Ag Cross reactivity -- single ab may react with an epitope that didn't stimulate production How are specificity and cross-reactivity related? Lower specificity = increased cross reactivity What is the difference between a monoclonal antibody and a polyclonal antibody? Polyclonal less specific than monoclonal Polyclonal may cross react Monoclonal = 1 type of Ab directed to 1 epitope Briefly outline how monoclonal antibodies are produced. Infect animal with Ag Remove spleen and take out B cells Mix B cells with myeloma cells = immortality What is a "Hybridoma" and how is it selected for? Fusion of B-cell and cancer cell Myeloma cells don't survive as they don't produce HGPRT Grow on HAT media -- Cancer-cancer cells can't survive and B cell -- B cell die What is "HAT" media? What is in it and how does it work? Hypoxanthine, Aminopterin, Thymidine Aminopterin stops de novo pathway Cancer -- cancer will not grow due to lack of HGPRT B cell -- B cell die (not immortal) Hybridomas will grow Give 3 uses for monoclonal antibodies Primary Ab in assays Detecting tumour Ag CD ID on surface of WBC Bacteria and virus ID What is a "humanised" monoclonal antibody? Antibodies from non-human species whose protein sequence have been modified to increase similarity to human Ab A diagram of human structure Description automatically generated What is Rituximab and describe what is it used for. Chimeric monoclonal antibody directed against CD20 CD20 expressed by B cells Used to remove B cells B cell type cancer **Immune response to infectious disease** List three functions of antibodies against viruses? - Neutralisation of virus -- Ab bind to viral particles and block binding to host cells - Opsonisation -- ab tag viruses for destruction by immune cells - Activation of complement system -- MAC formation and lysis of virally infected cells Which innate and adaptive cell/s can directly kill virus-infected cells? Innate - NK cells -- NK cells detect Ab coated cells via Fc receptors, release of toxic granules Adaptive -- CD8+ T cells -- APCs phagocytose infected cells and present viral ag to CD4+ and CD8+ cells, CD8+ activated to kill cells via perforin and granzyme B Which helper T cell subset plays a role in responses to extracellular fungi? Th17 Briefly explain the difference between antigenic shift and antigenic drift Shift -- Virus previously adapted to another animal enters new animal and adapts to circulate in new host (creates new virus) Drift -- Ag changes from accumulation of mutations (new variants/strains) List three mechanisms by which virus-infected cells are eliminated 1. NK cells kill infected cells lacking MHC Class I 2. NK cells kill infected cells by detecting Ab bound to cell (ADCC) 3. Cytotoxic CD8+ T cells kill infected cells via Ag presented on MHC class I Briefly explain the role of type I interferons in anti-viral responses Enhancing immune cell function -- activate various immune cells (NK and macrophages) Promote adaptive immunity -- promote activation of dendritic cells, enhancing APC, and stimulating development of CD8+ cytotoxic T cells Which helper T cell subset plays a role in responses to *Plasmodium sp*? Th1 produces IFN-y which activates macrophages. It also enhances CD8+ T cell repsonse Briefly explain the adaptive immune response against *Schistosomasp* Th2 response produces IL-4 and IL-13, activate eosinophils, basophils and mast cells IgE and IgG Ab produced (ADCC) **Immunisation** Describe three points of evidence for why vaccines rely on antibodies Neutralisation of pathogen Passive immunisation -- transfer of Ab confers protection Memory B cell responses -- Long lasting Ab production after vaccination Briefly explain the process by which a vaccine leads to immunity 1. Antigen introduction 2. Innate immune activation -- Dendritic cells and APCs 3. Antigen presentation to T cells 4. Activation of T cells and B cells -- CD4+ and CD8+ T cells, B cells differentiate into plasma cells which produce Ab 5. Formation of memory cells Describe three differences between live/attenuated and non-live vaccines Live attenuated - Longer lasting - Fewer doses - Live pathogen that's been weakened - Mimic natural infections Non-live - Short lasting - More doses required - Killed or inactive pathogens - Stimulate humoral response List three potential methods to improve immune responses with vaccines 1. Adjuvants -- promote inflammation, enhance Ag presentation, stimulate specific immune pathways 2. Increased spacing -- 8 weeks improves memory response 3. Starting Ag valency/affinity and b cell receptor activity 1. True or False - mRNA vaccines enter the nucleus False - Antibodies produced by B cells help prevent infection True - Adjuvants are used to enhance immunogenicity True 3.