Immunology 1: Introduction to ImmunologyExplain the importance of immunology for human healthFunction of the immune system: to identify and eliminate harmful microorganisms and substances Mechanisms of action:1. Distinguishing ‘self’ from ‘non-self’ proteins2. Identifying ‘danger’ signals (e.g. from inflammation) The immune system must make a compromise between removing the pathogen and causing accidental damage to the host (immunopathology)Failure of proper function of the immune system function may lead to: Persistent/fatal infections Allergy Autoimmune disease Transplant rejectionThe advent of vaccines resulted in a significant increase in human health (e.g. vaccines for diphtheria, polio and measles)Generation times: time taken for reproduction Pathogens have much shorter generation times than hosts (i.e. humans) The host exerts selection on the pathogen and the pathogen exerts selection on

the host The pathogen replicates and can evolve much faster than the host The host relies on a flexible and rapid immune response with a degree of non-

specificity

Outline the basic principles of immune responses, and the timescales in which they occur

Innate immunity Acquired immunity

Involves pre-formed cells and molecules Involves clonal selection

Fast response Slow response

Non-antigen specific: i.e. limited specificity Antigen specific: i.e. high degree of specificity

Innate immunity: an early phase of the response of the body to possible pathogens, characterised by a variety of non-specific mechanisms and pattern recognition receptors; it does not generate memory Anatomical/physical barriers: Skin Mucous membranes: produce mucous; cilia propel the mucous along epitheliaPhysiological barriers: Low pH (in the stomach) Secretion of lysozyme (in the tears)

Interferons Antimicrobial peptides Complement

Components of the innate immune system

Cellular components: granular leukocytes Humoral components

Natural killer cells (NK) Acute-phase proteins

Macrophages (mononuclear phagocytes) Cytokines

Neutrophils (polymorphonuclear leukocytes or PMN) Complement

Basophils/mast cells

Eosinophils

Granulocytes: Neutrophils (PMN): multi-lobed nucleus; phagocytic; 50-70% of circulating

leukocytes Eosinophils: bi-lobed nucleus; act against parasites; 1-3% of circulating leukocytes Basophils: release granules containing histamines, serotonin or prostaglandins; not

phagocytic; less than 1% of circulating leukocytes Cytokines: small proteins which carry messages between cellsMechanism of cytokine action:1. A stimulus induces cytokine-producing cells to secrete cytokines2. Cytokines bind to a receptor on the surface of a target cell3. This stimulates gene activation which has biological effects: cytokines stimulate

proliferation of lymphocytesFunctions of the innate immune system: Buys time while the acquired immune system is mobilised Stimulates the acquired immune response (e.g. via cytokines and complement)Acute phase inflammatory response: occurs in response to tissue damage (e.g. due to infection or trauma)Effects: Fever (rise in body temperature) Increased production of acute phase proteins, mainly by the liver:

o C-reactive protein and serum amyloid protein: bind to molecules found on the cell wall of some bacteria and fungi

o Mannan-binding lectin: binds to mannose residues- these are not found on mammalian cells

o These acute phase proteins direct phagocytes and complement to combat the infectious agent as part of the innate immune response

Acquired/adaptive immunity: the response of antigen-specific lymphocytes to antigen; it involves the development of immunological memory; adaptive responses can increase upon repeated exposure to the potential pathogen Antigen specific Can form memory Requires priming Effector arms: include B lymphocytes, T lymphocytes and antibodies

Components of the acquired immune system

Cellular components Humoral component

T lymphocytes Antibodies

B lymphocytes

Dendritic cells

(Basophils/mast cells)

(Eosinophils)

Define the terms antigen, antibody, B lymphocyte, T lymphocyte, active and passive immunity, primary and secondary immune responsesAntigen: a molecule which reacts with antibodies or T cells Antibodies bind to the epitope region of the antigen T cell receptors bind to peptide fragments of the antigen Immunogen: an antigen which is sufficient to induce an immune response by itselfAntibody: an immunoglobulin molecule which binds specifically to the immunogen that induced its synthesis Antibodies are produced by B lymphocytes and found in the bloodstream and body

fluids Antibodies are the humoral component of the acquired immune response Types of immunoglobulin (Ig): IgG, IgA, IgM, IgE, IgDIgG: monomer 75% of serum Ig Bivalent Can be transferred across the placenta Longest serum half-life Contributes to the secondary immune responseIgA: dimer Tetravalent: 2 molecules are held together by a J chain Found in body secretions Contains a secretory piece to protect it from the action of hydrolytic enzymes in

secretionsIgM: pentamer (star-shaped)

10% of serum Ig Multivalent (decovalent): 5 molecules are held together by a J chainIgE: monomer Bivalent Involved in allergic response and defence against parasites Bound to basophils/mast cells: it triggers the release of histamines IgD: monomerAntibodies are specific to a particular antigen since the antigen-binding region (Fab region) on the antibody is complementary to the epitope (antibody-binding region) on the antigenMechanisms of antibody action against viruses: 1. Neutralisation: the antibody binds to a virus and prevents its attachment to the

host cell2. Opsonisation: the virus-antibody complex is phagocytosed by macrophages3. Complement-mediated lysis of enveloped viruses4. Antibody-dependent cell-mediated cytotoxicity (ADCC): this is mediated by NK cellsLymphocytes: mononuclear agranular leukocytes Antigen-specific: lymphocytes express antigen receptors on their surface which are

specific to a particular antigen Form 20-40% of circulating leukocytes and 99% of cells in lymph fluid Lymphocyte precursors are produced in the bone marrow by self-renewing

haematopoietic stem cells Lymphocytes consist of bone marrow-derived and thymus-derived cells Subsets of lymphocytes are distinguished by unique cell-surface markers

B lymphocytes: bone marrow-derived lymphocytes Mature in the bone marrow Recognise free antigen in the body fluids Surface markers include: CD19 and class II MHC molecules B cells produce 1 clone of antibody The antibody may be bound to the surface of the B cells or it may be secreted by

the B cells The Fab (antigen-binding) region of an antibody is unique to a particular antigen

When an antigen binds to a surface antibody, this promotes the B cell to secrete antibodies

T lymphocytes: thymus-derived lymphocytes Mature in the thymus Recognise antigen presented at the cell surface by MHC molecules Surface markers include: CD3, CD4 and CD8 TH cells (helper T cells): express the CD4 co-receptor on their surface TC cells (cytotoxic T cells or CTLs): express the CD8 co-receptor on their surface Regulatory T cells: a subset of TH cells which suppress the immune responseNaïve lymphocytes: have never encountered their cognate antigen and hence have never responded to itMemory lymphocytes: the product of an immune response; they allow their specific receptor to remain in the pool of lymphocytes in the body Active immunity: the induction of an immune response by the introduction of antigenPassive immunity: immunity gained without antigen introduction (i.e. by transfer of antibody or immune serum into a naïve recipient)Primary response: the response made by naïve lymphocytes when they first encounter their cognate antigenNaïve lymphocytes are activated by antigen to ensure proliferation of a lymphocyte with the appropriate receptor for the antigenAfter the antigen has been removed, a few lymphocytes with the cognate antigen remain as memory cells; the remaining lymphocytes dieSecondary response: the response made by memory lymphocytes when the reencounter their cognate antigenThe secondary response is more effective than the primary immune response: it is quicker, greater in magnitude and lasts longer than the primary response

Outline the concept of clonal selection, and its role in immune responsesImmune memory (secondary response): a response which occurs upon the 2nd or subsequent exposure to a particular antigenLymphocyte antigen receptors: T and B lymphocytes express unique surface antigen receptors which bind to

proteins Each cell has a particular antigen receptor which recognises and binds to the

epitope of its cognate (associated) antigen There are many copies of the antigen receptor on the cell surface Lymphocytes circulate constantly between the blood and tissues via the lymph

The thoracic duct is the final lymphatic vessel which reintroduces lymph into the subclavian vein

B cell antigen receptor (BCR): a surface immunoglobulin (i.e. a membrane-bound antibody)

T cell antigen receptor: the T cell receptor (TCR) consists of 2 protein chains Generation of clonal diversity in lymphocytes:

During development of T and B lymphocyte precursors in the bone marrow: random genetic recombinations occur within each cell among multiple copies of immunoglobulin genes or TCR genes

This generates diversity of clones of lymphocytes Each clone of lymphocytes has a different antigenic specificity (i.e. it is unique to a

particular antigen) Each naïve lymphocyte is the precursor of a genetically identical clone of daughter

lymphocytes Principle of clonal selection: when the surface antigen receptor of a T or B lymphocyte recognises and binds to its cognate antigen, it stimulates that particular clone of cells to proliferate The surface antigen receptor of a T or B lymphocyte recognises and binds to its

cognate antigen This leads to selection and activation of a lymphocyte clone Clonal expansion occurs: a clone of lymphocytes with identical antigenic

specificities is produced Most of the recently proliferated lymphocytes die at the end of the primary

immune response due to lack of antigen, lack of cytokines or activation-induced cell death

Some proliferated lymphocytes survive as memory cellsIn other words: T and B cells produced in the primary lymphoid organs are released into the

peripheral lymphoid pool Those that meet their cognate antigen proliferate and produce effector and

memory cells; those that do not meet their cognate antigen die The size of the peripheral lymphoid pool is regulated by homeostatic mechanismsAntigen presentation: Antibodies and BCRs bind directly to the antigen TCRs can only recognise processed antigen (peptide) which is bound to major

histocompatibility complex (MHC) proteins on the cell surface Human leukocyte antigens (HLA): the MHC molecules which occur in human cells 2 types of MHC molecules: class I MHC and class II MHC Class I MHC molecules: occur on the surface of all cells Class II MHC molecules: only occur on the surface of antigen presenting cells

(APCs)APCs include: dendritic cells, macrophages and B lymphocytes

Immune response: T cell activation: occurs in the secondary lymphoid organs (e.g. lymph nodes) Antigens are taken up by APCs and transported from tissues into secondary

lymphoid organs where they meet T cellsTC cells (CTLs): kill infected cells TC cells recognise virally infected cells via the peptides presented by class I MHC

molecules

They kill infected cells by injecting granules containing lethal enzymes into the cells

Antibodies: recognise and label infected cells for destruction

Understand the roles of natural selection and the physical organization of the immune system in its functionOrganisation of the lymphatic system: Lymphatic vessels: venules, veins and ducts Lymphatic tissues: nodules, nodes, tonsils and Peyer’s patches Lymphatic organs: spleen and thymusLymphoid organs: organised tissue where lymphocytes interact with non-lymphoid cells; site of initiation and maturation of the adaptive immune responsePrimary lymphoid organs: site of maturation of lymphocytes Bone marrow: site of B cell maturation Thymus: site of T cell maturationSecondary lymphoid organs: lymph nodes, spleen (white pulp) and mucosa-associated lymphoid tissue (MALT)Lymphocytes and APCs recirculate through lymphatic vessels from tissues via lymph nodes or the spleen into the blood

Immunology 2: Immune Cells and OrgansName the primary and secondary lymphoid organs and briefly differentiate between their functionsPrimary lymphoid organs: the site of lymphopoiesis (production of lymphocytes) Bone marrow: the site of haematopoiesis and B cell maturation Thymus: the site of T cell maturationSecondary lymphoid organs: the site where antigen, antigen presenting cells and mature lymphocytes come together to initiate an immune response Spleen Lymph nodes MALT (mucosal-associated lymphoid tissue) Secondary lymphoid organs have special vascular adaptations to recruit lymphocytes from the blood

Draw simple diagrams to illustrate the structure of the thymus, lymph node, spleen, Peyer’s patch, and indicate the changes that occur after stimulation by antigenThymus: A bi-lobed structure which is located in the thorax Each lobe is organised into lobules Each lobule contains histologically defined regions of cortex and medulla Cortex: contains immature thymocytes, some of which undergo selection to

become mature thymocytes in the medulla Medulla: contains mature thymocytes Only ~5% of cells exit the thymus and enter the peripheral T cell pool Atrophies with age: areas of active T cell maturation are replaced by adipose tissueSimple diagram of a human thymic lobe:

Bone marrow: Produces haematopoietic stem cells and B cells Haematopoietic stem cells destined to become T cells migrate to the thymus Differentiation of B cells is centripetal with stem cells under the bone; the most

mature B cells are found towards the centre of the bone marrow In the foetus: the liver and spleen are the first active sites of haematopoiesis;

bones become active subsequently to the liver and spleen

In adults: active sites of haematopoiesis include spongy regions at the end of long bones, vertebral bones, the sternum, ribs and flat bones of the cranium and pelvis

Lymph nodes: Diameter: 1-15 mm Round or kidney-shaped Have an indentation at the hilus where blood vessels (BV) enter and leave the

lymph node Lymph enters the lymph node through several afferent vessels (A) Lymph leaves the lymph node through one efferent vessel (E) at the hilus As lymph passes through a lymph node, phagocytes remove free antigens Cortex: rich in B cells Paracortex: rich in T cells Simple diagram of a lymph node:

A: afferentE: efferentBV: blood vesselSpleen: Contains 2 main types of tissue: Red pulp: acts as a general filter for antigens in the blood White pulp: the lymphoid tissue; it initiates the major responses to blood-borne

antigensPeriarterial lymphatic sheath (PALS): concentric areas of lymphoid tissue which surround the central arteriole The PALS region nearest the central arteriole is rich in T cells Lymphoid follicles: rich in B cells; occur periodically along the PALS Marginal zone: the primary entry site of B and T cells into the white pulp;

surrounds the PALSSimple diagram of a part of the spleen:

Diagram of a part of the spleen:

Individuals who do not have a spleen are highly susceptible to infections with encapsulated bacteriaEpithelium: the first line of defence against infection The skin and mucosal layers form a physical barrier with a very large surface area Epithelia are heavily defended by MALT and the cutaneous immune systemMALT: an aggregate of lymphoid tissue which does not have a tough outer capsule Occur predominantly in the lamina propria and submucosal areas of the GI,

respiratory and genito-urinary tracts Examples of MALT: tonsils; appendix; Peyer’s patchesPeyer’s patches: organised regions of lymphoid tissue found in the wall of the gut Predominantly contain B cells Contain germinal centres during immune responses Simple diagram of a Peyer’s patch:

Cutaneous immune system:Consists of 2 parts: the epidermis and the dermis

Epidermis: contains Langerhans cells (epidermal dendritic cells) and intraepidermal lymphocytes

Dermis: contains T lymphocytes, macrophages and dermal dendritic cells

Outline the re-circulation of lymphocytesNaïve lymphocytes: lymphocytes which have matured and have not yet encountered antigenThere are many naïve T and B lymphocytes with different specificities (i.e. they recognise different antigens); however there may only be a limited amount of antigenLymphocyte recirculation increases the likelihood that naïve lymphocytes will encounter their cognate antigenRecirculation of naïve lymphocytes: Naïve lymphocytes constantly circulate from the blood into secondary lymphoid

organs: they leave blood vessels through high endothelial venules (HEVs) HEVs: specialised sections of post-capillary venules

Naïve lymphocytes migrate from lymph nodes into efferent lymphatic vessels They eventually return to the blood via the thoracic duct In case of an infection: lymphocytes which recognise the infectious agent remain in

the lymphoid tissue in lymph nodes, where they proliferate and differentiate

Explain the use of CD (cluster of differentiation) markers for discrimination between lymphocytesLymphocytes: small cells with agranular cytoplasm and a large nucleusSubdivided into 2 groups:1. B lymphocytes: mature in the bone marrow2. T lymphocytes: mature in the thymusB cells and T cells cannot be differentiated under a microscope using conventional stainsCD markers: an internationally recognised systematic nomenclature for cell surface molecules which is used to discriminate between haematopoietic cells; more than 300 CD markers exist

Compare and contrast phenotypic characteristics of B and T cellsT cells: Produced by the bone marrow; mature in the thymus All T cells express CD3 90% express αß TCRs

o 2/3 of these express CD4o 1/3 of these express CD8

10% express γδ TCRs2 subsets of T cells: 1. CD4+: T helper cells and regulatory T cells

Effector function: secrete cytokines2. CD8+: CTLs

Effector functions: lyse infected cells; secrete cytokines Thymic output (production of mature T cells by the thymus) decreases with age

since the activity of the thymus steadily decreases after puberty TCRs recognise processed antigen presented by MHC molecules at the surface of a

cell B cells: Produced by and mature in the bone marrow Express CD19 and CD20 Express MHC class II molecules which present antigen to T helper cells Effector function: produce and secrete antibodies BCRs (surface immunoglobulins) recognise intact, free antigen present in blood and body fluids

T cells B cells

Recognise… Processed antigen presented at the cell surface by MHC molecules Intact, free antigen

Antigen receptor TCR: either αß or γδ BCR (surface

immunoglobulin/antibody)

Express… All T cells express CD3αß T cells express CD4 or CD8

CD19 and CD20

Site of maturation Thymus Bone marrow

Give examples of antigen presenting cells and their locationsAntigen presenting cells (APCs): cells which present processed antigen (peptide fragments derived from proteins) to effector T cells to initiate the acquired immune response

APC Location Present antigen to

Activated dendritic cells

Widely spread (e.g. in the skin and mucosal tissue) Include Langerhans cells which are present below the skin

T cells

Activated B cells Lymphoid tissue T cells

Activated macrophages Lymphoid tissue T cells

Follicular dendritic cells Lymph node follicles B cells

Immunology 3: Innate Immunity Characteristics of innate immunity: Present from birth Broad specificity: uses pattern recognition receptors to recognise pathogen

associated molecular patterns (PAMP); does not use antigen-specific receptors No immunological memory: i.e. it is not enhanced upon second exposure to a

particular pathogen Includes both cellular and humoral components Provides a rapid response Cooperates with and directs (subsequent) adaptive immunity

Innate immunity Innate & adaptive immunity Adaptive immunity

Cellular components

Neutrophils Monocytes/macrophages B lymphocytes

NK cells Dendritic cells T lymphocytes

Mast cells/basophils

Humoral components

Anti-microbial peptides Complement Antibodies

Cytokines

Briefly describe the functions of the important phagocytic cells: neutrophils, monocytes/macrophagesPhagocytes: engulf invading organisms, try to kill them and release signals to alert other cells General mode of action of phagocytes:1. The pathogen is engulfed by the phagocyte via formation of pseudopodia around

the pathogen2. The pseudopodia fuse to form a phagosome around the pathogen 3. A lysosome fuses with the phagosome to form a phagolysosome4. The pathogen is killed by the action of hydrolytic enzymes contained within the

lysosome 5. Certain peptides from the degraded pathogen are released from the phagocyteNeutrophils (PMN leukocytes): granular leukocytes (granulocytes) Multi-lobed nucleus Present abundantly in the circulation; make up 50-70% of circulating leukocytes Short life-span: to limit damage to normal tissues Die by apoptosis (programmed cell-death) Circulate in the blood and migrate out into tissues to the site of damage/infection

when required They are the first cells to be recruited to the site of tissue infectionMode of action of neutrophils:

1. Migrate from circulation to the site of tissue infection2. Bind to the pathogen at the site of infection3. Phagocytose the pathogen4. Kill the pathogenMigration of neutrophils from circulation into tissues: diapedesis and chemotaxisDiapedesis: passage of cells through adjacent endothelial cells into tissues Chemotaxis: directed movement of cells along a chemokine gradient to the site of infectionChemokines: chemotactic cytokines which attract and direct cells to the site of infection 1. Endothelial cells which line the blood vessels express integrin ligands on their

surface2. Neutrophils express selectin ligands on their surface3. Activated macrophages at the site of tissue infection secrete alarm cytokines

which signal infection4. Cytokines stimulate endothelial cells which line local blood vessels to express

selectins on their surface5. Selectins bind to selectin ligands on the surface of neutrophils: this is a weak

interaction and causes neutrophils to slow down and roll along the blood vessel6. Inflammatory signals stimulate neutrophils to express integrins on their surface7. Integrins bind to integrin ligands on the surface of endothelial cells: this is a strong

interaction and causes neutrophils to stop rolling8. Chemokines stimulate neutrophils to migrate through the endothelial cell layer to

the site of inflammation Phagocytosis of pathogens by neutrophils: Neutrophils are highly phagocytic cells: this is a key component of host defence When many neutrophils are involved in phagocytosis an abscess filled with pus is

formed Phagocytosis is much more effective following opsonisation Opsonisation: the process by which pathogens are coated with opsonins to facilitate phagocytosis Opsonins: molecules which can bind to antigens and be bound by phagocytes Opsonins include antibodies and complementMechanism of neutrophils binding opsonins: 1. IgG opsonises the pathogen by binding to cognate antigens on the surface of a

pathogen 2. This activates the complement system3. C3b opsonises the pathogen by binding to the surface of the pathogen4. IgG binds to the Fc receptor and C3b binds to the CR1 receptor on the surface of a

neutrophil5. This activates the neutrophil and the pathogen is phagocytosed Killing of pathogens by neutrophils: (following phagocytosis)

Neutrophils have multiple killing mechanisms; the respiratory burst is the most important of theseOxygen-independent mechanisms: Granules, containing hydrolytic enzymes, fuse with phagolysosomes and kill

phagocytosed pathogens Antimicrobial peptides (e.g. defensins) Oxygen-dependent mechanisms: Respiratory burst: activated neutrophils produce toxic metabolites which damage

pathogenso Hydrogen peroxide, superoxide anions, singlet oxygen and hydroxyl radicalso Reactive nitrogen intermediates (nitric oxide)

Neutrophil deficiency is associated with infections caused by extracellular bacteria and fungi

Monocytes: Leukocytes which enter the bloodstream from the bone marrow Precursors to macrophages and dendritic cells Leave the circulation through pores between adjacent endothelial cells lining the

blood vessels, enter tissues and mature into macrophages Secrete alarm cytokines, particularly: IL-1, IL-6, IL-8 and TNF-αMacrophages: Less abundant than neutrophils Long life-span relative to neutrophils Dispersed throughout peripheral tissues and beneath all exposed tissues Produce cytokines to signal infection to recruit other immune system cells to the

site of infection Function as APCs: macrophages present processed antigen to helper T cellsMode of action of macrophages: Express pattern recognition receptors which recognise PAMP Become activated when PAMP bind to macrophage receptors Activated macrophages secrete cytokines to recruit other cells to the site of

infection Phagocytose and kill pathogens to which they bind

Define cytokines and describe their general propertiesCytokines: small soluble molecules which facilitate communication between cells of the immune system Small secreted proteins which are involved in cell-cell communication Generally act locally Powerful biological effects, even at low concentrations Short-lived When a cytokine binds to its receptor: a change in gene expression occurs (e.g. cytokines

stimulate proliferation of lymphocytes)

Type of cytokine Function

Interleukins (IL) Facilitate communication between leukocytes (IL is a generic name for a cytokine)

Interferons (IFN) Have anti-viral effects

Chemokines Required for chemotaxis (i.e. to recruit cells to the site of infection)

Growth factors Required for the development of cells of the immune system

Cytotoxic cytokines Can induce cell death; e.g. tumour necrosis factor (TNF)

Cytokines secreted by activated macrophages

Function

IL-1 Alarm cytokine which induces the fever response

TNF-α Alarm cytokine

IL-6 Stimulates production of acute phase proteins in the liver

IL-8 Chemokine: stimulates chemotaxis of neutrophils to the site of tissue infection

IL-12 Directs adaptive immunity; activates NK cells

Bacterial septic shock: a systemic infection caused by bacterial endotoxinsEffects: Secretion of alarm cytokines (TNF-α and IL-1) by activated macrophages Increased vascular permeability due to cytokines This leads to hypotension severe drop in blood pressure Death occurs in 10% of casesDendritic cells: recognise pathogens and secrete cytokines Found in the skin and near mucosal epithelia Express pattern recognition receptors which recognise PAMP Become activated when PAMP bind to dendritic cell receptors Activated dendritic cells secrete cytokines which stimulate chemotaxis of

monocytes to enter tissues and mature into dendritic cells Activated dendritic cells capture pathogens and migrate to local lymph nodes to

present antigens to the adaptive immune system

Define complement, list its major functions, and draw a simple diagram of the complement pathwaysComplement system: a cascade of serum enzymes activated by the presence of a pathogen Plays a major role in innate and antibody-mediated immunity Consists of a series of ~30 proteins and glycoproteins with a relatively high serum

concentration (3-4 mg/ml) Enzyme cascade system which produces a rapid, highly amplified response Components of the complement system are mainly produced in the liver and also

by monocytes and macrophages Must be activated before it can function: there are 3 mechanisms for activation3 mechanisms for activation of the complement system:1. Classical pathway: antibody-dependent activation; activated by antibody-antigen

complexes2. Alternative pathway: antibody-independent activation; direct activation by the

pathogen surface

3. Lectin pathway: antibody-independent activation by lectins which bind to carbohydrates only found on pathogens

All of the pathways converge at the activation of C3: C3 C3a + C3b C3 leads to the final Common Pathway The Common Pathway ends with the formation of the membrane attack complex

(MAC)

Functions of the complement system: Lytic pathway: complement causes cell lysis by formation of the MAC Opsonisation: C3b opsonises the surface of pathogens which facilitates

phagocytosis Anaphylatoxins: complement fragments which as chemokines (these include C3a

and C5a)Control of the complement system: Components of the complement system are labile (i.e. they have a short half-life) Components of the complement system are diluted in biological fluids Specific regulatory proteins help inhibit complement activation: these include

circulating and membrane-bound proteins

Describe a typical inflammatory response to a localised infection involving recruitment of neutrophils, and phagocytosis and killing of bacteria

Briefly outline the events involved in a systemic acute phase responseMast cells: Located beneath all exposed surfaces (like macrophages)

o Mucosal mast cells: present in the lungso Connective tissue mast cells: present in the skin, peritoneal cavity and near

blood vessels Protect against parasitic infection by degranulation:

o Mast cells have granules which contain active chemicals, such as histamineo The mast cell releases the contents of its granules onto the parasite to kill it

Recognise, phagocytose and kill opsonised bacteria Secrete inflammatory mediators, including histamine and cytokines Anaphylatoxins activate mast cells and trigger mast cell degranulation Activated mast cells lead to vasodilation and increased vascular permeability Local acute inflammatory response: 1. Macrophage activation2. Cytokine secretion3. Endothelium activation4. Leukocyte extravasation and chemotaxis5. Phagocytosis and killing by neutrophils 6. Complement activation7. Opsonisation8. Mast cell degranulation9. Increase in vascular permeabilitySystemic acute phase response: Often accompanies a local inflammatory response, 1-2 days later Alarm cytokines (IL-1, IL-6 and TNF-α) induce the acute phase response Alarm cytokines affect the hypothalamus, adrenal cortex, liver and bone marrowEffects of the acute phase response: Fever Increased leukocyte production (leukocytosis) Production of acute-phase proteins by the liverAcute-phase proteins: C-reactive protein (CRP):

o Binds to the C polysaccharide of pneumococcuso Activates complement o The CRP level may increase up to 1000 fold

Mannan-binding lectin (MBL): binds to mannose residueso Opsonises pathogens for phagocytosis by monocyteso Activates complement via the lectin pathway

Serum amyloid A: similar to CRP Complement components Fibrinogen: involved in clotting

Outline the phenotype and functions of natural killer (NK) cellsNatural killer cells: Large granular lymphocytes Cytotoxic Circulate in the blood, spleen and liver; make up 5-10% of peripheral circulating

lymphocytes

Activated NK cells secrete cytokines (interferon-γ) Lyse target cells Antibody dependent cell cytotoxicity (ADCC): when Fc receptors on NK cells bind to

antibodies, the cytotoxic ability of NK cells is stimulated Important in defence against tumour cells and viral infections (especially Herpes):

NK cells induce apoptosis in these cells Do not have antigen-specific receptors (unlike T and B lymphocytes) Express both activating and inhibitory receptors Killing depends on a balance of different signals:

o Ligation of inhibitory NK receptors inhibition of target cell killingo Ligation of activating NK receptors target cell killing

Immunology 4: Antibodies Describe with the aid of a simple diagram the immunoglobulin molecule, identifying the antigen-binding site (Fab) and Fc portions of the moleculeAntibody: a protein that is produced in response to an antigen and has the property of binding specifically to the antigen that induced its formation Antibodies constitute the immunoglobulin class of proteins Antibodies are a large family of soluble glycoproteins B lymphocytes produce antibodies Individuals have many different antibodies (>107); antibody-deficiency is life-

threatening Secondary effector functions of the antibody-antigen complex:

o Complement activation (via the classical pathway)o Opsonisation: this facilitates phagocytosis of the pathogeno Cell activation via binding of the Fc region of the antibody to Fc receptors on

the surface of certain cells (e.g. macrophages)IgG structure: Symmetrical structure: composed of 2 light chains (Lc) and 2 heavy chains (Hc)

held together by disulphide bonds Each IgG molecule has 2 identical Fab regions which can bind to antigens

The Fab regions of a particular antibody can bind to a specific antigen Each IgG molecule has an Fc region which can bind to Fc receptors on the surface

of certain cells The IgG molecule is flexible due to the presence of a hinge region The hinge region allows the each of the 2 Fab arms to bind to an antigen

Ig treated with

No. of fragments produced Ratios of each type of fragment

Papain 3 2 Fab fragments : 1 Fc fragment

Pepsin 2 1 F(ab)2 fragment : many small fragments derived

from Fc

Mercaptoethanol 2 1 Hc : 1 Lc

Heavy and light chains: Consist of constant domains (CH or CL) and variable domains (VH or VL) Heavy chain structure: 1 variable domain (VH) + 3 or more homologous constant

domains (CH) Light chain structure: 1 variable domain (VL) + 1 constant domain (CL)

Briefly describe the properties of the antigen-binding siteProperties of the antigen-binding site (Fab region): A relatively large, flat surface with undulations Composed of the variable domains of heavy and light chains (i.e. the VH and VL

domains) Each variable domain has 3 hypervariable regions The 3 hypervariable regions form the complementarity determining regionsEpitope: the site on an antigen which is bound by the Fab region of an antibody There is a high degree of complementarity between an Fab region and an epitope

Distinguish between antibody affinity and avidityAntibody affinity: the strength of the total non-covalent interactions between a single antigen-binding site of an antibody and a single epitope on its cognate antigen Antibody + antigen antibody-antigen complex

Each individual antibody-antigen interaction is relatively weak and has a low affinity since the interactions are non-covalent

Forces involved in antibody-antigen binding include: hydrogen bonds; ionic bonds; hydrophobic interactions; van der Waals interactionsAntibody avidity: the overall strength of multiple interactions between an antibody with multiple antigen-binding sites and a complex antigen with multiple epitopes Avidity is a better measure of binding capacity in biological systems Polyvalent antibodies have a higher avidity than monovalent/bivalent antibodiesCross-reactivity: an antibody elicited in response to one antigen can also recognise a different antigenExamples of cross-reactivity:1. Vaccination with cowpox induces antibodies which are able to recognise smallpox2. ABO blood group antigens (glycoproteins on red blood cells): antibodies made

against microbial antigens on normal gut flora may cross-react with ABO blood group antigens

List the immunoglobulin classes and sub-classes in man. Describe their functions and relate these to their individual structureIsotypes: immunoglobulin structures which are present in all members of a species E.g. kappa and lambda light chains; the different heavy chains; Ig classes and

subclassesAllotypes: allelic polymorphisms of an Ig molecule which vary in the population; allotypes are not present in all members of a species; allotypes are usually detected by antibodies E.g. gamma chain; kappa chainImmunoglobulin classes: The constant region of the heavy chain of an antibody determines its class and

function Immunoglobulins may have either a κ or a λ light chain; a given Ig has the same

type of light chain since the Ig molecule must be symmetricalDifferent classes of antibodies differ in the constant regions of their heavy chains:

Ig class IgG IgA IgM IgE IgD

Heavy chain γ α μ ε δ

Light chain κ or λ

Ig class IgG IgA

Ig subclass IgG1 IgG2 IgG3 IgG4 IgA1 IgA2

Heavy chain γ1 γ2 γ3 γ4 α1 α2

IgG: Present in the blood and extracellular fluids

The most abundant immunoglobulin The most important immunoglobulin in defence generally: IgG neutralises toxins

and viruses Exists as a monomer (which has 2 Fab regions) Actively transported across the placenta: this provides passive immunity to the

foetus Activates complement via the classical pathway (mainly IgG1 and IgG3) Opsonises pathogens for phagocytosis: phagocytes have Fc receptors that

recognise the Fc chain 4 subclasses of IgG: variability of the subclasses is mainly located in the hinge

region and effector function domains (i.e. the Fc region) (Stimulates ADCC in NK cells)IgA: Major secretory immunoglobulin: IgA has an important role in seromucous

secretions The 2nd most abundant immunoglobulin In the blood: exists as a monomer (which has 2 Fab regions) In secretions: exists as a dimer (which has 4 Fab regions)

o Dimer structure: 2 IgA monomers are held together by a J chain and a secretory component

o The secretory component is added to the molecule during its passage through epithelial cells out into the secretions

o It protects IgA from acids and enzymes found in secretions (e.g. the GI tract) Secretory IgA neutralises toxins and blocks the infectivity of infectious agents Protects mucosal surfaces from bacteria, viruses and protozoa 2 subclasses of IgA: IgA1 predominates in the blood; IgA2 predominates in

secretions (Secreted in the milk: IgA coats the baby’s intestinal mucosa and protects against

ingested pathogens) (Does not activate complement)IgM: Mainly present in the blood (80%) Exists as a pentamer (which has 10 Fab regions)

o Pentamer structure: 5 monomers are held together by a J chaino The 5 Fab pairs allow it to bind strongly to the surface of bacteria: this causes

agglutination The 1st antibody to be produced after exposure to an antigen; it is involved in the

primary antibody response Active in the blood Multiple Fab sites compensate for its low affinity; therefore it has a relatively high

avidity Efficient at agglutination (clumping together of antigens) Activates complement (via the classical pathway)

IgE: Present in extremely low concentrations Produced in response to parasitic infections and in allergic diseases

o Defends against parasiteso Causes allergies and anaphylactic shock

Fc receptors on mast cells and basophils have a high affinity for the Fc region of IgE Cross-linking by antigen triggers mast cell activation and histamine release: this is

important in protection against certain parasitic infections (Allergic response):

On 1st exposure to an allergen: o Many IgE antibodies are produced o Fc receptors on the surface of mast cells bind to the Fc region of the IgE

antibodiesOn 2nd exposure to the allergen:o IgE antibodies which are bound to the surface of mast cells bind to the allergeno This causes cross-linking (clustering) of many IgE antibodies which brings many

Fc receptors close to each othero Degranulation occurs: the mast cell releases granules containing histamine into

tissuesIgD: The least well characterised immunoglobulin Extremely low serum concentrations (it is not really found as a free antibody) Surface IgD is expressed early in B cell development Involved in signalling during development and activation of B cells

Selective distribution of immunoglobulins: Blood: IgG and IgM ECF: IgG Secretions: IgA (dimer) Foetus (via placental transfer): IgG (maternal) Below the epithelium: IgE (associated with mast cells) Brain: lacks immunoglobulinsFunctions of antibodies:In defence: Targeting and recognising infectious organisms Recruitment of secondary effector mechanisms Neutralisation of toxins Removal of antigens Inducing passive immunity in newborns (IgG)In medicine: Ig levels are used in diagnosis and monitoring (e.g. a viral infection leads to

synthesis of specific antibodies) Pooled antibodies are used for passive therapy and protection (i.e. prophylaxis) In laboratory science: Many diagnostic and research applications

Immunology 5: B lymphocytesAdaptive immune response: Develops after exposure to antigen Requires time to develop 2 arms: humoral and cell-mediatedB lymphocytes: Derived from haematopoietic stem cells in the bone marrow Main effector cells of humoral immunity: activated B lymphocytes secrete

antibodies Mature in the bone marrow in the absence of antigen Migrate into circulation (blood and lymph) and into lymphoid tissues Activation and differentiation occur in peripheral lymphoid tissues in the presence

of antigenB cell maturation: antibody-independent phase1. Stem cell pro-B cell2. Pro-B cell pre-B cell3. Pre-B cell immature B cell4. Immature B cell mature B cell: self-reactive immature B cells are destroyedB cell antigen receptors (BCRs): confer specificity to a particular antigen Many identical BCRs are present on the surface of a mature B cell Therefore each B cell is specific to a particular antigen since all the BCRs on a

given B cell recognise the same antigen BCRs have a unique antigen-binding site (Fab region) which binds to the epitope of

an antigen BCRs are made before the B cell encounters antigenBCR structure: transmembrane protein complex BCRs consist of a membrane-bound immunoglobulin and 2 associated

heterodimers Each heterodimer is composed of Igα and Igß, linked by a disulphide bond Heterodimers contain an immunoglobulin-fold structureN.B. in contrast to BCRs, antibodies are free immunoglobulins which are not bound to the membrane of a B lymphocyte since they lack the protein sequence which anchors BCRs to the surface of a B cellBCR signalling: The membrane-bound Ig of a BCR recognises the epitope of its cognate antigen: it

must communicate this to the nucleus of the B cell The cytoplasmic tail extending from the heavy chain of the membrane-bound Ig

into the interior of the B cell is too short to signal The cytoplasmic tails of Igα and Igß are long enough to interact with intracellular

signalling molecules

Briefly outline the principles of immunoglobulin (Ig) gene rearrangement in the generation of diversityThere is an almost infinite number of potential antigensThe immune system must be able to respond to all these potential antigens; therefore it requires a large repertoire of cells which have specific receptors that can recognise all potential antigensImmunoglobulin gene rearrangement: exclusive to B and T lymphocytes Each Ig of a BCR is composed of heavy chains and κ and λ light chains Each chain is encoded by a gene which is assembled from gene segments Genes encoding each type of chain are present on different chromosomes During B cell maturation the gene segments are rearranged and combined This generates the diversity of the lymphocyte repertoire Only B cells which successfully rearrange their genes will mature and enter

circulationHeavy chain synthesis: chromosomes which encode the heavy chain have multiple copies of V, D, J and C gene segments Light chain synthesis: chromosomes which encode the heavy chain have multiple copies of V, J and C gene segmentsProcess of gene rearrangement:1. Rearrangement of germline DNA B cell DNA

Each B cell chooses one of each kind of segment at random and joins them together by deleting the DNA sequences in between them to form B cell DNAThe unused DNA sequences are deleted by being looped out and removed

2. Transcription of B cell DNA primary RNA transcript Transcription only occurs if there is a productive rearrangement of gene segments (i.e. the B cell DNA encodes a functional chain)

3. Splicing of primary RNA transcript mature mRNA4. Translation of mature mRNA functional chain polypeptide5. The functional chain is transported to the cell surface6. BCRs are made at the cell surface by assembly of heavy and light chains

Describe the process of stimulation of individual B cells to divide and secrete antibody such as to generate immunity to a particular antigen (clonal selection)Clonal selection of B cells: antigen-dependent phase A naïve B cell is activated when its BCRs bind to their cognate antigen and when it

receives co-stimulation (see below) Naïve B cell: a B cell which has not yet encountered antigen

The activated B cell proliferates to produce a clone of cells with identical BCRs All of the clonally expanded B cells have the same specificity (i.e. they express

BCRs which recognise the same antigen) The clonally expanded B cells secrete antibodies into the bloodstream Finally when the pathogen has been eliminated most of the B cells die

Outline the differences in antibody production during primary and secondary immune responsesA naïve B cell cannot be activated by encountering antigen alone; it requires a co-stimulatory signal in addition to encountering antigen2 pathways of B cell activation:1. Thymus-independent activation: microbial constituents directly supply the co-

stimulatory signal2. Thymus-dependent activation: T helper cells supply the co-stimulatory signalThymus-independent activation: Thymus-independent antigens directly activate B cells without the help of T cells Therefore antibody formation can be induced in people with no thymus and no T

cells The co-stimulatory signal is supplied by the microbial constituent or by an

accessory cellThymus-dependent activation: Thymus-dependent antigens require T helper cells to activate B cellsAntigen recognition and presentation by B cells:1. A BCR recognises and binds to its cognate antigen2. The BCR-antigen complex is internalised by the B cell3. The internalised antigen is degraded into peptides by hydrolytic enzymes 4. The peptides associate with class II MHC molecules to form a class II MHC-peptide

complex5. The class II MHC-peptide complex is transported to the surface of the B cell for

expression6. T helper cells with complementary TCRs recognise the complex and secrete

cytokines which bind to the B cell and activate itT helper cell dependent activation: 1. When BCRs bind to antigens they cross-link (cluster) on the surface of the B cell:

this co-stimulates B cells to up-regulate expression of class II MHC molecules and B7 proteins

2. Antigens are internalised, processed and presented by B cells as above3. T helper cells recognise their cognate antigen: this co-stimulates and activates T

helper cells 4. Activated T helper cells express CD40L5. CD40L interacts with CD40 on the surface of B cells: this co-stimulates B cells to

express cytokine receptors on their surface6. B7 interacts with CD28 on the surface of T helper cells: this co-stimulates T helper

cells to secrete cytokines which bind to cytokine receptors on the surface of B cells7. This stimulates B cells to proliferate and differentiate to form plasma B cells which

secrete antibodies into the bloodstreamClass switching: only occurs in mature B cells which have encountered antigen When naïve B cells are activated they mainly produce IgM antibodies As B cells mature they can change the class of antibody which they make

The class of an antibody is determined by the constant region of its heavy chain The gene segments which encode the constant regions of the heavy chain are

located adjacent to each other on the same chromosome Special switching signals are located between constant region gene segments:

they allow class switching to occur Cytokines made by T helper cells influence class switching: Proliferation cytokines stimulate activated B cells to proliferate

E.g. IL-2, IL-4, IL-5 Differentiation cytokines stimulate proliferating B cells to differentiate into different

classesE.g. IL-2, IL-4, IL-5, IFN-γ, TGF-ß

Immunological memory: once the immune system has recognised and responded to a particular antigen it exhibits memory for that antigenImmunological memory is a consequence of clonal selection Primary immune response: induced by initial exposure to antigen Involves naïve B cells which mainly produce IgMSecondary immune response: induced by subsequent exposure to the same antigen Involves the clonally expanded population of memory B cells which mainly produce

IgG Memory B cells only produce antibodies when they are activated by T helper cells The secondary immune response is more faster and greater than the primary

immune response Antibodies produced by the secondary immune response have a higher affinity for

antigen

Differentiate between monoclonal and polyclonal antibodiesAn antigen has multiple epitopes; therefore a mixture of antibodies is produced in response to a single antigen (polyclonal response) Monoclonal antibodies: derived from a single clone of B cellsPolyclonal antibodies: derived from many different clones of B cells

Immunology 6: T-Lymphocytes and Antigen RecognitionOutline the origins and functions of T lymphocyte subsetsT lymphocytes: Destroy intracellular pathogens Recognise foreign antigen via a specific T cell receptor (TCR) on their surface TCRs recognise processed antigen (i.e. small peptide fragments derived from

larger proteins of a pathogen) presented by an MHC molecule on the surface of the infected host cell

T cell receptors (TCRs):TCRs consist of 2 polypeptide chains: they are either αß or γδ

αß TCRs: composed of an α chain and a ß chain; present on the surface of the majority of T cells

γδ TCRs: composed of a γ chain and a δ chain; present on the surface of a small subset of T cells

α ß TCRs : Recognise an MHC-peptide complex on the surface of a cell TCRs on a given T cell recognise peptides associated either with MHC class I

molecules or with MHC class II molecules Structure of each chain: variable region, constant region, hinge, transmembrane

region and cytoplasmic tailThe α chain and ß chain are held together by a disulphide bond at the hinge

Signalling: The cytoplasmic tails of the α and ß chains are too short to signal T cells express the CD3 signalling complex The CD3 complex is composed of 4 proteins: γ, δ, ε and ζ CD3 proteins are anchored in the cell membrane; they have cytoplasmic tails

which are long enough to signalAntigen recognition: there are 2 major populations of T cells which express either CD4 or CD8 co-receptors on their surface Class I restricted: express CD8 co-receptors; recognise peptide presented by MHC

class I Class II restricted: express CD4 co-receptors; recognise peptide presented by MHC

class II Functions of co-receptors: Recognise antigen and bind to the appropriate MHC molecule Increase the avidity of the interaction between a T cell and its target cell Important in signallingInteraction with target cells: CD8+ cells (CTLs): cytotoxic (kill target cells); secrete cytokines Induce apoptosis in the target cell

CD4+ cells (T-helper cells): secrete cytokines Recruit the effector cells of innate immunity Help activate macrophages Amplify the CTL and B cell responsesT lymphocytes only recognise processed antigen presented by MHC molecules at the cell surface:1. CD8+ CTLs: recognise and kill virus-infected cells2. CD4+ Th1 cells: recognise and activate macrophages which have phagocytosed

bacteria3. CD4+ Th2 cells: recognise and activate antigen-specific B cellsThymus: Movement of T cells: 1. Progenitor T cells produced by the bone marrow enter the thymus from the blood2. T cells migrate to the cortex of the thymus, where they are known as immature

thymocytes3. T cells migrate from the cortex to the medulla, where they are known as mature

thymocytes4. Mature thymocytes leave the thymus and enter the circulation Development of T cells: 1. Progenitor T cells which enter the thymus don’t express CD4, CD8 or a TCR (i.e.

CD4- CD8- TCR-)2. T cells undergo gene rearrangement and express low levels of pre TCR: this

consists of a ß chain and a surrogate αTCR (i.e. CD4- CD8- pre TCR+)3. T cells become double positive as they migrate through the cortex: they express

both CD4 and CD8 co-receptors in addition to TCRs (i.e. CD4+ CD8+ TCR+)4. T cells become single positive as they move from the cortex into the medulla: they

express either CD4 or CD8 in addition to TCRs (i.e. CD4+ TCR+ or CD8+ TCR+)Gene rearrangement: the gene encoding each chain (α and ß) is formed by recombination of germline DNA to produce rearranged DNA ß chain: rearranged DNA is formed by recombination of V, D, J and C fragments of

germline DNA α chain: rearranged DNA is formed by recombination of V, J and C fragments of

germline DNASelection: only 5% of thymocytes survive selection 1) Following gene rearrangement, T cells which express pre TCR undergo selection: If the ß chain is functional: T cells survive selection and develop to form double

positive T cells If the ß chain is not functional: T cells die by apoptosis 2) Double positive T cells undergo positive selection in the cortex: Useless T cells whose TCRs cannot recognise self MHC-peptide complexes die by

apoptosis Useful T cells whose TCRs recognise self MHC-peptide complexes weakly survive

selection

3) T cells whose TCRs which survive positive selection undergo negative selection in the medulla: Dangerous T cells whose TCRs recognise self MHC-peptide complexes strongly die

by apoptosis Selection results in mature T cells which recognise self MHC-peptide complexes but not self antigens

Briefly describe the structure and distribution of major histocompatibility complex (MHC) class I and class II moleculesMHC: the major histocompatibility complex A group of tightly linked genes which are important in specific immune responses Discovered by experiments looking at the genetic basis of transplant rejection in

mice Found in all vertebrates MHC molecules are highly polymorphic glycoproteins which present processed

antigen to T lymphocytes 2 types of MHC molecules: class I and class IIHLA (human leukocyte antigens): encoded by the human MHC Human MHC genes have various functions, including immune-related functions The human MHC is polygenic: i.e. it consists of several genes for both class I and

class II MHC proteins Each individual inherits 2 MHC alleles at a single locus, 1 from each parent Expression of genes is co-dominant: i.e. both maternal and paternal genes are

expressed Human MHC genes are highly polymorphic: i.e. there are many alleles of the genes

which encode MHC proteins MHC haplotype: a group of MHC alleles which are linked on one chromosome Different people have different immune responses due to polymorphism of MHC

genes MHC alleles are not randomly distributed; they segregate with race due to

exposure to different infections in different regions

MHC class I molecules: Transplantation antigens: control transplant acceptance/rejection Expressed on the surface of most nucleated cells in the body at varying levels Expression levels may be altered by cytokines or during infectionGene expression: Each MHC class I molecule is composed of 1 heavy chain and 1 light chain

The heavy chain is an α chain which has 3 regions: α1, α2 and α3The light chain is ß2-microglobulin

There are 3 MHC class I genes on each chromosome: HLA-A, HLA-B and HLA-C Therefore each individual has 6 MHC class I genes Each MHC class I gene encodes a heavy chain which pairs with the light chain Peptide binding: MHC class I molecules accommodate peptides 8-10 amino acids in length The binding groove of the MHC molecule is closed at both ends The anchor amino acids which bind to the peptide are clustered at the ends of the

binding grooveMHC class II molecules: Regulatory: control the ability of T cells to mount an immune response Usually expressed only on the surface of antigen presenting cells (dendritic cells, B

lymphocytes and macrophages) Expression levels may be regulated by cytokinesGene expression: Each MHC class II molecule is composed of 2 heavy chains which are of a similar

lengthHeavy chain 1 is an α chain which has 2 regions: α1 and α2 Heavy chain 2 is a ß chain which has 2 regions: ß1 and ß2

MHC class II proteins are encoded by genes in the HLA-D regionPeptide binding: MHC class II molecules accommodate peptides which are 13 amino acids in length

or greater The binding groove of the MHC molecule is open at both ends The anchor amino acids which bind to the peptide are spaced along the binding groove

MHC structure: MHC class I MHC class II

Peptide-binding region α1 and α2 α1 and ß1

Ig-like region α3 and ß2-microglobulin α2 and ß2

CD binding site α3: binds to CD8 ß2: binds to CD4

Transmembrane region

Cytoplasmic region

Outline the mechanisms by which antigen presenting cells (APCs) process and present antigens2 mechanisms of antigen presentation: 1. Endogenous antigens: presented to MHC class-I restricted CD8+ T cells by MHC

class I moleculesVirus-infected cells present to CTLs (T-killer cells) via MHC class I molecules

2. Exogenous antigens: presented to MHC class-II restricted CD4+ T cells by MHC class II moleculesMacrophages which have ingested bacteria present to Th1 cells via MHC class II moleculesB cells which have bound to their cognate antigen present to Th2 cells via MHC class II molecules

Two pathways of antigen presentation are required because: Antigens in different locations require different responses Each pathway presents antigen from different locations to different T cell subsetsEndogenous antigen presentation by MHC class I molecules: 1. Endogenous antigens are cut up into peptides by proteasomes 2. Peptides are transported into the rER by TAP transporters for processing

TAP: transporter associated with antigen processing 3. Peptides bind to the grooves of MHC class I molecules4. MHC I-peptide complexes move from the rER to the Golgi complex within vesicles5. MHC I-peptide complexes are transported from the Golgi complex to the cell

surface for displayExogenous antigen presentation by MHC class II molecules: 1. An MHC class II molecule is formed by association of α and ß chains in the

cytoplasm2. The class II MHC molecule is injected into the rER; an invariant chain occupies its

binding groove3. The MHC-invariant chain complex is transported from the rER to an endosome in

the cytoplasm4. Exogenous antigens are phagocytosed into the cell and enclosed within a

phagosome 5. The phagosome fuses with the endosome 6. Enzymes within the endosome cut up exogenous antigens into peptides 7. A peptide replaces the CLIP at the groove of the MHC class II molecule

CLIP: class II associated invariant chain peptide (the piece of the invariant chain which is associated with the MHC binding groove)

8. The MHC II-peptide complex is transported from the Golgi complex to the cell surface for display

Compare and contrast antigen recognition by B and T lymphocytes and by CD4+ and CD8+ T lymphocytes

B lymphocytes T lymphocytes

Have surface BCRs (surface Ig receptors) Have surface TCRs

Recognise intact extracellular antigen directly Recognise processed antigen presented at the cell surface by MHC molecules

Immunology 7: Effector T-lymphocytesFunctions of T cell mediated immunity: Detection and elimination of intracellular pathogens (i.e. viruses, bacteria,

intracellular parasites) Elimination of altered cells (i.e. tumour cells) Lymphocyte recirculation: Lymphocytes recirculate from the blood and lymph to lymphoid organs They enter lymph nodes across high endothelial venules (HEVs) in the cortex

HEVs: specialised areas in post-capillary venules where there are high endothelial cells which allow lymphocytes to exit the blood

This increases the likelihood that naïve lymphocytes will encounter antigen and become activated

Naïve T cells: mature recirculating T cells which have not yet encountered their cognate antigen Only recirculate in secondary lymphoid organs; must be activated to enter non-

lymphoid tissue Migration of T cells into secondary lymphoid organs is mediated by receptors

presented on the surface of the T cells (e.g. L-selectin) Activated in secondary lymphoid organs (i.e. lymph nodes) Effector T cells: mature T cells which have encountered their cognate antigen and proliferated and differentiated into cells which exert immune effector functionsCD8+ CTLs: class I restricted; target virus-infected cells Recognise peptides of intracellular pathogens presented by MHC class I molecules

on the surface of target cells Cytotoxic: secrete granules which kill target cells CD4+ Th1 cells: class II restricted; target macrophages Recognise peptides presented by MHC class II molecules on the surface of target

cells Activate macrophages and cause inflammation Secrete cytokines: IL-2, IFN-γ and TNF-ßCD4+ Th2 cells: class II restricted; target B cells Recognise peptides presented by MHC class II molecules on the surface of target

cells Stimulate B cells to differentiate into plasma B cells which secrete antibodies Secrete cytokines: IL-4, IL-5, IL-6, IL-10 and IL-13Target cells: cells which are acted on by T cellsActivation of naïve T cells: Naïve T cells are activated by cross-linking of TCRs by MHC-peptide complexes on the T cell surfaceActivation of naïve T cells requires 2 signals:1. Antigen recognition: TCRs must recognise their cognate antigen presented by the

appropriate MHC molecules

2. Co-stimulation: co-stimulatory molecules on the surface of an APC (e.g. B7 proteins) must ligate CD28 receptors on the surface of the T cell

Co-stimulation of naïve T cells amplifies the signal sent to the nucleus by engaged TCRs; therefore fewer TCRs need to be cross-linked to activate the T cell in the presence of co-stimulationEffector T cells are less dependent on co-stimulation than naïve T cellsT cell activation: 1. Naïve T cells enter lymph nodes across HEVs 2. Naïve T cells monitor antigen presented by APCs (activated dendritic cells and

macrophages) in lymph nodes3. Naïve T cells are activated when they encounter their cognate antigen and receive

co-stimulation (Naïve T cells which do not encounter their cognate antigen leave the lymph node through lymphatic vessels and continue recirculation)

4. Activated naïve T cells proliferate and differentiate into a clone of effector T cells with the same specificity (i.e. they express the same TCR on their surface and are specific to the same antigen)

5. Effector T cells enter the circulation and migrate to the site of antigen in peripheral tissues

6. Effector T cells are activated when they encounter their cognate antigen 7. Activated effector T cells exert effector immune functionsResting T cells: express a moderate-affinity IL-2 receptor consisting of ß and γ chains Activated T cells: express a high-affinity IL-2 receptor consisting of α, ß and γ chains; secrete IL-2 IL-2 secreted by activated T cells binds to the IL-2 receptor and stimulates T cell

proliferation

Outline the importance of antigen presenting cells in the induction of T lymphocyte responsesAntigen presenting cells (APCs): express high levels of MHC molecules and co-stimulatory molecules which are required for T cell activation Co-stimulation: usually involves B7 proteins on the surface of APCs which ligate

CD28 receptors on the surface of T cellsAPCs include: Activated dendritic cells (DCs) Activated macrophages Activated B cellsActivated dendritic cells: DCs are distributed below exposed surfaces Langerhans cells: a subset of dendritic cells which are found below the epidermis Immature DCs in uninfected peripheral tissues: express low levels of MHC

molecules and B7 proteins; therefore they cannot function effectively as APCs When there is a microbial invasion of peripheral tissues immature DCs are

activated by:

o Cytokines released by other immune system cells (e.g. TNF) and/or chemicals released by dying cells

o Ligation of their pattern-recognition receptors (e.g. Toll-like receptors) by PAMP Activated DCs travel to lymph nodes, mature and activate naïve T cellsActivation of DCs: 1. Immature DCs become activated when they phagocytose foreign antigen in

peripheral tissues 2. Activated DCs leave the peripheral tissue and migrate to secondary lymphoid

tissues (the nearest lymph node)3. Activated DCs mature as they travel to lymph nodes4. Activated DCs become mature DCs in the lymph nodes: they express high levels of

both MHC-peptide complexes and co-stimulatory molecules Activated macrophages: Dispersed throughout exposed surfaces Inactivated macrophages: express low levels of MHC and co-stimulatory molecules;

therefore they cannot function effectively as APCs Activated macrophages: express sufficient levels of MHC and co-stimulatory

molecules to function effectively as APCs Macrophages are activated by:

o Cytokines (e.g. IFN-γ)o Ligation of their pattern-recognition receptors (e.g. Toll-like receptors) by PAMP

Activated macrophages do not migrate; they re-stimulate activated T cells at the site of infection in peripheral tissues

Activated B cells: Naïve B cells: express low levels of class II MHC molecules and B7 proteins;

therefore they cannot function effectively as APCs Naïve B cells are activated by activated T helper cells??? Activated B cells: express high levels of class II MHC molecules and B7 proteins;

therefore they can function effectively as APCs for T helper cells Activated B cells concentrate MHC II-peptide complexes on their cell surface for

presentation to T helper cells Memory B cells play a major role as APCs either late in the initial infection or early

in subsequent infections by the same pathogen

Explain the different requirements for the activation of naïve and memory T lymphocytesImmunological memory: The immune system recognises and responds to antigen; it can also remember the

same antigen upon subsequent encounters Memory may confer life-long immunity to many infections It is the basis for successful vaccination It is the most important consequence of adaptive immune responses Memory responses are characterised by a faster and stronger immune response

which eliminates the pathogen and prevents infection

T cell memory: In contrast to B cells, T cells do not undergo class switching (i.e. the TCR remains

the same) or affinity maturation (the process whereby BCRs increase their affinity for antigen)

Memory T cells are different from naïve T cellsGeneration of memory T cells: Activated naïve T cells proliferate and differentiate into either effector T cells or central memory T cells: Effector T cells travel to the site of infection in peripheral tissues After the infection most effector T cells die by apoptosis Effector memory T cells: some effector T cells remain in the tissues Central memory T cells remain in the secondary lymphoid organs and the bone

marrow Central memory T cells activate quickly during subsequent attack and mature into

effector T cells2 subsets of memory T cells: Expression of CD45RA allows differentiation between naïve T cells and memory T cells Naïve T cells express CD45RA; whereas memory T cells do not express CD45AExpression of CCR7 (a chemokine receptor) allows differentiation between memory T cells Expression of CCR7 instructs memory T cells to remain in secondary lymphoid

organsEffector memory T cells (TEM) are CCR7- CD45RA-

They display immediate effector functionCentral memory T cells (TCM) are CCR7+ CD45RA-

They lack immediate effector function; they differentiate into CCR7- effector cells after secondary stimulation

Briefly outline the function of T helper cells in relation to the cytokines they produce Th1 and Th2 cells differentiate from a common precursor: naïve CD4+ T cellsActivation of T helper cells: Requirements for activation of T helper cells: Recognition of the cognate antigen presented by MHC class II molecules on the

surface of an APC Ligation of CD28 receptors on the surface of the T helper cell by B7 proteins on the

surface of an APCT helper cell activation: Naïve CD4+ T cells are activated when they encounter their cognate antigen

presented by MHC class II molecules on the surface of an APC and receive co-stimulation from the same APC

Activated T cells proliferate to form a clone of cells which have the same specificity Proliferated T helper cells differentiate and mature into effector T helper cells

(either Th1 or Th2 cells) which have a particular cytokine profile

Cytokine secretion by effector T cells: Effector T helper cells direct a local immune response against a particular pathogen by secreting the appropriate profile of cytokines: Th1 cytokines: direct production of cells and antibodies which are effective against

viral and bacterial infections Th2 cytokines: direct production of cells and antibodies which are effective against parasitic

and mucosal infections

Effector T cell

Cytokine Effector function

Th1 cells

IFN-γActivates macrophagesStimulates B cells to secrete IgG which is good at opsonisation and at fixing complement

TNF Activates macrophages and NK cells

IL-2 Stimulates proliferation of CTLs and NK cells

Th2 cellsIL-4

Stimulates growth of B cellsStimulates B cells to secrete IgE which causes degranulation of mast cells

IL-5Stimulates growth and differentiation of eosinophilsStimulates B cells to secrete IgA

Cytokine profiles of activated T helper cells can be reinforced or changed by cytokines at the site of infection: At the site of a bacterial or viral infection activated macrophages secrete IL-12:

this influences T helper cells to secrete Th1 cytokines At the site of a parasitic infection there is a high concentration of IL-4: this

influences T helper cells to secrete Th2 cytokines Cytokines secreted by committed T helper cells (i.e. effector T helper cells) influence uncommitted Th0 cells (i.e. newly activated T helper cells) to secrete the same profile of cytokines: Th1 cells secrete IFN-γ: this activates macrophages; activated macrophages

secrete IL-12 which influences Th0 cells to secrete Th1 cytokines Th2 cells secrete IL-4: this influences Th0 cells to secrete Th2 cytokinesCommitted T helper cells secrete cytokines which result in positive and negative feedbackPositive feedback: Th1 cells secrete IL-2: this stimulates proliferation of Th1 cells Th2 cells secrete IL-4: this stimulates proliferation of Th2 cellsNegative feedback: Th1 cells secrete IFN-γ: this inhibits proliferation of Th2 cells Th2 cells secrete IL-10: this inhibits proliferation of Th1 cells

Describe the effector functions of T lymphocytes including cell-mediated cytotoxicity, macrophage activation, delayed type hypersensitivity and T/B lymphocyte cooperation Effector functions of T helper cells: Effector T helper cells coordinate the immune response against infections caused by intracellular pathogens Effector T helper cells have 2 main functions:1. To direct the local immune response (i.e. at the site of infection) by cytokine

secretion2. To help B cells and CTLsCell-mediated cytotoxicity: CTL precursors (CTL-Ps): naïve CTLs which exert no cytotoxic effector function (i.e. they cannot kill) CTL-Ps must be activated to differentiate into effector CTLs which are functionalInitial activation requires an interaction between a CTLp and an APC; it is T helper cell-independentGeneration of CTLs:1. A CTLp is activated when it encounters its cognate antigen presented by MHC class

I molecules on the surface of an APC and receives co-stimulation from the same APC

2. Activated CTL-Ps migrate to the site of infection in peripheral tissues

CTL-Ps require an external supply of IL-2 to proliferate in peripheral tissues3. Th1 cells secrete IL-2 at the site of infection: this stimulates activated CTL-Ps to

proliferate4. Proliferated CTL-Ps differentiate into effector CTLs which exert effector cytotoxic

functionEffector cytotoxic function of CTLs: Effector CTLs become polarised when they encounter their cognate antigen

presented by MHC class I molecules on the surface of a target cell (e.g. virus-infected cells)

Polarisation results in redistribution of the cytoskeleton and cytoplasmic components within CTLs: granules migrate to the site of TCR-MHC I-peptide interaction at the plasma membrane

CTLs release their granules at the site of cell-cell contact after recognition of the target cell

CTLs kill a target cell by inducing apoptosis: 1. The CTL recognises and binds to its cognate antigen presented by MHC class I

molecules on the surface of a virus-infected cell2. The CTL programs the target cell for death by inducing DNA fragmentation3. The CTL migrates to a new target cell 4. The original target cell dies by apoptosis 2 pathways of cell-mediated cytotoxicity:1. Granules 2. Fas-FasL interactionsGranules: contain cytotoxic effector molecules which trigger apoptosis Perforin: perforin molecules polymerise to form a pore in the plasma membrane of

the target cellPerforin helps a CTL to deliver granzymes into the cytoplasm of the target cell

Granzymes: serine proteases which induce apoptosis once they are in the cytoplasm of the target cell

Granulysin: induces apoptosis in the target cell Fas-FasL interactions: CTLs have Fas ligands (FasL) on their surface which can bind to Fas proteins on the

surface of a target cell Fas-FasL interactions induce apoptosis in the target cellCTLs also release cytokines which contribute to host defence by amplifying the immune response: IFN-γ: inhibits viral replication; activates macrophages TNF-α and TNF-ß: synergise with IFN-γ receptorsN.B. CTLs are recycledMacrophage activation: Macrophages must interact with Th1 cells in order to be activatedRequirements for macrophage activation:

TCR-MHC II-peptide interaction: a TCR on the surface of a Th1 cell recognises and binds to its cognate antigen presented by MHC class II molecules on the surface of a macrophage

CD40-CD40L interaction: CD40L on the surface of a Th1 cell ligates CD40 on the surface of a macrophage

Cytokine interaction: Th1 cells secrete cytokines (IFN-γ) which bind to IFN-γ receptors on the surface of a macrophage

Cytokine mediated interactions between naïve CD4+ T cells and macrophages: APCs secrete IL-12: this activates naïve CD4+ T cells and stimulates them to

proliferate and differentiate into a clone of Th1 cells Th1 cells secrete IFN-γ: this activates macrophagesDelayed type hypersensitivity (DTH): an inflammatory reaction in which T helper cells recognise a specific pathogen and secrete cytokines that activate and recruit cells of innate immunity to kill the pathogenFunctions: Primary role in defence against intracellular pathogens by eradication of the

pathogen If the pathogen is not eradicated, it is contained by chronic stimulation and

granuloma formation Hypersensitivity: if the antigen is not a microbe, DTH produces tissue injury

without protection as in contact hypersensitivity (CHS) DTH may be protective as well as pathological2 phases of DTH:1. Sensitisation phase2. Effector phaseTuberculin hypersensitivity: Clinical appearance: firm red swelling of the skin which is maximal 48-72 hours

after injection Histological appearance: there is a dense dermal infiltrate of leukocytes and

macrophages T/B lymphocyte cooperation: T helper cell dependent activation of B cells requires cell-cell contact and cytokinesSee “Immunology 5” Beneficial and deleterious actions of effector T cells: Clearance of the pathogen: effector T cells recognise antigenic peptide derived

from foreign pathogensEffector T cells may cause pathological reactions: Autoimmunity: effector T cells recognise antigenic peptide derived from self

proteins Rejection (of transplants): effector T cells recognise antigenic peptide derived from

proteins of the transplant donorRegulatory T cells: T cells which regulate the activation or effector functions of other T cells A subset of CD4+ T cells

Have a relatively high affinity for MHC II-self peptide complexes: it may be necessary to maintain tolerance to self antigens

Control immune responses: suppress the immune response to self antigenso Secrete cytokines (IL-10 and TGF-ß) which inhibit T cell responseso Cell-cell contact inhibits T cell responses

Immunology 8: Host Defence Overview Name the important components of the immune system

Summarise and give examples of the roles taken by: physical or chemical boundaries; mechanical removal; innate immune responses

Define and contrast: innate and adaptive/acquired immunity; humoral and cellular immunity; attributes of specific immunological memory

Appreciate special features of immune reactions at mucosal sites

Understand the sequence and timing of events during acute infection

List defence mechanisms operative against viruses and bacteria