Samenvatting hoofdstuk 1 – 5 uit:
Basic and clinical immunology
Peakman M, Vergani D
1e druk 1997

Chapter 1: Anatomy and cells of the immune system

CELLS OF THE IMMUNE SYSTEM
Bone marrow is source of precursor cells (fetal life; liver too).
Haemopoiesis = process by which all cells that circulate in the blood arise and mature.
Pluripotent haemopoietic stem cell = is capable of giving rise to all blood cell lineages.
White blood cells: granulocytes, monocytes and lymphocytes.

Granulocytes = 65% of white cells
0,5-1% basophils (blue staining)
3-5% eosinophils (red-staining)
90-95% neutrophils (unstained)
Polymorphonuclear cell = multilobed nuclei of granulocytes (= neutrophils/eosinophils).
G. circulate in blood and migrate into the tissues. Mast cells are fixed in the tissues and have an unidentified precursor.

Monocytes and dendritic cells = 5-10% of white cells
Monocytes stay for +/- 24 hrs in the blood, after which they enter the tissues and stay there as macrophages.
Characteristics: bigger, single nucleus en abundant granular cytoplasm.
Specialised forms: alveolar macrophages (lung), Kupffer cells (liver), mesangial cells (kidney), microglial cells (brain) and osteoclasts (bone)
Dendritic cells: bone marrow derived. Function in activation and priming of lymphocytes. Specialised form = follicular dendritic cell

Lymphocytes = 25-35% of white cells
Bone marrow derived. Two subtypes: B and T (ratio 1:5). They have highly specialised glycoprotein molecules on their surface. Location: blood, lymphoid organs and sites of chronic inflammation.
· B lymphocytes: differentiate within the bone marrow (also liver during fetal life). Function = recognition of antigens through antibodies (= surface receptors). Fixed in the tissues they mature in plasma cells and secrete antibodies.
· T lymphocytes: their involvement with the thymus is early in life and critical for acquiring the ability to distinguish self and non-self (= protection against infection) => graft rejection. They also make a telling contribution to B lymphocyte function.
Thymus = lymphoid organ

Natural killer cells
Capable of lysing virus-infected cells and tumour cells. A population of cells defined morphologically as large granular lymphocytes (LGL) also have NK function.

ORGANS OF THE IMMUNE SYSTEM
Lymphocytes recirculate on average in 1-2 days. It is a highly regulated process of immune surveillance, controlled according to cell type and anatomy.
· Primary lymphoid organs = bone marrow and thymus: development and maturation.
· Secondary lymphoid organs = lymph nodes and spleen: maturation and development of immunity. MALT = mucosa-associated lymphoid tissue (respiratory tract). GALT = gut-associated lymphoid tissue (gut).


Primary lymphoid organs
Both microenvironment within the marrow and the influence of soluble mediators that act as colony stimulation factors determine the celltype in which the pluripotent stem cell develops.
The thymus is an immunological ‘black box’. It develops itself in the sixth week of fetal life. By exposure to the immature cells of thymic epithelial and macrophage-derived, they develop into immature T lymphocytes.

Secondary lymphoid organs
Lymph nodes
Lymphocytes enter the lymph nodes either through the lymphatics or from the blood:
· Afferent lymphatics -> subcapsular marginal sinus -> cortex -> medulla -> efferent lymphatics -> thoracic duct -> venous system.
· Blood -> large cuboidal endothelial cells on high endothelial venules (HEV)
Primary follicles (in cortex) are at rest.
Secondary follicles arise following stimulation of a local immune response: germinal centre enlarges and B lymphocytes proliferate and differentiate.
Paracortex = T lymphocytes + interdigitating dendritic cells
Medulla has medullary cords of lymphoid cells (plasma cells)
Lympho’s have specific ‘homing’ receptors for the HEVs -> organisation
GALT = Peyer’s patches + isolated lymphoid follicles = lack a capsule and afferent lymphatics.

Spleen
White pulp = lymphoid tissue
Red pulp = reticular tissue and sinuses bathed in blood
Arteries entering the white pulp are surrounded by the periarterial lymphatic sheath (PAL; T zone), which contains follicles and germinal centres (B zone).
In the red pulp, blood in the splenic venous sinuses interact with the marginal zone (macrophages), next to the PAL.
Function: lymphoid organ (infectious agents + antigen-antibody complexes -> immune response), filter bed (removal of effete and defective red and white blood cells -> macrophages eat them) and reclamation site (resources are recycled -> ‘green’ pulp)

Chapter 2: Innate immunity 1: physical and humoral protection

FREEDOM FROM THE BURDEN OF DISEASE
Definition of the immune system = identify self and recognise non-self.
We are born with some immunity and the rest may be acquired during life. Some specific responses may be retained in an ‘immunological memory’.

TYPES OF IMMUNITY
Innate immunity
= immunity present at birth = present for life, has no specificity and no memory.
Useful in protection against:
-pyogenic organisms
-fungi
-multicellular parasites
Three components:
· physicochemical: barriers like skin, mucosae, secretions and cilia.
· humoral = immunologically active factors: complement, opsonins (C-reactive protein) and proteolytic enzymes (-> chapter 2)
· cellular: neutrophil, eosinophil, mast and NK cell (-> chapter 3)

Acquired immunity
Absent at birth, has specificity and memory = adaptive (-> chapter 4 & 5)

COMPLEMENT
An antibody is specific for antigens on a target (acquired immunity). A complement complements the action of the antibody in destruction of organisms.
Complement is a (40-) protein cascade. Components are made in the liver.
Three pathways: alternative, classical and common/membrane attack pathway.

Complement proteins
Most are soluble. They circulate in an inactive state. After activation they catalyse the conversion of the next component -> amplification -> cell/bacterial lysis + production of pro-inflammatory mediators + solubilisation of antigen-antibody complexes.
There’s a standard nomenclature (Table 2.1, page 12).

Complement activation
Occurs through two distinct pathways: classical and alternative and stimulate the final common pathway. The first two have a distinct enzyme cascade that culminate in the cleavage of C3 and C5. They are triggered by different substances and through different initiation mechanisms.

COMPLEMENT PATHWAYS
Alternative pathway = primitive + part of the innate immune system.
Classical pathway = recent + combines with antibodies -> acquired immune system.

The classical pathway
Activated by an immune complex (= antigen + antibody) (and by aggregated immunoglobulins, DNA and C-reactive protein) which binds with complement C1. C1 = C1qr2s2. This cleaves C4 and C2 to form the classical pathway C3 convertase C4b2a. Following C3 cleavage, the C5 convertase is formed, which cleaves C5. Biologically active fragments C4a and C3a are generated.

The alternative pathway
Consists of a ‘tick-over’ mechanism: a continuous, slow reaction sequence, accelerated by activators. C3 'tick-over’ generates C3b, C3bB and C3bBb (by factor D) = alternative pathway C3 convertase, which in turn cleaves C3. The tick-over is accelerated if the active enzymes are stabilised on bacterial cell walls, or if more C3b is produced from the classical pathway, or by impetus of factor P (properdin). The alternative pathway C5 convertase C3bBb3b is generated.

The membrane attack pathway
The C5 convertases generate C5b and the pro-inflammatory C5a. C5b67 binds the target cell membrane and with addition of C8 and a C9 polymer the membrane attack complex (MAC) is formed. Holes are made into the membrane and death results through osmotic lysis.

Control mechanisms
By lability of the active molecules (C3a, C4a and C5a). Dilution of activated molecules lessens their potential impact.
Circulating proteins:
-C1-inhibitor: blocks C1
-Factor I: degrades C3b, destroys C4b (enhanced if bound with C4-binding protein = C4bp)
-Factor H: binds with C3b to accelerate factor I.
-Protein S and SP-40,40: bind C5b67 complex -> inactivity.
-Carboxypeptidase N: inactivates C3a, C4a and C5a.
Membrane bound proteins:
-Membrane attack complex inhibitory factor = CD59 = cluster of differentiation 59: avoids accidental insertion by MACs into a lympho or host cell.
-Decay accelerating factor (DAF): competes for C4b, thus inhibiting formation of the classical pathway C3 convertase

Complement receptors = CR
CR-1 binds C4b and enhances factor I (like C4bp). There are two main groups of CRs: CR-1 to CR-4 and receptors for C4a, C3a and C5a.

BIOLOGICAL ACTIVITIES GENERATED BY COMPLEMENT ACTIVATION
Opsonisation
= coating of bacteria or other pathogens and facilitating their removal. C3b has an opsonic activity.

Cell recruitment and activation
C4a, C3a and C5a are anaphylatoxins -> role in anaphylaxis in activating mast cells and basophils.
C5a and (lesser) C3a are also chemotactic = the ability to attract cells (neutrophils).

Cell lysis
Through complete complement activation.

Removal of immune complexes
Immune complexes are potentially harmful because of inciting complement activation. Large complexes can become insoluble and fixed in the tissues.
Complement can maintain immune complexes in solution and can expedite their removal from the circulation. C3b-coated complexes attach to cells’ CR-1 (immune adherence) on erythrocytes, who transport them to the liver and spleen, where they are released and taken up by macrophages.

OTHER FACTORS IN HUMORAL IMMUNITY
C-reactive protein (CRP) is produced in the liver and binds certain bacterial cell wall components and then activates complement through the classical pathway, independently of antibody. It’s blood levels rise within hours of the start of an infective or inflammatory process -> useful in monitoring this and their response to treatment.
Fibronectin: binds staphylo- and streptococci + macrophages and monocytes -> clearance. Especially used in disease monitoring in premature babies.
Lysozyme: bactericidal enzyme secreted in saliva, tears and other body fluids and present in neutrophil granules.

Chapter 3: Innate immunity 2: cellular mechanisms
Neutrophils: kill and remove bacteria and fungi.
Eosinophils: role in controlling infection with multi cellular parasites, such as worms.
Mast cells: are involved in allergy.
Basophils: unknown.
All of them do something by releasing their granules.
Division between innate and acquired immune system is artificial.

NEUTROPHILS
The multilobed nucleus is important to make rapid transit from the blood through tight gaps in the endothelium. Concentration: 2-7 x 109 /litre. The half-life in blood is 6 hrs and in tissues 1-2 days.

Neutrophil granules
Two main types: primary or azurophilic granules (appear during development in the bone marrow) and secondary or specific granules (appears later; 3x more common).
Releasing granules intra- or extracellularly follows in response to several stimuli:
· The products of bacterial cell walls: N-formylated peptides (FMLP) -> bind to neutrophil receptors.
· Complement proteins: iC3b -> bind to receptors.
· The leukotriene (LTs) group of lipid mediators (from the lipooxygenase pathway of arachidonate metabolism): LTB4.
· Cytokines: NAP-1 (neutrophil activation protein-1; = IL-8), TNF-alfa (tumour necrosis factor-alfa) and GM-CSF (granulocyte-monocyte colony stimulating factor).
Primary granules:
· Myeloperoxidase: microbicidal enzyme.
· Collagenase + elastase: break down fibrous structures in extracellular matrix -> progress through the tissues.
· Cathepsin G: cidal to Gram+ and Gram- organisms and some Candida species.
· Cathepsin B, D and E: also bactericidal.
Secondary granules:
· contain pre-synthesised receptors for some of the molecules capable of activating them -> enhancing the directional nature of the response -> important in chemotaxis
· assortment of proteins with unknown function
· Others: Lactoferrin: decreases cell surface charge -> enhancing cell adhesion; it also helps in generating the *OH, a microbicidal toxin.

Neutrophil activation
Stimuli: C5a, LTB4, FMLP and IL-8 -> are released at sites of infection and inflammation.
Neutrophils must migrate to the relevant site in the tissues. This requires organisation:
Tissue damage results in release of mediators with profound effects on vessel walls (endothelial cells lining the post-capillary venules) -> dilatation with increase ‘leakiness’ and reduction of blood flow rate.
Neutrophils have a tendency to adhere lightly to the endothelium: ‘rolling’.
Substances from damaged tissues or bacteria have a direct effect on endothelial cells: become ‘sticky’ -> adhesion of neutrophils.
Rolling and adhesion involves adhesion molecules. Ligand and counter-ligands are up-regulated on both rolling neutrophils and endothelium -> adhesion.

Adhesion molecules
Three main families of surface proteins: selectin, integrin and intercellular adhesion molecules (ICAMs). Many are named using the CD nomenclature.

Selectins
Their N-terminal domain resembles various lectins (molecules with affinity for sugar moieties).
· P-selectin (on a platelet/ endothelium) -> bind leukocytes
· E-selectin (on endothelium) -> bind leukocytes
· L-selectin (on a leukocyte) -> bind endothelium

Integrins
Heterodimers with an alpha and beta chain. Two families: beta-1 (T cell function) and beta-2 (adhesion all leukocytes) integrins.
Beta-2 integrins have the same beta chain (CD18) and three different alpha chains (CD11a-c).
· CD11a/CD18 = Leukocyte function associated antigen-1 (LFA-1) on lympho’s, monocytes and neutrophils.
· CD11b/CD18 = Macrophage-1 (Mac-1) or complement receptor-3 (CR-3) on granulocytes and monocytes.
· CD11c/CD18 = p150/95 or complement receptor-4 (CR-4) on tissue macrophages.

Intercellular adhesion molecules (ICAMs)
ICAMs are singlechain molecules that form part of the immunoglobulin gene superfamily of molecules. It is expressed on a variety of cells involved in immune processes, as well as on endothelium. It’s a ligand for integrins and also the receptor for rhinoviruses (cause the common cold).

Neutrophil migration
Constitutive expression = permanent expression. The interaction between selectins and their ligands is a weak adhesive force, but it provides the first step in neutrophil migration -> slow down and move along the margin of the endothelium = margination.
Rolling = process of proceeding of the marginating pool along the endothelium.
Now further adhesive mechanisms are needed -> activating signals for neutrophils and endothelium: iC3b, FMLP, LTB4 and cytokines as IL-8 (= stimuli for releasing granules too).
Neutrophil activation has two effects:
· Functional upregulation of adhesion molecules (LFA-1 and Mac-1).
· Secondary granules release fresh adhesion molecules onto the neutrophil surface.
On the endothelium ICAM-2 and –1 is constitutively expressed and induced by various mediators (interferon-gamma, IL-1 and TNF).
The induction of the integrin/ICAM system locally in inflamed tissues increases neutrophil-endothelium affinity by many times -> neutrophils stop and pass through the endothelium = diapedesis. The neutrophil is drawn to the point of highest concentration of the activating signals.

Chemotaxis
= the directed movement of a cell along a gradient of increasing concentration of the chemoattractant. It is used by neutrophils to migrate. Chemoattractants: C5a complement component, FMLP, IL-8 and LTB4.
When a chemotactic factor binds at one pole of the cell:
· the cell releases granules, to up-regulate receptors
· the cell extends its membrane and cytoplasm into pseudopodia
The leading pseudopodium anchors itself and the remainder of the cytoplasm is drawn up. Etc.

Phagocytosis
Neutrophils can ingest more than one bacterium or fungus at once. An abscess filled with pus (dead or dying neutrophils) can be formed. A ‘sterile’ abscess can be formed if the process is inflammatory but not infective (foreign body).
For phagocytosis opsonins are needed. They coat the targets and enhance the neutrophil in engulfment. Neutrophils have receptors for opsonins on their surface and are also activated by the opsonins.
Opsonins: C3b, C-reactive protein and antibody (specific immune system).
Phagocytosis: pseudopodia extend to surround the target and meet and fuse to form a phagosome. The phagosome fuses with neutrophil granules, releasing their digestive and toxic contents. Occasionally, granule contents may be discharged into the external milieu, leading to tissue damage = ‘reverse phagocytosis’.

Neutrophil killing
When the killing of bacteria and fungi is impaired, the individual suffers recurrent, often fatal infections.
Killing is the result of two microbicidal routes:
· oxygen-dependent route:
- Respiratory burst (use of O2): results in toxic metabolites: O2-, H2O2, 1O2 and *OH. The main constituents, found in the secondary granules, are a membrane bound complex of three components: flavin adenine dinucleotide (FAD), quinolone and cytochrome b558.
- Hydrogen peroxide-myeloperoxidase-halide system: results in toxic metabolites: chlorine (Cl2) and hydroxyl ions (OH-).
· oxygen-independent route: microbicidal enzymes such as lysozyme and cathepsins (->from primary granules).

EOSINOPHILS
3-5% of granulo’s in blood. But there are many more in the tissues at epithelial surfaces for several weeks. Staining is result of cationic proteins with affinity for eosin. Main role: protection against multicellular parasites such as worms (helminths) by the release of toxic, cationic proteins. They also contribute to allergic disease (asthma).

Eosinophil granules
Two main types:
· Specific: predominate. Contain four types of cationic proteins: major basic protein (MBP; toxic to helminths), eosinophil cationic protein (ECP; toxic to helminths and bactericidal), eosinophil neurotoxin (toxic to helminths) and eosinophil peroxidase (catalyses a similar reaction as myeloperoxidase).
· Primary: mostly unknown.
· Other: lipid vesicles which can swiftly release LTC4 and LTD4. LTC4 and platelet activating factor (PAF) can produce changes in airway smooth muscle and vasculature -> allergic reactions.

Eosinophil activation
By a variety of mediators. They have specific receptors for C3b/C4b (CR-1), iC3b (CR-3), C5a and LTB4, for different classes of antibody: IgE (allergy) and IgA (protection of mucosal surfaces) and for cytokines: IL-3, IL-5 (growth factor for eosinophils) and GM-CSF. After binding they are activated.

Eosinophil adhesion
Similar to neutrophils, but eosinophils alone appear to possess very late antigen-1 (VLA-1 = integrin), which binds vascular cell adhesion molecule-1 (VCAM-1) on the endothelium.

Eosinophils in host defence
They employ complement and antibody-guided local release of toxic cationic proteins onto the surface of helminths (no phagocytosis and intracellular degranulation!).
They overlap with the acquired immune response (interaction) and synthesise and express CD4 and HLA-DR (associated with antigen-specific cell-mediated responses) -> this cell is more sophisticated than the neutrophil (together with longer life, cytokines secretion and cell activation state).

MAST CELLS AND BASOPHILS
Two main features: they have histamine-containing granules and they have high-affinity receptors for IgE (i.c.w. eosinophils).

Mast cell and basophil granules
Two categories:
· pre-formed mediators: histamine: blood half-life of <5 min (effects on blood vessels and bronchial smooth muscle). Injected into the skin: ‘wheal and flare’ or ‘triple’ response:
- reddening (arterioles dilate and post-capillary venules contract),
- increased vascular permeability -> leakage of plasma fluid -> swelling (wheal).
- acting directly on local axons with inducement of more widespread vascular changes (flare).
· synthesised de novo
Effect differs according to the stimulus and site, from a localised wheal and flare to anaphylactic shock.

Mast cell and basophil activation
By antibody class IgE (allergic disease), anaphylatoxins C3a, C4a and C5a (basophils and lung mast cells) and FMLP (basophils).

Chapter 4: Acquired immunity: antigen receptors
During first encounter between host and microbe, the immune system identifies and learns the distinctive structural features of it -> specific recognition. Effector responses can be initiated and a memory bank can be formed, so that next time the reaction is more quickly and with a greater initial force.
=> specificity, memory and variable intensity.
The innate and acquired immune responses can complement each other (antibodies).

ANTIGENS AND ANTIBODIES

Antigens
= structures which generate an anti- response by the immune system. Three elements that are used in binding and recognition of antigens:
· Antibodies: generated by B-lympho’s and plasma cells. Have recognition sites for part of an intact antigen. The interaction is shape and conformation dependent. Can bind soluble antigens. Epitope = part of the antigen with which the antibody interacts = antigenic determinant (determines which antibody will bind).
· T cell receptors: differences: interacts with a short segment of amino acids derived from the intact antigen by proteolysis = peptide antigen = T cell epitope. Cannot bind soluble antigens directly -> they must be held and presented by MHCs.
· Major histocompatibility complex (MHC) molecules: hold the peptide antigen enclosed within a groove -> is recognised by the T cell receptor.
Antigens interact with these three through non-covalent forces (reversible). There’s a dynamic equilibrium between the dissociated and complex form. Affinity = the concentration (= dissociation constant (Kd)) of antigens allowing half the antibodies to be complexed and half to remain in solution. The smaller the number the higher the affinity. Order: antibody > MHC molecules > T cell receptor. Antibodies have at least 2 sites for binding and also a flexible hinge -> increases the overall strength of attachment = avidity.

Antibodies
= soluble glycoproteins that belong to immunoglobulins.
Functions and uses:
1. In host defence:
· targeting infective organisms
· recruitment of damaging host effector mechanisms
· neutralisation of toxins
· removal of foreign antigens from circulation
2. In clinical medicine:
· diagnosis/monitoring a disease
· administration of pooled antibodies passively for host therapy/protection
3. In laboratory science: diagnostic and research applications.
Activation of the following immune effector functions:
· complement activation
· stimulation of phagocytosis and killing by polymorphonuclear cells.
· recruitment of killer cells
· activation of mast cells

The many roles and uses of antibodies
Antibodies act as a guidance system, focusing effectors onto appropriate targets. So antibodies provide target specificity for the innate immune system.
Characteristic features of antibodies:
1. Two modes of expression:
- synthesised and displayed on B lympho’s as receptors.
- exported as soluble proteins by plasma cells.
2. Each antibody monomer has two identical antigen-binding sites.
3. The gene that codes for antibodies enables antibody structure to vary enormously -> 1011 different antigen-binding sites.
4. Apart from the antigen-binding site, other parts can be modified to give the antibody molecule different effector functions.

IMMUNOGLOBULINS

Structure
Four polypeptide chains: 2 identical heavy (H) and 2 identical light (L) chains. Formula: H2L2.
Light chains: 2k or 2l chains.
Heavy chains: 2a, 2b, 2e, 2g or 2m chains.
Each H or L chain has a stable segment = C region -> hold effector functions; and also a variable segment = V region -> include the antigen-binding site.
The 2 H chains are linked together by 2 interchain disulphide bonds. Each H chain has a light chain attached to it by an interchain disulphide bond. Within the H and L chains, there are intrachain disulphide bonds, creating domains, which contain two layers of b-pleated sheet with 3 or 4 strands of antiparallel polypeptide chain. Many molecules in the immune system share a similar domain structure = immunoglobulin supergene family.
The domains are named according to which chain they are in, and numbered: VL, CL domain and VH, CH1-4. VL and VH domains = antigen-binding site. CH domains = major effector functions.
Hinge region = in the middle of the molecule for maximising the chances of binding 2 antigens at one time.
Cleaving the Ig molecule with papain produces 2 Fab fragments (fragment antigen binding) with antigen-binding ability and 1 Fc fragment (fragment cystallisable) with effector functions. Fc receptors = surface receptors for Ig molecules, that interact with the Fc fragment.
Cleaving the Ig molecule with pepsin produces a F(ab’)2 fragment with 2 antigen-binding sites, but no effector functions remaining.
The effector function part of the antibody can be varied by variation in the genes, producing the 5 different H chains -> giving rise to the major classes of Igs (Ig = whole immunoglobulin molecule): IgA, IgD, IgE, IgG and IgM and to the subclasses: IgG1-4 and IgA1-2.

Immunoglobulin classes and subclasses
Immunoglobulin G (IgG)
Most abundant (10g/l). Has 3 CH domains; monomer; 4 subclasses with variability in the hinge region and functional domain. The subclasses vary in their relative ability to perform some of the effector functions (IgG2 for response to bacterial polysaccharide) and in their serum concentration (Table 4.2; page 39). IgG is a major activator of the classical complement pathway (IgG1 and IgG3) and binding to C1q is greatly increased when IgG is complexed to antigen.
Three cellular receptors: Fcc R1, 2 and 3 -> used to bind, recruit and activate cells; also important for placental transfer of IgG (specific protection on the newborn)

Immunoglobulin A (IgA)
Next most abundant (1-4g/l). Distinctive in 2 ways:
1. Can occur as a monomer, but also as a dimer (2 IgA molecules joined by an J chain).
2. It is the major Ig secreted onto the external surfaces -> presence in secretions (saliva, bronchial fluid, gut secretions, tears, etc.) of secretory IgA. Achieved by attachment to poly-IgR (synthesised by epithelial cells lining mucosal surfaces) -> endocytosis, transport and secretion (of IgA and the secretory chain (= remnant of poly-IgR)). IgAs plays an important role in protection against bacterial, viral and protozoal infections of mucosae -> can activate the alternative pathway and is an opsonin, reacting with FcaR.

Immunoglobulin M (IgM)
Pentamer: 5 IgM monomers joined by the J chain. It is the first immunoglobulin synthesised in an antibody response (primary response) -> it has multiple functional domains (complement activator) and 10 antigen-binding sites. There’s not a high affinity for antigens, but the many sites compensate this.

Immunoglobulin D (IgD)
Serum concentrations are extremely low -> function unclear: surface expression on B cell lympho’s at an immature stage and the signalling on interaction with antigen in a lymphoid follicle is a critical part of B lympho activation.

Immunoglobulin E (IgE)
Largest: 4 CH domains. Serum concentration rises in response to parasitic infections, atopic individuals and type 1 hypersensitivity patients (allergy). Function: bind and activate mast cells. Mast cells have high affinity receptors: Fce R1. Antigen + IgE + FceR1 on mast cells causes vascular effects. B lympho’s and eosinophils have low-affinity receptors: FceR2 -> for regulation of IgE production.

IMMUNOGLOBULIN EXPRESSION

B cell surface expression
Binding of antigen to surface IgD is an early event in the B lympho cell cycle. In the mature B lympho antigen interaction with other Ig classes leads to internalisation -> antigen is broken down and presented to T lympho’s -> T lympho’s are activated -> immune response.

Isotypes, allotypes and idiotypes
Immunoglobulins can be characterised by their own ability to act as antigens: isotypes are epitopes that are present on all molecules of a class (e.g. IgE) or chain (e.g. e CH) type in a species; allotypes vary between individuals, idiotypes reflect variation in the antigen-binding sites of the immunoglobulin. The antigenic epitope within the antigen-binding site = idiotope. A collection of idiotopes = idiotype.

Primary and secondary antibody responses
Primary antibody responses are slow, mainly IgM and decline to low levels (IgG appears towards the end of the primary response). Repeated exposure to the antigen at a later time elicits a more rapid response to a higher peak level that declines to a higher baseline level; this secondary response is predominantly IgG. The secondary response is antigen-specific and demonstrates acquisition of memory. There’s also a higher intensity. IgG in the secondary response has a higher affinity for antigen: positive selection.

Haptens
= molecules that are too small to generate an antibody response. But coupled to a carrier, haptens may elicit production of antibodies that are able to bind the hapten directly, without the carrier.

Clonality in antibody responses
Each plasma cell produces a single antibody = monoclonal antibody; tumours formed from a single plasma cell will produce large quantities of this monoclonal antibody. A population of plasma cells will respond to different epitopes on macromolecular antigens giving rise to many clones of plasma cells and many antibody types: a polyclonal response. Some areas of antigens are targeted more frequently than others = dominant epitopes.

IMMUNOGLOBULIN GENES
The genetic system for antibodies have to generate a diversity for antigen binding and a limited choice of functional characteristics.
Generation of antibody diversity: a limited number of genes are available, which combine randomly. Organisation: each chain is encoded by a gene complex. Within the complex are segments: V genes (variable region), C genes (constant region), J genes (joining these two) and D genes (generating diversity).

Light chain genes
There’s no D segment for light chains. Number of combinations = V genes x J genes x C genes. k chain: 1000 different combinations. l chain: 150.

Heavy chain genes
Two differences:
1. It has D genes.
2. The different C genes, encoding the H chain isotypes, are located together, downstream from the other genes.
There are 250-1000 VH genes, 12 DH and 4 JH gene segments (= 48 000). When combined with L chains: more. Forming different antibody specificities through combining different genes randomly = combinatorial diversity.

Generation of immunoglobulin diversity
Two other additions to diversity:
-Junctional diversity = the random imprecision in the joining of the segments caused by loss or retention of nucleotides or codons, which results in frame-shifts.
-Somatic hypermutation = the occurrence of a single mutational change after the initial gene rearrangements, probably during class switching (from primary to secondary response).

Class switching
In class switching in B cells, the early expression of IgM gives way to the mature, activated expression of other classes of Igs -> achieves modulation of the functional capabilities determined by the antibody produced.

Affinity maturation
B cells with the highest affinity (by somatic hypermutation during class switching) will be stimulated to proliferate further = affinity maturation.

Genetic basis for immunoglobulin gene rearrangement and class switching
There’s an order in choosing the different genes. So that some genes are more often chosen than others.

T CELL RECEPTORS FOR ANTIGEN
T cell receptors = TCRs: have a basic molecule composed of 2 chains: these may be a/b (90%) or c/d . The chains of each type of TCR are divided into variable and constant domains. The T cell expresses only one form of TCR. The c/d TCR appears first on primitive T lympho’s in the thymus = TCR1. a/b TCR = TCR2.

Structure and function of the ab T cell receptor
A disulphide-bonded heterodimer of an a and a b chain. They’re also part of the immunoglobulin supergene family. They have hypervariable regions of the variable domains, forming CDRs 1-3. CDRs 1 and 2 interact with the a1 and a2 domains of the MHC molecule, whilst CDR3 interacts with the peptide within the groove.

Structure and function of the cd T cell receptor
Three types: a disulphide-linked heterodimer of c and d, a non-disulphide-linked heterodimer and a disulphide-linked cc homodimer

The T cell receptor genes
There’s a similar depth of diversity as that for antibodies. The genes are divided into two separate groups: for the variable an for the constant domain.

Genes for the a and b chains
Diversity for the a chain = V x J x C = 10 000. For the b chain = V x D x J x C = 4800 => diversity for an ab chain = 48 million.
The Ca and Cb genes do not appear to encode segments typical of secreted proteins -> secretion is functional not important.
There is no somatic hypermutation.

Genes for the c and d chains
Diversity for c chain = 32 and for d chain = 60 => total diversity = 1920.

Generation of T cell receptor diversity
TCRs vary in structure through the use of a large pool of gene segments for the receptor chains, random recombination of variable, diversity and joining genes and highly random imprecision in the joining process.

Chapter 5: The human leukocyte antigens
Collection of genes in general = major histocompatibility complex (MHC): contains a group of genes that code for proteins expressed on the surface of a variety of cell types. In humans = human leukocyte antigens = HLA system ( human MHC).
HLA molecules are involved in antigen recognition by T lympho’s by presenting antigen as a short peptide embedded within a physical groove.
Differences in HLA molecules between individuals are responsible for organ graft rejection. Possession of certain HLA genes was linked to greater susceptibility to particular diseases (MS, DM1 and ankylosing spondylitis).
The new HLA nomenclature is based on genotyping. But the old is also used.

THE MAJOR HISTOCOMPATIBILITY COMPLEX
The immune response
Law of MHC restriction: a cytotoxic T cell will only kill target cells that present both a specific antigen and the correct MHC molecule.

Structure of the HLA
HLA comprises 3 major classes (I, II and III ) of genes. Genes: CAPITAL ITALIC LETTERS; antigen molecules produced by them: CAPITAL ROMAN LETTERS; polypeptide chains: Greek letters a and b.

Class I region
Class I HLA genes: HLA-A to HLA-J, with HLA-A, HLA-B and HLA-C as best known. They encode the amino acid sequence of the class I a chains. The class I b chain is an invariant molecule = b2-microglobulin.
Function: presenting peptide antigens to T lympho’s.

Class II region
The class II genes have 3 major subregions: DP, DQ and DR -> for presentation of peptide antigens to T lympho’s. HLA-DM plays a critical role in loading peptide into the other class II HLA molecules.
There are different haplotypes for an individual, governing the number of DR molecule types produced.
Gene polymorphism = a large number of different alleles for DRB genes -> there is enormous potential for individuals to differ in the HLA genes they possess.
Molecules encoded by the MHC act further as:
- peptidases to cleave large protein antigens
- transporters, carrying antigenic peptides to the correct intracellular compartment.

Class III region
Contains several genes coding for complement components.