Immunology

• B and T lymphocytes derive from bone marrow stem cells
• B lymphocytes mature to plasma cells which produce antibody (humoral immune response)

• T lymphocytes produce lymphokines which are chemotactic and activate macrophages (cell mediated immune response)

• Lymph nodes:
• B cells reside within cortical follicles
• T cells reside in the paracortical zones

• The immune system recognises antigen through molecular interaction with specific molecules including:

• Antibody 
• The T-cell receptor (TCR)
• MHC molecule

• The majority of immune responses are T-cell dependent ie the effector response depends on the initial interaction between resting T cells and an APC

Immunoglobulins (antibodies)

• Y shape: 4 polypeptide chains held by disulphide bonds
• Pair of heavy chains and pair of light chains
• Each chain is a series of repeating units
• Light chains consist of variable domain and constant domain
• Either kappa or lambda light chains (does not impact on the immunoglobulin function)
• Heavy chains have one variable domain and a number of constant domains
• IgM and IgE have 4 constant domains
• IgG, IgD and IgA have 3
• The variable domain of antibodies results from gene segment arrangements
• This unique sequence is the idiotype
• The variable regions of the heavy and light chains form the Fab
• The constant-region gene determines the class or isotype of the immunoglobulin
• Each immunoglobulin unit has two fragment antigen binding sites (Fab): 
• The contact area of an antigen is the epitope
• The contact region of an antibody is the paratope

• The forces of interaction are electrostatic, hydrogen-bonding, hydrophilic bonding and Van der Waals bonds (non-covalent)

• There is a third fragment crystallisable (Fc) site which activates complement and binds mast cells and PMLs but cannot bind antigen. 

• Determines transplacental transfer
• B cells possess Fc receptors
• Antibody diversity is essential for an effective humoral response
• Germline encoded diversity (contributes the least to diversity)
• Different combinations of the V, D, and J segments in heavy chains
• Different sites for the D-J and V-DJ binding

• Nucleotide insertion at random (somatic mutation) into the DNA at segment joining points

• Junctional diversification: random loss and gain of nucleotides at the joining of V, D and J gene segments

• IgM:
• Primary immune response
• 6% of total Ig

• B-cell produces IgM then switches to produce antibody with the same specificity but of a different isotype

• Activates the classical complement pathway: most effective at fixing complement
• Third longest half-life
• IgG:
• Most common serum Ig (80% of circulating Ig in healthy persons)

• Predominates during secondary antibody response: specific IgG indicates a mature antibody response to repeated contact with an antigen

• Crosses the placenta
• Two antigen binding sites
• 4 subclasses: IgG1-4
• Activates the classical complement pathway
• Longest half-life
• IgA: 
• 10-15% of total serum Ig

• IgD:
• <1% of serum Ig
• B cell surface Ig
• IgE:
• 0.001% of serum Ig
• High affinity for mast cells

Major histocompatibility complex

• Genes on the short arm of chromosome 6 encoding surface glycoproteins important in ‘self’ recognition

• Note: T-cells cannot recognise free-antigen and only respond when it is presented with MHC

• Class I molecules (encoded by HLA A, B and C genes)
• A single transmembrane polypeptide with beta-2-microglobulin
• Present on all cells except erythrocytes (ie. all nucleated cells)

• Endogenous/intracellular antigens are combined with Class I MHC heavy chains (in the ER) and form a stable complex which is transported to the cell surface and presented to cytotoxic (CD8) T cells to indicate that the cell should be destroyed

• Class II molecules (encoded by HLA D gene)
• Comprise two polypeptide chains
• Found on macrophages and other APCs
• B lymphocytes
• Mononuclear phagocytes
• Dendritic cells
• Langerhan’s cells (epidermal dendritic cells)

• Also on endothelial and epithelial cells after interferon gamma or TNF-alpha stimulation (these become non-professional APCs)

• Exogenous/extracellular antigen is ingested via phagocytosis or endocytosis and combined with vesicles containing Class II MHC.

• Antigen proteins are degraded and the class II-peptide complex is presented to T-helper (CD4) cells on the cell surface.

• T-helper cells bind to the complex and stimulate IL-1 release from the APC which promotes T cell proliferation and differentiation. If the T cell is binding a complex on a B cell it promotes B cell proliferation and produce IL-4 and IL-5 which induce differentiation of B cells to plasma (producing IgM) and memory cells. Plasma cell maturation is driven by cytokines

• Class III molecules are a group of heterogenous structures including TNFs and complement components

• Presentation by the MHC molecule to the TCR requires binding of the peptide to the groove in the MHC molecule which requires some specificity but not as much as needed for the TCR which in turn is much less specific than antigen-antibody binding

TCRs

• T-cell receptors have a greater role in determining the specificity of the response to an antigen than the MHC genes

• Transmembrane dimers consisting of Ig-like domains: beta chain locus is analagous to the heavy chains and has V, D, J, and C segments; the alpha chain is analagous to the light chains

Cytokines

• Low molecular weight proteins secreted to act as intercellular messengers
• Mediate:
• Cell growth
• Inflammation
• Immunity
• Differentiation
• Repair
• Tumour necrosis factors are produced by macrophages and T cells
• Induce acute phase proteins
• Activate polymorphs and monocytes
• Induce interferon-gamma, IL1, IL6 and GM-CSF.
• Interleukins:
• Cytokines produced by T cells

• Some are pro-inflammatory, others suppress inflammatory cytokines (see below, IL-4, IL-10, IL-13)

• Function as growth and differentiation factors

• Interferons: produced mainly by lymphocytes (NK cells and T cells, B cells), macrophages, fibroblasts, endothelial cells

• Interferon-gamma is produced solely by T cells and NK cells
• Macrophage activator
• Inhibits Th2 clone proliferation
• Increases the function of APCs by enhancing MHC class I expression
• Interferon-alpha is produced by virally-infected leukocytes
• Anti-angiogenic  

• Interferon-beta is produced by virally-infected macrophages, fibroblasts, epithelial cells and others

• Activates NK cells 
• Encourages production of antiviral proteins 
• Stimulates reduced mRNA translation and viral/host mRNA in neighbouring cells 

• Interferons have anti-tumour activity and non-specific antiviral properties including increasing expression of class I HLA on cell surfaces and activation of NK and T-cells

Complement system

• Three main functions
• Opsonisation (C3b)
• Cytolysis of pathogenic organisms (C5-C9)
• Production of inflammatory mediators
• Chemotaxis (C3a and C5a)
• Antibody fixation
• Complement proteins are synthesised by hepatocytes and macrophages (extra-hepatic)
• There are 9 plasma proteins
• Three pathways of activations all sharing common terminal events: membrane attack complex
• Classical: activated by immune complexes. IgM and IgG can activate

• Alternative: activated by bacterial cell wall constituents eg LPS factor B, binding to C3b

• Lectin: activated by binding of serum lectin to mannose-containing proteins on bacteria or viruses

• Hydrolysis of C3 by C3 convertase is a major amplification step in all three pathways
• Membrane attack complex (MAC): can lyse a broad range of microorganisms
• Especially active against enveloped viruses and gram negative bacteria

• Gram positive bacteria are resistant to complement-mediated lysis due to thick peptidoglycan wall

• Forms pores within the lipid bilayer allowing ions and water to pass resulting in osmotic lysis

• Opsonisation is a more important function

• C3b, C5a and C4b are recognised by receptors on phagocytes to enhance phagocytosis of a coated pathogen

• C3a, C4a and C5a are anaphylatoxins: smooth muscle contraction and increased vascular permeability

• C3a and C5a mediate mast cell degranulation with histamine release

• Regulatory/inhibitory mechanisms prevent inappropriate activity: factor H and factor I, C1 inhibitor and sialic acid.

• C3 has the highest concentration in serum

Phagocytes

• Main phagocytes are macrophages and neutrophils

• Innate immune system phagocytes use membrane receptors to recognise patterns on pathogens including

• Fc component on immunoglobulin
• Complement components

• Note: binding of these receptors to these ligands increases the effectiveness of phagocytosis: opsonisation

• Phagocytosis of apoptotic cells and debris is important in the resolution of inflammation

• After internalising microbes, phagocytes release cytokines which amplify the adaptive immune system: IL1, IL6, TNF.

• Selectins and integrins facilitate phagocyte entry into tissues and from there they transmigrate via CD31 (platelet endothelial cell adhesion molecule or PECAM)

Macrophages

• Tissue form of monocytes: monocytes circulate in blood but macrophages do not
• Part of the innate immune system
• Functions
• Phagocytosis of foreign bodies, microbes or debris
• Pattern recognition receptors detect microbial structures

• Receptors for immunoglobulin and complement (which enhance phagocytosis via opsonisation)

• Production of cytokines: IL1, IL6, IL12 and TNF
• Antigen presentation: MHC class II is presented when the cells are active

Neutrophils

• Granulocytes

• Part of the polymorphonuclear leucocyte family (with basophils and eosinophils): multilobed nuclei

• Phagocytic
• Enter tissues in response to inflammation and IL8 in particular 

CD4 cells

• T helper cells
• Normally 600-1500 cells per mm3
• Contain gp120 envelope protein receptors
• Increased during treatment with HAART

• T-helper 1 cells: secrete IFN gamma and IL2 leading to B cell, NK and macrophage activation

• T-helper 2 cells: secrete IL3, 4, 5, 6 leading to mast cell and eosinophil activation

CD8 (cytotoxic) T cells

• Recognise antigen presented with Class I MHC

• Cytotoxic T cells are activated with the help of T-helper cells: Th1 cells release IL2 in response to binding antigen-MHC complexes and these cytokines stimulate proliferation and differentiation of cytotoxic T cell precursors

• These cells contain granules with perforin, TNF and serine protease which can be released between the T-cell and target cell

• Perforin inserts itself into the target cell membrane forming a membrane pore (similar to MAC) 

• Cytotoxic agents (eg. granzymes) then enter the cell and kill it by apoptosis via DNA fragmentation (different to MAC-mediated cell lysis)

• T-cell then disengages to attack another target

Apoptosis

• Programmed cell death
• Allows removal of specific cells without damage to surrounding structures

• Membrane integrity is maintained and lysosomes remain intact so that the cell components do not cause collateral damage

• No inflammatory response
• Chromatin condenses and DNA fragments
• The plasma membrane undergoes blebbing (zeiosis)

T and B cell interactions

• Immune responses are T cell dependent

• B cells generally need T cell assistance to achieve full activation including heavy chain class switching

• Class-switching: B-cells in primary follicles express IgM and IgD
• Following class-switching they can express IgG, IgA or IgE.
• CD40 and CD40L are needed as co-stimulants with T cells
• Deficiency of these can lead to impaired class switching
• “Thymus-independent” antigens can activate B cells without T cell assistance
• Eg. pneumococcal polysaccharide, LPS
• Primarily an IgM response
• Spleen is important in the immune response to these antigens

NK cells

• Distinct class of lymphocyte derived from lymphoid progenitors
• Important role in the early response to viral infections
• Recognised MHC Class I receptors on cells
• Ligand bindings inhibits cytolysis

• If the number or composition of MCH Class I on the cell surface is abnormal, eg. due to viral infection, then the NK cell is not inhibited and cell killing can occur

• Note: have cytotoxic activity without specific antigen receptors

• Recognise Fc fragments of antibody coating budding viruses (antibody-dependent cell-mediated cytotoxicity -ADCC)

• Release perforins and granzymes to induce apoptotic cell death
• Secrete IL-1 and GM-CSF
• Activity enhanced by IFM-gamma, IFN-beta and IL-12.

Dendritic cells

• Derived from stem cell precursors in the bone marrow and proliferate in response to cytokines

• Called Langerhans’ cells in the skin, conjunctiva, peripheral/limbal corneal epithelium

• Migrate to lymph nodes and spleen in response to stimulation and interact with T cells or may act as antigen traps which they present to B cells

• Short-lived cells

Secondary lymphoid organs (peripheral)

• Includes:
• Spleen
• Lymph nodes
• MALT
• Cutaneous immune system
• Liver
• T and B cells are segregated in these tissues
• B cells aggregate to form follicles
• Primary follicles are non-activated B-cells, expressing IgM and IgD
• Spleen
• Traps blood borne antigens and mounts immune response
• Red pulp: abundance of erythrocytes
• White pulp: numerous T cells
• Marginal zone: B-cell follicles and interdigitating dendritic cells, macrophages
• Lymph nodes: first lymphoid structure to encounter antigens
• Cortex: lymphocytes (mostly B cells), macrophages, dendritic cells
• Paracortex: T cells and interdigitating dendritic cells
• Medulla: lymphocytes and antibody secreting plasma cells

Type I hypersensitivity (humoral)

• “Allergic” reaction immediately following contact with an antigen (which would not normally cause such a response)

• Mast cell degranulation: IgE or C3a and C5a cross-linking on mast cell surfaces

• Histamine, 5HT, heparin, eosinophil chemotactics and platelet activating factors are released

• NB: histamine exerts negative feedback to inhibit mast cells

• Examples: anaphylaxis, allergic/atopic conjunctivitis

Type II hypersensitivity (humoral)

• Antibody-mediated reaction: IgG/IgM activating the complement pathway

• “Frustrated” phagocytes release their lysosomal contents causing tissue damage

• Eg. Rhesus incompatibility, ABO incompatibility, ocular cicatricial pemphigoid (antibody production against basement membranes) and hyperacute graft rejection reactions

Type III hypersensitivity (humoral)

• Immune complex deposition in tissues stimulating complement activation

• Persistent infections eg. viral hepatitis; and autoimmune diseases eg. rheumatoid arthritis

• Arthus reaction: intradermal injection of antigen into individual with high circulating antibody levels due to previous immunisation, leads to acute inflammatory reaction when antigen-antibody complex is deposited, peaking within 4-10 hours

• Wessely ring: ring infiltrates in the corneal stroma associated with ulcers etc. “immune ring”. Causes

• Neomycin
• Corneal foreign bodies
• Contact lens reaction
• Examples: scleritis

Type IV hypersensitivity (cellular)

• Cell-mediated (Th1 cells): recruitment of pre-sensitized immune cells. Not antibody mediated.

• Take more than 12 hours to develop
• Jones-More type: basophils stimulation
• Contact dermatitis: mononuclear cells
• Graft-versus-host disease following transplantation

• Corneal graft rejection. Risk factors

• Young age
• Repeat grafts
• Large grafts
• Preoperative stromal vascularisation
• Loose/broken sutures
• Active inflammation
• Tuberculin type: Heaf test

• Granulomatous reaction: eg. persistent antigen in macrophages such as tuberculosis or sarcoidosis

• Stevens-Johnson syndrome. Common precipitants
• Sulphonamides, penicillin, phenytoin
• Viruses: HSV, HIV
• Malignancy 
• Marginal keratitis

• Phlyctenulosis conjunctivitis: associated with staphylococcus aureus in developed countries and mycobacterium tuberculosis in developing countries

• Limbal small round nodules with vessel engorgement

Type V hypersensitivity

• Considered a subtype of type II
• Antibody reaction with cell surface receptors stimulates or suppresses cell function
• Examples
• Myasthenia gravis
• Graves’ disease