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

IgA is the main Ig in the conjunctiva and other external secretions (important for mucosal immunity)

2 subclasses: IgA1 and IgA2
Activates the alternative complement pathway
Second longest half-life

IgD:

<1% of serum Ig
B cell surface Ig

IgE:

0.001% of serum Ig
High affinity for mast cells

IgE is important in the response to helminth parasites

Shortest half-life

Functions

Opsonisation
Neutralization of toxins
Trigger the complement cascade

## 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

Interleukins
IL-1	Macrophages, B cells, monocytes, dendritic cells	T cell proliferation
IL-2	CD4 (T helper) cells	Growth and differentiation of the T cell response (primary chemical stimulus for T cell proliferation)
IL-3	T helpers, mast cells, NK cells	Differentiation of myeloid progenitors
IL-4	T-helper cells	Proliferation and differentiation. Suppression of IL-1 and other inflammatory cytokines
IL-5	T-helpers, mast cells	Eosinophil activation
IL-6	Macrophages, T-helpers, B cells	B-cell differentiation to plasma cells, antibody secretion, acute phase protein synthesis
IL-7	Bone marrow stromal cells	Differentiation and proliferation of lymphoid progenitors
IL-8	Macrophages, lymphocytes, epithelial and endothelial cells	Neutrophil chemotaxis
IL-10		Suppress inflammatory cytokines
IL-12		Stimulating Th1 response
IL-13		Suppress inflammatory cytokines

## 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