immunology immunoglobulins - overview

“Completely central to modern

immunology is antibody and

immunoglobulin”

Sir MacFarlane Burnet 1972

1870 - 1961

“Rabbits could be immunized against RBCs

from another species resulting in haemolysis”

Jules Bordet 1898

Nobel prizes for Medicine

Emil von Behring 1901 Serum therapy

Jules Bordet 1919 Antibody / complement

MacFarlane Burnet / Medawar 1960 Clonal selection theory, transplantation tolerance

Edelman / Porter 1972 Ig structure

Benacerraf / Dausset / Snell 1980 HLA

Jerne / Kohler / Milstein 1984 Monoclonal antibodies

Tonegawa 1987 Antibody diversity

Murray / Donnall-Thomas 1990 BMT

Doherty / Zinkernagal 1996 HLA

Steinman 2011 DCs - HLA

Synopsis• Basic structure

– Ig domains/superfamily

– Ig classes

• Structural diversity – VDJ recombination

– somatic hypermutation

• Structure-function correlations – Ig classes and normal function

– disease

• Uses

IMMUNOGLOBULIN (Ig)SUPERFAMILY

Ig domain/fold70-110 aa

SANDWICH OFTWO -SHEETS

Bork P, 1994

self- stabilising/ folding unit via disulphide bridges between conserved cysteine residues

forms a barrel-like shape

Immunoglobulin superfamily evolution

• expressed in jawed vertebrates from

500 million years ago

• one third of cell surface receptors

CELL-CELL INTERACTIONself & non-self

CELL-ADHESION - ORGANOGENESIS

IMMUNE RECOGNITION

neural cell adhesion molecule

CD56 – homophilic binding

Molecule function/category Examples

Antigen receptors ANTIBODIES/ IMMUNOGLOBULINS; TCR

Antigen presenting molecules MHC I; MHC II; 2 microgloulin

Co-receptors CD4; CD8; CD19

Antigen receptor accessory molecules

CD3-γ, -δ and -ε chains; CD79a; CD79b

Co-stimulatory or inhibitory molecules

CD28; CD80; CD86

Receptors on Natural killer cells Killer-cell immunoglobulin-like receptors (KIR)

Adhesion molecules Neural Cell Adhesion Molecule (NCAM/ CD56); CD2; SIGLEC family (CD22, CD83); CTX family (CTX, JAMs, BT-IgSF, CAR, VSIG, ESAM); Intercellular adhesion molecules (ICAMs); vascular cell adhesion molecules (VCAM-1)

Cytokine and growth factor receptors

Others

IL-1R; PDGFR; IL-6R; M-CSF-R; c-kit

thymocyte differentiation antigen-1 (Thy-1); sodium channel subunit beta-1 precursor

•2 identical heavy chains (isotypes: m g

Subclasses have different glycosylation & disulphide bridges due to allelic variation mainly on the heavy chain (allotypes)

•2 identical light chains ( l

Fab – combines variable regions of heavy and light chains – Ag binding

Fc – crystalizable / constantFragment – binds C’ and immune cells

• PROTOTYPE, default Ig• first Ig produced in response to antigen challenge• also exists as a membrane bound receptor on naïve B cells• in serum forms a pentamer• powerful fixer of complement

Why is IgM first?Why switch?

AFFINITY: strength of the binding between a single paratope of an antibody and a single antigenic epitopeIgM – low affinity, IgG high affinity

VALENCY: number of epitopesIgM - decavalent (10), IgG – divalent (2)

AVIDITY: total affinity of an antibody with more than one binding site

•the predominant Ig in serum•in humans there are 4 subclasses•subclasses vary in disulphide bonds and glycosylation sites•IgG is the only Ig to cross the placenta•arises from a mechanism known as class switching from IgM producing activated B-cells

Notarangelo LD et al, 2006

Hyper IgM syndrome

• high IgM, low or absent IgG and IgA

• absent specific antibody production

• risk of bacterial infections

• treat with immunoglobulin replacement (BMT for X-linked form)

Davies & Thrasher, Br J Dermatol, 2010

•serum IgA has a similar monomeric structure to IgG•in secretions it forms a dimer•its heavy chain is slightly larger at 60K•two subclasses: IgA1 and IgA2•as with IgM, polymerisation is initiated and stabilised by J chain•the predominant isotype in breast milk•important for mucosal immunity?

IgM IgG IgA IgE

Complement activation

+++ ++ - -

Binds to neutrophils - +++ _ _

Binds to mast cells/ basophils

- - - +++

Crosses the placenta - ++ - -

Present in breast milk - + ++ -

Immunoglobulin

diversity• Ig required for recognition of myriad of Ags

– 10 billion different antibodies

• number of genes limited– 30 l V genes, 300 V genes, 1,000 heavy V genes

• somatic mutation - VDJ recombination• somatic hypermutation - point mutations within

hypervariable regions

Schroeder & Cavacini, JACI, 2010

VDJ recombination

Bassing CH et al, 2002

Hypervariable regions

Complementarity determiningRegions (CDR) – antigen binding specificity

activation-induced (cytidine) deaminase

(AID)

Kavli B et al, 2007; Neuberger & Rada, JEM, 2006

Uracil

Cytosine

Uracil DNA glycosylase

B-cellclonal

selection

ANTIBODY PREVENT INFECTION BY

PROMOTING THE UPTAKE OF ENCAPSULATED

BACTERIA BY PHAGOCYTES

OPSONISATION

ANTIBODY PREVENT INFECTION BY

BLOCKING THE UPTAKE OF BACTERIAL TOXINS

AND VIRUSES BY TARGET CELLS

Mechanism of action

TOXIN

VIRUS

ANTIBODY

ACTIVATESCLASSICAL COMPLEMENT CASCADE

• opsonisation

• membrane attack complex

NephelometryGk nephele = cloudmeasures Igs, C’ components•depends on light scatter properties of Ag/Ab complexes•add antisera of component to be measured complexes turbidity•check absorbance at 840nm

AdvantagesHigh throughputRapid, automated

DisadvantagesHigh initial set up costInterference if turbid sera/lipemia/Freeze thawing

Immunoglobulin (IVIG)

• Therapeutic preparation of normal human IgG obtained from pools of plasma from 3,000 to 100,000 healthy blood donors (Cohn et al, 1946)

• Diverse repertoire of IgG molecules that possess a wide spectrum of antibacterial and antiviral specificities

• Specificity may vary depending on population from which it is drawn

Immunoglobulin therapy

• Replacement therapy – IV or SC– 1o 2o immunodeficiencies

• Passive immunisation– e.g. tetanus, rabies, hepatitis A&B, VZV, RSV

• Monoclonal antibody therapy– selective cytotoxic: e.g. B-cells – rituximab, T-cells – anti-CD3, cancers– anti-inflammatory: e.g. TNF – infliximab, IL-1 anakinra

Laboratory uses – monoclonal antibodies

• Identification/localisation of cellular protein– FACS– immunoflurescence– immunohistochemistry

• Identification of soluble proteins– immunoprecipitation– Western blot– ELISA

Summary

– Immunoglobulins are critical in biology• cell adhesion – self to self recognition• Immunity – self to non-self recognition

– Diversity is generated by enzymes which cut and paste DNA

• defects are associated with propensity to infections (immunodeficiency) and to cancer

– Immunoglobulins are used widely in laboratory and clinical medicine

• monoclonal antibodies form the basis of ELISA, flow cytometry, biologic therapies