Immunology Immunoglobulins - Overview
Transcript of 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