B cells and antibodies Flashcards
Antibody structure
Formed from pairs of heavy and light chains held together by covalent (disulphide bridges) & non-covalent bonds.
- DiS bridge between CL and CH1
The 5 main classes of antibody (A, D, E, G, M) can be distinguished by their heavy chains denoted α, δ, ε, γ, μ
Chains are constructed from series of structurally related immunoglobulin fold domains- may be constant or variable.
Highly variable antigen binding (variable) domains give molecule specificity
Constant domains allow antibody to interact with other immune system effector cells & molecules e.g macrophages, complement.
Flexibility to interact with antigen arises at:
- Hinge region between CH1 and CH2
- V-C junction.
Light chains
One constant (CL), one variable (VL) domain. Present as one of two types: λ and κ
Heavy chains
One variable domain (VH)
Three (IgG, IgA, IgD) or four (IgM, IgE) heavy domains (CH1, CH2 etc)
Fab fragment
Contains 1 antigen binding site.
VL, CL, VH and CH1.
Antigen binding site
Complementary determining regions?
Formed by interaction between heavy & light chain variable domains
Variation is concentrates in 3 complementary determining regions:
- CDR3: most variable, variation due to sequence variation in heavy chain: in diversity gene segments (27), junctional gene segments (6), and in junctions between rearranged V-D-J segments
- CDR1 and CDR2: variation due to sequence variation in heavy chain variable gene segments (65)
Processes generating antibody diversity:
1) Different heavy & light chain combinations
2) Selection of different heavy V, D and J segments
3) Junctional diversity due to variable addition & loss of nucleotides at VDJ junctions
4) Somatic hypermutation. Point mutations are introduced into heavy & light chain variable regions (mutation rate is ~1/1000 base pairs per cell division). Antibodies with increased affinity are selected for by affinity maturation.
Heavy chain rearrangement
Intervening genomic DNA is lost.
1st: D -> J joining
2nd: V -> DJ joining
Can occur on both chromosomes but if a functional heavy chain is generated then rearrangement on other chromosome is prevented: allelic exclusion.
A single B cell can only express 1 heavy chain.
Controlled by recombinases that recognise converted heptamer & nonomer sequences located adjacent to V, D and J segments.
Variation in CDR3
Due mainly to
Sequence variation in heavy chain…..
- diversity gene segments (D): 27
- junctional gene segments (J): 6)
Junctional diversity due to variable addition & loss of nucleotides at rearranged heavy chain VDJ junctions:
- addition by TdT (N-nucleotide addition)
- addition due to recombination mechanism (P-nucleotide addition)
- deletion of nucleotides
Variation in CDR1 and 2
Due mainly to sequence variation in heavy chain variable gene segments (V): ~65
Light chain rearrangement
Light chains have no diversity segments
κ chain rearranges first, then if unproductive the λ chain.
Controlled by recombinases that recognise converted heptamer & nonomer sequences located adjacent to V, D and J segments.
Affinity maturation
In a primary response, B cells with receptors for antigen internalise, process & present it to T helper cells.
T helper cells activate the B cells to produce antibody, generally of relatively low affinity. Antibody can bind & clear the antigen.
Only B cells with receptors of the highest affinity for antigen will continue to capture & present antigen to T helper cells- these cells with receive T help, & be stimulated to proliferate & predominate
Isotype switching
Mature B cells with rearranged VDJ region segments are initially expressed with μ and γ constant regions- producing IgM and IgD immunoglobulin isotypes.
IgM and IgD are co-expressed, generated by alternative splicing. Each B cell can also change the constant region isotype by isotype/ class switching.
Generation of BCR and soluble antibody
By alternative splicing: Choice of different poly adenylation sites (before or after TM region) generates soluble or membrane bound immunoglobulin with identical specificities.
IgM
Mean serum level: 1.5 mg/ml
Half life in serum: 10 days
No Fc receptor but activates complement v efficiently, & is recognised by the C3b receptor
Present in secretions: mucus, etc.
IgG
Transfer?
Subclasses: IgG1, igG2, IgG3, IgG4
Mean serum level (G1-4): 9-3-1-0.5 mg/ml
Half life in serum (G1-4): 21-20-7-21 days
Placental transfer: 1 > 3 > 2, not 4
Activation of complement: 3 > 1 > 2, not 4
Binding to (Fc) macrophage: 1 and 3
Present in milk.
Maternal IgG is transported across the placenta directly into the fetal bloodstream, provides high level protection vs pathogens common in the environment at birth.
(NB contrast to humans: rodents & ruminants acquire IgG postnatally from colostrum via an IgG specific receptor in the gut)/
IgA
Transport? Functions?
Subclasses: IgA1, IgA2 (apparently no functional difference between)
Mean serum level: 3 mg/ml
Half life in serum: 6 days.
Weakly activates complement. Binds to (Fc) macrophage
Present in secretions:
Most imp Ig in protecting mucosal surfaces.
- Receptor for polymeric Ig recognises the J chain region of IgA & transports it across the epithelial cell to the mucosal surface.
In milk provides imp defence for newborn infants. Remains in the gut, prevents attachment of bacteria & toxins to gut epithelia.
IgD
Mean serum level: 0.03 mg/ml
Half life in serum: 3 days
IgE
Mean serum level: 5X10{-5] mg/ml
Half life in serum: 2 days
Binds to (Fc) macrophage Binds v efficiently to mast cells.
Fc receptors
= receptors specific for the Fc region of different antibody isotypes.
Determine most of the effector functions of antibodies.
- deliver antibody to different anatomical sites
- link antigen to molecules/ cells that effect its destruction.
High affinity Fc receptors (e.g FcγRI, Kd~10{-9} ) bind monovalent antigen/antibody complexes
Low affinity Fc receptors (e.g FcγRII and III, Kd~10[-6]) only bind multivalent antigen/antibody complexes.
FcγRI
High affinity for IgG
Distribution: macrophages, activated PMN
Function: phagocytosis.
FcγRII
Low affinity for IgG
Subtypes a and b
- a: distribution macrophages & neutrophils, function = phagocytosis
- b: distribution B lymphocytes, function = antibody regulation.
FCγRIII
Low affinity for IgG
Subtypes a and b
- a: distribution NK cells, macrophages. Function = ADCC
- b (GPI linked): distribution neutrophils, function phagocytosis.
Also present on mast cells & basophils- crosslinking signals degranulation & release of inflammatory mediators.
Opsonisation
Role of FcγR
Role of C3b receptors
= coating of the surface of an antigen to enhance phagocytosis.
Receptors for Ig (FcγR) and complement (C3b) can bind antibody & complement-opsonised antigen.
FcγR are present on macrophages & neutrophils, recognise Fc region of IgG esp IgG1. Receptor engagement -> phagocyte activation, enhanced antigen uptake & degradation.
IgM has no Fc receptor, but is recognised by C3b receptor.
Binding of antigen/antibody complexes by C3b receptors present on RBCs delivers complexes to liver & spleen for removal by macrophages.
ADCC
Ligation of FCγRIII (= low affinity Fc receptor. Cannot be triggered by free Ig, requires antigen/antibody complexes to provide multiple Fc regions for recognition) on NK cells triggers release of cytoplasmic granules containing lytic enzymes.
Eosinophils mediate unique type of ADCC vs helminths.
- IgE binds to surface of worms, is recognised by FcεRI.
- release granules containing proteins that are toxic to helminths.
