l6-7 - antibody diversity
TRANSCRIPT
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Learning Objectives
Describe the pathway of B-cell development with respect to immunoglobulinRelate antibody diversity to antibody structureUnderstand how gene re-arrangement leads to antibody diversityKnow what mechanisms there are to increase diversity further and whyUnderstand how this knowledge can be used commercially/therapeutically (brief)
Introduction
All of the diversity in terms of antigen binding occurs in the variable heavy and variable light regions which
together form the antigen binding site
It is not just antibodies that contain the immunoglobulin fold, other molecules of the immune system thatbelong to the immunoglobulin superfamily also have it
MHCT-cell receptors
As there is conserved structure between these molecules, many of the processes that provide
immunoglobulin diversity also apply to other molecules in the immunoglobulin superfamily
B-cell Development
Stem cellp
mature B-cell that expresses different subtypes of antibodiesMany steps along the way
There are two distinct parts of B-cell development:
1. The first part takes place in the bone marrow and is antigen independent
2. The second occurs in the peripheral lymphoid organs and involves interaction with antigens
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History
The one gene p one mRNA p one protein hypothesis could not explain antibody diversity due to the huge
number of different antibodies produced.
The heavy and light chains have a variable N-terminus and a constantC-terminus
Isotopes were found with the same antigenic specificity but differentC-terminal heavy chains
Germ-line theory All sequences are encoded by the genome
Somatic-variation theory Small number of genes p large number of products
By mutation and/or recombination
Dreyer & Bennett (1965)
Variable (V) and constant (C) genes are encoded on separate genesGene products come together later on to form a single polypeptideRejects the one gene p one protein hypothesis1000s ofVgenes, a single CgeneNo direct evidence, could not be proven until molecular biology advanced
Tonegawa & Hozumi (1976-1987)
Used restriction endonucleases to prove gene re-arrangement was occurringUsed mouse embryonic and adult mouse myeloma cells
oImmortal cells that produce antibodiesProbed with radioactive mRNA for a specific location on the geneRan blots to see if probe changed location to another fragment
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Multi-gene Organisation:
Cloning and sequencing showed that this is a very complex process
There are two loci on the genome for light chains
O (kappa)P (lambda)
There is one locus for heavy chains
All are multi-genes
The light chains have multiple variable genes, as do the heavy chains.
The heavy chains have diversity regions.
There are also a number of pseudogenes (), which will lead to non-functional products
LightChain
In mice, most of the diversity comes from the O light chain
In humans diversity is equal between O&P
Within each light chain the majority of the diversity originates from the VH
HeavyChain
Much more diversity
In mice and humans there are manyVH, fewer DHand even less JH
A range ofC-gene isotypes
P, O and the heavy chain genes are all on different chromosomes
This suggests that in evolutionary history there was a common gene that was duplicated
Generation of Diversity:
Recombination is the first step towards diversity
e.g. in a O light chain, a random VO joins with a random JO
~85VOv ~5 JO = 425 possibilities
Once the sequences join, all information between them is permanently lost
From that point on, the B-cell in question can only make that combination
This does not occur through splicing
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LightChain
Secretion signal is cleaved
Diversity lies in the VO/P region
Light chain = VJC(O/P)
One re-arrangement followed by splicing
L = leader sequence (secretion signal) found before every variable domain
V= Variable
J = Joining
C= Constant
D = Diversity (heavy chain only)
CDR3, the most diverse of the three CDRs is found at the junction between Vand J sequences
It is the mostC-terminal / nearest the 3 endCDR1/2 are found in the Vregion and have less variation(1/3 along and 2/3 along respectively)
The variation here is only from choosing differentVsequences
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HeavyChain
Two re-arrangements (recombination events)
1. Recombination between D & J
Heavy chain has extra diversity from the D sequences (more diversity in the heavy chain)2. Recombination between V& DJ
Again, all information between the combined sequences is lost
Differential splicing occurs to produce the different classes of antibodies
IgM/ IgD etc.Membrane-bound or free
They all have the same specificity
CDR3 in heavy chains is found in the VDJ border region
Incredibly diverse CDRregion due to the large numbers of possible V, D & J sequences available
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Recombination Signal Sequences (RSS):
Sequencing revealed conserved sequences flanking the V, D ad J regions
Two types
Two-turn (23bp): Heptamer 23bp NonamerOne-turn (12bp): Nonamer 12bp Heptamer
Each type is surrounded by two conserved sequences
Palindromic heptamer (7nt) on one sideAT-rich nonamer (9nt) on the other
This provides directionality which is key for recombination or else there will be bad products
The RSSs are found at the following locations only:
3V5 J3/5 D
Recombination will only occur between a one-turn and a two-turn
The RSSs are arranged differently in the P, O and heavy to prevent formation of strange products
Evolution has caused the right sequences to face the right direction in the correct location
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V-D-JRecombination:
RAG (Recombination Activating Genes)
RAG-1/-2 are involved
Only occurs in lymphoid cells
Two mechanisms
1. Deletional joining
Coding regions have the same orientationExcision product is circular with RSS and intervening DNA
2. Inversional joining
Coding sequences are in opposite directionsDNA is not lost, it is inverted
Depends on which way the RSSs are pointing
1. Enzymes align the two RSSs forming a
synapse.
2. Enzymes cleave one strand only.
Cleavage is specific
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3. Hairpin forms and another break occurs in
the DNA, making it a ds break. This leaves a
free 3-OHgroup and a phosphate at the end
of each strand, which will form a
phosphodiester bond and a loop.
4. Hairpins are critical for generating
extra diversity. The hairpin is cleaved
at a random location, then gaps are
filled with extra nucleotides (additions
of P-nucleotides). Different cleavage
positions generate different
overhangs.o diversity.
5. Ligation occurs using ds break
repair enzymes (DSBR)
In heavy chains, non-coded
nucleotides are added by terminal
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transferase
An enzyme that adds nucleotides to a 3-OHThis increases diversity
In light chains the sequences are simply joined
Junctional Flexibility:
The dsDNA break is precise at the RSS/coding junction, so it does not generate diversity
The final joins on the other hand are imprecise
The following processes generate diversity:
Variation in hairpin cutting to generate P-nucleotidesTrimming coding sequencesVariation in N-nucleotide additionFlexibility in coding sequence joiningJunctional diversity
Non-productive re-arrangement of both alleles will result in B-cells being killed (apoptosis)
A non-productive re-arrangement will include a premature stop codon1/3 V-J /V-D-J are productive (due to averages, not reading frames)
1/9 pre-B cells leave the bone marrow to mature into immunocompetent B-cells
V- & N-nucleotide addition:
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Allelic Exclusion:
B-cells are diploid, two different copies ofO etc.
Maternal orpaternal genes are re-arranged
The genes are chosen randomly
Could be paternalO and maternalHfor example
To prevent a B-cell from having more than one antibody type,
allelic exclusion takes place, the cell prevent the other alleles
from being expressed.
A productive rearrangement of the heavy chain will result in a
signal that inhibits rearrangement of the other allele. It will also
stimulate rearrangement of the O allele.
Is there was a non-productive rearrangement due to a frameshift, the cell will stimulate rearrangement of the
other allele. If this works, the next step will be stimulated. If it fails again the cell will die, as 2/2 alleles have
been non-productive.
This continues as shown below. Heavy chain allele 1 p 2pO1 p k2pP1 pP2p death
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O is preferred overP
Even with all of these fall-backs, only 11% of B-cells can fully mature
All values are ~ Heavy Light (O) Light (P)
V 50 40 30
D 25 0 0J 5 5 5
Possible combinations 6250 200 120
Total heavy / light chain associations 1,000,000+
Similar number for mice
Recombination results in ~106different specificities
Somatic mutation results in 109 variants (another 1000v increase in diversity)
Sources of variation in the CDRs:
CDRs need to be diverse as they are in contact with the antigen
CDR1 Vsequence, somatic hypermutation
CDR2 Vsequence, somatic hypermutation
CDR3 Vsequence, somatic hypermutation, junctional flexibility, P-/N-nucleotide addition
Somatic mutation & hypermutation
The average affinity of antibodies during the humoral immune response increases
TheirKddecreases, meaning the binding is stronger most of the complex is in complex form, not the free
form.
This occurs during affinity maturation
Studied by immunising a rabbit with a hapten-protein complex that is recognised by its immune system. The
researchers then followed the rabbit anti-hapten antibodies over a few weeks. The hapten was DNP
(dinitrophenol). They noticed that sequences were changing in the antibodies and that they had higher
affinities for the hapten.
The rate of mutation in this gene region was 10-3/bp/division
The increased rate of mutation was found in germinal centres such as lymph nodes
This is a million times higher than normal
Effectively 1 mutation per 2 cell divisions
The mutations are concentrated in the variable domain, mostly in the CDRs
Antigen stimulated B-cells migrate to germinal centres (collections of lymphocytes)
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Activated B-cells are known as centroblastsThis is where the mutations take place (centroblasts pcentrocytes)
There will be some B-cells with high affinity antibodies, some with low affinity
Follicular dendritic cells present antigens to the centrocytes
Antibodies on centrocytes will bind to the presented antigens
This process selects for high affinity binders
The different centrocytes will compete to bind with the presented antigensOnly those with high affinity for the antigen will be able to bindWhen they bind they get selected, and getT-cell helpSelected centrocytesp plasmablastsp plasma cells or memory cellsThe plasma cells then go on to produce antibodies
The cells that do not get help will die
Class Switching:
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After receiving help, antibody class switching can occur
A switch recombinase facilitates the switch at a switch region upstream of the CH
Mechanism is unknown, cytokines are involved
Interleukin-4 will stimulate: C (IgM)p CK1 (IgG) or CI (IgE)Different cytokine will stimulate formation of different Igs, depending on requirements
A circular excision product is generated
AID (activation-induced cytosine deaminase) is a key mediator
Also involved in somatic hypermutation (SHM)If this gene is knocked out then there will be no SHMor class switchingThe enzyme is RNA and maybe DNA editingDeaminates Cp U in RNA, leading to repairMechanism is not clear
Product is IgE
Antibodies can be membrane-bound or secreted
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This is determined byC-terminal sequences
C-terminus depends on class of antibody
Occurs by alternative splicing
S-segment + polyA signal
orNo S-segment, M1 + M2 + polyA
The C-terminus of a secreted form is very hydrophilic
No TMregions
The C-terminus of a membrane form contains some hydrophilic portions with a large, membrane-spanning
hydrophobic portion.
Mature B-cells will only express membrane Ig, wheras differentiated plasma cells express secreted Ig.
Different polyA sites will result in differential splicing, and ultimately different Ig locations
Synthesis, Assembly and Secretion:
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Diversity and variability leads to expression problems
Plasma cells can make 1000 antibodies / sec (fast)
Synthesised on the rough ER
Leader sequence is cleaved once the chains enter the ER
Dummy light chains are removed
IgMassembles as HL, then dimerises
IgG assembles as HH, the Ls are added
Enzymes catalyse the formation of disulphide bridges, glycosylation etc.
Chaperones facilitate folding
BiP (immunoglobulin binding protein) binds to unfolded Igs and aids folding, if it cannot fold they areubiquitinated and degraded by proteasomes
Antibody with TMsegment will sit in the membrane of a secretory vesicle, and will later fuse with membrane.
Secreted Igs are released by exocytosis.
Igs are only glycosylated on an Asp in the CH2 domain (in the Golgi)
Important and complex but we dont need to know details
Ig Gene Transcription:
Antibody promoters are very strong, and there are cancers associated with them, where cell cycle / gene
regulation proteins are moved into the antibody locus, and are highly expressed.
Also have enhancers and silencers to regulate transcription
Various transcription factor binding sites here and there
oct-1/-2 are conserved octamer sequences that are specific to B-cellsThis allows B-cell only expression
Differs between O /P /H
Enhancers are short acting sequences; they need to be brought close to the promoter in order to have a
function. The silencers will act at a longer distance
The RNApol II promoters are upstream of each V-gene
Ig expression is low until after re-arrangement, when the enchancers are brough closer to the promoter,
resulting in a 10,000v increase in expression
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T-cell Gene Re-arrangement and Diversity:
A similar process to that of Igs
rag-1/-2 recombinase mediated
If rag molecules are knocked out, the mmune system will be compromised as it hasa fundamental rolein creating diversity in Igs andT-cell receptors
Ig gene expression is switched off in T-cells
Numbers / letters are different, but the process is very similar
Production of a variable T-cell receptor
Variability is lower than that of Igs
No need to learn in detail
Application of Ig Genes:
Re-arranged genes can be cloned, then added to vectors
The vectors can be transfected into myeloma cells (immortal, cancerous B-cells)
These cells will then express the antibody of choice
This allows production of monoclonal and chimeric antibodies (e.g. mouse VL/Hhuman CL/H)
Antibody libraries can be constructed
Summary
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Pre-B cells have germ line DNA but mature B-cells have lost DNA and can only make one specificantibody
Recombination allows diverse repertoire in antibody responseRecombination occurs in class switchingSplicing accounts for membrane or secreted antibodySomatic mutation results in 1000-fold more diversity and allows the affinity of antibody to be fine-
tuned in germinal centres
Antibody genes now easily manipulated for biotechnology and drug research