L6-7 - Antibody Diversity

8/7/2019 L6-7 - Antibody Diversity

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Immunology

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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

Documents

L6-7 - Antibody Diversity