Immunology Antibody Diversity

Learning Objectives

 Describe the pathway of B-cell development with respect to immunoglobulin

 Relate antibody diversity to antibody structure
 Understand how gene re-arrangement leads to antibody diversity
 Know what mechanisms there are to increase diversity further and why
 Understand 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 that belong to the immunoglobulin superfamily also have it
 MHC
 T-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 cell  mature B-cell that expresses different subtypes of antibodies

 Many 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  one mRNA  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 constant C-terminus
Isotopes were found with the same antigenic specificity but different C-terminal heavy chains

Germ-line theory – All sequences are encoded by the genome

Somatic-variation theory – Small number of genes  large number of products

 By mutation and/or recombination

Dreyer & Bennett (1965)

 Variable (V) and constant (C) genes are encoded on separate genes
 Gene products come together later on to form a single polypeptide
 Rejects the ‘one gene  one protein’ hypothesis
 1000’s of V genes, a single C gene
 No direct evidence, could not be proven until molecular biology advanced

Tonegawa & Hozumi (1976-1987)

 Used restriction endonucleases to prove gene re-arrangement was occurring
 Used mouse embryonic and adult mouse myeloma cells
o Immortal cells that produce antibodies
 Probed with radioactive mRNA for a specific location on the gene
 Ran 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

  (kappa)
  (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 (ψ), which will lead to non-functional products

Light Chain
In mice, most of the diversity comes from the  light chain
In humans diversity is equal between  & 
Within each light chain the majority of the diversity originates from the
VH

Heavy Chain
Much more diversity
In mice and humans there are many VH, fewer DH and even less JH
A range of C-gene isotypes

,  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  light chain, a random V joins with a random J
~85 V  ~5 J = 425 possibilities

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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
Light Chain

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Secretion signal is cleaved

Diversity lies in the V/ region
Light chain = VJC(/)
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 V and J sequences
 It is the most C-terminal / nearest the 3’ end
CDR1/2 are found in the V region and have less variation (1/3 along and 2/3 along respectively)
 The variation here is only from choosing different V sequences

Heavy Chain

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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 CDR region 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 – Nonamer
 One-turn (12bp): Nonamer – 12bp – Heptamer

Each type is surrounded by two conserved sequences

 Palindromic heptamer (7nt) on one side
 AT-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:

 –3’ V
 –5’ J
 –3’/–5’ D

Recombination will only occur between a one-turn and a two-turn

The RSSs are arranged differently in the ,  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-J Recombination:
RAG (Recombination Activating Genes)
RAG-1/-2 are involved
Only occurs in lymphoid cells

Two mechanisms

1. Deletional joining
 Coding regions have the same orientation
 Excision product is circular with RSS and intervening DNA

2. Inversional joining
 Coding sequences are in opposite directions
 DNA 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’-
OH group 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.  diversity.

5. Ligation occurs using ds break repair enzymes (DSBR)

In heavy chains, non-coded nucleotides are added by terminal transferase

 An enzyme that adds nucleotides to a 3’-OH
 This 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-nucleotides

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 Trimming coding sequences

 Variation in N-nucleotide addition
 Flexibility in coding sequence joining
 Junctional 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 codon
1/3 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:

Allelic Exclusion:
B-cells are diploid, two different copies of
 etc.
Maternal or paternal genes are re-arranged
The genes are chosen randomly
Could be paternal  and maternal H for 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.

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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  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  2  1  k2  1  2  death

 is preferred over 
Even with all of these fall-backs, only 11% of B-cells can fully mature

All values are ~ Heavy Light () Light ()

V 50 40 30
D 25 0 0
J 5 5 5
Possible combinations 6250 200 120

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Immunology Antibody Diversity

Total heavy / light chain associations 1,000,000+

Similar number for mice

Recombination results in ~106 different specificities

Somatic mutation results in 109 variants (another 1000 increase in diversity)

Sources of variation in the CDRs:

CDRs need to be diverse as they are in contact with the antigen

CDR1 – V sequence, somatic hypermutation

CDR2 – V sequence, somatic hypermutation
CDR3 – V sequence, somatic hypermutation, junctional flexibility, P-/N-nucleotide addition

Somatic mutation & hypermutation

The average affinity of antibodies during the humoral immune response increases
Their Kd decreases, 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)
 Activated B-cells are known as centroblasts
This is where the mutations take place (centroblasts  centrocytes)
 There will be some B-cells with high affinity antibodies, some with low affinity

Follicular dendritic cells present antigens to the centrocytes

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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 antigens
 Only those with high affinity for the antigen will be able to bind
 When they bind they get selected, and get T-cell help
 Selected centrocytes  plasmablasts  plasma cells or memory cells
 The plasma cells then go on to produce antibodies

The cells that do not get help will die

Class Switching:
After receiving
help, antibody class switching can occur

A switch recombinase facilitates the switch at a switch region upstream of the

CH

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Mechanism is unknown, cytokines are involved

 Interleukin-4 will stimulate: Cμ (IgM)  C1 (IgG) or C (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 SHM or class switching
 The enzyme is RNA and maybe DNA editing
 Deaminates C  U in RNA, leading to repair
 Mechanism is not clear

Product is IgE

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Antibodies can be membrane-bound or secreted

This is determined by C-terminal sequences
C-terminus depends on class of antibody

Occurs by alternative splicing…

S-segment + polyA signal
or No S-segment, M1 + M2 + polyA

The C-terminus of a secreted form is very hydrophilic

 No TM regions

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

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Synthesis, Assembly and Secretion:

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
IgM assembles 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 are ubiquitinated and degraded by proteasomes

Antibody with TM segment 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 CH 2 domain (in the Golgi)
 Important and complex but we don’t 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-cells
 This allows B-cell only expression
Differs between  /  / 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

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Ig expression is low until after re-arrangement, when the enchancers are brough closer to the
promoter, resulting in a 10,000 increase in expression

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 role in creating diversity in Igs and T-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 V L/H human CL/H)
Antibody libraries can be constructed

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Summary

 Pre-B cells have germ line DNA but mature B-cells have lost DNA and can only make one
specific antibody
 Recombination allows diverse repertoire in antibody response
 Recombination occurs in class switching
 Splicing accounts for membrane or secreted antibody
 Somatic 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

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