17 How and why Ig diversity was created

Dr. M. N. Gupta

1. Objectives

• To understand meaning of allotypes and idiotypes

• To understand various origins of diversity of antibodies

• To understand why “class switch” takes place?

• To explain its mechanism of diversity generation and class switching in the light of clonal selection theory.

2. Concept Map

3. Description

One of the many successes of the clonal selection theory has been to explain how various classes of Ig can be created against the same antigen.

We know that initially IgM is produced upon immunization and then the same B-lymphocyte switches over to synthesis of IgG.

The immunoglobulin diversity can be viewed from various perspectives.

To start with is a question of having unlimited B-repertoire response in terms of generating antibody in response to any antigen.

At another level, we have various classes of immunoglobulins produced with specificity towards the same antigen. We have discussed these various classes. The various classes of immunoglobulins reactive towards a common antigen are called isotypes (and subisotypes)

An immunoglobulin is also defined in terms of the presence of genetic markers called allotypes. The same Ig (same isotype against the same antigen) in different members of a species can contain different allotypes.

Allotypic markers are immunogenic i.e. if an Ig from one individual is injected into another individual (of the same species) which lacks that allotype, this individual will produce antibodies directed against that allotypic determinant. So, like ABO blood group antigens, allotypic determinants seggregate within a specie.

The allotypic variation arises from allelic forms (encoded by alleles or alternative genes at a single locus).

For example G1m (a) locus on IgG1 subtype: An individual can have Asp.Glu.Leu.Thr.Lys sequence on IgG1 molecule whereas another with a negative allotype will have Glu.Glu.Met.Thr.Lys. instead.

In human 25 Gm (marker on IgG) groups on the γ-heavy chain and 3 on the k-constant regions are known. Allotypes are also known on other animals such as mouse or rabbit. Allotypes have been recognized by using red cells coated with anti Rh bearing the Gm allotype and agglutinating these cells by mixing with anti Gm rheumatoid factor. The test sera are evaluated for the presence of the allotype by their capacity to break this agglutination.

The allotype are inherited in a mendalian fashion. The individuals may be homozygous or heterozygous. The alleles are expressed co dominantly. So, a fraction of Ig of an animal may have one allotype marker, rest of the fraction may have another.

Idiotypes

An antibody differs from another antibody in its specificity. This specificity arises from the sequence of amino acids in its antigen binding site, specially hypervariable regions discovered by Kabat’s group.

Antisera can be raised against an Ig which are directed against these determinants which characterize its specificity. Such determinants collectively are called idiotypes by Kunkel and Oudin. Kunkel and Oudin, of course at that time did not know the existence of or concept of hypervariable regions.

Private idiotype : Antibodies (raised against Ig) which react with just one Ig and none other are said to recognise private idiotype.

Some Ig have closely similar sequence and may share idiotypes. Such idiotypes are called public idiotypes or cross reacting idiotypes. (The antibodies raised against these cross reacts). Early data on public or private idiotypes were generated in case of anti-dextran Ig.

Anti idiotypic sera are useful in many applications such as:

•  identifying V regions

•  identifying specific immuno complexes in patient sera.

•  identifying VL type amyloid in patient secreting Bence Jones proteins.

•  Detection of residual monoclonal antibody after therapy.

After this, let us shift our attention to understand how any Ig is synthesized like any eukaryotic protein, its gene also has be discontinuous i.e. consist of introns and exons.

If a separate gene was responsible for each specific antibody, the nature of Ig structure presented a dilemma. How in each case, parts of the L-chains and H-chains remain constant? How this constancy was maintained in different genes.

In 1965, William Dreyer and Claude Bennet had proposed that these are multiple V gene along with a single C gene in the germ line. During differentiation, one of the V gene joins the C gene.

Dreqer and Bennet’s proposed in a way was very bold. It was known that integration of λ phage DNA involves splicing of the DNA. Discontinuity of genes for proteins from higher organisms was discovered much later.

The hypothesis also turned out to be correct. In 1976, Susumu Tonegawa discovered that V and C genes are at a distance in embryonic DNA but are closely associated in B-lymphocyte. It is worth noting that couple of technological developments had to be in place to enable Tonegawa’s lab to discover that

• Isolation of pure Ig mRNA had become possible

• Genomic isolation with restriction endonucleases was in place.

• Hybridization techniques enabled hybridization of mRNA specific for constant and variable regions.

Thus Dreyer and Bennett were ahead of their times. Tonegawa’s group moved on after this success to try to understand T-cell receptor structure. Susumu Tonegawa was awarded noble prize!

This finding that Ig chains are coded by multiple genes and by combining V gene with C gene was the first step towards understanding the source of the huge diversity of antibody structures.

In an individual B-cells with different antigen specificities are estimated to be in the range 106-108.

So, each individual will have these many genes just for antibody production. That is a very significant fraction of genome.

Just by creating Ig with many specificities with a single C gene reduced this requirement considerably.

What ultimately turned out to be the correct picture was slightly more complicated.

It was found that mouse have few hundred genes for VK and VH. Assuming 300 of each, the combination (300×300) would generate 9×104 different specificities. From where the extra diversity is coming as mice is expected much higher number of specific antibodies (if needed).

This actually related to another old question. Does the diversity of antibody generation have a somatic origin or genetic origin?

The information, so far indicated that it is genetic in nature, but the potential diversity inherent in the genome did not look adequate.

The next insight had to wait determination of sequence of cloned Ig genes from embryonic and myeloma cells.

Many leading labs – those of Tonegawa, Philip Lederer and Leroy hood (Leroy hood later moved out of academia and coined the term system biology which has now become a frontier area in biological sciences) collaborated.

The first new discovery was that V gene in embryonic cells did not seem to code for entire V region of L & H chains.

The germ line V gene codes till 95 amino acids residues whereas V region has 108 amino acid residues. So the next question was what codes for the 13 amino acid residues.

The missing encoding DNA segment turned out to be located near C-gene. As it joins V gene and C gene, it was called J gene. It was found that a number of J genes were there near the C- gene in the embryonic cell.

J genes have a special significance as they code for the last hypervariable region of the Fab. Hence, they contribute not just to enhancing the number of different antibody which can be produced but also directly to creation of diversity of specificities.

It was clear that the strategy which seems to be followed by the antibody producing cells during differentiation was that one of the many V genes combine with the one of the J gene and then joins the C gene.

250 V genes combining with 5 J gene will generate 1250 diverse complete V gene in the differentiated B-cell.

This leads to consideration amplification of the diversity of antibody specificities.

Hence the source of antibody diversity turned out to be both genetic as well as somatic.

The genetically diverse pool gets amplified by recombination which is somatic in nature.

It may be pointed out that even in fully differentiated B-cells, these segments are not joined. That takes place only in antibody producing plasma cells which is clonally selected by an antigen.

D (Diversity) genes code part of heavy chains and are source of further diversity

In the case of variable region of heavy chains (VH), another kind of gene segment required in addition to V segment, J segment and C region.

About 15 D segments were found between V segments and J segments. Just like J segment, these D segments also play an important role in determining the specificity of antibody which is coded by the complete gene. D segment is responsible for determining the third complementary determining region of the heavy chain.

The splicing is not always precise

The analysis of amino acid sequence and the corresponding base sequence data indicated that splicing between the chosen V segment and chosen J segment can be in different ways. The imprecise splicing becomes further source of diversity of variability in both L chains and H chains.

An enzyme terminal deoxyribonucleotidyl transferase is a polymerase which can insert extra nucleotides between VH and D. This is the further source of diversification at the third complementary determining region of the heavy chain. The enzyme is able to do it as though a polymerase, it does not require a template.

The enzymes which mediate the site specific recombination of V, D & J gene segments recognise flanking sequences around these segments. The recognition elements are palindromic heptamers with 23 basepair or 12 base pair spacers.

Recombination can occur between sites characterized by 23 base pair and 12 base pair but not between 23 base pair and 23 base pair (or 12 base pair and 12 base pair) V & J gene segments are flanked by 23 base pair types. Hence, V-V, J-J, V-J recombination cannot take place. It has to be V-D-J sequence. Only properly Ig gene is expressed.

While discussing clonal selection theory we had talked about allelic exclusion. In a particular cell only one member of an allelic pair of gene is expressed. Apparently only in one case, the correct recombination takes place and that is the complete Ig gene which is expressed. This ensures that a single B-cell produces Ig with same antigen binding site.

Formation of mRNA for L and H chains from primary transcripts

The mRNA for K chain has 1250 bases. It is a typical eukaryotic mRNA with untranslated regions at both ends, poly A tail and a leader sequence (which ensures that the corresponding polypeptide chain is secreted out of the cell ultimately). In addition to this the primary transcript has two introns. During processing of this primary transcript, these introns are removed.

Hence J gene seems to have two kinds of information. First to combine with V gene segment at the DNA level itself. Second, to ensure that down the line at mRNA transcript level J segment combines with C segment. This specific splicing information at two different levels is inherent in the sequence of J gene segment.

H-chain gene

The four domains of H chain are VH, CH1, CH2, CH3 and a hinge element between CH1 & CH2. The gene mapping confirms that these five components are encoded by different DNA segments.

Class Switch

We are now ready to understand that if need arises how the same B lymphocyte produces another isotype of Ig with same antigen binding specificity. This “class switch” definitely required when B-lymphocyte switches to production of IgG after the initial production of IgM. It turns out that the class switch involves only the CH regions. The L-chain gene is not involved nor is the VH region.

In embryonic mouse cells, CH gene segments of the 5 classes of Ig lie together next to J-Gene segments. This includes 4 subisotypes of IgG in mouse.

So the first to be formed is gene for µ chain (of IgM). The further processing which involves removal of intron sequences is done at the primary transcript of mRNA level as illustrated earlier for the L-chain.

The class switch however takes place at the DNA level itself. The evidence for this comes from comparison of restriction digests of DNA from both embryonic and melanoma cells.

This way, VHDJH remains same which ensures that antigen binging specificity remains identical.

However change in CH part changes the other effector functions of Ig molecules

Origin of diversity in other vertebrates

Not all vertebrates follow the same mechanism for the generation of diversity of antibodies. In sharks, H chains have repeats of VH-DH1-CH2-JH-CH and there is no possibility of recombination between different units. Hence the diversity is more limited

Chiken have only one VL, one J and one C segment gene. H chains also have only one VH and J segment. There are 15 DH segment but with very similar sequences.

How are chickens then able to mount effective immune response?

It is found that upstream of a VL gene segments, there are 25 sequences similar to the VL regions but lack a leader exon and a promoter region. The sections from these “pseudogenes” can be inserted into the VL regions. This is also a somatic event which is continuously created even after B-cells have left bursa. Similarly, about 100 VH pseudogenes produce diversity for VH segment.

Hence, the origin of diversity are:

•  Multiple V genes and J genes

•  Multiple possibility of V-J and V-O-J recombinations

•  Somatic mutations

•  Pseudogenes causing gene conversions in birds and possibly in other organisms.

For many decades scientists argued about whether antibody diversity has a genetic origin or somatic origin. As happens in case of many scientific controversies, both groups were partly right. It is both genetic and somatic in nature.

Summary

•  Isotypes, allotypes and idiotypes define the diversity in the structure of the immunoglobulins

•  Antibody diversity arises from multiple copies of V gene, J gene and D gene segments

•  Somatic recombination of these segments and imprecise splicing generates further diversity

•  Class switch retains antibody specificity and involves recombination again