An immunoglobulin (Ig) consists of 2 identical light chains (L) and 2 identical heavy chains (H) (for example IgG-type); at the three-dimensional level, an Ig chain consists of one N-terminal variable domain, V, and one (for an L chain) or several (for an H chain) C-terminal constant domain(s), C.

The cells of the B line synthesize immunoglobulins. They are either produced at a membrane (on the surface of the B-lymphocytes) or are secreted (by the plasmocytes).

See also : IMGT Education - Fig 1

As soon as the main characteristics of the immunoglobulins were discovered, a number of questions arose:

A

• The antigens are highly varied; to be able to respond to them, the immunoglobulins must be equally diverse (there are 10¹¹ to 10¹² different Igs!), which corresponds to the diversity of the amino acids of the N-terminal parts of the L and H chains (i.e. to the variable domains).
• Does this reflect extreme diversity of the genes responsible for coding the immunoglobulins? (in line with the model of the germline theory: 1 gene = 1 Ig chain; in which case many genes would have to be implicated; they may arise from the duplication of ancestral genes; but the entire human genome would not suffice to encode all the immunoglobulins!).
• Does this reflect an accumulation of mutations? (in line with the model of the theory of somatic mutations: in this case, only a few genes would be implicated, but numerous somatic mutations would then have to take place to produce the diversity of the immunoglobulins produced; however this model would run counter to the generally accepted principles of genetics).
• Does this reflect a mechanism specific to the immunoglobulin genes?

B

• During its differentiation, a B cell, first produces membrane immunoglobulins on the surface of the B-lymphocyte, and then produces the immunoglobulins secreted by the plasmocyte. The amino acid sequence of the heavy chains of the membrane and secreted Igs differ only at their C-terminal end: are the same genes implicated in both cases?

C

• A B-cell first expresses the IgM at its surface and then, during its differentiation, may express another class of Ig (IgG, IgE or IgA) (this mechanism is known as an isotype switch): how does this switch occur? How can we explain that regardless of the immunoglobulin isotype produced, the same specific antigen variable domain (same idiotype) is expressed?

D

• A B-cell synthesizes a single type of heavy chain and light chain, even though its genome has 2 chromosomes (2 alleles) for each Ig locus; allele exclusion must therefore occur and a hemizygote phenotype is produced; how does this allele exclusion take place?

E

• Finally, if the variable regions do undergo mutations, why aren’t there any in the constant regions?
• Various methods used in molecular biology and gene cloning in the mouse and in human beings have been used to answer these questions; we will limit our discussion to human immunoglobulins.

2.1 Light chains (kappa or lambda)

2.1.1 Kappa chain: V-J rearrangements

• IGK (kappa) genes at 2p11 on chromosome 2.
• Multiple IGKV genes for the variable region, V (76 genes, of which 31 to 35 are functional); 5 IGKJ genes for the junctional region, J; a single IGKC gene for the constant region, C; the V, J and C genes are separated in the DNA of the genome (germline configuration of the Ig genes).
• These are multigene families (also see the section on the families of genes, in Globin genes "... of the duplications of the ancestor gene have succeeded each other, and the mutations of each of the genes have led to some degree of diversity. Many of these duplicated genes are functional ...").
• First the DNA is rearranged: this makes it possible to join 1 V and 1 J; the intermediate sequences are then deleted.
• The pre-messenger RNA is copied (transcription); this includes introns.
• Then comes splicing: the elimination of the introns from the pre-messenger RNA , to yield mature, messenger RNA.
• This is followed by protein synthesis (known as translation).
• N.B.: It is crucial not to confuse DNA rearrangements with RNA splicing.

Note:Only the genes for the immunoglobulins and T-receptors undergo DNA rearrangement.

See also : IMGT Education - Fig 2

• V-J rearrangements occur at the recombination signals (RS), which include a heptameric sequence (7 nucleotides) and a nonameric sequence (9 nucleotides), separated by a spacer.

Each IGKV gene is followed downstream (in the 3 position) by an RS consisting of a CACAGTG heptamer, and then by a 12-bp spacer, and then an ACAAAAACC nonamer.

Each IGKJ gene is preceded upstream (in the 5 position) by an RS consisting, between 5 and 3, of a GGTTTTTGT nonamer, a 23-bp spacer and a CACTGTG heptamer.

See also : IMGT Education - Fig 3

2.1.2 Lambda chain: V-J rearrangements

IGL (lambda) genes at the 22q11 position on chromosome 22; the V-J rearrangement mechanism is the same as that described for the IGK genes: the rearrangements take place between one of the 29 to 33 functional IGLV genes and a J gene; it should be noted that there are 4 to 5 functional IGLC genes, each of which is preceded by a IGLJ gene.

2.1.3 Allele exclusion and isotype

Allele exclusion can be explained in part by the timing of rearrangements, and partly by the surface expression of a functional immunoglobulin, which inhibits the rearrangements and therefore the expression of a second chain. Only one 14 chromosome and one 2 (or 22) chromosome are therefore productive (answer to question D).

2.2 Heavy chains

IGH (heavy) genes at 14q32 on chromosome 14.
There are 11 IGHC genes, 9 of which are functional (IGHM, IGHD, IGHG1, IGHG2, IGHG3, IGHG4, IGHA1, IGHA2 and IGHE) and correspond respectively to 9 heavy chain isotypes m, d, g1, g2, g3, g4, a1, a2 and e.

2.2.1. V-D-J rearrangements

DNA rearrangements between one of the 38 to 46 functional variable IGHV genes, one of the 23 functional diversity IGHD genes, and one of the 6 functional junction IGHJ genes: there are also some RSs, which are located downstream (in position 3) of the V genes, either side of the D genes and upstream (at 5) of the J genes. During V-D-J rearrangement, a junction is first formed between 1 D and 1 J, and then one between 1 V and the D-J complex.

See also : IMGT Education - Fig 4

Note: there are also 2 or 3 open reading frames for the D genes; each of which can code for 2 or 3 different peptide sequences. The V-D-J junctions are also characterized by nucleotide deletions (by an exonuclease) and by the random addition of nucleotides (by means of TdT, terminal deoxynucleotidyl transferase);the V regions which result are not, therefore, coded in the genome of the individual and considerably increase the diversity of the V-D-J junctions of the variable domains of the heavy chains of the immunoglobulins.

2.2.2.Isotype switching

• In the pre-B lymphocyte, a mu chain is first synthesized, because the constant IGHM gene (C) is located near to the V-D-J rearrangement. This mu chain is associated with the pseudo-light chain and the combination constitutes the pre-B receptor. The first complete Ig synthesized by the B-lymphocyte is an IgM, in which the mu chain is combined with a light kappa or lambda chain.
• During its differentiation, the B lymphocyte can express some other isotype or sub-isotype of Ig. This involves the replacement of an IGHC gene by another, as the result of DNA recombination (isotype switch), with the excision of the entire intermediate part of a deletion loop. This excision occurs at the switch sequences (role related to that of the RSs).
• The usual sequence is then as follows: synthesis of pre-messenger RNA, splicing of the introns, resulting in mature RNA, and then protein synthesis.
• This explains why:
  • 1) a B-lymphocyte can at first synthesize an IgM and then, during its differentiation, an IgG (IgG1, IgG2, IgG3 or IgG4), an IgA (IgA1 or IgA2) or an IgE.
  • 2) that it retains the same V-D-J rearrangement and therefore the same antigen recognition site (idiotype) (answer to question C).

See also : IMGT Education - Fig 5 and IMGT Education - Fig 6

2.3 Membrane and secreted Igs

Alternative splicing of the pre-messenger RNA of the heavy chain can yield either a membrane heavy chain (membrane Ig of B lymphocytes), or a secreted heavy chain (plasmocyte secreted Ig), which retain the same V-D-J rearrangement (idiotype) and the same constant region (isotype) (answer to question B).

See also : IMGT Education - Fig 7

Note: the same mechanism (alternative splicing of a pre-messenger) expresses the IgMs and IgDs in the same B cell (situation in mature B cells leaving the bone marrow and reach the lymph nodes via the circulation).

3.1 Germline diversity: multigene families

• Germline diversity depends on the number of genes at each locus. These are families of genes, offering the possibility of a choice between similar? functional sequences. Possible intergene recombinations permit the long-term evolution of the locus with duplication or deletion of the genes.
• These genes undergo intragene conversions and recombinations, leading to mixing and diversity (polymorphism) between individuals.
• The presence of several open reading frames, in the case of IGHD genes, further increases the possibility of choice between similar functional sequences.

3.2 Diversity due to DNA rearrangements

• Combination diversity - in the mathematical sense of the term - permits the potential synthesis of a million immunoglobulins. The IGH genes permit the synthesis of about 6000 heavy chains, the IGK or IGL genes of about 160 light chains, which is equivalent to about a million possible combinations 6 x 10 ³ x 160).

• In addition to this, during the rearrangements of the IGH of the heavy chains, the acquisition of the N regions, and using one or other of the reading frames for the D genes at the V-D-J junctions, and during the IGK or IGL rearrangements of the light chains, flexibility of the V-J junctions. These mechanisms contribute to increasing the diversity by a factor 10³ to 10⁴ (potential synthesis of 10⁹ Ig chains).

3.3 Diversity as a result of somatic hypermutations

Finally, somatic mutations are extremely numerous (somatic hypermutations) and produce very targeted characterization of the rearranged V-J and V-D-J genes of the Ig, but their mechanism of onset is not yet known. AID (activation-induced cytidine deaminase) may be implicated both in the occurrence of the mutations and the switch mechanism. The mutations appear during the differentiation of the B lymphocyte in the lymph glands and contribute to increasing the diversity of the Igs by a further factor of 10³, which makes it possible to achieve a potential diversity of 10¹² different Igs (answer to question A).

These different mechanisms of diversity make it possible to obtain 10¹² different immunoglobulins, capable of responding to the several million known antigens (answer to question A).

The number of different Igs is in fact limited by the number of B cells in a given species.

For further details, see: IMGT