Antibody Diversification And Synthesis
In contrast to the MHC and the T-cell receptor, the existence of the antibody, or immunoglobulin (Ig), molecule has been known for over 100 years and its basic structure for about 50, which makes it one of the most studied and best understood molecules in biology.
The two-chain multidomain structure characteristic of MHC and T-cell receptors is seen here in a slightly more complex form, a typical Ig molecule being made up of four chains: a pair of heavy chains and a pair of light chains (for structural details see Fig. 14). Two main kinds of diversity are found within these chains: in the constant regions of the heavy chains are the variations that classify Ig molecules into classes and subclasses with different biological effects, while the much more extensive variations in the variable regions (blue in the figure) are responsible for the shape of the antigen-binding site and thus of the antigen specificity of the Ig molecule.
Within B lymphocytes, the genes for Ig heavy and light chains are put together by a process of rearrangement at the DNA level followed by further excisions in the mRNA, very much as in T cells with their receptor, one important difference being that in B, but not T, cells, further somatic mutation in the variable regions can occur. Finally, the polypeptide chains are synthesized on ribosomes, similar to other proteins, assembled and exported – some to reside on the cell surface as receptors and others to be secreted into the blood as antibody.
Ig Immunoglobulin; the name given to all globulins with antibody activity. It has replaced the old term ‘gamma globulin’ because not all antibodies have gamma electrophoretic mobility.
Igκ, Igλ, IgH Three genetic loci on different chromosomes (see Fig. 47), which code for the light chain (κ, λ) and heavy (H) chain of the Ig molecule. A typical Ig molecule has two H chains and two L chains, either both κ or both λ.
Germline This denotes those genes in the ova and sperm giving rise to successive generations, which can be regarded as a continuous family tree stretching back to the earliest forms of life. Mutations and other genetic changes in these genes are passed to subsequent generations and are what natural selection works on. Changes that occur in any other cells of the body are ‘somatic’ and affect only the individual, being lost when death occurs. This includes the changes in the DNA of B lymphocytes that lead to the formation of the Ig molecule. The antibody germline genes have presumably been selected as indispensable, and many of them have been shown to code for antibodies against common bacteria, confirming that bacterial infection was prob- ably the main stimulus for the evolution of antibody.
V Variable region genes. Their number ranges from two (mouse λ chain) to about 350 (mouse κ chain; the numbers shown in the figure are for the human). The greatest variation is found in three short hypervariable regions, which code for the amino acids that form the combining site and make contact with the antigen. V genes are classified into families on the basis of overall sequence similarity.
C Constant region genes. In the light chains, these code for a single domain only, but in the heavy chains there are three or four domains, numbered CH 1, 2, 3, (4). Which of the eight (mouse) or nine (human) C genes is in use by a particular B lymphocyte determines the class and subclass of the resulting Ig molecule (IgM, IgG, etc.; see Fig. 14).
J Joining region genes, coding for the short J segment. Note that in the κ and H chains, the different J genes lie together while in the λ chain each C gene has its own. In primitive vertebrates there are repeated V–J–C segments, which restricts the number of possible combinations.
D region genes are found only on IgH, where they provide additional possibilities for hypervariability.
Gene rearrangement occurs in the Ig genes of B lymphocytes in a similar way to the TCR genes of T lymphocytes. First, the intervening segments of DNA (‘introns’) between the V and J (and D if present) genes are excised in such a way as to bring together one particular V and one J gene. The excision and joining is an imprecise process, generating even more diversity, but also resulting in many B cells failing to produce a proper Ig molecule, and therefore dying during development. Once a correctly formed Ig molecule has been produced by rearrangement on one chromosome, the Ig locus on the other chromosome is switched off (‘allelic exclusion’), thus ensuring a B cell mosome is switched off (‘allelic exclusion’), thus ensuring a B cell only ever expresses antibody of one specificity. This unique process of DNA rearrangements is catalysed by a complex of enzymes, many of which are involved in DNA repair functions in other cells. However, the first cleavage of DNA that initiates the recombination event is catalysed by two specialized enzymes, RAG-1 and RAG-2 (recombination activating genes). These enzymes are expressed only in developing B and T cells, and knocking out these genes in mice results in a complete absence of B or T cells.
Class switching can occur within the individual B cell by further excisions of DNA, which allow the same VDJ segment to lie next to a different C gene, leading to antibodies with the same specificity for antigen but a different constant region (see Fig. 14). This allows the same antigen to be subjected to various different forms of attack. The decision which class or subclass to switch to is largely regulated by cytokines released locally by helper T cells; thus, IL-4 favours IgE, IL-5 IgA, IFNγ IgG3, etc. (these examples are from the mouse).
Somatic mutation After they have been activated by antigen and T cells, B cells migrate into the germinal centres (see Fig. 19). Here they undergo extensive rounds of replication. In addition, individual B cells introduce point mutations into their immunoglobulin genes, a process known as somatic hypermutation, which requires cytidine deaminase, an enzyme that chemically modifies individual bases on the DNA. After mutation, the B cells with the highest affinity are selected to survive, and become part of the memory lymphocyte populations. In this way, successive exposures to antigen select for ever higher affinity antibodies, a process known as affinity maturation.
CD19 One of the molecules (the complement receptor CR2 is another) that need to be bound in order to fully activate the B cell, thus playing a ‘coreceptor’ role somewhat analogous to CD4, CD8 and CD28 on the T cell. CD19 is also a convenient ‘marker’ for B cells, because it is not expressed on other types of cell.
Origins of diversity Four features of antibody contribute to the enormous number of possible antigen-binding sites and thus of antibody specificities: (i) gene rearrangement allows any V, D and J genes to become associated; (ii) a heavy chain can pair with either a κ or λ light chain; (iii) V–D and D–J joining is imprecise, allowing the addition or removal of a few DNA bases; and (iv) mutations are introduced into the V genes of an individual B cell after antigen stimulation (this would be a case of somatic mutation, see above). Because of all these possibilities it is difficult to put a number on the size of the Ig repertoire, but it may be as high as 1010. Note that diversity within the MHC is generated in a quite different way, individuals having only one or two of the allelic variants of each gene. The members of a species differ from each other much more in their MHC genes and molecules than in their Ig and T-cell receptors, of which they all have a fairly complete set with only minor inherited differences.
Igα, Igβ Two molecules that form a link between cell-surface Ig and intracellular signalling pathways, analogous to CD3 on the T cell. intracellular signalling pathways, analogous to CD3 on the T cell.