IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 8. Sources of Diversity in Antigen-Recognizing Structures

SOMATIC MUTAGENESIS

Mutations in heavy and light chain genes occur following antigenic stimulation

Somatic mutations, which can occur throughout an individual's lifetime, serve as an additional source of antibody diversity. This concept was convincingly argued many years ago. As noted above (Fig. 8.5), the majority of mouse λ1 light chains possess a nearly identical sequence with a few substitutions in the CDRs, yielding a total of eight sequence variants. However, only a single Vλ1 germline segment encoding the basic Amino Acid Sequence taken as the prototype has been found in the mouse genome. Consequently, all λ1 light chain variants must arise through somatic mutations in the form of single nucleotide substitutions. Products of somatic mutations have also been identified among κ-type light chains and heavy chains of certain isotypes.

Further support for somatic mutagenesis as a source of antibody diversity came from studies of the Gene family encoding Antibodies against phosphorylcholine. Out of 19 fully sequenced V_H segments, 10 shared an identical nucleotide sequence designated as the "prototype," while the remaining nine exhibited 1 to 8 substitutions. Only a single prototype sequence was found in the germline genome, which unequivocally points to somatic mutations as the cause of its other variants (Fig. 8.15). Notably, all somatic mutation-derived variants of V regions were found exclusively within IgA and IgG molecules, suggesting that they are somehow linked to immunoglobulin isotype switching. Presumably, owing to improved complementarity toward the antigen, mutant sequences confer selective advantages. When assessed for phosphorylcholine binding, variants generated via somatic mutation display higher affinity than antibodies encoded by the germline V_H segment.

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Fig. 8.15. Amino acid sequence of V_H regions from Monoclonal Antibodies against phosphorylcholine (five samples each of IgM and IgG) compared with the Introduction/19.html">Primary Structure of the germline V_HT15 gene product. Positions where residues are identical in both sequences are highlighted in yellow and dark yellow; Amino Acid Substitutions are shown in pink; hypervariable regions (HV1, HV2) are also indicated. As can be seen, mutational substitutions of amino acid residues occurred exclusively in IgG molecules, within both the hypervariable and framework Regions of the V_H domains.

It is well established that the DNA region encoding the variable domains of immunoglobulin molecules can exhibit a heightened susceptibility to mutation. For example, V_H genes from two distinct samples of anti-phosphorylcholine antibodies of the T15 idiotype display numerous nucleotide substitutions compared to the germline sequence (3.8% mutated NUCLEOTIDES in the V_H gene of protein M167). Mutations were found in both introns and exons of the V_H gene, but not in adjacent DNA regions, indicating a specific hypermutability of the entire V gene locus (Fig. 8.16). These somatic mutations occur within germinal centers of lymphoid tissue, where antigen drives the Selection (survival and proliferation) of Cells producing high-affinity antibodies. This process is covered in more detail in Chapter 11. It depends on both T cells and cellular elements residing within the germinal centers themselves. Athymic mice lack T cells and germinal centers, and consequently fail to exhibit affinity maturation of antibodies against the antigen. Frogs possess T cells but do not form germinal centers and (like athymic mice) lack antibody affinity maturation.

Fig. 8.16. Genes encoding the synthesis of two monoclonal antibody samples of the T15 idiotype against phosphorylcholine. Black vertical lines indicate positions where nucleotide substitutions (mutations) were detected relative to the germline sequence. A significant number of mutations are present in the introns and exons of both genes, particularly within the second hypervariable region, HV2. In contrast, no mutations were detected in the constant region genes.

Thus, antibody diversity arises through the interplay of several mechanisms. The first is the generation of a vast array of V genes via recombination of V, J, and D segments. Additional diversity is introduced by inaccuracies in the joining of gene segments. It is noteworthy that the First and Second hypervariable regions of V domains are entirely encoded by germline nucleotide sequences. The broad diversity of CDR2 is initially hardwired into The Genome, whereas CDR1 Variability is generated by somatic mutagenesis, and CDR3 primarily through recombination. Because theoretically any light chain can pair with any heavy chain, their combinatorial joining expands antibody diversity almost limitlessly (Fig. 8.17). Presumably, less than 5% of the total nucleotide sequence variability results from somatic hypermutation. However, up to 90% of B cells express V_H genes that have undergone somatic mutation.

Fig. 8.17. Each of the sources of antibody diversity can function in concert with any other, so the capacity to generate various specificities multiplies at each successive stage of immunoglobulin production.