Somatic Hypermutation Adds Diversity in Already Rearranged Gene Segments

All the antibody diversity described so far stems from mechanisms that operate during formation of specific variable regions by gene rearrangement. Additional antibody diversity is generated in rearranged variable-region gene units by a process called somatic hypermutation. As a result of somatic hypermutation, individual nucleotides in VJ or VDJ units are replaced with alternatives, thus potentially altering the specificity of the encoded immunoglobulins.

Normally, somatic hypermutation occurs only within germinal centers (see Chapter 11), structures that form in secondary lymphoid organs within a week or so of immunization with an antigen that activates a T-cell-dependent B-cell response. Somatic hypermutation is targeted to rearranged V-regions located within a DNA sequence containing about 1500 nucleotides, which includes the whole of the VJ or VDJ segment. Somatic hypermutation occurs at a frequency approaching 10~3 per base pair per generation. This rate is at least a hundred thousand-fold higher (hence the name hypermutation) than the spontaneous mutation rate, about 10~8/bp/generation, in other genes. Since the combined length of the H-chain and L-chain variable-region genes is about 600 bp, one expects that somatic hypermutation will introduce at least one mutation per every two cell divisions in the pair of VH and VL genes that encode an antibody.

The mechanism of somatic hypermutation has not yet been determined. Most of the mutations are nucleotide substitutions rather than deletions or insertions. Somatic hypermutation introduces these substitutions in a largely, but not completely, random fashion. Recent evidence suggests that certain nu-cleotide motifs and palindromic sequences within VH and VL may be especially susceptible to somatic hypermutation.

Somatic hypermutations occur throughout the VJ or VDJ segment, but in mature B cells they are clustered within the CDRs of the VH and VL sequences, where they are most likely to influence the overall affinity for antigen. Following exposure to antigen, those B cells with higher-affinity receptors will be preferentially selected for survival. This result of this

(a) P-nucleotide addition Hairpin

IAG) CATS=

Cleavage of hairpin 71 generates sites for the addition of P-nucleotides

TTITCGA rri-

Repair enzymes add complementary nucleotides

(b) N-nucleotide addition

Hairpin

cAT^

Cleavage of hairpin 71 generates sites for the addition of P-nucleotides

TTITCGA rri-

ATAT J-

TdT adds N-nucleotides Repair enzymes add complementary nucleotides n TCGAAGTTATAm-

DAGCTTCAATAT J_-

FIGURE 5-13

P-nucleotide and N-nucleotide addition during joining. (a) If cleavage of the hairpin intermediate yields a double-stranded end on the coding sequence, then P-nucleotide addition does not occur. In many cases, however, cleavage yields a single-stranded end. During subsequent repair, complementary nucleotides are added, called P-nucleotides, to produce palin-

dromic sequences (indicated by brackets). In this example, four extra base pairs (blue) are present in the coding joint as the result of P-nucleotide addition. (b) Besides P-nucleotide addition, addition of random N-nucleotides (light red) by a terminal deoxynu-cleotidyl transferase (TdT) can occur during joining of heavy-chain coding sequences.

differential selection is an increase in the antigen affinity of a population of B cells. The overall process, called affinity maturation, takes place within germinal centers, and is described more fully in Chapter 11.

Claudia Berek and Cesar Milstein obtained experimental evidence demonstrating somatic hypermutation during the course of an immune response to a hapten-carrier conjugate. These researchers were able to sequence mRNA that encoded antibodies raised against a hapten in response to primary, secondary, or tertiary immunization (first, second, or third exposure) with a hapten-carrier conjugate. The hapten they chose was 2-phenyl-5-oxazolone (phOx), coupled to a protein carrier. They chose this hapten because it had previously been shown that the majority of antibodies it induced were encoded by a single germ-line VH and VK gene segment. Berek and Milstein immunized mice with the phOx-carrier conjugate and then used the mouse spleen cells to prepare hybridomas secreting monoclonal antibodies specific for the phOx hapten. The mRNA sequence for the H chain and k light chain of each hybridoma was then determined to identify deviations from the germ-line sequences.

The results of this experiment are depicted in Figure 5-14. Of the 12 hybridomas obtained from mice seven days after a primary immunization, all used a particular VH, the VH Ox-1 gene segment, and all but one used the same VL gene segment, VK Ox-1. Moreover, only a few mutations from the germ-line sequence were present in these hybridomas. By day 14 after primary immunization, analysis of eight hybridomas revealed that six continued to use the germ-line VH Ox-1 gene segment and all continued to use the VK Ox-1 gene segment. Now, however, all of these hybridomas included one or more mutations from the germ-line sequence. Hybridomas analyzed from the secondary and tertiary responses showed a larger percentage utilizing germ-line VH gene segments other than the VH Ox-1 gene. In those hybridoma clones that utilized the VH Ox-1 and VK Ox-1 gene segments, most of the mutations were clustered in the CDR1 and CDR2 hypervariable regions. The number of mutations in the anti-phOx hybridomas progressively increased following primary, secondary, and tertiary immunizations, as did the overall affinity of the antibodies for phOx (see Figure 5-14).

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Essentials of Human Physiology

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Responses

  • Abi
    Does p nucleotide addition occur in the light chains?
    7 years ago
  • Jana Weisz
    Which enzyme generates p nucleotides?
    7 years ago
  • nea
    Where does somatic hypermutation occur?
    6 years ago

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