9. B cell development and memory

24.8 Somatic mutation generates additional diversity

Key terms defined in this section
Hybridoma is a cell line produced by fusing a myeloma with a lymphocyte; it continues indefinitely to express the immunoglobulins of both parents.
Somatic mutation is a mutation occurring in a somatic cell, and therefore affecting only its daughter cells; it is not inherited by descendants of the organism.

Comparisons between the sequences of expressed immunoglobulin genes and the corresponding V gene segments of the germline show that new sequences appear in the expressed population. We have seen that some of this additional diversity results from sequence changes at the V-J or V-D-J junctions. However, other changes occur upstream at locations within the variable domain; they represent somatic mutations induced specifically in the active lymphocyte.


A probe representing an expressed V gene segment can be used to identify all the corresponding fragments in the germline. Their sequences should identify the complete repertoire available to the organism. Any expressed gene whose sequence is different must have been generated by somatic changes.


One difficulty is to ensure that every potential contributor in the germline V gene segments actually has been identified. This problem is overcome by the simplicity of the mouse λ chain system. A survey of several myelomas producing λ 1 chains showed that many have the sequence of the single germline gene segment. But others have new sequences that must have been generated by mutation of the germline gene segment.


To determine the frequency of somatic mutation in other cases, we need to examine a large number of cells in which the same V gene segment is expressed. A practical procedure for identifying such a group is to characterize the immunoglobulins of a series of cells, all of which express an immune response to a particular antigen.


(Epitopes used for this purpose are small molecules Xhaptens Xwhose discrete structure is likely to provoke a consistent response, unlike a large protein, different parts of which provoke different antibodies. A hapten is conjugated with a nonreactive protein to form the antigen. The cells are obtained by immunizing mice with the antigen, obtaining the reactive lymphocytes, and sometimes fusing these lymphocytes with a myeloma [immortal tumor] cell to generate a hybridoma that continues to express the desired antibody indefinitely.)


In one example, 10 out of 19 different cell lines producing antibodies directed against the hapten phosphorylcholine had the same VH sequence. This sequence was the germline V gene segment T15, one of four related VH genes. The other 9 expressed gene segments differed from each other and from all 4 germline members of the family. They were more closely related to the T15 germline sequence than to any of the others, and their flanking sequences were the same as those around T15. This suggested that they arose from the T15 member by somatic mutation (Kim et al., 1981).


Sequence changes extend through and beyond the V gene segment. They take the form of substitutions of individual nucleotide pairs. The variation is different in each case. It represents ~3 V15 substitutions, corresponding to <10 amino acid substitutions in the protein. Only some of the mutations affect the amino acid sequence, since others lie in third-base coding positions as well as in nontranslated regions (for review see French et al., 1989; Kocks and Rajewsky, 1989).


The large proportion of ineffectual mutations suggests that somatic mutation occurs more or less at random in a region including the V gene segment and extending beyond it. There is a tendency for some mutations to recur on multiple occasions. These may represent hotspots as a result of some intrinsic preference in the system.


Somatic mutation requires the enhancer that activates transcription at each Ig locus. There is a correlation between the occurrence of transcription and the induction of mutations, but the molecular basis for somatic mutation remains unknown.


Somatic mutation occurs during clonal proliferation, apparently at a rate ~10 V3 per bp per cell generation. Approximately half of the progeny cells gain a mutation; as a result, cells expressing mutated antibodies become a high fraction of the clone.


In many cases, a single family of V gene segments is used consistently to respond to a particular antigen. Upon exposure to an antigen, presumably the V region with highest intrinsic affinity provides a starting point. Then somatic mutation increases the repertoire. Random mutations have unpredictable effects on protein function; some inactivate the protein, others confer high specificity for a particular antigen. The proportion and effectiveness of the lymphocytes responding is increased by selection among the lymphocyte population for those cells bearing antibodies in which mutation has increased the affinity for the antigen.




Reviews
French, D. L., Laskov, R., and Scharff, M. D. (1989). The role of somatic hypermutation in the generation of antibody diversity. Science 244, 1152-1157.
Kocks, C. and Rajewsky, K. (1989). Stable expression and somatic hypermutation of antibody V regions in B-cell developmental pathways. Ann. Rev. Immunol. 7, 537-559.

Research
Kim, S., Davis, M., Sinn, E., Patten, P., and Hood, L. (1981). Antibody diversity: somatic hypermutation of rearranged VH genes.. Cell 27, 573-581.



Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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