Targeted somatic hypermutation in cultured B cell lines

Abstract

Using the NSO in vitro system that was established in our lab several years ago, we find that not only variable (V) regions of transfected Ig genes undergo relatively high rates of mutation, but that the constant (C) regions of transfected Ig genes also undergo mutation at rate that may be as high as V region. Further study shows that these constant region mutations are the result of large deletions within the constant region. The characteristics of these constant region deletions resemble those reported in human heavy chain disease. In the discussion, we address the issue of whether the mechanism of constant region deletion is related to that of variable region mutation.;The interpretation of the data gathered from our NSO in vitro system is complicated by the fact that NSO is not from the stage of B cell differentiation that normally undergoes somatic hypermutation; and there are multiple copies of transfected Ig genes at ectopic locations in these cells. To solve these problems, we switched from NSO to Ramos, an EBV- human Burkitt's lymphoma cell line that constitutively undergoes a high rate of V region mutation. By isolating mutant Ramos cells that were unable to produce IgM antibody due to the introduction of nonsense mutations in the V regions of their endogenous mu heavy chain locus, we identified mutating subclones of Ramos. We studied reversion of these nonsense stop codons by monitoring the reappearance of surface IgM. Our data from the Ramos in vitro system supports the proposal that somatic mutation primarily targets G.C base pairs. These cells provide us with a chance to study somatic mutation in a single copy, endogenous heavy chain gene in vitro. This system should now allow us to study the regulation and targeting of the somatic mutational process, and to address the relationship between the V and C region mutation. (Abstract shortened by UMI.).