The generation of antibody diversity is a complex and highly regulated process. Immunoglobulin (Ig) gene rearrangement, to form functional variable (V) region genes (which bind antigen) is the major way this diversity is initially generated, and occurs before antigen is encountered. Additional diversity arises during an immune response by somatic mutation and antigen selection of mutated antibodies with higher antigen affinity. The goal of this proposal is to study, in transgenic mice, how expression of chimeric immunoglobulins composed of rearranged murine variable region genes and human constant (C) region genes, affects the rearrangement of endogenous variable region genes and whether such chimeric genes undergo somatic mutation and affinity maturation. Initial studies will be done on mice expressing only a chimeric Ig heavy chain; later studies will be done on mice expressing chimeric heavy and light chains. Two types of chimeric transgenes will be used. Both the heavy and the light chain murine variable regions are derived from a hybridoma making an anti-dansyl response (dansyl is a hapten, which is a small, simple molecule that contains only one antigenic determinant). The heavy chain constructs consist of a murine heavy chain anti-dansyl V region and a human gamma-1 C region, with the murine V/H promoter, intron enhancer, and plus or minus the 3' alpha enhancer. The light chain construct contains a murine light chain anti-dansyl V region and a human kappa light chain C region, with the V/L promoter, intron enhancer, and 3' enhancer. Since the murine V regions are specific for a known antigen, we can study both somatic mutation and affinity maturation in these mice. In particular, we can ask whether a human gamma-1 constant region can send three different types of signals to murine B lymphocytes; one to shut down endogenous V gene rearrangement, a second to initiate somatic mutation and a third to mediate antigen selection. The specific questions to be asked are: 1) does expression of a human gamma-1 constant region inhibit rearrangement of endogenous heavy chain V genes; 2) do the transgenes undergo somatic mutation in these animals; in other words, can the human gamma-1 gene signal the B lymphocyte that it has encountered antigen, and in the presence of T lymphocyte help turn on somatic mutation, and are the transgenes then mutated; and 3) do the transgenes undergo affinity maturation; are cells expressing mutated transgenes with increased affinity for dansyl selectively stimulated to proliferate, resulting in increased antibody affinity over time after immunization.
