Claudia Berek

Mutation drift and repertoire shift in the maturation of the immune response.

The halltnark of the immune response is its specificity and the specificity is directly correlated with the affinity of the antigen-antibody interaction. The requirement for high affinity antibodies may be more important than specificity alone, since antibodies are designed to detect soluble antigens which are sometimes capable of inflicting great harm at very low concentrations (e.g. toxins). This may not be required by. or may even be a disadvantage to T-cell responses where the affinity for the ligand involves interactions of the T-cell receptor not only with antigen, but also with other molecules, e.g. those involved in MHC restriction (Yague et al. 1985. Dembic et al. 1986). T cells therefore may not have developed the equivalent of the elaborate mechanism which is the object of this paper. During the course of an antigen-specific immune response, the affinity of the serum increases with time, a phenomenon commonly referred to as maturation of the response (Jerne 1951, Siskind & Benaceraff 1969). Such a maturation results from specific alterations of the structure of the antibody molecules (Steiner & Eisen 1967). What is the precise nature of these alterations, which are the root of the production of high affinity antibodies? There is no doubt that somatic mutation contributes to antibody diversity (Weigert et al. 1970. Bernard et al. 1978, Griffiths et al. 1984). There are many reasons to believe that a mechanism of hypermutation operates within restricted stretches of the DNA to further diversify the genes encoding the antibody molecules (Kim et al. 1981, Gearhart & Bogenhagen 1983). This mutational drift is, however, not the full extent of the change. Major changes in the antibody structures involved result from a shift in the antigen-specific B-cell repertoire over the course of the immune response. In the primary response the most frequent B-cell clones already expressing antibody molecules with a relatively

The Dynamic Nature of the Antibody Repertoire

Antibody production by an animal in response to an immunogen is dictated by the repertoire of B cells at the time of stimulation. The repertoire of B cells, however, is continuously changing. This change is brought about by the dynamic character of the repertoire, and by the antigenic experience of the animal. The naive repertoire is formed by genetic recombination events which are independent of antigenic stimulation. Since the size of the B-cell repertoire only allows the expression of a fraction of the genetic repertoire at any given time, only a random sample of the total potential is available. This constitutes the raw material that must be capable of providing initial recognition to any antigenic challenge to which animals are subjected. This raises the question of how an animal discriminates between self and non-self antigens, an aspect of the response we will not discuss here. We want to restrict ourselves to points in which our experience of the anti-oxazolone response gives informative data. We will therefore concentrate our attention on describing the way in which the antigenic experience influences the available B-cell repertoire. Our general ideas on the subject will be discussed in the context of the model presented in Fig. 1. We will argue that antigensensitive B cells from both the naive and the memory repertoire are modified through hypermutation and selection following each round of antigenic stimulation, so as to continuously refme the pool of memory B cells. One of the important conclusions we draw from otir experiments is that the antigetiic stimulation of memory cells leads to further processes of hypermutation and selection, functioning through a highly organized and purpose-developed procedure, probably operating in the germinal centers (see Fig. 1). We wish to argue that the development of such an elaborate procedure, so far unique to antibody-producing B cells, arose in response to the evolutionary advantage

Dideoxy mRNA Sequencing as a Method to Analyze Antibody Diversity

The antigenic specificity of an antibody (Ab) molecule is determined by the amino acid sequence of the light and heavy chain variable regions. We have analyzed the extent to which various possible mechanisms for generating Ab diversity contribute in an immune response to a simple antigen, the hapten 2-phenyloxazolone. In order to identify the relative contributions of the different mechanisms which can generate Ab diversity (e.g. the use of different germ-line V genes, different combinations of V, D and J gene segments and the role of somatic point mutations), nucleotide sequence information was required. We therefore analyzed the V region expression of a series of oxazolone-specific hybridomas by sequencing the mRNA isolated from these cells. The major advantage of this technique is the speed and ease with which the required sequence information can be generated.