The adult murine B-cell repertoire is extraordinarily diverse and consists of greater than 107 unique antibody specificities. The extent and specificity of this expressed repertoire appears to be primarily a product of genetic and environmentally-induced regulatory processes. The overall goal of this proposal is to examine these variables with an antigen which cross-reacts with the normal flora of the host, the gram negative bacterium, Salmonella typhimurium and will be accomplished through the completion of 3 specific aims. First, the splenic B-cell repertoire specific for S. typhimurium will be analyzed in germfree (GF) mice using a modification of the splenic fragment assay developed in this laboratory for conventional mice (CV). These studies will permit an estimate of the size of the repertoire at the B-cell precursor level, a determination of the isotype distribution pattern of the antibodies produced by the precursors, and a fine specificity analysis of the antibody products. The splenic salmonella-specific B-cell repertoire of GF mice which have been re- associated with either one or more members of the normal flora will also be analyzed. Secondly, hybridoma technology will be employed to study the utilization of immunoglobulin VH/VL gene families in pre-immune GF and CV mice and in mice deliberately exposed to antigen and will permit a comparison of primary/memory salmonella-specific responses. Thirdly, the effect of chronic antigen exposure on B-cell repertoire development will be evaluated by examination of the pre- and post-immune repertoires in the Peyer's Patches of GF and CV mice. These studies will be the first to examine the regulation of the expressed B-cell repertoire in a system which has not been manipulated by environmentally cross-reactive epitopes. Also, since we will be able to re-associate GF mice with one or more members of the normal flora, we will have the unique opportunity to systematically analyze the events which help to shape the expressed repertoire. Moreover, these studies should provide insights into the establishment of a critically important subset of the B-cell repertoire, that which is specific for a pathogenic bacterium. The proposed studies derive medical significance from the fact that an increased understanding of the variables which regulate repertoire expression may permit manipulation of the system, either to enhance the immune response by the host, or to inactivate host responses. Clearly, a knowledge of genetic and environmental variables should facilitate an understanding of the mechanisms.
