This is a proposal to investigate the orderly diversification of the murine B cell specificity repertoire during ontogeny in both conventional and autoimmune strains. Initially, this will be accomplished by analyzing the specificities and idiotypes of antibodies elicited from neonatal B cells from conventional mice responsive to the antigenic determinant (4-hydroxy-3,5-dinitrophenyle)acetyl (NNP). Previously, distinct B cell clonotypes specific for NNP have been found to be expressed in a temporally regulated manner during neonatal development in Ighb bearing mice. The in vitro splenic focus assay will be employed in order to antigen stimulate individual B cells or their precursors from the spleen and liver of neonatal mice of various ages. The monospecific antibodies thus elicited will be analyzed with respect to their fine specificity for antigen and idiotype and the frequency of B cells within the developing neonatal repertoire which possess these specificities will be assessed. Hybridomas which secrete antibodies representative of the neonatal B cell repertoire will also be constructed; these will provide a complimentary means to examine the repertoire of antigen responsive B cells during ontogeny. The heavy and light chain variable region genes expressed by these neonatal hybridomas will be analyzed by restriction enzyme analysis on Southern blots, by assignment to particular variable region gene families, and, ultimately, by sequence analysis. These experiments will provide insight into the mechanisms responsible for the temporal acquisition of the B cell repertoire during ontogeny and the relationships among the variable region genes which encode antibody specificities acquired at different ages. These studies will be extended to include inbred strains, such as the NZB and BXSB mice, which develop spontaneous autoimmune disease. Such strains exhibit unusual patterns of B cell development which include an early, precocious development of B lineage cells during ontogeny followed by a diminution in the proportion of B cell progenitors in the bone marrow of adults. The influence of the abnormal B cell development apparent in these strains upon the diversification and expression of the B cell repertoire both during ontogeny and in the mature and generative (pre-receptor) B cell pools of adults will be assessed. These experiments will constitute the first comprehensive study of the development of the B cell repertoire in the autoimmune strains and will contribute to an understanding of the immune defects present in autoimmunity.
