This is a proposal to study the differentiation of B lymphocytes and the process of diversification under defined conditions using an in vitro fetal organ culture system. The development of antigen responsive B cells in organ culture will be assessed at the clonal level using the splenic focus assay. We intend to study the developmental acquisition of the B cell repertoire in vitro using a variety of antigen probes as well as anti-idiotypes probes for following individual clonotypes, and compare it with in vivo findings. In addition, the ontogenetic appearance of functional B cells thus determined will be correlated with the ontogenetic appearance of cell surface markers. Since in the experiments described, newly developing B cells, which should be uninfluenced by the environment, will be used, the system will provide a unique opportunity for delineating genetic vs. environmental factors that may influence the expression of the immune repertoire. Thus, we plan to study the effects of anti-allotype, anti-idiotype, anti-light chain, mitogens, biologically active factors (e.g. T cell growth factors), "bursapoetin", and tolerogen on the development of the immune repertoire. Moreover, with the many probes we have available to assess ongoing diversification in organ culture, we intend to study the metabolic requirement of the diversification process by adding various metabolic inhibitors to the culture and determining their effect on the appearance of particular clonotypes. Finally, we will attempt to determine the importance of compartmentalization on B cell differentiation and antibody diversity by comparing the kinetics of appearance of various antigen reactive B cell clonotypes in fetal liver vs. fetal spleen organ cultures.