The proposed research is intended to extend our analyses of humoral immune responsiveness at the level of individual B cells isolated in fragment culture. Using fragment cultures derived from carrier-primed irradiated recipients of limiting dilutions of B cells from various sources, it has been possible to demonstrate that in this system: a) a majority of specific donor B cells do respond to give clones antibody forming cells, b) the parameters of both tolerance induction and stimulation including isotype control can be assessed in vitro and at the most sensitive level (responsiveness per B cell), c) the monoclonal antibodies produced are homogeneous and can be scrutinized as to isotype and clonotype (including isofocusing, idiotype, and possibly amino acid sequence), and d) B cells representing various sub-populations including fetal and neonatal B cells can be assessed for competence and diversity. Thus the methods are now available to characterize the adult B cell repertoire and its development and to attempt an evaluation of the genetic and environmental forces which determine its expression. In order to continue this analysis of the B cell repertoire, its acquisition and interactions, we propose these future investigations: A) We will extend our """"""""library"""""""" of identifiable murine clonotypes which currently includes several anti-PC specific clonotypes and neonatal DNP and TNP specific clonotypes to allow a more definitive characterization of these as well as a large variety of clonotypes specific for other determinants. B) We will attempt to increase the yield of monoclonal antibodies by transforming antibody forming cells and attempt to internally label monoclonal antibodies to facilitate defined analyses. C) Finally, we will extend our analyses of the mechanism of B cell triggering, isotype expression (M yields T switch), and tolerance induction with particular emphasis on the role of a) receptor affinity for antigen, b) antigen form, c) specific syngeneic and allogeneic T cells, and d) idiotype specific suppression.
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