This proposal is to continue studies intended to define the molecular and cellular processes responsible for expression of the B cell repertoire and the mechanisms and consequences of antigenic triggering of B cells. Although it is clear that the combinatorial association of multiple available V gene segments, junction flexibility, and somatic mutation could give rise to an enormous variable region repertoire, several findings now indicate that these processes may not be totally random. This is exemplified by findings indicating that: a) certain clonotypes recurr within the newly generated, sIg-, bone marrow precursor cell population of normal adult mice at disproportionately high frequencies; b) the repertoire of fetal and neonatal precursor cells reflects preferential Vh gene segment utilization; and c) the precursor cell repertoire of aged mice includes high frequencies of novel clonotypes that are rare or absent in younger animals. We will continue to identify and analyze, at the molecular level, clonotypic expression that evidences such non-randomness in V region utilization in neonates and sIg- populations of adult and aged mice in an effort to delineate the mechanisms that control variable region generation and expression in the primary B cell repertoire. Studies will also be conducted to analyze the progenitors of secondary B cells, the parameters of their stimulation, and the expression of variable region genes and somatic mutations in these cells and thier clonal progeny. Comparisons of repertoire expression in environmentally naive sIg- vs mature sIg+ precursor cell populations have revealed that several clonotype sets are much more highly represented within the sIg- precursor cell pool. It is likely that such disparities evidence environmental down-regulation in repertoire expression as B cells mature. We will investigate further these disparities in order to define the contribution to mature B cell repertoire expression of environmental immunoregulatory processes and the mechanisms by which such processes operate. Finally, we have been able to establish several experimental systems which give great promise for enabling a detailed analysis of B cell triggering. We will utilize these approaches to further examine the membrane and intracellular requisites for tolerance induction vs stimulation, and the basis for differences in the affinity threshold of receptor-antigen interactions required to trigger immature vs mature precursor cells.
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