The acquisition of somatic mutations in rearranged variable region genes is a feature nearly unique to B-cells. In vivo, the process appears to occur in the tightly regulated microenvironment of a germinal center. in vitro models have only recently been described. This project is based on preliminary data obtained using in vitro systems for the culture of human B-cells, that demonstrate that the inclusion of activated T-cells in the B-cell cultures results in the acquisition of somatic mutations in rearrangements in virtually every viable B-cell by the termination of a 14 day culture. In contrast, human B-cells cultured in the presence of CD40 ligand and IL-4, but in the absence of T-cells, acquire a partial germinal center phenotype but either do not acquire somatic mutations or acquire them at a vastly reduced frequency. The goal of this application, using these in vitro systems, is to dissect the cellular and molecular interactions occurring between B-cells and T-cells that result in the enhanced acquisition of somatic mutations. The investigators hypothesize that the accumulation of mutations in B-cells in vitro is dependent upon a T-cell signal. The data are consistent with the hypothesis that the proximal signal permitting the accumulation of somatic mutations is delivered by one or more, possibly novel, T-cell ligands whose expression occurs during the course of the in vitro culture period. The investigators postulate that either as a result of anti-CD3 activation directly, or as an event subsequent to interaction with B-cells, T-cells upregulate surface expression of this putative ligand. The primary goal of this project is to identify, clone and characterize this T-cell molecule.
