MECHANISM OF HUMAN B CELL SELECTION AND V GENE DIVERSIFICATION

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Antigenic selection and the associated process of V gene hypermutation have a major influence on the antibody repertoire. These processes act at a stage in B-cell development also critical for the most common forms of human lymphomas. This project characterizes the corresponding cellular and molecular processes using two unique experimental systems developed in the previous grant period. A viral antigen, HIV gp120, was found to selectively bind and activate a large B-cell subset through conserved sequences expressed by the VH3 Ig gene family, through apparent interaction with novel non-CDR framework sites. During clinical HIV infection, the VH3 B-cell population is initially expanded and activated, followed by a selective clonal deletion paralleling the loss of CD4 T cells. These findings can be conceptualized as a precedent for superantigens of the B-cell lineage. The corollary of this concept is that the biologic disturbance of gp120- binding B-cell clones may be an important factor in the humoral immune dysfunction and elevated risk of B-cell lymphomagenesis in these individuals. In the proposed project, we will first define the structural basis of the gp120-VH3 Ig interaction. The antibody motifs and gp120 epitopes required for binding will be defined, and selective peptide or protein antagonists of this interaction will be generated. Second, we will determine the cell biologic processes accounting for HIV- induced VH3 B-cell depletion. We will define their direct response to gp120, and the effectiveness of candidate VH3 depletion mechanisms. Third, we will evaluate the potential role of this superantigen interaction in HIV-associated lymphomagenesis by testing the prevalence of this VH3 Ig subset among HIV-associated versus sporadic lymphomas. A real-time assay for V gene hypermutation has been developed, based on a gene-specific PCR DNA repair assay which detects mutational activity in endogenous Ig VDJ segments of primary B-cells or cell lines. Our studies reflect the predominance of a gene conversion process which is selectively active in a sequence and stage-specific manner for VDJ segments in hypermutation-positive (germinal center) B cells. In the proposed work, our first goal is to ascertain the structural features of these events by genomic isolation and characterization of the gene conversion tracks and candidate donor genes. Second, the primary cell assay will be manipulated with enzyme specific inhibitors and a panel of immunologic stimuli to search for predominant biochemical and cell physiologic processes which may activate or silence V gene conversion. Third, the hypermutation assay will be used to screen and characterize germinal center B-cell lines with constitutive or inducible hypermutation activity.