The long-term objectives of the research are to understand B cell development, the mechanisms which regulate the antigen-specific stimulation of B cell subsets, and the mechanisms which influence the selective expression of the immunoglobulin (Ig) idiotypic repertoire by B cell subsets. The proposed research focuses on Ig gene usage and idiotypic expression by the B cells in neonatal mice, in mice expressing the X-linked immune deficiency gene (xid), and in normal adult mice whose B cell population is a continuum of heterogeneous cells. These B cell populations are of interest because the Ig repertoire available in neonatal mice for immunity is limited and restricted, and the X-linked immune deficiency syndrome of xid mice is similar to that of humans. A premise of the research is that antigen directs which B cell subsets are stimulated and thus influences the selective expression of the idiotypic repertoire. Therefore, several antigenic forms of (T,G)-A--L are used: the random polypeptide (T,G)-A--L (type 2), the defined peptide TyrTyrGluGluAla coupled to hemocyanin (type 1), and (T,G)-A--L coupled to bovine serum albumin (type 1).
The specific aims are (1) to examine the developmental expression of VH and VL genes specific for the type 1 and type 2 forms of (T,G)-A--L by neonatal B cells, (2) to determine whether neonatal, xid, and normal adult B cells use the same germline Ig genes in response to type 1 and 2 forms of (T,G)-A--L, (3) to examine the restriction versus heterogeneity of the primary versus secondary response of neonatal, xid, and normal adult B cells to these antigens, and (4) to determine whether neonatal, xid and normal adult B cells undergo the same levels of somatic diversification in their expressed Ig genes. The methodology includes the splenic focus assay for B cell cloning, production of monoclonal anti-idiotopic antibodies, idiotypic and idiotopic analysis by radioimmunoassay, and Ig gene analysis by Southern and Northern blots, by DNA cloning, and by RNA sequencing.
