The long term goals are to determine the mechanisms that regulate shifts in the expressed antibody repertoire during the generation of immunological memory. The proposed research focuses on two aspects of the repertoire shift observed during the maturation of the immune response to (T,G)-A--L: (1) the dominance in the primary response of B cells that recognize side- chain specific epitopes (GT+) and use the H10/VK1 germline gene combination and (2) the high frequency in the memory response B cells that recognize epitopes on the A-L backbone. To determine whether the H10/VK1 germline combination has an intrinsically high frequency in the preimmune repertoire, relative to other V-genes, its frequency will be measured in a cell lysate hybridization assay that uses probes specific for these V genes. A mutation/selection model for repertoire shift will be examined by measuring the affinities of primary and memory antibodies using H10/VK1; if selection has resulted in higher affinity memory antibodies, the heavy chains from some of these will be sequenced to determine whether/where mutations have occurred. Mutation/selection may also explain the higher frequency of A-L+ clones in the memory response. This will be determined by analyzing for mutations the heavy chains of A-L+ antibodies derived from clonal progeny (sibs) of a single precursor, which we have shown are in the pauciclonal A-L+ response of mice immunized as neonates. Since one of the clonal bursts uses a VGam3.8 gene, which is from a VH gene family of low complexity, its germline gene equivalent will be identified. A second model for repertoire shift, that primary and memory responses originate from two separate precursor lineages, will be tested. The frequency of GT+ vs A-L+ precursors in J11D-hi (primary lineage) and J11D-lo (memory lineage) B cells will be measured in the splenic focus assay. Adoptive transfer experiments will show whether the repertoire shift is seen with J11D-lo B cells in vivo, and the clonal frequencies of GT+ and AL-memory B cells in the transfer recipients with time after stimulation will be measured. Other adoptive transfer experiments, will (a) measure the need for continuous antigenic stimulation in the repertoire shift, and (b) show whether removal of the dominant primary B cell clonotype (H10/VK1) from the preimmune repertoire modulates the expressed primary and memory repertoire. These studies have relevance for health issues concerning modes of vaccination to establish a memory pool directed to different epitopes on the same immunogen.
