MOLECULAR AND KINETIC-ANALYSIS OF AN EPITOPE-SPECIFIC SHIFT IN THE B-CELL MEMORY RESPONSE TO A MULTIDETERMINANT ANTIGEN

Our previous studies showed that the primary and memory B cell respons es to the multideterminant antigen poly-(L-Tyr, L-Glu)-poly-D,L-Ala-po ly-L-Lys ((T,G)-A-L), differ. The primary response is dominated by ant ibodies binding side-chain epitopes; there is little antibody response to epitopes on the poly-D,L-Ala-poly-L-Lys backbone of (T,G)-A-L. In contrast, B cells producing A-L+ antibodies constitute approximately a third of the memory response to (T,G)-A-L. To determine the basis of this epitope-specific repertoire shift, we have examined the kinetics of expression of A-L+ B cells and antibodies after in vivo antigen pri ming and identified VH and Vkappa genes used by A-L+ hybridoma antibod ies derived from primary vs memory B cells. Kinetic studies, using the splenic focus assay, showed that the clonal frequency of A-L+ B cells remains low (<3% of (T,G)-A-L-specific B cells) 1 wk after Ag priming , increases (9%) by 2 wk, but does not reach the memory frequency (30% ) until at least 3 wk after immunization. Molecular analyses showed th at both the primary and memory A-L+ antibody responses are heterogeneo us, using different VH and Vkappa gene families as well as different g erm-line genes within a VH gene family. Both H and L chain gene sequen ces showed somatic mutations in primary as well as memory antibodies. Analysis of antibody binding patterns and somatic mutations in a set o f clonally related B cells that use a new germ-line VH gene in the VGA M3.8 family (VGK7, described here), showed a direct correlation betwee n somatic mutation and change in antibody binding specificity. Our res ults demonstrate how somatic mutation and Ag selection play a role in the development of the memory response to a multideterminant Ag. The d ata are discussed in the context of the single vs dual lineage models for memory B cell generation.