Primary antibody production and generation of B-cell memory are two essential components for protective immunity to pathogen. Our previous analysis of the primary and memory response to (T,G)-A--L showed that primary antibody secretion and memory generation are separable events: no primary antibodies are made to the AL epitope, however, AL-specific memory B-cells are generated, they constitute 20-30% of the anti-(T,G)-A--L memory response, and they produce antibody upon memory recall. The signals which determine whether the primary B-cell differentiates to primary antibody secretion or enter the memory pathway are not known. We hypothesized that the nature and strength of the signal transduced by the B-cell receptor (BCR) is key, and that low affinity BCRs is signal for entry to the memory pathway but not for Ig secretion. This hypothesis will be tested by examining the cellular and molecular outcomes of activating primary B-cells whose BCRs differ in epitope specificity and affinity, using VDJ knock-in mice made by recombination. The primary-AL knock-in will express the VDJ from a primary, AL-specific B-cell, the secondary AL knock-in will express that cell's mutated heavy chain (memory cell), and the primary-GT knock-in will express the VDJ from a primary GT-specific B-cell. The physiological outcome of activating these primary B-cells will be established, with emphasis on determining whether the primary-Al, primary-GT and secondary-AL B-cells can form extrafollicular foci, primary antibody forming cells germinal centers and memory B-cells, either in situ or after adoptive cell transfer. in vitro activation studies with these knock-in B-cells will establish whether antigen-specific stimulation leads to similar or different patterns of response, as measured by proliferation, surface phenotype, Ig secretion, and nuclear translocation of transcription factors. The role of Btk in signaling low affinity B-cells for primary antibody secretion vs. memory formation will be examined using xid-VDJ-knock-ins. The results of these studies will aid our understanding of the B-cell activation signals for antibody secretion and memory, and thus provide information helpful in the design of vaccines for multi-determinant pathogens.
