PROJECT SUMMARY/ABSTRACT B cell memory generated by antigen exposure consists of antibodies and memory B cells (MBCs). The human spleen is well recognized as a major MBC reservoir, but recent studies suggest that its role goes beyond MBC storage. There is evidence that MBCs in the spleen receive signals that modify phenotype and function. In addition, it has been proposed that the spleen serves as an archive of all MBC clones that disperse from sites of formation via the blood. Our objective is to perform the first antigen-specific evaluation of the spleen as an evolving MBC archive that represents an individual’s MBC response potential. We will use the influenza A virus hemagglutinin (HA) as a model antigen, since most adults have a large pool of HA-reactive MBCs modified by multiple exposures to variant HA strains and often retaining an imprint of early-life infection. We will test hypotheses about the composition a splenic HA-reactive MBC archive based on our current strong understanding of HA-specific B cell responses and MBC formation in humans. Experiments will be performed using a large collection of stored splenocytes from surgically removed trauma spleens. Under Aim 1, we will test the hypothesis that the size of splenic MBC populations reactive to HA strains and to the conserved HA stalk domain reflect early-life imprinting. The size of MBC populations will be determined by in vitro stimulation of splenic MBCs and measurement of secreted HA-reactive antibodies by multiplex binding assay. Association of HA reactivity with phenotypic MBC subsets will be analyzed by flow cytometry using 29 surface markers and a set of HA probes. Under Aim 2, we will test the hypothesis that an evolving splenic archive mostly contains large numbers of MBCs that are broadly HA cross-reactive. Individual HA+ MBCs will be index sorted and stimulated to induce antibody-secreting cell formation. Clonal antibodies in culture supernatants will be analyzed to determine HA reactivity and affinity. To evaluate the completeness of the splenic archive, Aim 3 studies will test the hypothesis that the archive contains families of clonally-related MBCs with ranges of somatic hypermutation and reactivities to HA strains recognized only in early life, chronological ranges of HAs that frequently include early HAs, and sets of contemporary HAs. Splenocyte samples shown under Aim 2 to contain MBCs with an early-life HA imprinting signature will be selected for multivariate single cell analysis. Immunoglobulin heavy chain variable gene sequencing, calculation of mutation frequencies, and evolutionary tree analysis of clonal clusters will be performed and correlated with HA reactivity profiles. We expect our investigation of the spleen as an archive of HA-reactive MBCs to extend our understanding of the spleen as a key determinant of an individual’s MBC-mediated immunity. This information is essential for a full appreciation of the potential consequences of splenectomy, disease ...