Project Summary Each individual elicits a diverse and complex mixture of different B cell clonal lineages after vaccination or infection, some of which are retained as memory B cells or plasma cells. However, the relationships between antibody specificity and affinity in determining which B cell lineages persist, which cellular compartments they occupy, and what future antigens they are able to recognize are not well understood. Detailed molecular understanding of the changes that decrease vaccine responses in old age has also been an elusive goal. We aim to overcome these barriers by studying a 5-year longitudinal cohort of healthy young adult and elderly human subjects vaccinated with seasonal influenza vaccine, as well as a cohort of deceased organ donors whose spleen, lymph nodes, bone marrow, and blood are sampled. We will compare the antibody repertoires and binding specificities of influenza-specific cells found in memory B cell pools compared to blood plasmablasts and a distinct CD71+ subset of ?activated B cells? in the longitudinal cohort, and memory B cells in lymph nodes and spleen compared to bone marrow plasma cell (BMPC) pools in the organ donors. By studying the influenza hemagglutinin (HA) specificity of unmutated ancestors of antibody lineages, and the mutated memory B cell, plasmablast, activated B cell and BMPC progeny, we will determine: the extent to which somatic mutation alters the HA specificity of later members of the clonal lineage; whether future vaccine responses can be predicted from an individual's memory B cell pool; whether BMPCs are subject to different selection criteria than the memory B cells in the lymph nodes, spleen and blood; and the impact of human aging on each of these critical components of humoral immunity. This knowledge will help to shape strategies for improved vaccines for influenza, as well as the whole range of emerging pathogens for which new vaccines need to be developed.