Project 2: Characterization of Virus-Specific Human B Cell Subsets in Lymphoid and Non-Lymphoid Tissues Project Summary Antibodies against influenza virus protect against infection and reduce morbidity and mortality. As a result, vaccines against influenza are designed to elicit antibodies against circulating strains of virus. Given that antibodies are made by B cells, it makes sense to characterize influenza-specific B cells in order to determine how they are selected, how they function and where they are maintained. However, most studies of influenza- specific B cells in humans are limited to B cells from peripheral blood, rather than those in lymphoid organs or the lung. Moreover, most studies characterize influenza-specific antibody-secreting cells (ASCs), which circulate for a short period after infection or vaccination. Consequently, we have minimal understanding of influenza-specific memory B cells or ASCs in human tissues. Thus, there is an urgent need to characterize the phenotypes and functions of influenza-specific B cells in human lymphoid and non-lymphoid tissues. Given this gap in knowledge, we are proposing experiments that will use ?B cell tetramers? to identify influenza-specific B cells responding to hemagglutinin (HA), nucleoprotein (NP) and non-structural-1 (NS1) proteins of influenza virus in blood, lymphoid tissues, lung and visceral adipose tissues of normal human donors. We will then determine how the phenotypes, transcriptional programs, functional properties, reactivities and affinities of those B cells differ between tissues and between B cells of different specificities. Importantly, we will use an automated, single-cell cloning and antibody-expression system to rapidly and efficiently generate recombinant, influenza-specific antibodies from sorted memory B cells in order to determine their affinity and cross-reactivity. Therefore, we believe that this project is highly innovative and will significantly advance our understanding of influenza-specific B cell biology in various human tissues.
