Molecular Analysis and Lineage Tracing of Influenza-Specific, Lung-Resident Memory B Cells

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ABSTRACT Our recently published data show that memory B cells elicited by influenza infection reside in both lymphoid tissues and the lung (Allie et al Nat Immunol 2019). Memory B cells in the lung do not recirculate and have properties, including broad cross-reactivity, that distinguish them from their lymphoid counterparts. We termed these cells lung-resident memory B cells or BRM cells. Influenza-specific BRM cells rapidly colonize the lung, where they reside in both the lung tissue and the lung airways for months. Upon challenge infection, lung BRM cells differentiate in situ and become antibody-secreting cells (ASCs) that contribute to secondary protection. Our data also show that antigen deposition in the lung is essential for BRM cell formation – perhaps because BRM cells are generated locally in inducible Bronchus-Associated Lymphoid Tissue (iBALT) or because BRM precursors generated in lymph nodes need to re-encounter antigen in the lung in order to become resident in that location. Taken together, these data suggest that BRM cells are an important component of immunity to influenza, however we have only a rudimentary understanding of where BRM cells come from, how they are selected, what antigens they react with and how they are recalled (or not) after vaccination or secondary infection. Our central hypothesis is that BRM cells in the lung are formed from a distinct subset of responding B cells, are uniquely selected for broad reactivity, and respond exclusively to respiratory antigens. To test this hypothesis, we will take advantage of single cell methods that allow us to define individual B cells by a combination of transcriptome (single cell RNseq), BCR clonotype (single cell BCRseq), DNA-barcoded antibodies to surface markers (CITEseq) and affinity/specificity/cross-reactivity of BCRs cloned and expressed as recombinant antibodies (single cell cloning). Using these methods to compare populations of influenza- specific B cells in the lung, draining lymph node, spleen and blood over time and after challenge infection or vaccination, we will be able to determine how memory B cells in various tissues (particularly the lung) are related to one another, the depth of their selection, the extent of their cross-reactivity and how they can be recalled by either vaccination or infection. This information will give us a clear path forward in designing vaccines that elicit broadly reactive BRM cells that home to the lung and provide long-lived immunity against a wide variety of influenza subtypes.