PROJECT SUMMARY Project 3: Evolution, dynamics and durability of B cell and antibody responses in lung transplantation Emerging data suggest that, like lung-resident memory T cells, memory B cells can reside in the lung without recirculating and act as first responders to pulmonary pathogens like influenza and SARS-CoV-2. We termed these cells lung-resident memory B cells or BRM cells. We also showed that lung-resident memory B cells require contact with antigen within the lung in order to initiate their residency program. Taken together, these data suggest that BRM cells are an important component of immunity to pulmonary pathogens. 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. Although lung-resident memory B cells are clearly generated in response to pulmonary infection, it is less clear whether they are generated in response to other types of antigens like auto-antigens or allo-antigens. Interestingly, many pulmonary diseases, like COPD, IPF and ILD, have an autoimmune component in some patients, perhaps as a consequence of persistent inflammation. Moreover, allo-reactive antibodies are often observed in lung transplant patients. Given that auto-antigens and allo-antigens are widely expressed in the lung tissue, it makes sense that lung-resident B cells will respond to these antigens in the lung. Our overall hypothesis is that allo-reactive, auto- reactive and pathogen-reactive B cells in lung allografts are primed and selected locally in the lung, and that the signals and cellular interactions involved in this process are different than those in conventional secondary lymphoid organs. To test this hypothesis, we will take advantage of single cell methods that allow us to define (and compare) individual B cells in blood, bronchalveolar lavage (BAL) fluid and lung tissue 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). We will also use high-dimensional antigen arrays of HLA alleles, auto-antigens, and virus-derived antigens to quantify the reactivity of antibodies in the blood and BAL fluid of lung transplant patients. Using these methods to compare populations of auto-reactive, allo-reactive and virus-reactive-specific B cells in the lung, BAL and blood over time and, in some cases, after infection, we will be able to determine how memory B cells in the lung are related to one another, the depth of their selection, the extent of their cross- reactivity and their ability to respond to local antigens. This information will be informative about the evolution of donor-specific antibodies in the context of lung transplant, the potential role of auto-antibodies in the lungs of t...