C96-05 Ex Vivo Lung Perfusion as a Novel Model System of the Lung Resident Humoral Immune Response to Influenza

Rationale Lung tissue resident humoral immunity is critical for protection from respiratory diseases like influenza and SARS-CoV2. Studies of non-transplanted explant tissues from human organ donors have identified cells transcriptionally similar to murine lung-resident B cells, however these analyses are limited to a single time point. Murine studies demonstrate that lung flu-specific B cells exhibit differential fates, migration properties and plasma cell differentiation potential as early as 24 hours post-infection, underscoring the importance of studying flu-specific B cells recalled to human lung over time after infection. Ex vivo lung perfusion (EVLP) is a clinical bridge to transplant modality that maintains human lung viability and has been used for immunologic, virologic and genomic studies. We hypothesized that EVLP can be adapted to facilitate dynamic studies of the humoral immune response to influenza that is recalled to the human lung longitudinally after infection. Methods To test this hypothesis, we placed N = 7 lungs on normothermic EVLP with matched blood as perfusate for 24 hours. We endobronchially challenged the right middle lobe of N = 7 human lungs with varying doses of California09 H1N1 virus diluted in Indigo Carmine blue [Fig 1A] and obtained bronchoalveolar lavage fluid every 6 hours. We used the left upper or lower lobe as an uninoculated control. We biopsied influenza-inoculated and uninoculated lung tissue for histologic and spatial transcriptomic studies. CD45+ cells were collected for scRNAseq. We used hemagglutinin (HA) B cell tetramers to enumerate influenza-specific or HA+ B cells in the lung parenchyma for B cell enriched flow cytometric, scRNAseq and spatial transcriptomic analyses (Visium). Results We identified cells staining positive for influenza nucleoprotein via immunohistochemistry at 12 hours after EVLP. These cells localized only to inoculated segments, suggesting a productive infection [Fig 1B, C]. We found an increase in total and cycling CD4 and CD8 T cells in BAL in inoculated over uninoculated lung segments, similarly suggesting a productive infection. We enumerated leukocytes recruited to lung parenchyma via flow cytometry that had sufficient viability for scRNAseq and spatial transcriptomic analyses (10XGenomics). Within the CD45+ scRNAseq data, we observe enrichment of CD8 T cell and NK cell populations in the inoculated sample, and identify distinct populations of memory and naïve B cells. Conclusions EVLP is a successful model to study human lung immune responses to viral pathogens, like flu. Future studies will characterize the B cell enriched scRNAseq and spatial transcriptomic properties of influenza inoculated versus uninoculated segments. This abstract is funded by: NIH