Characterizing Lung Resident B1 cells and their Impact on Lung Homeostasis during Development - PROJECT ABSTRACT Tissue resident innate immune cells contribute to lung homeostasis by orchestrating local immunity and regulating inflammation in a tissue-specific manner. Thus far, characterization of homeostatic mechanisms has been largely focused on myeloid populations, but the contribution of other innate immune cells to homeostasis is still in its infancy and requires further inquiry. A population of innate-like B cells, B1 cells, has been critically implicated in the innate immune response to inflammatory lung diseases and infections. Notably, B1 cells are a resident immune cell described only in the peritoneal cavity (PeC) and were thought to be recruited to the lungs during inflammation. In the PeC, B1 cells perform immunoregulatory functions essential for maintaining homeostasis including spontaneous secretion of large amounts of IL-10 and polyreactive, self-antigen directed ‘Natural’ antibodies (Abs). Recently, B1 cells have been identified as an immunoregulatory resident immune population in the small intestinal lamina propria (SI-LP). In this tissue, B1 cells comprise half of the plasma cell pool and secrete IgA to confer mucosal homeostasis. However, the functional role of B1 cells in developing lungs and contribution to homeostasis remain unknown. Our preliminary studies have identified a transcriptionally distinct B1-cell subset in scRNA-seq data from embryonic lungs that persists throughout aging. High-dimensional spectral flow-cytometry confirmed the presence of a phenotypically distinct B1 cell population, expressing the characteristic markers CD19, IgM, and CD43, in developing lungs and revealed a significant increase in B1 cells during alveolar development. Further, a subset of B1 cells in the lungs expressed IgA, analogous to B1 cells in the SI-LP. Thus, we have formulated the central hypothesis that B1 cells constitute a unique lung resident population with tissue-specific antibody repertoires that contribute to local homeostasis by secreting NAb and regulating the functions of AMs during tissue development. To test this hypothesis, we will complete a characterization of resident B1 cells in developing lungs. The experiments in Aim 1 will determine if lung B1 cells have distinct transcriptional signatures, phenotypic properties, and tissue-specific Ab repertoires compared to other mucosal and non-mucosal sites. This will be completed using CITE-sequencing, spectral flow cytometry and Enzyme Linked Immunosorbent Assays from B cells in distinct anatomical locations. Aim 2 will employ histological techniques and characterization of antibody production in the lungs to investigate whether B1 cells modulate alveolar macrophage phagocytosis of cell debris and pulmonary surfactant to regulate tissue homeostasis during lung development. Finally, we will evaluate the lungs of C57BL/6 and Cd19creBtkfl/fl (B1KO) mice, a B1 cell deficient strain, by flow cytometry and spatial sequencing to determine how B1 cells alter lung development. This highly impactful study will critically identify how a novel tissue resident B cell population impacts pulmonary innate immunity with broad implications in lung development and tissue homeostasis.
