Deciphering the Functional Role of Commensal-Induced T-DC Clustering along Sensory Nerves in the Lung

Abstract The airway mucosa is a primary site of the immune response to inhaled antigens. However, how immune homeostasis is maintained in this organ during continuous exposure to airborne particles and microbial antigens is not known. Previous studies have shown that the spatial organization of immune components in relationship to each other and to non-hematopoietic components in tissues is critical to their proper function, as is communication with commensal microbes. To interrogate such relationships in the lung, especially the possible connections between immune cells and the densely innervated pulmonary airway, 3D information is required. However, current methods for 3D imaging of optically cleared tissue do not provide the marker depth afforded by high-content 2D imaging. To overcome this constraint, we combined a fast hydrophilic tissue clearing technique, Ce3D, with the recently developed IBEX technique, and created Ce3D-IBEX. To date, up to 20 markers have been visualized in a single sample. Using Ce3D-IBEX, we identified a previously uncharacterized clustering of T cells and dendritic cells in proximity to the pulmonary sensory nerves in naïve mice. Agonistic denervation of the sensory nerves with resiniferatoxin reduced the number of clusters, and the cluster formation was disrupted in germ-free mice as well as OT-I and OT-II TCR-Tg mice. These findings indicate that the clustering requires sensory nerve-derived signals and TCR-mediated recognition of bacterial antigens. Future studies will be aimed at elucidating the functional role of the clusters in homeostasis and disease conditions and determining the molecular basis for neuro-immune communication that regulates cluster formation and activity. All studies were done with approval from the ICUC of the NIH/NIAID. This work was supported by the Intramural Research Program of NIAID, NIH.