Bitplane imaris v9

z-stacks images were rendered in 3D dimensions and quantitatively analyzed using Bitplane Imaris v9.5 software package (Andor Technology PLC, Belfast, N. Ireland). Individual IL5⁺ and IFNγ⁺ lymphocytes were annotated using the Imaris spots function based on the fluorescent reporter signal and using the Ortho slicer function to visualize size, morphology, and nuclear staining (DAPI). 3D reconstructions of alpha smooth muscle actin (αSMA)-labeled structures were performed using Imaris surface function and based on the morphological characteristics of orientation, location, and SMA content to subdivide blood vessels types and airways (Dahlgren et al., 2019 (link)). Three dimensional distances between lymphocytes and SMA⁺ surfaces were calculated using the Imaris Distance Transform Matlab XTension and volumetric decile calculations were performed using a Matlab-based Imaris XTension. Adventitial lymphocytes were defined as spots localized <100μm from SMA⁺ smooth muscle surface and parenchymal lymphocytes as >100μm away from SMA⁺ surfaces.

3D image analysis was performed as previously described ^{12 (link),48 (link)}. Briefly, z-stacks images were rendered in 3D dimensions and quantitatively analyzed using Bitplane Imaris v9.5 software package (Andor Technology PLC, Belfast, N. Ireland). Individual IVCD45+/CD3+ and IVCD45−/CD3+ lymphocytes were annotated using the Imaris spots function based on the colocalization analysis. 3D reconstructions of alpha smooth muscle actin (aSMA)-labeled structures were performed using Imaris surface function and based on the morphological characteristics of blood vessel types and airways. Three dimensional distances between lymphocytes and SMA+ surfaces were calculated using the Imaris Distance Transform Matlab XTension and volumetric decile calculations were performed using a Matlab-based Imaris XTension. Adventitial lymphocytes were defined as spots localized <80-100mm from SMA+ smooth muscle surface and parenchymal lymphocytes as >80-100mm away from SMA+ surfaces.