PURPOSE: The immunological drivers of chronic lung allograft dysfunction (CLAD), the major barrier to long-term survival after lung transplantation, are poorly understood at a tissue level. Tissue imaging using mass spectrometry with laser ablation of regions of interest offers single-cell resolution of distinct immune cell populations and their spatial relationships and may improve our understanding of CLAD pathophysiology. METHODS: Lung tissue from 23 lung transplant recipients, 20 with and 3 without CLAD, was sectioned and stained with a 40-plex antibody panel before 81 regions of interest from airways, blood vessels and lung parenchyma were laser ablated. RESULTS: 190,851 individual segmented cells across 41 mm CONCLUSION: Imaging mass cytometry enables in-depth analyses of immune cell phenotypes in their local microenvironment. Using this approach, we identified major differences in cell populations in CLAD versus non-CLAD and in BOS versus RAS, with novel insights into the fibrotic progression of CLAD.