Immunophenotyping of tissue immune responses in Chronic Lung Allograft Dysfunction after lung transplantation

Abstract

Background Long-term survival after lung transplantation remains limited, mainly due to the development of chronic lung allograft dysfunction (CLAD), with its main phenotypes bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). The pathophysiology of CLAD is multifactorial, yet the exact immunological drivers are poorly understood. To investigate the immunological processes in CLAD, a systematic review was performed, followed by detailed immunophenotyping of human lung transplant tissue using imaging mass cytometry with laser ablation of regions of interest (ROIs). Methods Explanted lung tissue from 23 recipients, 20 with and 3 without CLAD, was sectioned and stained with a 40-plex antibody panel before 81 ROIs from airways, blood vessels and lung parenchyma were ablated. 190,851 single cells across 41 mm2 tissue were captured before 26 distinct immune and structural cell populations were identified and interrogated across CLAD phenotypes. Results The systematic review confirmed that alloreactive T and B cells, neutrophils and eosinophils are key drivers of CLAD. Our findings support this, with evidence of classical cellular (cytotoxic T cells) and humoral (B cells, especially plasma cells) immune responses, alongside infiltration of eosinophils. Within CLAD, BOS was characterised by increased γδ T cells and non-classical M2 macrophages, but RAS by an increase in Th1 cells and intermediate M2 macrophages. Fibrotic remodelling of airways and parenchyma was associated with common cell profiles; however, different profiles in RAS (M2 macrophages, Th1 cells) and in BOS (γδ T cells) were identified. Conclusion In-depth immunophenotyping of cells in their local tissue microenvironment identified major differences in CLAD versus non-CLAD and between BOS and RAS. The innate immune system appeared to be more activated in BOS, while more pronounced alloimmune and repair responses were observed in RAS. Our findings in the fibrotic progression of CLAD suggest γδ T cells and M2 macrophages in particular merit further investigation.