Spatial transcriptomics reveals differences in bronchus-associated lymphoid tissue composition in stable and...

Lung allograft rejection is determined by pathologist grading of transbronchial biopsies (TBBx) according to criteria established by the International Society of Heart and Lung Transplantation (ISHLT). Lymphocytic bronchiolitis (LB), described as mononuclear cells in bronchiolar submucosa, exhibits significant histologic overlap with bronchus-associated lymphoid tissue (BALT) leading to diagnostic uncertainty. Additionally, the role of BALT in tolerance and rejection is debated. We sought to characterize lymphoid aggregates in ISHLT acute cellular rejection (ACR) grades A0 or A3 biopsies to better understand the spectrum of BALT and rejection lesions.

TBBx were reviewed for the presence of BALT. Representative A0 and A3 biopsies were selected for spatial transcriptomics and multiplex immunofluorescence microscopy.

Spatial transcriptomics of allograft biopsies enabled unbiased identification of BALT and rejection lesions via neighborhood analysis. CXCL9/10⁺ T and PDL1/L2⁺ myeloid cell clusters were enriched in A0 BALT, while an NKG7⁺CD8⁺ T cell cluster was enriched in A3 BALT. Higher expression of CXCL9 and CXCL10 in the BALT correlated with A0 grade. Computational methods identified occult areas of lymphoid aggregates in rejecting lung parenchyma with similarity to rejection lesions. A3 BALT and rejection lesions showed substantial similarities. Immunofluorescence confirmed the key transcriptomics findings.

This study suggests a relationship between the immune microenvironment in BALT and graft rejection. Additionally, lymphoid infiltration in allografts graded A3 may be more widespread than apparent on Hematoxylin and Eosin (H&E) stain.