Abstract Rationale Lung transplantation is the only viable therapeutic option for patients with advanced lung diseases. However, half of lung transplant recipients develop chronic lung allograft dysfunction (CLAD) within five years, significantly limiting long-term survival. Interstitial macrophages are enriched within lymphocytic infiltrates in CLAD, yet their ontogeny and functional contributions to disease pathogenesis remain largely unknown. Given their strategic localization - allowing close interactions with endothelial cells, fibroblasts, and other immune cells - we hypothesized that donor-derived perivascular interstitial macrophages play a pivotal role in CLAD development and may represent a potential therapeutic target. Methods To investigate the cellular niches driving CLAD development and the role of perivascular interstitial macrophages, we performed single-cell spatial transcriptomic analysis (Xenium platform, 10x Genomics) on lung tissues from patients with CLAD and healthy donors. We also established an HLA-A2 single-mismatched CLAD mouse model to trace interstitial macrophage lineages, evaluated the therapeutic efficacy of a colony-stimulating factor 1 receptor (CSF1R) antagonist targeting these macrophages, and conducted single-cell spatial transcriptomic analysis on these lungs. Dimensionality reduction and cell clustering were performed using Uniform Manifold Approximation and Projection (UMAP) to visualize transcriptionally distinct cell populations. Differential cell abundance was assessed using Milo, which detects condition-dependent changes in cell populations based on transcriptionally similar neighborhoods. Spatially organized niches were comprehensively characterized using CellCharter, a computational framework that integrates cell identity, spatial localization, and neighborhood interactions to delineate tissue microenvironments. Results Using the Xenium platform, we identified 61 distinct cell types in human lungs and 57 in mouse lungs, uncovering an expanded repertoire of cell populations typically underrepresented in single-cell RNA-seq studies. Perivascular interstitial macrophages were distinguished from nine human and six mouse macrophage subtypes based on their unique transcriptional signatures and spatial localization. Notably, FOLR2-positive perivascular interstitial macrophages were markedly expanded within lymphocytic bronchiolitis-associated niches in both humans and mice, and their donor-derived origin was confirmed via HLA-A2 lineage tracing. Donor-derived perivascular interstitial macrophages exhibited distinct chemokine profiles and activation states compared with recipient-derived interstitial macrophages, suggesting that they uniquely promote the formation of disease-specific niches. Pharmacological inhibition of CSF1R synergistically reduced the number of both donor-derived perivascular and recipient-derived monocyte-derived interstitial macrophages in a murine CLAD model, alleviating lymphocytic bronchiolitis by disrupting their positive feedback interaction with lymphocytes. Conclusions Pathogenic donor-derived perivascular interstitial macrophages represent a promising therapeutic target in CLAD, and modulation of CSF1/CSF1R signaling may offer a novel treatment strategy for affected patients. This abstract is funded by: the Cell Science Research Foundation, the Cugell Fellowship, the Pulmonary Fibrosis Foundation Scholars Program, 5P01HL169188-02, 5R01HL153312-03
Suzuki et al. (2026) studied this question.