A single-cell transcriptomic atlas of the pigtail macaque lung

Abstract

Background: The lungs undergo substantial physiological and immunological changes during respiratory illness to meet the host's increased metabolic and oxygen demands. These responses require coordination among epithelial, endothelial, stromal, and immune cells throughout the respiratory zone, interstitium, and vasculature. Nonhuman primates (NHPs) are a valuable model for studying pulmonary biology as they have similar anatomic, physiologic, and immunologic correspondence to humans. Despite the widespread use of NHPs in pulmonary research, the cellular diversity and molecular characteristics of the pigtail macaque lung remain incompletely defined. Methods: Parenchymal lung tissue from five adult female pigtail macaques (Macaca nemestrina) was dissociated into single cells and profiled using 10x Genomics 3' single-cell RNA sequencing (scRNA-Seq). Next, ambient RNA was removed and quality control analyses performed. Samples were then integrated using scVI and scANVI. Cell types were labeled after unsupervised clustering and subclustering, followed by manual annotation using canonical marker genes. We compared lineages and cell types with the Level 4 Human Lung Cell Atlas (HLCA) using scArches reference mapping and with the Rhesus Immune Reference Atlas (RIRA) using Seurat label transfer. Results: The atlas includes 61,323 cells representing 37 cell types in epithelial, mesenchymal, endothelial, lymphoid, and myeloid populations. Annotated populations included both major and rare lung cell types, including alveolar type I and II cells, club and multiciliated cells, aerocyte capillary, and lymphatic endothelial cells, pre-terminal bronchiole secretory cells, CD4+ and CD8+ T cells, and several innate-like lymphocyte populations (gamma delta T cells, MAIT-like cells, innate-like CD8+ T cells, and group 3 innate lymphoid cells). There was strong concordance at HLCA annotation level 4, including AT1 and AT2 cells (96% and 93%), B cells (94 to 97%), CD4+ T cells (99 to 100%), classical and non-classical monocytes (84% and 85%), alveolar macrophages (80%), and mast cells (96%). Immune populations mapped to the RIRA with similar fidelity (alveolar macrophages and B cells, 99%). Conclusions: While studies of pandemic respiratory infections, vaccines, and therapeutics rely on NHP disease models, this atlas provides a unique reference for epithelial, endothelial, stromal, and immune cell states in the pigtail macaque lung. We found that single-cell populations from the pigtail macaque lung are highly similar to those in the human lung at the lineage level. This reference will enable studies of pathogenesis and vaccine and drug efficacy in a highly translational model of the human lung.