Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung at single cell resolution

At birth, the lungs rapidly transition from a pathogen-free, hypoxic environment to a pathogen-rich, rhythmically distended air-liquid interface. Although many studies have focused on the adult lung, the perinatal lung remains unexplored. Here, we present an atlas of the murine lung immune compartment during early postnatal development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed an orchestration of distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability.


Sample-size estimation
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Replicates
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Statistical reporting
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Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table For the single cell studies, each cell is a replicate in its own right. To minimize batch effects, two mice per time point and two plates per mouse were processed across several sequencing runs. Low quality cells were excluded by filtering out cells containing less than 50,000 reads and 400 genes per cell, as shown in the freely available code and stated in the Methods. Non-immune cells were filtered via unsupervised clustering as shown in the code. For the in situ validation studies, and the flow cytometry studies, each individual mouse was a separate biologic replicate. The number of mice used for the studies are detailed in the Figure Legends. Outliers were not removed, and the data was presented as whisker-dot plots to fully disclose the biologic variation between mice.
All statistical analyses are described in the Main text and in the Methods. N numbers and names of the statistical tests are also contained in the Figure Legends where applicable. The code and data to reproduce the analyses is freely available as stated in the Methods.
Female and male mice were randomly selected at each timepoint. Tissue for the in situ studies were obtained from both male and female mice from multiple litters to limit litterspecific effects. These details are included in the Methods. • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Please indicate the figures or tables for which source data files have been provided: The count and metadata tables as well as the scripts to generate the figures for the single cell data are all available online (on FigShare and GEO) as stated in the Methods.