LPL is required for engraftment into the alveolar space. (A) Schematic of competitive transfer experiment. Fetal monocytes were sorted from the lungs of congenically marked WT (CD45.1⁺/CD45.2⁺) and LPL^−/− (CD45.2⁺) neonatal pups. Precursors were mixed in equal proportions and transferred by intranasal (i.n.) administration into congenically marked WT (CD45.1⁺) neonatal pup recipients. Neonatal pups were employed as donors and recipients so as to evaluate engraftment and alveolar macrophage generation during the physiologically relevant developmental window. After 1 week, lungs were harvested and analyzed for alveolar macrophages derived from donor monocytes. (B) Representative flow cytometry from 1 recipient pup, demonstrating gating for mature alveolar macrophages and determination of origin. Flow cytometry plots showing controls for gating of CD45.1, CD45.2, and CD45.1/CD45.2 populations shown in supplemental Figure 2. (C) Percentage of alveolar macrophages derived from either WT (gray circles) or LPL^−/− (black circles) donor pups. Each symbol represents data from 1 recipient animal from 2 independent experiments. (D) Schematic of competitive transfer experiment in which alveolar macrophages were isolated from BAL fluid of congenically marked adult WT and LPL^−/− mice, mixed and coinjected intranasally into marked recipient PND1 pups. After 24 or ≥72 hours, lungs were harvested and analyzed for alveolar macrophages derived from donor mice. (E) Representative flow cytometric analysis of a recipient pup to evaluate presence of donor alveolar macrophages. As neonatal development of alveolar macrophages was actively ongoing in recipients, the proportion of transferred alveolar macrophages was low. (F) Ratios of LPL^−/−-derived:WT-derived alveolar macrophages recovered from recipient pups at the indicated times after adoptive transfer. We delivered more alveolar macrophages from LPL^−/− mice to ensure detection of LPL^−/−-derived cells after 24 hours.