Development of lung epithelial cells occurs in close interaction with surrounding cells, including mesenchymal cells, whilst the role of immune cells during lung formation is less clear. Therefore, we determined the co-development of- and interactions between epithelial and immune cells during the pseudoglandular and canalicular stages in human fetal lungs at post-conception week (pcw) 6, 8, 10, 13 and 18. Thereto, paraffin-embedded human fetal lung sections were analyzed using a 31-marker imaging mass cytometry panel.

We observed that, during the early pseudoglandular stage, keratin-8 (KRT8⁺) epithelial structures appeared which developed into KRT8⁺EpCAM⁺ budding structures. Stretched luminal structures were aligned by KRT8⁺D2-40⁺ cells, a marker for alveolar epithelial- and lymphatic endothelial cells. These stretched structures were surrounded by ?-smooth muscle actin-positive cells. We next visualized CD45⁺ immune cells. We observed that myeloid cells, including CD206⁺CD68⁺ macrophages (present at early pseudoglandular stages) and HLA-DR⁺ cells, dominated the developing pulmonary immune compartment. CD7⁺ innate lymphoid- and natural killer cells, CD20⁺ B cells, and CD66b⁺ neutrophils accounted for relatively low proportions during pcw 6-18 but exhibited robust expansion over developmental age. Interaction analyses revealed HLA-DR⁺ cell accumulation near the KRT8⁺EpCAM⁺ structures.

In conclusion, we showed that immune cells are present in early lung developmental stages and that HLA-DR⁺ myeloid cells are in close interaction with epithelial structures, suggestive of the involvement of immune cells in lung epithelial cell maturation.