Identification of a novel subset of alveolar type 2 cells expanding following pneumonectomy, responding differently to Fgfr2b deletion and enriched in PD-L1

Abstract

Alveolar type 2 (AT2) cells are a heterogeneous population; where specialized AT2 subpopulations within this lineage exhibit stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage, which become activated during lung regeneration, is unknown. In this study SftpcCreERT2/+; tdTomatoflox/flox mice were used for the labelling of AT2 cells, and two distinct AT2 subpopulations were detected with low tdTomato level (TomLow) and high tdTomato level (TomHigh). TomLow express lower levels of AT2 differentiation markers, Fgfr2b and Etv5, while TomHigh, as bona fide mature AT2 cells, showed higher levels of Sftpc, Sftpb, Sftpa1, Fgfr2b, and Etv5. ATAC-seq analysis indicated that TomLow and TomHigh constitute two distinct cell populations with specific silencing of Sftpc, Rosa26 and cell cycle gene loci in TomLow. Upon pneumonectomy, TomLow but not TomHigh cells proliferated and upregulated the expression of Fgfr2b, Etv5, Sftpc, Ccnd1 and Ccnd2 compared to sham. This study revealed that, TomLow cells overexpress PD-L1, an immune inhibitory membrane receptor ligand, which was used by flow cytometry to differentially isolate these two AT2 sub-populations. In support, in the human lung, PD-L1 and HTII-280 antibodies by flow cytometry were employed to differentially sort mature AT2 (HTII-280-high, PD-L1-low) as well as an additional subpopulation of epithelial cells characterized by HTII-280-low and PD-L1-high. Altogether, in the first part of this study, a novel population of AT2 quiescent immature progenitor cells was identified in mouse that proliferates upon pneumonectomy and provided evidence for the existence of such cells in human. In the second part of this study, the function of Fgfr2b signaling in AT2 subpopulations was investigate using SftpcCreERT2/+; Fgfr2bflox/flox; tdTomflox/flox (Fgfr2b-cKO) mice. Intriguingly, TomLow and TomHigh responded differently to Fgfr2b deletion. Losing Fgfr2b expectedly resulted in Fgfr2b downregulation and consequently decrease in TomHigh cell percentage, whereas in TomLow cells led to Fgfr2b upregulation and their expansion. As a consequence of TomLow to TomHigh transition and replenishment of the lost TomHigh, no lung phenotype was observed. Interestingly, TomHigh cells isolated from Fgfr2b-cKO lungs lost the self-renewal capability in vitro. By contrast, TomLow cells as quiescent and undifferentiated cells displayed weak alveolospheres formation capacity during steady state, while their ability to form organoids was enhanced after Fgfr2b activation. This shed light on the significance of Fgfr2b signaling in AT2 cell self-renewal. Overall, in the second part of this study , our data demonstrated the existence of subsets of TomLow cells which are resistant to Fgfrb2 deletion and they are able to bypass the deletion, expand and replace the lost TomHigh cells.