Identification of a novel subset of alveolar type 2 cells enriched in PD-L1 and expanded following pneumonectomy

Abstract

Alveolar type 2 (AT2) cells are heterogeneous cells, with specialised AT2 subpopulations within this lineage exhibiting stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage that are activated during lung regeneration is unknown.Sftpc^CreERT2/+;tdTomato^flox/flox mice were used for the labelling of AT2 cells and labelled subpopulations were analysed by flow cytometry, quantitative PCR, assay for transposase-accessible chromatin using sequencing (ATAC-seq), gene arrays, pneumonectomy and culture of precision-cut lung slices. Single-cell RNA-sequencing (scRNA-seq) data from human lungs were analysed.In mice, we detected two distinct AT2 subpopulations, with low tdTomato level (Tom^Low) and high tdTomato level (Tom^High). Tom^Low cells express lower levels of the AT2 differentiation markers Fgfr2b and Etv5, while Tom^High, as bona fide mature AT2 cells, show higher levels of Sftpc, Sftpb, Sftpa1, Fgfr2b and Etv5 expression. ATAC-seq analysis indicates that Tom^Low and Tom^High cells constitute two distinct cell populations, with specific silencing of Sftpc, Rosa26 and cell cycle gene loci in the Tom^Low population. Upon pneumonectomy, the number of Tom^Low but not Tom^High cells increases and Tom^Low cells show upregulated expression of Fgfr2b, Etv5, Sftpc, Ccnd1 and Ccnd2 compared to Sham. Tom^Low cells overexpress programmed cell death 1 ligand 1 (PD-L1), an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two subpopulations. In the human lung, data mining of a recent scRNA-seq AT2 data set demonstrates the existence of a PD-L1 ^Pos population. Therefore, we have identified a novel population of AT2 quiescent, immature progenitor cells in mouse that expand upon pneumonectomy and we have provided evidence for the existence of such cells in human.

FIGURE 1

Identification of two subpopulations of AT2-lineage-labelled cells, named tdTomato^Low (Tom^Low) and tdTomato^High (Tom^High). a) Timeline of tamoxifen treatment of Sftpc^CreERT2/+;tdTomato^flox/flox mice (n=4). d: day. b) Representative flow cytometry of EPCAM^Pos population selection and identification of Tom^Low (8.5%) and Tom^High (44.2%) populations based on the tdTomato level. c) Percentage of Tom^Low (16.3%) and Tom^High (83.7%) in total tdTomato^Pos cells. d) PCR on genomic DNA isolated from fluorescence-activated cell sorting (FACS)-based sorted Tom^Low and Tom^High cells from Sftpc^CreERT2/+;tdTomato^flox/flox mice for Stop codon. e) Timeline of tamoxifen treatment of Sftpc^CreERT2/+;tdTomato^flox/+mice and f) representative flow cytometry analysis of Tom^Low (7.3%) and Tom^High (38.7%). g) The pie chart shows the percentage of Tom^Low (15.3%) and Tom^High (84.7%) in total tdTomato^Pos cells. h) PCR on genomic DNA isolated from FACS-based sorted Tom^Low and Tom^High cells from Sftpc^CreERT2/+;tdTomato^flox/+ mice. i) Quantitative PCR analysis of FACS-based sorted Tom^Low and Tom^High cells for the expression of Tomato, Fgfr2b, Etv5, Sftpc, Sftpb and Sftpa1. dCT: delta cycle threshold. Data are presented as mean±sem. *: p<0.05; **: p<0.01; ***: p<0.001.

FIGURE 2

Assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis on fluorescence-activated cell sorting (FACS)-based sorted tdTomato^Low (Tom^Low) and tdTomato^High (Tom^High) populations. a) Timeline of tamoxifen treatment of Sftpc^CreERT2/+;tdTomato^flox/flox mice (n=3). ATAC-seq was carried out on FACS-based sorted Tom^Low and Tom^High subpopulations. d: day. b, c) Coverage heat maps of Tom^Low and Tom^High, displaying genome-wide regions of differential open chromatin peaks in Tom^Low versus Tom^High. Tom^Low chromatin is less open and transcriptionally less active compared to Tom^High. Ctrl-low shows the peaks related to open chromatin regions in Tom^Low. Ctrl-high shows the peaks related to open chromatin regions in Tom^High. Common shows peaks detected in both Tom^Low and Tom^High subpopulations. d) ATAC-seq analysis of peaks based on the cut-offs shows 3605 upregulated in Tom^High (false discovery rate (FDR)<0.05, log₂(FC)>0.585, base mean>20), 3512 upregulated in Tom^Low (FDR<0.05, log₂(FC)>0.585, base mean>20) and 3878 non-regulated (base mean>20, FDR>0.5, log₂(FC) between −0.15 and 0.15), which means 32% and 32.2% of the genome is differently accessible in Tom^Low and Tom^High, respectively. e) ATAC-seq histogram of average read coverage at the Rosa26 locus shows distinct ATAC-seq peaks at the promoter and denser chromatin in Tom^Low compared to Tom^High in this locus. Representative peaks of Tom^Low and Tom^High are the average of three independent samples. f) Quantification of peaks at the Rosa26 locus. g) ATAC-seq profile at Sftpc locus shows distinct ATAC-seq peaks at the promoter and denser chromatin in Tom^Low compared to Tom^High. Representative peaks of Tom^Low and Tom^High are averages of three independent samples. h) Quantification of peak regions of Sftpc locus. The ATAC-seq data have been normalised for sequencing depth and the scale on the y-axis was chosen for optimal visualisation of peaks. i) ATAC-Heat-pVAI Z-score, Pearson, average heat map based on the ATAC-seq data of top cell cycle-related genes differentially regulated in Tom^Low and Tom^High. FDR: the significance of results by Benjamini–Hochberg correction of multiple tests (n=3).

FIGURE 3

Different colony-forming capabilities of tdTomato^Low (Tom^Low) and tdTomato^High (Tom^High) cells. a) Representative flow cytometry shows the gating strategy of CD31^Neg CD45^Neg EPCAM^Neg population and further selection of SCA1^Pos resident mesenchymal cells from C57BL/6 lungs (upper plot), as well as the selection of Tom^Low and Tom^High from the EPCAM^Pos population from Sftpc^CreERT2/+;tdTomato^flox/flox(lower plot). Mesenchymal cells were co-cultured with Tom^Low and Tom^High separately (n=3). d: day. b) Representative alveolospheres from Tom^Low and Tom^High (n=3). Scale bar: 100 μm. c) Representative SFTPC and RAGE immunofluorescence staining of alveolospheres after 14 days in culture. Scale bar: 50 μm. d) Quantification of colony-forming efficiency (CFE) and e) alveolospheres size in Tom^High and Tom^Low (n=3). Data are presented as mean±sem. *: p<0.05.

FIGURE 4

Expansion of tdTomato^Low (Tom^Low) but not tdTomato^High (Tom^High) following pneumonectomy (PNX). a) Schematic representation of the PNX and Sham models. b) Representative flow cytometry analysis of Tom^Low and Tom^High populations 7 days after PNX and Sham, and quantification of c) Tom^High and e) Tom^Low percentages in EPCAM^Pos population between Sham and PNX groups (n=4). f) Quantitative PCR (qPCR) analysis of fluorescence-activated cell sorting (FACS)-based sorted Tom^Low population for expression of Fgfr2b, Etv5, Sftpc, Ki67, Ccnd1 and Ccnd2. g) qPCR analysis of FACS-based sorted Tom^High population for expression of Fgfr2b, Etv5, Sftpc, Ki67, Ccnd1 and Ccnd2. Data are presented as mean±sem. dCT: delta cycle threshold. *: p<0.05; **: p<0.01; ***: p<0.001.

FIGURE 5

Characterisation of the fate of tdTomato^Low (Tom^Low) cells in precision-cut lung slices (PCLS). a, b) Representative flow cytometry analysis of Tom^Low and tdTomato^High (Tom^High) before processing the lungs for PCLS in freshly generated PCLS (a) and PCLS after 20 h culture (b). c) Visualisation of the Tom^Low in PCLS at t=0, t=96 h and t=240 h. Arrows indicate the formation of cell clusters. Scale bars: 250 μm (low magnification) and 50 μm (high magnification). d) Analysis of apoptosis with representative terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) immunofluorescence staining on the PCLS (scale bar: 50 μm) and quantification of e) tdTomato^Pos TUNEL^Pos cells out of total cells, f) TUNEL^Pos cells out of total cells and g) tdTomato^Pos TUNEL^Pos cells out of tdTomato^Pos cells (n=4). Data are presented as mean±sem. h) Analysis of proliferation with representative EdU immunofluorescence staining on the PCLS (scale bar: 50 μm) and quantification of i) tdTomato^Pos EdU^Pos cells out of total cells, j) EdU^Pos cells out of total cells and k) tdTomato^Pos EdU^Pos cells out of tdTomato^Pos cells (n=4). Data are presented as mean±sem. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 6

PD-L1 is a specific surface marker enriched in tdTomato^Low (Tom^Low) cells. a) Validation of gene array data by quantitative PCR (qPCR). d: day. b, c) Quantification of the expression levels of Cd33 (b) and Pd-l1 (c) in Tom^Low compared to tdTomato^High (Tom^High). Data are presented as mean±sem. dCT: delta cycle threshold. ***: p<0.001. d) Representative PD-L1 immunofluorescence staining on Tom^Low and Tom^High cytospin cells. Scale bar: 50 μm. e) Representative flow cytometry analysis of PD-L1^Pos population in Tom^Low and Tom^High. f) Representative PD-L1 immunofluorescence staining on the lung sections. Scale bar: 10 μm.

FIGURE 7

Identification of AT2 PD-L1^Pos cells in the lungs of wild-type mice. a) Representative flow cytometry analysis of wild-type mice lungs shows the gating strategy of EPCAM^Pos CD49f^Inter population, followed by negative selection of AT2 cells with the exclusion of AT1 PDPN^Pos cells. The third plot is a representative flow cytometry analysis of AT2 cells based on the use of the PD-L1 marker. b–d) Quantitative PCR analysis of fluorescence-activated cell sorting (FACS)-based sorted PD-L1^Pos and PD-L1^Neg cells for expression of Fgfr2b (b), Etv5 (c) and Sftpc (d) (n=4). Data are presented as mean±sem. **: p<0.01; ***: p<0.001; ****: p<0.0001. e) Representative flow cytometry analysis of SFTPC and PD-L1 co-staining. FSC: forward scatter; dCT: delta cycle threshold.

FIGURE 8

Identification of PD-L1^Pos cells in human AT2 single-cell RNA-sequencing (scRNA-seq) data set. a) UMAP plot of the initial AT2 cell cluster indicates the presence of five different AT2 sub-clusters. b) UMAP and c) violin plots showing PD-L1 enrichment in the AT2-PD-L1^High sub-cluster. d) UMAP and violin plots showing that the AT2-PD-L1^High sub-cluster displays low levels of ETV5, SFTPC and AXIN2 and high levels of TM4SF1. Green arrows indicate AT2 PD-L1^High and lavender arrows indicate AT2-proliferative. e) Model for tdTomato^Low (Tom^Low) and tdTomato^High (Tom^High) cells in homeostasis and after pneumonectomy/injury. Tom^Low cells are quiescent in the adult lung during homeostasis but are activated after injury. They acquire proliferative capabilities and differentiate into mature AT2 cells.